3D-Bioprinting and Decellularization

**69**

**Chapter 5**

*and Falguni Pati*

**Abstract**

human welfare.

**1. Introduction**

extracellular matrix (dECM), dECM bioink

Tissue-Specific Bioink from

Bioprinting of Tissue Constructs

*Sriya Yeleswarapu, Shibu Chameettachal, Ashis Kumar Bera* 

3D bioprinting brings new aspirations to the tissue engineering and regenerative medicine research community. However, despite its huge potential, its growth towards translation is severely impeded due to lack of suitable materials, technological barrier, and appropriate validation models. Recently, the use of decellularized extracellular matrices (dECM) from animal sources is gaining attention as printable bioink as it can provide a microenvironment close to the native tissue. Hence, it is worth exploring the use of xenogeneic dECM and its translation potential for human application. However, extensive studies on immunogenicity, safety-related issues, and animal welfare-related ethics are yet to be streamlined. In addition, the regulatory concerns need to be addressed with utmost priority in order to expedite the use of xenogeneic dECM bioink for 3D bioprinted implantable tissues for

**Keywords:** 3D bioprinting, xenogeneic tissues and organs, xenogeneic decellularized

The field of tissue engineering centers on development of tissues that are capable to regenerate and has a capacity to restore the damaged organs both structurally and functionally [1, 2]. Scaffolds that are developed to serve this purpose should be able to provide cell attachment sites and allow cell proliferation and migration while maintaining its structural and mechanical integrity [2]. Along with this, the placement and uniform distribution of cells in the scaffold play a major role to determine its functional efficiency [3]. This precise positioning of multiple cell types in an organized manner can be achieved with 3D bioprinting [4]. Plenty of natural materials, such as gelatin [5, 6], alginate [7–9], collagen [10, 11], and synthetic materials like polycaprolactone (PCL) [12–16] and polyethylene glycol (PEG) [17–22], come in handy while printing a structure. Although the abovementioned natural materials are biocompatible, disadvantages such as mechanical instability, limited degradability, restricted cell proliferation, and differentiation challenged researchers to investigate more on natural materials [23–25]. As a result, human organ/tissue specific extracellular matrix (ECM) emerged as a best source to develop a functional tissue in laboratory conditions [23, 26, 27]. Yet, the major

Xenogeneic Sources for 3D

#### **Chapter 5**

## Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs

*Sriya Yeleswarapu, Shibu Chameettachal, Ashis Kumar Bera and Falguni Pati*

#### **Abstract**

3D bioprinting brings new aspirations to the tissue engineering and regenerative medicine research community. However, despite its huge potential, its growth towards translation is severely impeded due to lack of suitable materials, technological barrier, and appropriate validation models. Recently, the use of decellularized extracellular matrices (dECM) from animal sources is gaining attention as printable bioink as it can provide a microenvironment close to the native tissue. Hence, it is worth exploring the use of xenogeneic dECM and its translation potential for human application. However, extensive studies on immunogenicity, safety-related issues, and animal welfare-related ethics are yet to be streamlined. In addition, the regulatory concerns need to be addressed with utmost priority in order to expedite the use of xenogeneic dECM bioink for 3D bioprinted implantable tissues for human welfare.

**Keywords:** 3D bioprinting, xenogeneic tissues and organs, xenogeneic decellularized extracellular matrix (dECM), dECM bioink

#### **1. Introduction**

The field of tissue engineering centers on development of tissues that are capable to regenerate and has a capacity to restore the damaged organs both structurally and functionally [1, 2]. Scaffolds that are developed to serve this purpose should be able to provide cell attachment sites and allow cell proliferation and migration while maintaining its structural and mechanical integrity [2]. Along with this, the placement and uniform distribution of cells in the scaffold play a major role to determine its functional efficiency [3]. This precise positioning of multiple cell types in an organized manner can be achieved with 3D bioprinting [4]. Plenty of natural materials, such as gelatin [5, 6], alginate [7–9], collagen [10, 11], and synthetic materials like polycaprolactone (PCL) [12–16] and polyethylene glycol (PEG) [17–22], come in handy while printing a structure. Although the abovementioned natural materials are biocompatible, disadvantages such as mechanical instability, limited degradability, restricted cell proliferation, and differentiation challenged researchers to investigate more on natural materials [23–25]. As a result, human organ/tissue specific extracellular matrix (ECM) emerged as a best source to develop a functional tissue in laboratory conditions [23, 26, 27]. Yet, the major

limitation for this best material is its availability [28–30]. The next alternative source of ECM is to use from other species that are anatomically, physiologically, and metabolically similar to the recipient such as nonhuman primates (like apes, monkeys, and porcine) [31–33]. However, due to the risk of infections from nonhuman primates to human patients and organs from apes, baboons are abandoned, and hence pig became a suitable candidate as an organ donor for humans [33]. There is growing interest of xenogeneic ECM material as printable bioink (biomaterial formulation used for bioprinting) in the field of bioprinting due to easy access and the availability in required quantity. A process termed decellularization allows maximum removal of cellular content while retaining the ECM components from the native animal tissue to reduce the chance of immune rejection when implanted in the patient [29]. The first ever reported *in vivo* study of decellularized tissue was reported in 1991 by Krejci et al. [34], where human decellularized skin was used in mouse model. In 1995, Badylak's group used decellularized xenogeneic small intestinal submucosa for Achilles tendon repair [35]. Later, a number of decellularized ECM (dECM)-based devices are introduced, e.g., human dermis, porcine urinary bladder, porcine small intestine submucosa, and porcine heart valves [36] (**Figure 1**; for details refer to **Table 1**). In the recent past, there are several preliminary reports demonstrating the use of animal-derived dECM in the form of bioinks for developing functional tissues [27, 37]. Not only high cellular viability, these dECM-based constructs also showed enhanced differentiation and proliferation of cells into specific cell types when embedded in tissue-specific ECM [23, 27, 38]. Apart from the need to develop a fully functional construct, the foremost reason for not implanting these structures into human beings is due to high risk of xenotoxicity. Other species, being the source of material for the scaffold that has to be transplanted into human, have to undergo several stringent laws and clear all the clinical trials and ethical concerns. In this book chapter, discussion on the status of xeno-sourced dECM-based bioprinting, including the few reported preclinical studies, is included. The processing steps for dECM preparation and associated

#### **Figure 1.**

*An upright triangle representing number of decellularied xeno-transplants that are being tested at various stages viz in vitro lab experiments, animal and human trials.*

**71**

*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs*

synoviocyte, human adipose derived stem cells

derived stem cells, rat adipose derived stem cells

stem cells

cells

derived mesenchymal stem cells

hepatocyte

human fetal liver cells-hepatocytes, stellate cells

cardiocytes, rat aortic endothelial

pluripotent stem cell-derived multipotential cardiovascular progenitor cells

Stromal cells derived from bone marrow of adult male mice

murine hematopoietic support endothelial cell line

cells

Liver Balb/c Mice

**Source Tissue Cell types Recipient Result Reference**

*In vitro* culture

Rat myocardial infarction model

*In vitro*  culture

*In vitro*, culture and *in vivo* porcine model

*In vitro* culture

*In vitro* culture

*In-vitro*, culture and *in vivo* porcine model

*In vitro* culture

*In vitro* culture

*In vitro* culture

*In vitro*, culture and *in vivo* Yorkshire porcine

• Production od synovial fluid with hyaluronic

• Stem cells expressed endothelial marker • Increased vascular formation in the myocardial tissue

• Clinically relevant vascularized bioengineered liver

• Maturation of hepatic like tissue

• *In vitro* maturation of liver with albumin secretion, urea synthesis and cytochrome P450 expression

• *In vitro* maturation of liver with albumin secretion, normal metabolic parameter

• Increasing of left and right ventricular

• Contraction after 8 days of *In vitro* culture

• Engineered heart tissues exhibited spontaneous contractions, generated mechanical

• Matrix from decellularized fibrotic lungs support prolonged growth of cells • Decellularized lungs that are diseased can significantly affect the cell growth and differentiation

• Unseeded implanted scaffolds sustained blood pressure, renal ultrastructure maintained

pressure

forces • Drug responsive

• Myocardial maturation [128]

[126]

[127]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136]

acid

*DOI: http://dx.doi.org/10.5772/intechopen.89695*

Porcine Pericardium Human sheath

Porcine Myocardium Porcine adipose

Porcine Myocardium Human embryonic

Porcine Liver Rat endothelial

Rat Liver Adult rat

Porcine Liver Second trimester

Rat Heart Rat neonatal

Mice Heart Human induced

Mice Lungs Mesenchymal

Porcine Kidney Immortalized

Balb/c Mice


*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs DOI: http://dx.doi.org/10.5772/intechopen.89695*

*Xenotransplantation - Comprehensive Study*

limitation for this best material is its availability [28–30]. The next alternative source of ECM is to use from other species that are anatomically, physiologically, and metabolically similar to the recipient such as nonhuman primates (like apes, monkeys, and porcine) [31–33]. However, due to the risk of infections from nonhuman primates to human patients and organs from apes, baboons are abandoned, and hence pig became a suitable candidate as an organ donor for humans [33]. There is growing interest of xenogeneic ECM material as printable bioink (biomaterial formulation used for bioprinting) in the field of bioprinting due to easy access and the availability in required quantity. A process termed decellularization allows maximum removal of cellular content while retaining the ECM components from the native animal tissue to reduce the chance of immune rejection when implanted in the patient [29]. The first ever reported *in vivo* study of decellularized tissue was reported in 1991 by Krejci et al. [34], where human decellularized skin was used in mouse model. In 1995, Badylak's group used decellularized xenogeneic small intestinal submucosa for Achilles tendon repair [35]. Later, a number of decellularized ECM (dECM)-based devices are introduced, e.g., human dermis, porcine urinary bladder, porcine small intestine submucosa, and porcine heart valves [36] (**Figure 1**; for details refer to **Table 1**). In the recent past, there are several preliminary reports demonstrating the use of animal-derived dECM in the form of bioinks for developing functional tissues [27, 37]. Not only high cellular viability, these dECM-based constructs also showed enhanced differentiation and proliferation of cells into specific cell types when embedded in tissue-specific ECM [23, 27, 38]. Apart from the need to develop a fully functional construct, the foremost reason for not implanting these structures into human beings is due to high risk of xenotoxicity. Other species, being the source of material for the scaffold that has to be transplanted into human, have to undergo several stringent laws and clear all the clinical trials and ethical concerns. In this book chapter, discussion on the status of xeno-sourced dECM-based bioprinting, including the few reported preclinical studies, is included. The processing steps for dECM preparation and associated

*An upright triangle representing number of decellularied xeno-transplants that are being tested at various* 

*stages viz in vitro lab experiments, animal and human trials.*

**70**

**Figure 1.**


**73**

tory restrictions are also discussed.

**2. Immunogenicity against dECM**

*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs*

Acellular patch Acute

Acellular scaffolds Human

**Source Tissue Cell types Recipient Result Reference**

myocardial infarction rat model

*In-vivo* rodent model

study 4 male children

study, 9 patients

Human study

• Firm attachment and integration with the infarcted region • Neovascularization within 1 week, contraction of left ventricle wall and cardiac functional parameters improved significantly

• Intact liver capsule with porous acellular lattice structure with cell supportive behaviour • No immunogenicity observed

• 3 Children died of graft rupture • Severe inflammation • Significant calcific deposits

• No cell repopulation of porcine matrix

• Acute and chronic transmural inflammation • Graft failure with aneurysmal dilation and thrombosis in complex arteriovenous

conduits

airway

• Immediate functional

• No immunogenic reaction

[146]

[147]

[56]

[148]

[149]

benefits in terms of immuno-compatibility, possible immunological reactions during xenotransplantation, importance of xenografts, ethical concerns, and regula-

Xenotransplantation may be the best way to alleviate the burden of allograft organ shortage from the last decade. The most enormous barrier to xenotransplantation is the immunological rejection which de-emphasizes this technique. The profound immunological rejection happens by both antibody-mediated immune response as well as cell-mediated innate or adaptive immune response. Several carbohydrate antigens have been identified that could act as targets for human natural

*DOI: http://dx.doi.org/10.5772/intechopen.89695*

Porcine Liver Hepatoblastoma

(SynergraftTM)

Bovine Ureter graft Acellular scaffolds Human

Trachea Patient epithelial

and MSC derived chondrocyte

*Various decellularized xeno derived organs that are used in in vitro, animal and human studies.*

(HepG2)

Porcine Myocardium Slice

Porcine Heart valves

Human (allograft)

**Table 1.**


*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs DOI: http://dx.doi.org/10.5772/intechopen.89695*

#### **Table 1.**

*Xenotransplantation - Comprehensive Study*

Porcine Kidney *Mice* embryonic

Porcine Pancreas Human amniotic

Rats Spinal cord Acellular scaffolds

Porcine Brain iPSC derived

Porcine Skin Human dermal

Porcine Cornea Rabbit corneal

Porcine Cornea and limbus

Female ICR mice stem cells

cells

Pancreas Acinar AR42J and

lines

fluid derived stem

beta MIN-6β cell

neural progenitor cells (NPCs)

fibroblasts

keratocytes, epithelial, endothelial

Acellular scaffolds *In vivo*

Porcine Cornea Acellular Cornea *In-vitro*

for in-vivo, NIH3T3 cells for in-vitro studies

**Source Tissue Cell types Recipient Result Reference**

*In vitro* culture

*In vitro* culture

*In-vitro* culture and *in vivo* Spraguedawley Rats

*In-vitro*, culture and *in vivo* mice model

*In-vitro* culture

rabbits

*In vitro* culture

rabbit model

• Reseeded scaffold showed HGF and VEGF levels similar to native kidney

• Acellular pancreas supported stem cell and pancreatic islets

growth • Could serve as a platform for bioengineering pancreas to treat diabetes mellitus

*Mice* model • Strong up-regulation of insulin gene

(*in vivo*)

• Induce the regeneration of injured nerves

• Enhanced adhesion and proliferation of cells (*in vitro*)

• NPC expressed neural markers in brain matrix gel (*in vitro*), • Formation and assembling of larger microscale fibril like structure in gel

(*in-vivo*)

• In-vivo good biocompatibility, • Translucent cornea within 8 weeks • Implants integrated into rabbit cornea without rejection

signs

vimentin

• Gene ontology showed skin morphogenesis, epidermis development

• Epithelial cells showed high expressions of CK3, spindle shape keratocytes displayed

• Corneal transparency and epithelial integrity with no graft rejection • Basal epithelial cell matured to limbal epithelial cells

[137]

[138]

[139]

[140]

[141]

[142]

[143]

[144]

[145]

**72**

*Various decellularized xeno derived organs that are used in in vitro, animal and human studies.*

benefits in terms of immuno-compatibility, possible immunological reactions during xenotransplantation, importance of xenografts, ethical concerns, and regulatory restrictions are also discussed.

#### **2. Immunogenicity against dECM**

Xenotransplantation may be the best way to alleviate the burden of allograft organ shortage from the last decade. The most enormous barrier to xenotransplantation is the immunological rejection which de-emphasizes this technique. The profound immunological rejection happens by both antibody-mediated immune response as well as cell-mediated innate or adaptive immune response. Several carbohydrate antigens have been identified that could act as targets for human natural antibodies to inhibit immune rejection; these include Galα1-3Galβ1-4GlcNAc (referred to as α1,3Gal), Hanganuziu-Deicher (H-D) antigen, Tn, Forssman antigen, Sda antigen, etc. [39, 40]. Two antibody-mediated processes are hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR), which attack mainly the vascular system of graft tissue. HAR is mediated by natural antibodies against α-1,3Gal epitope, present in vascular endothelium of mammals except for humans, or their most recent ancestors, the Old World monkeys [31, 41]. α-1,3Gal epitope is expressed in other organisms, because of increased human interaction with these animals; anti α1,3Gal is being developed in human sera. When it binds to its antigen determinant site of anti α1, 3Gal, it activates the complement system and coagulation system to reject the graft within minutes to hours. HAR is histologically characterized by the presence of interstitial hemorrhage edema and thrombosis in small blood vessels. The depletion of α1,3Gal antibody or complement inhibition may be the best strategies to prevent HAR. But early attempts to reduce antibody by injecting a competitive antagonist of α1,3Gal antigen were unsuccessful [42] because AHXR can reject graft with a very low concentration of α1,3Gal antibody after several days or weeks. On the other hand, non-alpha Gal antigens Hanganuziu-Deicher (H-D) antigen and Sda antigen are present in vascular endothelium and on the surface of erythrocyte of all mammals except humans. The antibody against these H-D and Sda antigens is responsible for HAR and AHXR reaction via activation of complement (classical pathway) and coagulation system in α1,3Gal transferase gene knockout (GalT-KO) pigs [40, 43, 44]. The complement can also be activated via alternative pathway by islets transplantation and cause instant blood-mediated inflammatory reaction (IBMIR), resulting in an early rejection of transplanted islets [45]. The most successful approach to prevent antibody-mediated xenograft rejection is (i) transgenic pigs that express human complement regulatory protein that inhibits antibody-mediated complement activation [46] and (ii) pigs with a knockout α1,3Gal transferase gene [47, 48]. The elimination of α1,3Gal epitope extended the survival of xenograft to 2–6 months [43]. On the other hand, combination of both strategies at a time has increased the graft survival. Recently significant prolongation of graft survival was documented more than 900 days in a pig-to-baboon cardiac xenograft from α1,3Gal transferase knockout, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM) [49, 50]. The strength of cellular rejection of xenotransplantation remains uncertain, because of difficulty in avoiding HAR and AHXR.

Xenografts are more prone to rejection when compared to allografts due to the antibodies produced by T-cells dependent activated B-cells. Inclusion of T-cell suppressive treatment significantly prolonged the survival rate (>400 days) of xenograft, where natural antibody-mediated immune rejection was suppressed [49–51]. The initial immune reaction by HAR and AHXR produced pathogen-associated molecular patterns (PAMPs) which activate the innate immune system, such as NK cells, macrophages, and neutrophils. Overcoming these barriers needs severe and sustained exposure to immune-suppressive drugs, which is very much harmful to host tissue.

All biologists are focusing on cells and intracellular contents and their regulation to escape from immune reaction, but the scenario has changed after Hauschka and Konigsberg's work in 1966 [52]. It was reported that only the ECM can differentiate myoblast to myotube formation. As the ECM has inbuilt tissue-specific matrix composition and topological cues, it may be an ideal scaffold for the use in tissue engineering. Both antibody-mediated and innate immune responses trigger by the specific receptor present on their respective target cells and inflammatory molecules like TNF, IFN, and different cytokines released upon activation of specific

**75**

*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs*

cells. Decellularization is the best strategies to evade immune reaction by removing cells as well as receptors present on their surface membrane. Unfortunately, the implantation of decellularized allograft into a human produced the mixed type of result of compatibility and recipient immune response. In spite of all the hurdles, some early clinical success of ECM scaffold was achieved [53, 54], but a low level of immune reaction was identified by some group. The heart and lung xenotransplantation working group in the National Heart, Lung, and Blood Institute (NHLBI) has identified xenogeneic immune response against ECM to be a major problem to use in clinical medicine [55]. Cryopreserved human allografts are extensively used in cardiac valve reconstruction; immunologic response of these allografts has been investigated by several groups to activate the anti-HLA antibody. Hawkins et al. reported that HLA class I and II antigens reduced by 99% in the decellularized human allograft, and postoperation reactive antibody levels of HLA class I or II did not increase in children up to 12 months [56]. The inhibition of the immunomodulatory effect of decellularized tissue is obtained mainly by the removal of predominantly alpha-gal epitope along with other non-gal antigen in vascular endothelium and by removal of MHC class I and II molecules during decellularization. Although the donor-derived MHC class I became undetectable at the time of decellularization, it again reached measurable value following implantation (host-derived MHC class I) and is vascularized with host tissue [57, 58]. The underlying mechanism of decellularization on host immune response remains to be determined. Due to low or zero levels of MHC class I and II, T-cell proliferative response as well as B-cell activation is inhibited, and the anti-inflammatory effect can be seen *in vitro*, which results in the reduction of IL-2 and IFN-γ as well. As there is no MHC class antigen-presenting receptor, T cell does not recognize the foreign antigen, and T-cell-mediated immune response is suppressed. But the elevation of IL-10 fails to conclude the underlying mechanism because it has the only source from activated T cell, B cell, and macrophages [58]. It is reported that M2 phenotype in the graft prevents rejection of the xenogeneic donor tissue; however, the mechanism of macrophage activation to release IL-10 remains unknown. Till now, it is not well understood which protein and in which way decellularized xenogeneic material promotes immune reaction. The decellularized tissue may expose new protein, and the decellularization protocol may also have a significant impact on the response of human mononuclear cells [59]. Rieder et al. [60] reported that decellularized vascular wall elicited more immune cell proliferation than native equivalent, and hence, it proved the above hypothesis. It also hypothesized that opsonization would be the way of inflammation response and can occur through preformed antibody or binding of unspecific plasma protein to the surface. In genetically modified organism, (pig) alpha-gal epitope is knocked out, and it does not elicit immune response in decellularized tissue, but in unmodified xenogeneic tissue, some amount of alphagal antigen may be retained, and that could be enough to stimulate immunogenic response. However, further study is needed to find out the mechanism of immune

*DOI: http://dx.doi.org/10.5772/intechopen.89695*

response with regard to decellularized matrices.

**2.1 Strategies to resolve immune reaction against xenogeneic DECM**

Xenogeneic dECM has a huge potential to be used in tissue engineering and regenerative medicine; some early enthusiastic studies in animal and clinical trials using decellularized tissues resulted in severe inflammatory reaction, fibrous overgrowth, and tissue destruction [61–64]. Despite all these immunological reactions, in recent years xenogeneic biomaterials are being used in abdominal surgery [65–67]. There have been some early studies, where glutaraldehyde cross-linking in native matrix inhibits immune response by the modification of surface area of tissues

#### *Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs DOI: http://dx.doi.org/10.5772/intechopen.89695*

cells. Decellularization is the best strategies to evade immune reaction by removing cells as well as receptors present on their surface membrane. Unfortunately, the implantation of decellularized allograft into a human produced the mixed type of result of compatibility and recipient immune response. In spite of all the hurdles, some early clinical success of ECM scaffold was achieved [53, 54], but a low level of immune reaction was identified by some group. The heart and lung xenotransplantation working group in the National Heart, Lung, and Blood Institute (NHLBI) has identified xenogeneic immune response against ECM to be a major problem to use in clinical medicine [55]. Cryopreserved human allografts are extensively used in cardiac valve reconstruction; immunologic response of these allografts has been investigated by several groups to activate the anti-HLA antibody. Hawkins et al. reported that HLA class I and II antigens reduced by 99% in the decellularized human allograft, and postoperation reactive antibody levels of HLA class I or II did not increase in children up to 12 months [56]. The inhibition of the immunomodulatory effect of decellularized tissue is obtained mainly by the removal of predominantly alpha-gal epitope along with other non-gal antigen in vascular endothelium and by removal of MHC class I and II molecules during decellularization. Although the donor-derived MHC class I became undetectable at the time of decellularization, it again reached measurable value following implantation (host-derived MHC class I) and is vascularized with host tissue [57, 58]. The underlying mechanism of decellularization on host immune response remains to be determined. Due to low or zero levels of MHC class I and II, T-cell proliferative response as well as B-cell activation is inhibited, and the anti-inflammatory effect can be seen *in vitro*, which results in the reduction of IL-2 and IFN-γ as well. As there is no MHC class antigen-presenting receptor, T cell does not recognize the foreign antigen, and T-cell-mediated immune response is suppressed. But the elevation of IL-10 fails to conclude the underlying mechanism because it has the only source from activated T cell, B cell, and macrophages [58]. It is reported that M2 phenotype in the graft prevents rejection of the xenogeneic donor tissue; however, the mechanism of macrophage activation to release IL-10 remains unknown. Till now, it is not well understood which protein and in which way decellularized xenogeneic material promotes immune reaction. The decellularized tissue may expose new protein, and the decellularization protocol may also have a significant impact on the response of human mononuclear cells [59]. Rieder et al. [60] reported that decellularized vascular wall elicited more immune cell proliferation than native equivalent, and hence, it proved the above hypothesis. It also hypothesized that opsonization would be the way of inflammation response and can occur through preformed antibody or binding of unspecific plasma protein to the surface. In genetically modified organism, (pig) alpha-gal epitope is knocked out, and it does not elicit immune response in decellularized tissue, but in unmodified xenogeneic tissue, some amount of alphagal antigen may be retained, and that could be enough to stimulate immunogenic response. However, further study is needed to find out the mechanism of immune response with regard to decellularized matrices.

#### **2.1 Strategies to resolve immune reaction against xenogeneic DECM**

Xenogeneic dECM has a huge potential to be used in tissue engineering and regenerative medicine; some early enthusiastic studies in animal and clinical trials using decellularized tissues resulted in severe inflammatory reaction, fibrous overgrowth, and tissue destruction [61–64]. Despite all these immunological reactions, in recent years xenogeneic biomaterials are being used in abdominal surgery [65–67]. There have been some early studies, where glutaraldehyde cross-linking in native matrix inhibits immune response by the modification of surface area of tissues

*Xenotransplantation - Comprehensive Study*

antibodies to inhibit immune rejection; these include Galα1-3Galβ1-4GlcNAc (referred to as α1,3Gal), Hanganuziu-Deicher (H-D) antigen, Tn, Forssman antigen, Sda antigen, etc. [39, 40]. Two antibody-mediated processes are hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR), which attack mainly the vascular system of graft tissue. HAR is mediated by natural antibodies against α-1,3Gal epitope, present in vascular endothelium of mammals except for humans, or their most recent ancestors, the Old World monkeys [31, 41]. α-1,3Gal epitope is expressed in other organisms, because of increased human interaction with these animals; anti α1,3Gal is being developed in human sera. When it binds to its antigen determinant site of anti α1, 3Gal, it activates the complement system and coagulation system to reject the graft within minutes to hours. HAR is histologically characterized by the presence of interstitial hemorrhage edema and thrombosis in small blood vessels. The depletion of α1,3Gal antibody or complement inhibition may be the best strategies to prevent HAR. But early attempts to reduce antibody by injecting a competitive antagonist of α1,3Gal antigen were unsuccessful [42] because AHXR can reject graft with a very low concentration of α1,3Gal antibody after several days or weeks. On the other hand, non-alpha Gal antigens Hanganuziu-Deicher (H-D) antigen and Sda antigen are present in vascular endothelium and on the surface of erythrocyte of all mammals except humans. The antibody against these H-D and Sda antigens is responsible for HAR and AHXR reaction via activation of complement (classical pathway) and coagulation system in α1,3Gal transferase gene knockout (GalT-KO) pigs [40, 43, 44]. The complement can also be activated via alternative pathway by islets transplantation and cause instant blood-mediated inflammatory reaction (IBMIR), resulting in an early rejection of transplanted islets [45]. The most successful approach to prevent antibody-mediated xenograft rejection is (i) transgenic pigs that express human complement regulatory protein that inhibits antibody-mediated complement activation [46] and (ii) pigs with a knockout α1,3Gal transferase gene [47, 48]. The elimination of α1,3Gal epitope extended the survival of xenograft to 2–6 months [43]. On the other hand, combination of both strategies at a time has increased the graft survival. Recently significant prolongation of graft survival was documented more than 900 days in a pig-to-baboon cardiac xenograft from α1,3Gal transferase knockout, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM) [49, 50]. The strength of cellular rejection of xenotransplantation remains uncertain, because of difficulty in avoiding HAR

Xenografts are more prone to rejection when compared to allografts due to the antibodies produced by T-cells dependent activated B-cells. Inclusion of T-cell suppressive treatment significantly prolonged the survival rate (>400 days) of xenograft, where natural antibody-mediated immune rejection was suppressed [49–51]. The initial immune reaction by HAR and AHXR produced pathogen-associated molecular patterns (PAMPs) which activate the innate immune system, such as NK cells, macrophages, and neutrophils. Overcoming these barriers needs severe and sustained exposure to immune-suppressive drugs, which is very much harmful to

All biologists are focusing on cells and intracellular contents and their regulation to escape from immune reaction, but the scenario has changed after Hauschka and Konigsberg's work in 1966 [52]. It was reported that only the ECM can differentiate myoblast to myotube formation. As the ECM has inbuilt tissue-specific matrix composition and topological cues, it may be an ideal scaffold for the use in tissue engineering. Both antibody-mediated and innate immune responses trigger by the specific receptor present on their respective target cells and inflammatory molecules like TNF, IFN, and different cytokines released upon activation of specific

**74**

and AHXR.

host tissue.

that inhibit the interaction with peripheral blood mononuclear cell (PBMC) and in turn T-cell activation [68]. But the problem of glutaraldehyde fixation is that it can change the tissues' topology and promote their degradation by calcification [69]. The natural cross-linking product quercetin, a plant flavonoid pigment, may be more effective, which increases mechanical strength and reduces immunogenicity [70].

#### **3. Importance of xenografts in dECM-based bioprinting**

Organs in the human body are extremely complex structures consisting of multiple cell types arranged in defined spatial organization, with varied ECM composition. It is due to this balanced and organized compositions that organs achieve perfect functionality [71]. Any disruption to this native structure alters the functionality of the organ drastically. The demand for organ transplantation is increasing exponentially due to the rise in traumatic injuries and changes in lifestyle, while the supply of organs increased marginally over time. The demand for organ transplantation is estimated to further rise with the advancements in diagnostics leading to early detection of diseases [72]. Researchers all over the world have been striving hard to find alternative strategies to reduce this gap for many years, using a combination of many materials along with cells [73]. As a result, researchers developed comparatively simple organs using tissue engineering approaches, such as artificial skin [74], cartilage [75], and trachea [76] that display a part or nearly full functionality of the particular tissue. Xenotransplantation is another promising approach that was started in early 1920s and has a potential to serve as a temporary measure to save patient's life in the absence of allogenic organ [77]. Nevertheless, the barriers such as graft failure due to immune reaction [63] and infections from the graft to the patient prevent the acceptance of xenotransplantation as a treatment option. Consequently, an emerging technique, 3D bioprinting, revolutionized the field of tissue engineering and regenerative medicine exhibiting its potential to develop complicated organs [78]. To fabricate a scaffold, this technique uses materials that are biocompatible and cells that are tissue-specific, while the best biomaterial to develop a tissue that eventually goes to human body is the material derived from that specific tissue, viz., ECM, as it can provide reseeded cells with local tissue environment [23]. This property of tissue-derived material can anchor cells and provides sufficient biochemical and mechanical cues allowing them to proliferate and differentiate to those tissue-specific lineages which ultimately aid in complex tissue formation [79, 80]. Ideally, autologous tissues are expected not to illicit an immune response after implantation, thus reducing the chance of organ rejection. However, due to the lack of sufficient autologous tissue, allogeneic tissues are chosen for transplantation. Allogeneic tissues also suffer from rejection from the host due to antibody-mediated rejection or T-cell movement into the allograft [81]. Genetic dissimilarity between donor and recipient turns out to be the main cause to induce immune response and eventually rejection of the graft [81]. Hence, the process of decellularization when applied on allogeneic tissues reduces the amount of genetic material, thereby allowing graft survival in the host [82]. But, the final yield of material after all the processing of tissue is very low and is insufficient for printing a higher volume 3D structure. Because of which, considering patient's own tissue or tissue from the same species for development of bioink is not practical. The very next alternative that researchers explored was to obtain tissue source from other species and use its matrix as a bioink for tissue development [23]. The concept of using other species (porcine) tissue as a source of material for humans emerged due to the anatomical and physiological similarities between both the species [83, 84]. Apart from the cellular content, organs are rich in the noncellular component, i.e.,

**77**

*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs*

ECM [85]. In almost all the tissues, ECM proteins are produced by the resident cells [85, 86]. Many macrolevel molecules, growth factors, and fibrillar proteins in varied quantities constitute this considerable volume of the tissue [85]. Polysaccharides and proteins such as glycosaminoglycans (GAGs), hyaluronan, collagen, fibronectin, laminin, and elastin are the major ECM components in an organ [85]. These ECM components allow cell adhesion and cell migration, provide biochemical and mechanical properties, and impart elasticity that helps cells to obtain morphological orientation and physiological functionalities. Of all the ECM components mentioned, collagen is the most abundant protein which almost covers 30% of the protein content present in multicellular organisms [85, 86]. In vertebrates, as many as 28 different types of collagen are recognized with 46 distinct polypeptide chains, and the sources of collagen are abundantly available from marine animals to animals that live on land [87]. The main role of this profoundly available protein is to provide mechanical strength, maintain cellular adhesion, and support migration and other cellular functionalities that direct mature tissue formation [85]. To develop tissues like bone [88], skin substitutes [89], small intestine tissue [90], skeletal muscle tissue [91], collagen that is extracted from xenogeneic sources has been used extensively in research works. Elastin is another ECM component that connects with collagen to provide elasticity to the tissue. It is due to this close association; elastic nature of tissue is being maintained. To develop constructs *in vitro*, along with the exposing cells to abundant proteins, enough mechanical properties are to be provided [85]. Hence, it is necessary to include elastin components into the engineered scaffold which imparts mechanical properties to the tissue. By combining the proteins, viz., different types of collagen and elastin, a reasonable amount of work has been done on blood vessel engineering, heart valve development, tissue-engineered vascular grafts, musculoskeletal tissues, cartilage, and skin engineering [92]. The other fibrous protein that contributes for organization of ECM and is responsible for cell functionality such as cellular attachment is fibronectin. Scaffolds that are functionalized with fibronectin enhanced properties such as cell adhesion [93, 94], promoting elastin deposition [95]; cellular migration responsible for tumor metastasis [96, 97] has been reported in literature. When it comes to engineering a tissue *in vitro* using 3D bioprinting, the material should be biocompatible as well as print friendly. Components of ECM such as collagen, elastin, and fibrin were explored for them to be used as bioinks either separately or in combination with one another in 3D bioprinting technology. The potential of collagen as bioink was displayed for developing human skin model with keratinocytes and fibroblasts [98], cartilage tissue engineering [99], 3D collagen-based cell blocks that exhibited

osteogenic activity [100], and osteochondral mimicking structures [101] and in bone regeneration applications [102]. The use of fibrinogen as a bioink was also reported for developing cartilage [103, 104] and vascular grafts [105]. The immune response to xenogeneic collagen in human models was reported to be not adverse, and in most of the cases, the presence of antibodies for xeno-derived collagen was due to byproducts during acceptance of implanted graft by host [106]. It is also reported *in vitro* experiments conducted with collagen and elastin derived from porcine and bovine did not trigger immune cells nor trigger proliferation of isolated B and T cells [107]. Nonetheless, to mimic native tissue environment for enhanced cellular functionality, a combination of all the proteins and macromolecules is required. Hence, instead of using all the macromolecules separately in varied amounts, researchers started using ECM of the tissue for tissue engineering and 3D bioprinting applications (**Figure 2**), thereby providing all the necessary cues to the reseeded cells in essential amounts. For better acceptance of the 3D printed structure with ECM, decellularization of animal tissue is done to remove the maximum cellular content prior to 3D printing process. This reduces the chances of xenogeneic

*DOI: http://dx.doi.org/10.5772/intechopen.89695*

#### *Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs DOI: http://dx.doi.org/10.5772/intechopen.89695*

ECM [85]. In almost all the tissues, ECM proteins are produced by the resident cells [85, 86]. Many macrolevel molecules, growth factors, and fibrillar proteins in varied quantities constitute this considerable volume of the tissue [85]. Polysaccharides and proteins such as glycosaminoglycans (GAGs), hyaluronan, collagen, fibronectin, laminin, and elastin are the major ECM components in an organ [85]. These ECM components allow cell adhesion and cell migration, provide biochemical and mechanical properties, and impart elasticity that helps cells to obtain morphological orientation and physiological functionalities. Of all the ECM components mentioned, collagen is the most abundant protein which almost covers 30% of the protein content present in multicellular organisms [85, 86]. In vertebrates, as many as 28 different types of collagen are recognized with 46 distinct polypeptide chains, and the sources of collagen are abundantly available from marine animals to animals that live on land [87]. The main role of this profoundly available protein is to provide mechanical strength, maintain cellular adhesion, and support migration and other cellular functionalities that direct mature tissue formation [85]. To develop tissues like bone [88], skin substitutes [89], small intestine tissue [90], skeletal muscle tissue [91], collagen that is extracted from xenogeneic sources has been used extensively in research works. Elastin is another ECM component that connects with collagen to provide elasticity to the tissue. It is due to this close association; elastic nature of tissue is being maintained. To develop constructs *in vitro*, along with the exposing cells to abundant proteins, enough mechanical properties are to be provided [85]. Hence, it is necessary to include elastin components into the engineered scaffold which imparts mechanical properties to the tissue. By combining the proteins, viz., different types of collagen and elastin, a reasonable amount of work has been done on blood vessel engineering, heart valve development, tissue-engineered vascular grafts, musculoskeletal tissues, cartilage, and skin engineering [92]. The other fibrous protein that contributes for organization of ECM and is responsible for cell functionality such as cellular attachment is fibronectin. Scaffolds that are functionalized with fibronectin enhanced properties such as cell adhesion [93, 94], promoting elastin deposition [95]; cellular migration responsible for tumor metastasis [96, 97] has been reported in literature. When it comes to engineering a tissue *in vitro* using 3D bioprinting, the material should be biocompatible as well as print friendly. Components of ECM such as collagen, elastin, and fibrin were explored for them to be used as bioinks either separately or in combination with one another in 3D bioprinting technology. The potential of collagen as bioink was displayed for developing human skin model with keratinocytes and fibroblasts [98], cartilage tissue engineering [99], 3D collagen-based cell blocks that exhibited osteogenic activity [100], and osteochondral mimicking structures [101] and in bone regeneration applications [102]. The use of fibrinogen as a bioink was also reported for developing cartilage [103, 104] and vascular grafts [105]. The immune response to xenogeneic collagen in human models was reported to be not adverse, and in most of the cases, the presence of antibodies for xeno-derived collagen was due to byproducts during acceptance of implanted graft by host [106]. It is also reported *in vitro* experiments conducted with collagen and elastin derived from porcine and bovine did not trigger immune cells nor trigger proliferation of isolated B and T cells [107]. Nonetheless, to mimic native tissue environment for enhanced cellular functionality, a combination of all the proteins and macromolecules is required. Hence, instead of using all the macromolecules separately in varied amounts, researchers started using ECM of the tissue for tissue engineering and 3D bioprinting applications (**Figure 2**), thereby providing all the necessary cues to the reseeded cells in essential amounts. For better acceptance of the 3D printed structure with ECM, decellularization of animal tissue is done to remove the maximum cellular content prior to 3D printing process. This reduces the chances of xenogeneic

*Xenotransplantation - Comprehensive Study*

that inhibit the interaction with peripheral blood mononuclear cell (PBMC) and in turn T-cell activation [68]. But the problem of glutaraldehyde fixation is that it can change the tissues' topology and promote their degradation by calcification [69]. The natural cross-linking product quercetin, a plant flavonoid pigment, may be more effective, which increases mechanical strength and reduces immunogenicity [70].

Organs in the human body are extremely complex structures consisting of multiple cell types arranged in defined spatial organization, with varied ECM composition. It is due to this balanced and organized compositions that organs achieve perfect functionality [71]. Any disruption to this native structure alters the functionality of the organ drastically. The demand for organ transplantation is increasing exponentially due to the rise in traumatic injuries and changes in lifestyle, while the supply of organs increased marginally over time. The demand for organ transplantation is estimated to further rise with the advancements in diagnostics leading to early detection of diseases [72]. Researchers all over the world have been striving hard to find alternative strategies to reduce this gap for many years, using a combination of many materials along with cells [73]. As a result, researchers developed comparatively simple organs using tissue engineering approaches, such as artificial skin [74], cartilage [75], and trachea [76] that display a part or nearly full functionality of the particular tissue. Xenotransplantation is another promising approach that was started in early 1920s and has a potential to serve as a temporary measure to save patient's life in the absence of allogenic organ [77]. Nevertheless, the barriers such as graft failure due to immune reaction [63] and infections from the graft to the patient prevent the acceptance of xenotransplantation as a treatment option. Consequently, an emerging technique, 3D bioprinting, revolutionized the field of tissue engineering and regenerative medicine exhibiting its potential to develop complicated organs [78]. To fabricate a scaffold, this technique uses materials that are biocompatible and cells that are tissue-specific, while the best biomaterial to develop a tissue that eventually goes to human body is the material derived from that specific tissue, viz., ECM, as it can provide reseeded cells with local tissue environment [23]. This property of tissue-derived material can anchor cells and provides sufficient biochemical and mechanical cues allowing them to proliferate and differentiate to those tissue-specific lineages which ultimately aid in complex tissue formation [79, 80]. Ideally, autologous tissues are expected not to illicit an immune response after implantation, thus reducing the chance of organ rejection. However, due to the lack of sufficient autologous tissue, allogeneic tissues are chosen for transplantation. Allogeneic tissues also suffer from rejection from the host due to antibody-mediated rejection or T-cell movement into the allograft [81]. Genetic dissimilarity between donor and recipient turns out to be the main cause to induce immune response and eventually rejection of the graft [81]. Hence, the process of decellularization when applied on allogeneic tissues reduces the amount of genetic material, thereby allowing graft survival in the host [82]. But, the final yield of material after all the processing of tissue is very low and is insufficient for printing a higher volume 3D structure. Because of which, considering patient's own tissue or tissue from the same species for development of bioink is not practical. The very next alternative that researchers explored was to obtain tissue source from other species and use its matrix as a bioink for tissue development [23]. The concept of using other species (porcine) tissue as a source of material for humans emerged due to the anatomical and physiological similarities between both the species [83, 84]. Apart from the cellular content, organs are rich in the noncellular component, i.e.,

**3. Importance of xenografts in dECM-based bioprinting**

**76**

#### **Figure 2.**

*Schematic representing the process of 3D bioprinting, in vitro maturation and transplantation of tissues developed from animal tissue derived decellularized extracellular matrix.*

rejection in human body. In the next section, the use of dECM as a bioink for 3D printing applications is discussed.

#### **4. Current status of the xenografts application in bioprinting**

The process of decellularization dates to 2000s, wherein organs such as skin, vascular tissue, and bladder were decellularized. In 2014, it was first shown that after decellularization process, the ECM that is devoid of cellular material could be used as a bioink for 3D printing applications [23]. In the recent past, almost all the organs have been subjected to the process of decellularization and used for 3D bioprinting. With 3D bioprinting of decellularized organs such as the heart, liver, cartilage, adipose tissue, skeletal muscle, skin, etc., researchers have demonstrated the potential of dECM-based constructs in terms of cell compatibility, cell

**79**

*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs*

attachment, migration, and proliferation. Decellularized heart matrix derived from porcine showed an enhanced expression of myosin heavy chain [23] and expression of transcription factors by cardiac progenitor cells [108]. The functionality of 3D engineered heart, developed from decellularized rat heart, was also demonstrated in one study [109]. Similarly, decellularized liver matrix from porcine exhibited consistent secretion of urea and albumin up to 14 days of culture [110] and higher levels of markers suggesting hepatocyte maturation [27]**.** Early adipogenic marker and lipoprotein lipase were notably observed in human-derived decellularized adipose tissue [23]**.** However, there is a need of further *in vitro* experiments on decellularized matrices, to completely replicate the complex geometry of the organs. With the current state of art, the *in vitro* models can be tested for immune response in animal models. For any biological material that is being implanted should contain as less as 50 ng/mg of DNA content for not eliciting the immune response in host body. To ensure this low level of nucleic acid content, the process of decellularization of xeno tissues must be stringent and harsh. Detergents such as SDS and Triton X served as chemical agents to remove the maximum DNA content from tissues in decellularization process. Using chemical treatment, acceptable level of DNA content was achieved in almost all the tissues decellularized so far. Apart from DNA nuclear material, Gal epitopes present in animals are also found to be responsible for acute implant rejection [23]. There are few reports from literature wherein 3D dECM scaffolds have been implanted in animal models to understand the host response. In one study, scaffolds that were fabricated using decellularized adipose tissue derived from porcine were implanted into mice. Due to significant reduction in DNA content and gal epitopes, the ECM grafts showed no signs of inflammation or necrosis. Also, there was formation of neo-adipose tissue with mature adipocytes supporting adipogenesis and acceptance of a xenograft [111]. Porcine-derived skin was also subjected to decellularization to show its potential in skin tissue engineering. Using chemical such as trypsin/EDTA and Triton X, the decellularized skin matrix was digested to form bioink, and a skin substitute was printed. This, when implanted into the wound of 10 mm in mice, accelerated wound healing was observed when compared to control groups. Further, immunofluorescence staining showed early differentiation markers for epithelial tissue and CD-31 signifying re-epithelialization and vascularization, respectively [112]. The reported results exhibit the acceptance of xeno-derived dECM-based 3D bioprinted scaffolds by the host tissue. This is made possible due to the stringent chemicals and enzymes involved in decellularization process. Nevertheless, much more studies and experiments both *in vitro* and *in vivo* must be done for using these scaffolds as

*DOI: http://dx.doi.org/10.5772/intechopen.89695*

replacement of deceased parts in the human.

related issues, and (3) animal welfare issues [113].

**5. Regulatory facets of xeno dECM-based tissue transplantation**

Although the prospective benefits are unquestionable, the use of xenogeneic products in human health care raises a number of issues; hence it has to be controlled strictly by the regulatory bodies to avoid complications. The duty of regulatory bodies is to regulate the indiscriminate use of animal-sourced material intended for human health application. The challenges include (1) the potential risk of transmission of infectious agents from source animals, (2) informed consent

From the preclinical testing, the regulations are made strict for the human welfare before use in clinical trials. In general, enough studies have to be performed for safety characterization of therapeutic agents including the efficacy or the activity and the toxicity or undesired effects to the host system. This type of potential clinical

#### *Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs DOI: http://dx.doi.org/10.5772/intechopen.89695*

attachment, migration, and proliferation. Decellularized heart matrix derived from porcine showed an enhanced expression of myosin heavy chain [23] and expression of transcription factors by cardiac progenitor cells [108]. The functionality of 3D engineered heart, developed from decellularized rat heart, was also demonstrated in one study [109]. Similarly, decellularized liver matrix from porcine exhibited consistent secretion of urea and albumin up to 14 days of culture [110] and higher levels of markers suggesting hepatocyte maturation [27]**.** Early adipogenic marker and lipoprotein lipase were notably observed in human-derived decellularized adipose tissue [23]**.** However, there is a need of further *in vitro* experiments on decellularized matrices, to completely replicate the complex geometry of the organs. With the current state of art, the *in vitro* models can be tested for immune response in animal models. For any biological material that is being implanted should contain as less as 50 ng/mg of DNA content for not eliciting the immune response in host body. To ensure this low level of nucleic acid content, the process of decellularization of xeno tissues must be stringent and harsh. Detergents such as SDS and Triton X served as chemical agents to remove the maximum DNA content from tissues in decellularization process. Using chemical treatment, acceptable level of DNA content was achieved in almost all the tissues decellularized so far. Apart from DNA nuclear material, Gal epitopes present in animals are also found to be responsible for acute implant rejection [23]. There are few reports from literature wherein 3D dECM scaffolds have been implanted in animal models to understand the host response. In one study, scaffolds that were fabricated using decellularized adipose tissue derived from porcine were implanted into mice. Due to significant reduction in DNA content and gal epitopes, the ECM grafts showed no signs of inflammation or necrosis. Also, there was formation of neo-adipose tissue with mature adipocytes supporting adipogenesis and acceptance of a xenograft [111]. Porcine-derived skin was also subjected to decellularization to show its potential in skin tissue engineering. Using chemical such as trypsin/EDTA and Triton X, the decellularized skin matrix was digested to form bioink, and a skin substitute was printed. This, when implanted into the wound of 10 mm in mice, accelerated wound healing was observed when compared to control groups. Further, immunofluorescence staining showed early differentiation markers for epithelial tissue and CD-31 signifying re-epithelialization and vascularization, respectively [112]. The reported results exhibit the acceptance of xeno-derived dECM-based 3D bioprinted scaffolds by the host tissue. This is made possible due to the stringent chemicals and enzymes involved in decellularization process. Nevertheless, much more studies and experiments both *in vitro* and *in vivo* must be done for using these scaffolds as replacement of deceased parts in the human.

#### **5. Regulatory facets of xeno dECM-based tissue transplantation**

Although the prospective benefits are unquestionable, the use of xenogeneic products in human health care raises a number of issues; hence it has to be controlled strictly by the regulatory bodies to avoid complications. The duty of regulatory bodies is to regulate the indiscriminate use of animal-sourced material intended for human health application. The challenges include (1) the potential risk of transmission of infectious agents from source animals, (2) informed consent related issues, and (3) animal welfare issues [113].

From the preclinical testing, the regulations are made strict for the human welfare before use in clinical trials. In general, enough studies have to be performed for safety characterization of therapeutic agents including the efficacy or the activity and the toxicity or undesired effects to the host system. This type of potential clinical

*Xenotransplantation - Comprehensive Study*

rejection in human body. In the next section, the use of dECM as a bioink for 3D

*Schematic representing the process of 3D bioprinting, in vitro maturation and transplantation of tissues* 

The process of decellularization dates to 2000s, wherein organs such as skin, vascular tissue, and bladder were decellularized. In 2014, it was first shown that after decellularization process, the ECM that is devoid of cellular material could be used as a bioink for 3D printing applications [23]. In the recent past, almost all the organs have been subjected to the process of decellularization and used for 3D bioprinting. With 3D bioprinting of decellularized organs such as the heart, liver, cartilage, adipose tissue, skeletal muscle, skin, etc., researchers have demonstrated the potential of dECM-based constructs in terms of cell compatibility, cell

**4. Current status of the xenografts application in bioprinting**

*developed from animal tissue derived decellularized extracellular matrix.*

printing applications is discussed.

**78**

**Figure 2.**

risks constitutes an important component of an FDA regulation. Transfer of animal microorganisms to the recipient with the graft during xenograft transplantation is another major concern for regulatory authorities [114]. There are reports that HIV, hepatitis B and C, Creutzfeldt-Jakob disease, and rabies can be transmitted between humans during transplantation. It is also proved that contact between animals and humans during animal husbandry and from pets or food products can lead to zoonotic infections. So, the use of animal cells, tissues, and organs in any forms keeps the public health at risk with known and unknown infections. Hence it is advised to go for thorough screening for all kind of possible zoonotic infections by following the standard protocol [113]**.** Moreover, the risk of these microorganisms or virus getting adapted to human-to-human transmission is also a major factor that has to be considered, which might be a concern for general population [115]. When it comes to cross-species whole organ transplantation, there is unavoidable transfer of endogenous retrovirus that is existing in the genome of all porcine cells into the patient receiving the organ. However, there exists no documentation regarding the transfer of these viruses in humans who are exposed to pig organs [116], probably due to the lack of long-term observation.

Preclinical studies provide valuable insight into the safety issues before being used in the human volunteers. Animal welfare is a major concern during the application of xenogeneic products in humans. Since animals' welfare is a major ethical issue, it is considered by regulatory bodies before approving any product of animal origin for clinical use.

Also, during the clinical trial stages or in long term, the volunteers or the patients and the close contacts should be educated about the chance of infectious disease risks and about how to manage those risks. Moreover, such counseling should also be continued for long term as some infection may take years to get manifested. Also, lifelong surveillance is advised by FDA irrespective of the status of the implant or graft or other xenotransplantation product.

Conversely, 3D bioprinted *in vitro* organs and tissues that are being developed using dECM are expected not to pose potential threat to recipients. This is because the cell and nucleus materials are being removed from the tissue using harsh chemicals during the process of decellularization. However, the regulatory bodies ensure that xenotransplantation is allowed only when there are evidences that show nearzero chance of recipient getting infected and informed consent, and acceptance for lifelong postoperative care from the patient was collected [116]. Nevertheless, stringent regulations will be required from regulatory bodies to monitor the pros and cons for a longer duration.

#### **6. Ethical and safety concerns**

There are numerous challenges and hurdles being faced for translating xenogeneic products to the clinical level. Though the potential of tissue- or organ-derived bioink for 3D bioprinting is getting proved and accepted, to reach human level it must overcome ethical concerns apart from dealing with technological and regulatory challenges. The opinions expressed on ethics behind using xeno-derived material for humans are based on the source of material and the consequence after transplants, which are already mentioned in the regulatory facets [117]. There are few groups who argue that the primary idea of using animal organ into human is unethical, while few claiming that the detrimental outcomes after the transplant are unacceptable [118]. The apprehension on the outcomes of the xenotransplantation seems valid as there are reports in the literature suggesting that patients who received the animal organs survived only for a short span [77]. The use of animal

**81**

**7. Future perspective**

*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs*

animal organs for transplantation to serve humans is yet to be understood.

We believe that the severity of some disease conditions will be able to justify the use of xenogeneic therapeutic options, but the risk and benefits must be evaluated and concluded at the earliest. The most important concern, infectious disease transmission, including the chance of latent viral infections, must be studied in a larger picture including all possible disease transmissions. Though studies are limited, severe immunological reactions are not reported by using decellularized bioinks till date indicating its future potential in regenerating organs and tissue. Large

organ in patient started in the twentieth century. Organs such as liver, heart from baboon [119], and kidney from chimpanzee [120] were transplanted to patients who survived for a very short lifespan ranging from 20 to 195 days after the implantation [77]. Immune rejection is the primary reason for failure of the graft [77]. Apart from immune response from the host, there are insufficient scientific evidences about the risk of transmission of pathogens that are passive in animal species [117]. Though it is proven that these microorganisms that are existing in animal species are not harming them, it could be fatal when they enter other species [117]. It is ethical to have an informed consent from the patient, not only regarding the transplantation but also about all the further complications that could arise due to the foreign material being placed inside [117, 121]. With xeno-organ transplantation, the risk of animal virus and microorganisms entering human body is expected to rise [121]. Apart from this, there are a lot many unknown viruses that are hosted by animal species whose effects are not at all predictable [117]. Hence, the recipient should also be informed about the risks and preventions that he/she must take posttransplantation, restricting his freedom [121, 122]. Further, to increase the success rate of transplants, recipients are constantly under the influence of immunosuppressant drugs, which would enhance his chances of other infections [117]. However, immunological reactions are not reported much after using dECM 3D bioprinted constructs. Additionally, one has to justify whether the amount that is being spent on xenotransplantation research for translation to clinical level is really worth, as it can help a relatively smaller group of people. Furthermore, for animal welfare, there are animal-related ethical issues which are considered important similar to human ethical issues [123]. Some groups believe that, the use of animals to fulfill human needs is strongly unethical, while few accept that if the benefits surpass the degree of suffering of animals, then there is no harm to use animal organs for saving human life [124]. Almost all the vertebrates suffer and perceive pain in a similar way [121]. Producing transgenic animals for organ transplantation also received criticism, as during this process, much more pain and suffering is imposed on animals due to multiple experiments in succession. In order to reduce the chance of viral transmissions, these transgenic animals are quarantined and kept in isolation [121]. Hence, the supports for animal welfare argue that the animals that undergo genetic engineering technique will be deprived of its natural habitat and are forced to live in a secluded place with pain and agony [117]. Will this suffering of an animal be the guarantee that its organ is successfully put into use remains as an unanswered question. Apart from ethics, religious feelings also come into play. A pig that is considered to have similar genetic and physiological traits similar to human [125] is considered unclean in many religions but is considered as a versatile model in biomedical research. On the other hand, if the benefits and safety of xenotransplantation is proven for human well-being, dealing with animal ethics could be vindicated. Nevertheless, how well the community approves and agrees to the use of transgenic

*DOI: http://dx.doi.org/10.5772/intechopen.89695*

#### *Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs DOI: http://dx.doi.org/10.5772/intechopen.89695*

organ in patient started in the twentieth century. Organs such as liver, heart from baboon [119], and kidney from chimpanzee [120] were transplanted to patients who survived for a very short lifespan ranging from 20 to 195 days after the implantation [77]. Immune rejection is the primary reason for failure of the graft [77]. Apart from immune response from the host, there are insufficient scientific evidences about the risk of transmission of pathogens that are passive in animal species [117]. Though it is proven that these microorganisms that are existing in animal species are not harming them, it could be fatal when they enter other species [117]. It is ethical to have an informed consent from the patient, not only regarding the transplantation but also about all the further complications that could arise due to the foreign material being placed inside [117, 121]. With xeno-organ transplantation, the risk of animal virus and microorganisms entering human body is expected to rise [121]. Apart from this, there are a lot many unknown viruses that are hosted by animal species whose effects are not at all predictable [117]. Hence, the recipient should also be informed about the risks and preventions that he/she must take posttransplantation, restricting his freedom [121, 122]. Further, to increase the success rate of transplants, recipients are constantly under the influence of immunosuppressant drugs, which would enhance his chances of other infections [117]. However, immunological reactions are not reported much after using dECM 3D bioprinted constructs. Additionally, one has to justify whether the amount that is being spent on xenotransplantation research for translation to clinical level is really worth, as it can help a relatively smaller group of people. Furthermore, for animal welfare, there are animal-related ethical issues which are considered important similar to human ethical issues [123]. Some groups believe that, the use of animals to fulfill human needs is strongly unethical, while few accept that if the benefits surpass the degree of suffering of animals, then there is no harm to use animal organs for saving human life [124]. Almost all the vertebrates suffer and perceive pain in a similar way [121]. Producing transgenic animals for organ transplantation also received criticism, as during this process, much more pain and suffering is imposed on animals due to multiple experiments in succession. In order to reduce the chance of viral transmissions, these transgenic animals are quarantined and kept in isolation [121]. Hence, the supports for animal welfare argue that the animals that undergo genetic engineering technique will be deprived of its natural habitat and are forced to live in a secluded place with pain and agony [117]. Will this suffering of an animal be the guarantee that its organ is successfully put into use remains as an unanswered question. Apart from ethics, religious feelings also come into play. A pig that is considered to have similar genetic and physiological traits similar to human [125] is considered unclean in many religions but is considered as a versatile model in biomedical research. On the other hand, if the benefits and safety of xenotransplantation is proven for human well-being, dealing with animal ethics could be vindicated. Nevertheless, how well the community approves and agrees to the use of transgenic animal organs for transplantation to serve humans is yet to be understood.

#### **7. Future perspective**

We believe that the severity of some disease conditions will be able to justify the use of xenogeneic therapeutic options, but the risk and benefits must be evaluated and concluded at the earliest. The most important concern, infectious disease transmission, including the chance of latent viral infections, must be studied in a larger picture including all possible disease transmissions. Though studies are limited, severe immunological reactions are not reported by using decellularized bioinks till date indicating its future potential in regenerating organs and tissue. Large

*Xenotransplantation - Comprehensive Study*

lack of long-term observation.

and cons for a longer duration.

**6. Ethical and safety concerns**

origin for clinical use.

risks constitutes an important component of an FDA regulation. Transfer of animal microorganisms to the recipient with the graft during xenograft transplantation is another major concern for regulatory authorities [114]. There are reports that HIV, hepatitis B and C, Creutzfeldt-Jakob disease, and rabies can be transmitted between humans during transplantation. It is also proved that contact between animals and humans during animal husbandry and from pets or food products can lead to zoonotic infections. So, the use of animal cells, tissues, and organs in any forms keeps the public health at risk with known and unknown infections. Hence it is advised to go for thorough screening for all kind of possible zoonotic infections by following the standard protocol [113]**.** Moreover, the risk of these microorganisms or virus getting adapted to human-to-human transmission is also a major factor that has to be considered, which might be a concern for general population [115]. When it comes to cross-species whole organ transplantation, there is unavoidable transfer of endogenous retrovirus that is existing in the genome of all porcine cells into the patient receiving the organ. However, there exists no documentation regarding the transfer of these viruses in humans who are exposed to pig organs [116], probably due to the

Preclinical studies provide valuable insight into the safety issues before being used in the human volunteers. Animal welfare is a major concern during the application of xenogeneic products in humans. Since animals' welfare is a major ethical issue, it is considered by regulatory bodies before approving any product of animal

Also, during the clinical trial stages or in long term, the volunteers or the patients and the close contacts should be educated about the chance of infectious disease risks and about how to manage those risks. Moreover, such counseling should also be continued for long term as some infection may take years to get manifested. Also, lifelong surveillance is advised by FDA irrespective of the status

Conversely, 3D bioprinted *in vitro* organs and tissues that are being developed using dECM are expected not to pose potential threat to recipients. This is because the cell and nucleus materials are being removed from the tissue using harsh chemicals during the process of decellularization. However, the regulatory bodies ensure that xenotransplantation is allowed only when there are evidences that show nearzero chance of recipient getting infected and informed consent, and acceptance for lifelong postoperative care from the patient was collected [116]. Nevertheless, stringent regulations will be required from regulatory bodies to monitor the pros

There are numerous challenges and hurdles being faced for translating xenogeneic products to the clinical level. Though the potential of tissue- or organ-derived bioink for 3D bioprinting is getting proved and accepted, to reach human level it must overcome ethical concerns apart from dealing with technological and regulatory challenges. The opinions expressed on ethics behind using xeno-derived material for humans are based on the source of material and the consequence after transplants, which are already mentioned in the regulatory facets [117]. There are few groups who argue that the primary idea of using animal organ into human is unethical, while few claiming that the detrimental outcomes after the transplant are unacceptable [118]. The apprehension on the outcomes of the xenotransplantation seems valid as there are reports in the literature suggesting that patients who received the animal organs survived only for a short span [77]. The use of animal

of the implant or graft or other xenotransplantation product.

**80**

population studies are required to rule out the possibilities of rejection. A welldefined animal source is also required as species close to humans are not preferred. The animal husbandry conditions must be defined and should start dedicated farms isolated from other animals and be monitored regularly to avoid unexpected or non-listed diseases. Moreover, an unquestionable monitoring system for animal welfare conditions is also important during the raise in the use of xeno-products in human.

### **8. Conclusion**

The tissue-derived decellularized extracellular matrice bioink is the latest trend in the field of 3D bioprinting. The 3D bioprinted constructs from xenogeneic dECM are yet to be studied and analyzed extensively. However, the immune response to xenogeneic collagen, the major dECM-derived bioink component, in human models is not induced by any complicated immune reactions in the host. Though studies are in progress, the 3D bioprinted constructs with xenogeneic dECM bioink are least studied for safety and efficacy despite immune reactivity studies. The animal welfare-related issue is untouched. The initial studies using xenogeneic decellularized matrices are tempting; therefore it is worth to speculate that 3D bioprinting with xenogeneic dECM can revolutionize the field of regenerative medicine.

### **Acknowledgements**

This work is financially supported by the Early Career Research (ECR) grant (ECR/2015/000458), awarded by the Science and Engineering Research Board, Department of Science and Technology, Government of India and the Ramalingaswami Fellowship (BT/RLF/Re-entry/07/2015), awarded by the Department of Biotechnology, Government of India.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Sriya Yeleswarapu, Shibu Chameettachal, Ashis Kumar Bera and Falguni Pati\* Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Sangareddy, Telangana, India

\*Address all correspondence to: falguni@iith.ac.in

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**83**

10.1038/srep13427

*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs*

[8] You F, Wu X, Zhu N, Lei M, Eames BF, Chen X. 3D printing of porous cell-laden hydrogel constructs

for potential applications in cartilage tissue engineering. ACS Biomaterials Science & Engineering. 2016;**2**:1200-1210. DOI: 10.1021/

acsbiomaterials.6b00258

[9] Xiong R, Zhang Z, Chai W, Huang Y, Chrisey DB. Freeform dropon-demand laser printing of 3D alginate and cellular constructs. Biofabrication. 2015;**7**(4):045011. DOI:

10.1088/1758-5090/7/4/045011

[10] Yoon H, Lee J, Yim H, Kim G, Chun W. Development of cell-laden 3D scaffolds for efficient engineered skin substitutes by collagen gelation. RSC Advances. 2016;**6**(26):21439-21447

[11] Lee W, Debasitis J, Lee V, Lee J, Fischer K, Edminster K. Multi-Layered Culture of Human Skin Fibroblasts and Keratinocytes through Three-Dimensional Freeform Fabrication. Biomaterials. 2009. DOI: 10.1016/j.

[12] Daly AC, Cunniffe GM, Sathy BN, Jeon O, Alsberg E, Kelly DJ. 3D bioprinting of developmentally

inspired templates for whole bone organ engineering. Advanced Healthcare Materials. 2016;**5**:2353-2362. DOI:

[13] Kim YB, Lee H, Yang GH, Choi CH, Lee DW, Hwang H, et al. Mechanically reinforced cell-laden scaffolds formed using alginate-based bioink printed onto the surface of a PCL/alginate mesh structure for regeneration of hard tissue. Journal of Colloid and Interface Science. 2016;**461**:359-368. DOI: 10.1016/j.jcis.2015.09.044

[14] Merceron TK, Burt M, Seol YJ, Kang HW, Lee SJ, Yoo JJ, et al. A 3D bioprinted complex structure for

biomaterials.2008.12.009

10.1002/adhm.201600182

*DOI: http://dx.doi.org/10.5772/intechopen.89695*

[1] Mandrycky C, Phong K,

mrc.2017.58

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s41598-018-33245-w

ma11112199

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#### **References**

*Xenotransplantation - Comprehensive Study*

human.

**8. Conclusion**

**Acknowledgements**

**Conflict of interest**

population studies are required to rule out the possibilities of rejection. A welldefined animal source is also required as species close to humans are not preferred. The animal husbandry conditions must be defined and should start dedicated farms isolated from other animals and be monitored regularly to avoid unexpected or non-listed diseases. Moreover, an unquestionable monitoring system for animal welfare conditions is also important during the raise in the use of xeno-products in

The tissue-derived decellularized extracellular matrice bioink is the latest trend in the field of 3D bioprinting. The 3D bioprinted constructs from xenogeneic dECM are yet to be studied and analyzed extensively. However, the immune response to xenogeneic collagen, the major dECM-derived bioink component, in human models is not induced by any complicated immune reactions in the host. Though studies are in progress, the 3D bioprinted constructs with xenogeneic dECM bioink are least studied for safety and efficacy despite immune reactivity studies. The animal welfare-related issue is untouched. The initial studies using xenogeneic decellularized matrices are tempting; therefore it is worth to speculate that 3D bioprinting with xenogeneic dECM can revolutionize the field of regenerative medicine.

**82**

**Author details**

Sangareddy, Telangana, India

\*Address all correspondence to: falguni@iith.ac.in

provided the original work is properly cited.

Department of Biotechnology, Government of India.

The authors declare no conflict of interest.

Sriya Yeleswarapu, Shibu Chameettachal, Ashis Kumar Bera and Falguni Pati\* Department of Biomedical Engineering, Indian Institute of Technology Hyderabad,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

This work is financially supported by the Early Career Research (ECR) grant (ECR/2015/000458), awarded by the Science and Engineering Research Board, Department of Science and Technology, Government of India and the Ramalingaswami Fellowship (BT/RLF/Re-entry/07/2015), awarded by the

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[7] Ahn S, Lee H, Lee J, Yoon H, Chun W, Kim GH. A novel cell-printing method and its application to hepatogenic differentiation of human adipose stem cell-embedded mesh structures. Scientific Reports. 2015;**5**:13427. DOI: 10.1038/srep13427

[8] You F, Wu X, Zhu N, Lei M, Eames BF, Chen X. 3D printing of porous cell-laden hydrogel constructs for potential applications in cartilage tissue engineering. ACS Biomaterials Science & Engineering. 2016;**2**:1200-1210. DOI: 10.1021/ acsbiomaterials.6b00258

[9] Xiong R, Zhang Z, Chai W, Huang Y, Chrisey DB. Freeform dropon-demand laser printing of 3D alginate and cellular constructs. Biofabrication. 2015;**7**(4):045011. DOI: 10.1088/1758-5090/7/4/045011

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[12] Daly AC, Cunniffe GM, Sathy BN, Jeon O, Alsberg E, Kelly DJ. 3D bioprinting of developmentally inspired templates for whole bone organ engineering. Advanced Healthcare Materials. 2016;**5**:2353-2362. DOI: 10.1002/adhm.201600182

[13] Kim YB, Lee H, Yang GH, Choi CH, Lee DW, Hwang H, et al. Mechanically reinforced cell-laden scaffolds formed using alginate-based bioink printed onto the surface of a PCL/alginate mesh structure for regeneration of hard tissue. Journal of Colloid and Interface Science. 2016;**461**:359-368. DOI: 10.1016/j.jcis.2015.09.044

[14] Merceron TK, Burt M, Seol YJ, Kang HW, Lee SJ, Yoo JJ, et al. A 3D bioprinted complex structure for

engineering the muscle-tendon unit. Biofabrication. 2015;**7**(3):035003. DOI: 10.1088/1758-5090/7/3/035003

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[19] Elomaa L, Pan C, Shanjani Y, Malkovskiy A, Seppälä JV, Yang Y. Three-dimensional fabrication of cellladen biodegradable poly(ethylene glycol-co-depsipeptide) hydrogels by visible light stereolithography. Journal of Materials Chemistry B. 2015;**3**(42):8348-8358. DOI: 10.1039/ c5tb01468a

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[26] Kim B, Kim H, Gao G, Jang J. Decellularized extracellular matrix: A step towards the next generation source for bioink manufacturing. Biofabrication. 2017;**9**(3):034104. DOI: 10.1088/1758-5090/aa7e98

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[34] Krejci N, Cuono C, Langdon RC, McGuire J. In vitro reconstitution of skin: Fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis. Journal of Investigative

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2014;**32**(2):462-484. DOI: 10.1016/j.

Xenotransplantation: Current status and a perspective on the future. Nature Reviews Immunology. 2007;**7**(7):519.

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*Tissue-Specific Bioink from Xenogeneic Sources for 3D Bioprinting of Tissue Constructs DOI: http://dx.doi.org/10.5772/intechopen.89695*

bioink for three-dimensional cell printing-based liver tissue engineering. Biomacromolecules. 2017;**18**:1229-1237. DOI: 10.1021/acs.biomac.6b01908

*Xenotransplantation - Comprehensive Study*

engineering the muscle-tendon unit. Biofabrication. 2015;**7**(3):035003. DOI: 2015;**10**:1568-1577. DOI: 10.1002/

[21] Maiullari F, Costantini M, Milan M, Pace V, Chirivì M, Maiullari S, et al. A multi-cellular 3D bioprinting approach for vascularized heart tissue engineering based on HUVECs and iPSC-derived cardiomyocytes. Scientific Reports. 2018;**8**(1):12532. DOI: 10.1038/

[22] Liu J, He J, Liu J, Ma X, Chen Q, Lawrence N, et al. Rapid 3D bioprinting

of in vitro cardiac tissue models using human embryonic stem cellderived cardiomyocytes. Bioprinting. 2019;**13**:e00040. DOI: 10.1016/j.

[23] Pati F, Jang J, Ha D, Sw K,

Phie JW, Kim DH, et al. Printing threedimensional tissue analogues with decellularized extracellular matrix bioink. Nature Communications. 2014;**5**:3935. DOI: 10.1038/ncomms4935

[24] Wu Z, Su X, Xu Y, Kong B, Sun W, Mi S. Bioprinting three-dimensional cell-laden tissue constructs with controllable degradation. Scientific Reports. 2016;**6**:42274. DOI: 10.1038/

[25] Lee HJ, Kim YB, Ahn SH, Lee JS, Jang CH, Yoon H, et al. A new approach for fabricating collagen/ECM-based bioinks using Preosteoblasts and human adipose stem cells. Advanced Healthcare Materials. 2015;**4**:1359-1368. DOI:

10.1002/adhm.201500193

10.1088/1758-5090/aa7e98

[26] Kim B, Kim H, Gao G, Jang J. Decellularized extracellular matrix: A step towards the next generation source for bioink manufacturing. Biofabrication. 2017;**9**(3):034104. DOI:

[27] Lee H, Han W, Kim H, Ha DH, Jang J, Kim BS, et al. Development of liver decellularized extracellular matrix

biot.201400635

s41598-018-31848-x

bprint.2019.e00040

srep24474

10.1088/1758-5090/7/3/035003

[15] Lee JW, Choi Y-J, Yong W-J, Pati F, Shim J-H, Kang KS, et al. Development of a 3D cell printed construct considering angiogenesis

for liver tissue engineering. Biofabrication. 2016;**8**:15007. DOI: 10.1088/1758-5090/8/1/015007

[16] Kang H, Lee S, Ko I, Kengla C, Yoo JJ, Atala A. A 3D bioprinting system

[17] Das S, Pati F, Choi Y-J, Rijal G, Shim J-H, Kim SW, et al. Bioprintable, cell-laden silk fibroin-gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of threedimensional tissue constructs. Acta Biomaterialia. 2014;**11**:233-246. DOI:

10.1016/j.actbio.2014.09.023

bioprinting technology. Tissue

DOI: 10.1089/ten.tea.2011.0543

[19] Elomaa L, Pan C, Shanjani Y, Malkovskiy A, Seppälä JV, Yang Y. Three-dimensional fabrication of cellladen biodegradable poly(ethylene glycol-co-depsipeptide) hydrogels by visible light stereolithography. Journal of Materials Chemistry B. 2015;**3**(42):8348-8358. DOI: 10.1039/

[20] Gao G, Yonezawa T, Hubbell K, Dai G, Cui X. Inkjet-bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging. Biotechnology Journal.

[18] Cui X, Breitenkamp K, Finn MG, Lotz M, D'Lima DD. Direct human cartilage repair using three-dimensional

Engineering. Part A. 2012;**18**:1304-1312.

to produce human-scale tissue constructs with structural integrity. Nature. 2016;**34**(3):312. DOI: 10.1038/

nbt.3413

**84**

c5tb01468a

[28] Lu H, Hoshiba T, Kawazoe N, Chen G. Autologous extracellular matrix scaffolds for tissue engineering. Biomaterials. 2011;**32**:2489-2499. DOI: 10.1016/j.biomaterials.2010.12.016

[29] Porzionato A, Stocco E, Barbon S, Grandi F, Macchi V, De Caro R. Molecular sciences tissue-engineered grafts from human decellularized extracellular matrices: A systematic review and future perspectives. International Journal of Molecular Sciences. 2018;**19**(12):4117. DOI: 10.3390/ijms19124117

[30] Cheng C, Solorio L, Alsberg E. Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering. Biotechnology Advances. 2014;**32**(2):462-484. DOI: 10.1016/j. biotechadv.2013.12.012

[31] Yang Y, Sykes M.

Xenotransplantation: Current status and a perspective on the future. Nature Reviews Immunology. 2007;**7**(7):519. DOI: 10.1038/nri2099

[32] Gutierrez K, Dicks N, Glanzner WG, Agellon LB, Bordignon V. Efficacy of the porcine species in biomedical research. Frontiers in Genetics. 2015;**6**:293. DOI: 10.3389/fgene.2015.00293

[33] Levy MF. Animal organs for human transplantation: How close are we? Baylor University Medical Center proceedings. 2000;**13**:3-6. DOI: 10.1080/08998280.2000.11927634

[34] Krejci N, Cuono C, Langdon RC, McGuire J. In vitro reconstitution of skin: Fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis. Journal of Investigative Dermatology. 1991;**97**(5):843-848. DOI: 10.1111/1523-1747.ep12491522

[35] Badylak SF, Tullius R, Kokini K, Shelbourne KD, Klootwyk T, Voytik SL, et al. The use of xenogeneic small intestinal submucosa as a biomaterial for Achille's tendon repair in a dog model. Journal of Biomedical Materials Research. 1995;**29**:977-985. DOI: 10.1002/jbm.820290809

[36] Badylak S, Freytes D, Gilbert TW. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomaterialia. 2009;**5**(1):1-3. DOI: 10.1016/j.actbio.2008.09.013

[37] Lin P, Chan WCW, Badylak SF, Bhatia SN. Assessing porcine liverderived biomatrix for hepatic tissue engineering. Tissue Engineering. 2004;**10**:1046-1053. DOI: 10.1089/ ten.2004.10.1046

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[142] Won JY, Lee MH, Kim MJ, Min KH, Ahn G, Han JS, et al. A potential dermal substitute using decellularized dermis extracellular matrix derived bio-ink. Artificial Cells, Nanomedicine, and Biotechnology. 2019;**47**:644-649. DOI: 10.1080/21691401.2019.1575842

[143] Du L, Wu X. Development and characterization of a full-thickness acellular porcine cornea matrix for tissue engineering. Artificial Organs. 2011;**35**:691-705. DOI: 10.1111/j.1525-1594.2010.01174.x

[144] Pang K, Du L, Wu X. A rabbit anterior cornea replacement derived from acellular porcine cornea matrix, epithelial cells and keratocytes. Biomaterials. 2010;**31**(28):7257-7265. DOI: 10.1016/j. biomaterials.2010.05.066

[145] Huang M, Li N, Wu Z, Wan P, Liang X, Zhang W, et al. Using acellular porcine limbal stroma for rabbit limbal stem cell microenvironment reconstruction. Biomaterials. 2011;**32**(31):7812-7821. DOI: 10.1016/j. biomaterials.2011.07.012

[146] Shah M, KC P, Zhang G. In vivo assessment of Decellularized porcine myocardial slice as an Acellular cardiac patch. ACS Applied Materials & Interfaces. 2019;**11**:23893-23900. DOI: 10.1021/acsami.9b06453

[147] Mirmalek-Sani S, Sullivan D, Zimmerman C, Shupe TD, Petersen BE. Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue. The American Journal of Pathology. 2013;**183**(2):558-565. DOI: 10.1016/j.ajpath.2013.05.002

[148] Spark JI, Yeluri S, Derham C, Wong YT, Leitch D. Incomplete cellular depopulation may explain the high failure rate of bovine ureteric grafts. The British Journal of Surgery. 2008;**95**:582- 585. DOI: 10.1002/bjs.6052

[149] Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, et al. Clinical transplantation of a tissue-engineered airway. Lancet. 2008;**372**:2023-2030. DOI: 10.1016/ S0140-6736(08)61598-6

**95**

**Chapter 6**

Liver

**Abstract**

tive processes.

**1. Introduction**

Optimization of a

Decellularization/

*Hongyu Zhang and Lianhua Bai*

Recellularization Strategy for

Transplantable Bioengineered

*Quanyu Chen, Xiaolin You, Jiejuan Lai, Shifang Jiang,* 

The liver is a complex organ that requires constant perfusion for the delivery of nutrients and oxygen and the removal of waste in order to survive. Efforts to recreate or mimic the liver microstructure via a ground-up approach are essential for liver tissue engineering. A decellularization/recellularization strategy is one of the approaches aiming at the possibility of producing a fully functional organ with in vitro-developed construction for clinical applications to replace failed livers, such as end-stage liver disease (ESLD). However, the complexity of the liver microarchitecture along with the limited suitable hepatic component, such as the optimization of the extracellular matrix (ECM) of the biomaterials, the selection of the seed cells, and development of the liver-specific three-dimensional (3D) niche settings, pose numerous challenges. In this chapter, we have provided a comprehensive outlook on how the physiological, pathological, and spatiotemporal aspects of these drawbacks can be turned into the current challenges in the field, and put forward a few techniques with the potential to address these challenges, mainly focusing on a decellularization-based liver regeneration strategy. We hypothesize the primary concepts necessary for constructing tissue-engineered liver organs based on either an intact (from a naïve liver) or a partial (from a pretreated liver) structure via simulating the natural development and regenera-

**Keywords:** tissue engineering, decellularization, recellularization, thrombogenicity,

The liver is the largest internal organ in the human body, accounting for approximately 2–5% of the total body volume [1, 2]. Physiologically, the liver possesses over 500 different functions [3] and any severe damage could be life-threatening, such as that caused by ESLD, including acute liver failure and chronic liver disease.

hemocompatibility, partial hepatectomy transplantation

### **Chapter 6**

## Optimization of a Decellularization/ Recellularization Strategy for Transplantable Bioengineered Liver

*Quanyu Chen, Xiaolin You, Jiejuan Lai, Shifang Jiang, Hongyu Zhang and Lianhua Bai*

### **Abstract**

The liver is a complex organ that requires constant perfusion for the delivery of nutrients and oxygen and the removal of waste in order to survive. Efforts to recreate or mimic the liver microstructure via a ground-up approach are essential for liver tissue engineering. A decellularization/recellularization strategy is one of the approaches aiming at the possibility of producing a fully functional organ with in vitro-developed construction for clinical applications to replace failed livers, such as end-stage liver disease (ESLD). However, the complexity of the liver microarchitecture along with the limited suitable hepatic component, such as the optimization of the extracellular matrix (ECM) of the biomaterials, the selection of the seed cells, and development of the liver-specific three-dimensional (3D) niche settings, pose numerous challenges. In this chapter, we have provided a comprehensive outlook on how the physiological, pathological, and spatiotemporal aspects of these drawbacks can be turned into the current challenges in the field, and put forward a few techniques with the potential to address these challenges, mainly focusing on a decellularization-based liver regeneration strategy. We hypothesize the primary concepts necessary for constructing tissue-engineered liver organs based on either an intact (from a naïve liver) or a partial (from a pretreated liver) structure via simulating the natural development and regenerative processes.

**Keywords:** tissue engineering, decellularization, recellularization, thrombogenicity, hemocompatibility, partial hepatectomy transplantation

#### **1. Introduction**

The liver is the largest internal organ in the human body, accounting for approximately 2–5% of the total body volume [1, 2]. Physiologically, the liver possesses over 500 different functions [3] and any severe damage could be life-threatening, such as that caused by ESLD, including acute liver failure and chronic liver disease.

In modern times, the failure of solid organs, such as ESLD caused by injury or disease, has become a major challenge in clinics [4]. According to the U.S. Centers for Disease Control and Prevention (https://www.cdc.gov/), in 2014, 38,170 people died of ESLD. Currently, orthotopic liver transplant (OLT) is an ideal therapy for ESLD. However, a shortage of liver organ donors severely limits OLT usage. The Department of Health and Human Services in the United States has estimated that (https://optn.transplant.hrsa.gov) 22 people on the National Transplant Waiting List die each day, while one person is added to the waiting list every 10 min. Additionally, people fortunate enough to receive an organ transplantation have to suffer from the lifelong use of immunosuppressants against chronic rejection. Therefore, new technologies are eagerly needed to create a transplantable liver [5]. Tissue engineering is a mixed field that aims to fabricate functional organs in vitro [6]. Over the decades, great progress has been achieved in the laboratory, and even some livers have been used in clinics [7]. Tissue engineering by using a decellularization/recellularization strategy, which maintains the architecture, vascular system, and ECM components, has been shown to be a promising tool for solid organs, such as liver.

Liver tissue engineering by using decellularization/recellularization strategy (**Figure 1**) involves biomimicking the architecture and physiological features of the native liver. The procedure generally needs three major components: a scaffolding platform, seed cells, and a 3D microenvironment. Despite the numerous advances over the years, it is still an enormous challenge to fabricate a liver organ [8]. Generating liver organ-specific 3D structure scaffold to keep as much as original biochemical, physiochemical, and biomechanical ECM microenvironment is the one of the main hurdles in liver engineering field [9]. Such physiological 3D structure also plays a remarkable role in influencing seeded cell long-term survival and complex liver tissue mass formation [10]. To achieve this, scientists have been working with different scaffolding systems for liver tissue engineering. Studies have shown that a construction strategy based on a combination of a decellularized naïve liver matrix and recellularization with seed cells has led to constructs that match human organs in size and structure. However, the present constructs still only fulfill

#### **Figure 1.**

*The decellularization/recellularization strategy in liver tissue engineering. Mammals donor-derived livers undergo a process of decellularization to obtain decellularized liver scaffolds (DLS) (step a–c), and then recellularized seed cells are placed onto the scaffolds (step d–f). Finally, the recellularized scaffolds are placed into 3D culture conditions in a bioreactor to construct liver-like tissues or organs with an overall structure and vasculature (step g).*

**97**

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered...*

partial functions of the liver. The preservation of a functional ECM during decellularization, cellular differentiation [11], and a lack of endothelial-lying vascular networks limits the long-term functional integration of constructs after in vivo transplantation. As techniques continue develop, some methods with the potential to overcome these challenges should be explored in the near future, which will further boost the development of a tissue-engineered liver with improved functions. In this chapter, we have tried to focus on the possibility of liver tissue engineering by using a decellularization/recellularization strategy and to describe the current advancements made in the field to address a possible clinical transplantation.

The term "biomaterials" traditionally means a nonliving substance used for a medical purpose. As the technology of biomaterials developed, the definition expanded to include substances to control the biological environment of cells and tissues for increased compatibility with a host to allow for colonization, proliferation, and differentiation of cells while maintaining their specific morphologies, configurations and avoiding immunological rejection. Based on the increasing knowledge of ECM biology, scaffold biomaterials can be grouped as synthetic materials, natural materials, or a decellularized matrix [12]. Moreover, modifications have been made to enhance the biologically active signals of scaffolds, leading

Biomaterials with required properties have been well studied from synthetic materials. For instance, a nanofibrous matrix made of poly and poly-embedded growth factors was transplanted into animals and restored cardiac regeneration by promoting vascularization [15]. Zawaneh et al. have reported the design of an injectable synthetic and biodegradable polymeric biomaterial consisting of polyethylene glycol and a polycarbonate of dihydroxyacetone that is easily extruded through narrow-gage needles, biodegrades into inert products, and is well tolerated by soft tissues [16]. Those chemically and biologically modified synthetic materials could result in a better way to mimic and control seed cell responses [17, 18]. Another advantage of synthetic materials is their easier to predict and control the degradation of synthetic scaffolds. However, despite this wealth of knowledge, the ability of synthetic biomaterials to support cell attachment, or induce phenotypic expression is much lower than that of natural biomaterials [19–21]; thus, natural

Natural biomaterials include collagen, alginate, and chitosan. These types of biomaterials are inherently able to facilitate for seed cell attachment, proliferation, and functional differentiation, thus they hold significant promise for liver tissue engineering [23, 24]. However, traditional natural materials have poor inherent bioactivity, acidic byproducts, etc., and alone cannot rebuild the complex architecture of solid organs like liver. Other limitations include their unpredictable degradation kinetics; generally, weak mechanical strength, and risk of evoking an immune

Decellularized scaffolds (matrices) being natural biomaterials, which are deprived of cellular components while maintaining their original architecture and vascular system, have been widely studied and used in more complex tissue engineering [26]. In the case of liver tissue engineering, the use of decellularization/ recellularization strategy was inspired by a pioneer study of heart tissue engineering from the Ott group in 2008 [27]. After that, liver tissue engineered by using this approach has been fabricated [28–30]. Compared to those derived from other synthetic or natural biomaterial scaffolds, the decellularized liver scaffold (DLS)

to improved cell attachment, survival, and tissue formation [13, 14].

*DOI: http://dx.doi.org/10.5772/intechopen.89493*

**2. Decellularization-based scaffold biomaterials**

biomaterials have been extensively studied [22].

response [25], etc. also need to be considered.

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered... DOI: http://dx.doi.org/10.5772/intechopen.89493*

partial functions of the liver. The preservation of a functional ECM during decellularization, cellular differentiation [11], and a lack of endothelial-lying vascular networks limits the long-term functional integration of constructs after in vivo transplantation. As techniques continue develop, some methods with the potential to overcome these challenges should be explored in the near future, which will further boost the development of a tissue-engineered liver with improved functions. In this chapter, we have tried to focus on the possibility of liver tissue engineering by using a decellularization/recellularization strategy and to describe the current advancements made in the field to address a possible clinical transplantation.

#### **2. Decellularization-based scaffold biomaterials**

The term "biomaterials" traditionally means a nonliving substance used for a medical purpose. As the technology of biomaterials developed, the definition expanded to include substances to control the biological environment of cells and tissues for increased compatibility with a host to allow for colonization, proliferation, and differentiation of cells while maintaining their specific morphologies, configurations and avoiding immunological rejection. Based on the increasing knowledge of ECM biology, scaffold biomaterials can be grouped as synthetic materials, natural materials, or a decellularized matrix [12]. Moreover, modifications have been made to enhance the biologically active signals of scaffolds, leading to improved cell attachment, survival, and tissue formation [13, 14].

Biomaterials with required properties have been well studied from synthetic materials. For instance, a nanofibrous matrix made of poly and poly-embedded growth factors was transplanted into animals and restored cardiac regeneration by promoting vascularization [15]. Zawaneh et al. have reported the design of an injectable synthetic and biodegradable polymeric biomaterial consisting of polyethylene glycol and a polycarbonate of dihydroxyacetone that is easily extruded through narrow-gage needles, biodegrades into inert products, and is well tolerated by soft tissues [16]. Those chemically and biologically modified synthetic materials could result in a better way to mimic and control seed cell responses [17, 18]. Another advantage of synthetic materials is their easier to predict and control the degradation of synthetic scaffolds. However, despite this wealth of knowledge, the ability of synthetic biomaterials to support cell attachment, or induce phenotypic expression is much lower than that of natural biomaterials [19–21]; thus, natural biomaterials have been extensively studied [22].

Natural biomaterials include collagen, alginate, and chitosan. These types of biomaterials are inherently able to facilitate for seed cell attachment, proliferation, and functional differentiation, thus they hold significant promise for liver tissue engineering [23, 24]. However, traditional natural materials have poor inherent bioactivity, acidic byproducts, etc., and alone cannot rebuild the complex architecture of solid organs like liver. Other limitations include their unpredictable degradation kinetics; generally, weak mechanical strength, and risk of evoking an immune response [25], etc. also need to be considered.

Decellularized scaffolds (matrices) being natural biomaterials, which are deprived of cellular components while maintaining their original architecture and vascular system, have been widely studied and used in more complex tissue engineering [26]. In the case of liver tissue engineering, the use of decellularization/ recellularization strategy was inspired by a pioneer study of heart tissue engineering from the Ott group in 2008 [27]. After that, liver tissue engineered by using this approach has been fabricated [28–30]. Compared to those derived from other synthetic or natural biomaterial scaffolds, the decellularized liver scaffold (DLS)

*Xenotransplantation - Comprehensive Study*

organs, such as liver.

In modern times, the failure of solid organs, such as ESLD caused by injury or disease, has become a major challenge in clinics [4]. According to the U.S. Centers for Disease Control and Prevention (https://www.cdc.gov/), in 2014, 38,170 people died of ESLD. Currently, orthotopic liver transplant (OLT) is an ideal therapy for ESLD. However, a shortage of liver organ donors severely limits OLT usage. The Department of Health and Human Services in the United States has estimated that (https://optn.transplant.hrsa.gov) 22 people on the National Transplant Waiting List die each day, while one person is added to the waiting list every 10 min. Additionally, people fortunate enough to receive an organ transplantation have to suffer from the lifelong use of immunosuppressants against chronic rejection. Therefore, new technologies are eagerly needed to create a transplantable liver [5]. Tissue engineering is a mixed field that aims to fabricate functional organs in vitro [6]. Over the decades, great progress has been achieved in the laboratory, and even some livers have been used in clinics [7]. Tissue engineering by using a decellularization/recellularization strategy, which maintains the architecture, vascular system, and ECM components, has been shown to be a promising tool for solid

Liver tissue engineering by using decellularization/recellularization strategy (**Figure 1**) involves biomimicking the architecture and physiological features of the native liver. The procedure generally needs three major components: a scaffolding platform, seed cells, and a 3D microenvironment. Despite the numerous advances over the years, it is still an enormous challenge to fabricate a liver organ [8]. Generating liver organ-specific 3D structure scaffold to keep as much as original biochemical, physiochemical, and biomechanical ECM microenvironment is the one of the main hurdles in liver engineering field [9]. Such physiological 3D structure also plays a remarkable role in influencing seeded cell long-term survival and complex liver tissue mass formation [10]. To achieve this, scientists have been working with different scaffolding systems for liver tissue engineering. Studies have shown that a construction strategy based on a combination of a decellularized naïve liver matrix and recellularization with seed cells has led to constructs that match human organs in size and structure. However, the present constructs still only fulfill

*The decellularization/recellularization strategy in liver tissue engineering. Mammals donor-derived livers undergo a process of decellularization to obtain decellularized liver scaffolds (DLS) (step a–c), and then recellularized seed cells are placed onto the scaffolds (step d–f). Finally, the recellularized scaffolds are placed into 3D culture conditions in a bioreactor to construct liver-like tissues or organs with an overall structure and* 

**96**

**Figure 1.**

*vasculature (step g).*

mostly preserves the native complex liver ECM components, spatial microstructure, and perusable vascular architecture [31, 32] as more "biocompatible ways" for seed cells attaching and reorganizing on a complex 3D level [33]. Therefore, the DLS might have more favorable advantages than other scaffolds for clinical application although the biocompatibility signal between ECM of the DLS and seed cells is still unclear. Scientists have recellularized stem cells onto the natural 3D DLS and have found that these culturing cells not only survive better in the scaffold structure than their culturing in 2D environment but also differentiate into functional cells as well [34]. Zhang et al. seeded adult mouse liver hepatic stem/ progenitor cells onto the DLS that generated from naïve liver (nDLS) and cultured the complex in bioreactor, which formed a liver-like construction. Importantly, the nDLS/cell construction was able to repair a cirrhotic liver and even replace the failure liver [35].

Although many studies have been performed in the DLS field for liver tissue engineering [36–40], unfortunately, because of the nDLS being a lack of "active microenvironmental" support in existing ECM components, the optimization of the nDLS biomaterials become an important procedure for improving the skill of liver tissue engineering. Many protocols have been applied to modify the non-bioactive decellularized scaffolds. The application of a variety of growth factors [41] to promote the survival, proliferation, and differentiation of cells, like insulin-like growth factor 1 thought to promote hepatic cell differentiation from bone marrow-derived mesenchymal stem cells, vascular endothelial growth factor applied to enhance the vascularization of tissue-engineered tissues or organs. Additionally, the complex synergistic and antagonistic actions between different kinds of growth factors in vivo, more attention should be paid to the combined and sequential application of different growth factors. Consideration of optimizing the ECM of nDLS foir its behave like "naïve liver regenerative niche" might be a nice way to induce liver-like tissue formation spontaneously both in vitro and in vivo. Based on this, recently, Yang et al. has presented a very interesting experiment: the authors generated an acellular liver scaffold from pretreated naïve liver. They pretreated a naïve liver by performing a 30–55% partial hepatectomy, and the liver was maintained in vivo for 3–5 days until acute liver regeneration occurred, which allowed for the generation of the scaffold from the regenerative liver (rDLS) (**Figure 2**). These rDLS retain a variety of higher level of supporting growth factors for liver spontaneous regeneration as compared to that of nDLS, including their collagens, growth factors (HGF, TGF-α, IL-6, b-FGF, VEGF), glycosaminoglycans, antithrombotic proteins, and other matrix proteins [42]. Since the novel rDLS possesses a natural liver regenerative microenvironment, so-called "bioactive" ECM, it has shown more efficiency than nDLS in promoting primary hepatocyte survival and antithrombotic activity. Notably, when recellularized the rDLS with intrahepatic stem/progenitor cells and cultured them in 3D environment, a more likely liver organ was formatted as compared to the nDLS recellularized with the same stem/progenitor cells, after transplanted into recipients [42]. This pioneer study demonstrated that "bioactive" scaffolds of the rDLS obtained from a regenerative liver possess an advanced natural "active state niche" as compared to nDLS ("still state niche") for promoting primary hepatocyte survival, resistance to thrombosis, and liver-like organ construction. Other forms of bioactive factors are also involved in liver tissue engineering, like microRNAs, etc. [43, 44]. Furthermore, it needs to be mentioned that the advantage of highly conserved each specific ECM protein of decellularized scaffold among species of which the ECM are recognizable within and between species largely without immune rejection [45, 46] when properly processed to remove cellular antigens that would induce an immune rejection without damaging the ECM.

**99**

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered...*

**3. Seed cells response to the natural three-dimensional-decellularized** 

*Generation of a porcine decellularized liver scaffold from naïve livers and livers that had undergone partial hepatectomy (PHx). (A) Perfusion procedure for liver organ decellularization. (B) Blood-vessel tree of a decellularized scaffold from a naïve liver (nDLS). (C) Blood vessel tree of a decellularized scaffold from a* 

Cellular components are an integral part of any tissue engineering. In the case of the liver, it is important to find appropriate cells, such as hepatocytes or stem cells and to seed them into biomaterial scaffolds to regenerate liver tissues or organs [47]. Appropriate seed cells contain parenchymal such as hepatocytes, cholangiocytes, and supportive cells like liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and pit cells [48]. Hepatocytes account for 60–65% of a liver's cell population [1, 2, 49], which is important for liver tissue engineering. If it is difficult to obtain patientderived hepatocytes, along with challenging isolation, culture, and the low yields of these cells in vitro [50], stem cells are required for liver tissue engineering [51, 52]. Stem cells are generally grouped as embryonic stem cells (ESCs), somatic stem cells (SSCs), and inducible pluripotent stem cells (iPS) [53]. ESCs have a higher regenerative capacity and can be manipulated to differentiate into other cell types [54, 55]. For liver tissue engineering, ESCs are considered beneficial for the purpose of cell differentiation. For instance, epithelial cells differentiate from ESCs, which could cover the interior of vessels (arteries, veins, and capillaries) of DLS, and the interior of vessels is one of the major players of the angiogenesis process in physiological and pathological conditions involved in thrombus resistant effects. Due to the ethical problems with ESCs, tetratomics and expanded adult human hepatocytes [56], iPS are described as an alternative for adult human hepatocyte differentiation [57–61]. More studies about iPS are under active investigation at present [62], but dozens of publications regarding iPS-derived hepatic lineages have varied from report to report, making it difficult to compare the relative successes of the various modified protocols in enhancing hepatocyte differentiation [63, 64]. Moreover, cultured human hepatocytes often upregulate inappropriate immature markers, such as alpha-fetal protein (AFP). Consequently, any comparisons made to these altered adult hepatocytes may make the candidate immaturely appear more strongly functional than they truly are. Indeed, an examination of published accounts reveals that many protocols lead to fetal hepatocyte-like cells, but in some

*DOI: http://dx.doi.org/10.5772/intechopen.89493*

**liver biomaterial scaffold**

*partial hepatectomy (PHx) liver (rDLS).*

**Figure 2.**

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered... DOI: http://dx.doi.org/10.5772/intechopen.89493*

**Figure 2.**

*Xenotransplantation - Comprehensive Study*

failure liver [35].

mostly preserves the native complex liver ECM components, spatial microstructure, and perusable vascular architecture [31, 32] as more "biocompatible ways" for seed cells attaching and reorganizing on a complex 3D level [33]. Therefore, the DLS might have more favorable advantages than other scaffolds for clinical application although the biocompatibility signal between ECM of the DLS and seed cells is still unclear. Scientists have recellularized stem cells onto the natural 3D DLS and have found that these culturing cells not only survive better in the scaffold structure than their culturing in 2D environment but also differentiate into functional cells as well [34]. Zhang et al. seeded adult mouse liver hepatic stem/ progenitor cells onto the DLS that generated from naïve liver (nDLS) and cultured the complex in bioreactor, which formed a liver-like construction. Importantly, the nDLS/cell construction was able to repair a cirrhotic liver and even replace the

Although many studies have been performed in the DLS field for liver tissue engineering [36–40], unfortunately, because of the nDLS being a lack of "active microenvironmental" support in existing ECM components, the optimization of the nDLS biomaterials become an important procedure for improving the skill of liver tissue engineering. Many protocols have been applied to modify the non-bioactive decellularized scaffolds. The application of a variety of growth factors [41] to promote the survival, proliferation, and differentiation of cells, like insulin-like growth factor 1 thought to promote hepatic cell differentiation from bone marrow-derived mesenchymal stem cells, vascular endothelial growth factor applied to enhance the vascularization of tissue-engineered tissues or organs. Additionally, the complex synergistic and antagonistic actions between different kinds of growth factors in vivo, more attention should be paid to the combined and sequential application of different growth factors. Consideration of optimizing the ECM of nDLS foir its behave like "naïve liver regenerative niche" might be a nice way to induce liver-like tissue formation spontaneously both in vitro and in vivo. Based on this, recently, Yang et al. has presented a very interesting experiment: the authors generated an acellular liver scaffold from pretreated naïve liver. They pretreated a naïve liver by performing a 30–55% partial hepatectomy, and the liver was maintained in vivo for 3–5 days until acute liver regeneration occurred, which allowed for the generation of the scaffold from the regenerative liver (rDLS) (**Figure 2**). These rDLS retain a variety of higher level of supporting growth factors for liver spontaneous regeneration as compared to that of nDLS, including their collagens, growth factors (HGF, TGF-α, IL-6, b-FGF, VEGF), glycosaminoglycans, antithrombotic proteins, and other matrix proteins [42]. Since the novel rDLS possesses a natural liver regenerative microenvironment, so-called "bioactive" ECM, it has shown more efficiency than nDLS in promoting primary hepatocyte survival and antithrombotic activity. Notably, when recellularized the rDLS with intrahepatic stem/progenitor cells and cultured them in 3D environment, a more likely liver organ was formatted as compared to the nDLS recellularized with the same stem/progenitor cells, after transplanted into recipients [42]. This pioneer study demonstrated that "bioactive" scaffolds of the rDLS obtained from a regenerative liver possess an advanced natural "active state niche" as compared to nDLS ("still state niche") for promoting primary hepatocyte survival, resistance to thrombosis, and liver-like organ construction. Other forms of bioactive factors are also involved in liver tissue engineering, like microRNAs, etc. [43, 44]. Furthermore, it needs to be mentioned that the advantage of highly conserved each specific ECM protein of decellularized scaffold among species of which the ECM are recognizable within and between species largely without immune rejection [45, 46] when properly processed to remove cellular antigens that would induce an immune rejection without

**98**

damaging the ECM.

*Generation of a porcine decellularized liver scaffold from naïve livers and livers that had undergone partial hepatectomy (PHx). (A) Perfusion procedure for liver organ decellularization. (B) Blood-vessel tree of a decellularized scaffold from a naïve liver (nDLS). (C) Blood vessel tree of a decellularized scaffold from a partial hepatectomy (PHx) liver (rDLS).*

#### **3. Seed cells response to the natural three-dimensional-decellularized liver biomaterial scaffold**

Cellular components are an integral part of any tissue engineering. In the case of the liver, it is important to find appropriate cells, such as hepatocytes or stem cells and to seed them into biomaterial scaffolds to regenerate liver tissues or organs [47]. Appropriate seed cells contain parenchymal such as hepatocytes, cholangiocytes, and supportive cells like liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and pit cells [48]. Hepatocytes account for 60–65% of a liver's cell population [1, 2, 49], which is important for liver tissue engineering. If it is difficult to obtain patientderived hepatocytes, along with challenging isolation, culture, and the low yields of these cells in vitro [50], stem cells are required for liver tissue engineering [51, 52].

Stem cells are generally grouped as embryonic stem cells (ESCs), somatic stem cells (SSCs), and inducible pluripotent stem cells (iPS) [53]. ESCs have a higher regenerative capacity and can be manipulated to differentiate into other cell types [54, 55]. For liver tissue engineering, ESCs are considered beneficial for the purpose of cell differentiation. For instance, epithelial cells differentiate from ESCs, which could cover the interior of vessels (arteries, veins, and capillaries) of DLS, and the interior of vessels is one of the major players of the angiogenesis process in physiological and pathological conditions involved in thrombus resistant effects. Due to the ethical problems with ESCs, tetratomics and expanded adult human hepatocytes [56], iPS are described as an alternative for adult human hepatocyte differentiation [57–61]. More studies about iPS are under active investigation at present [62], but dozens of publications regarding iPS-derived hepatic lineages have varied from report to report, making it difficult to compare the relative successes of the various modified protocols in enhancing hepatocyte differentiation [63, 64]. Moreover, cultured human hepatocytes often upregulate inappropriate immature markers, such as alpha-fetal protein (AFP). Consequently, any comparisons made to these altered adult hepatocytes may make the candidate immaturely appear more strongly functional than they truly are. Indeed, an examination of published accounts reveals that many protocols lead to fetal hepatocyte-like cells, but in some

cases, the characterization reported is not sufficient to determine the fetal versus mature nature of the resulting differentiated hepatic cells. Given the seemingly fetal nature of iPS-derived hepatic cells produced to date, it is apparent that additional, careful modification of differentiation protocols is still required for further investigation before clinical implementation. Somatic stem cells could overcome the obstacles caused by ECSs, thereby making them more appropriate for liver tissue engineering [65, 66].

SSCs are composed of intrahepatic SSCs and extrahepatic SSCs. Bone marrow-, umbilical-, and fat tissue-derived mesenchymal stem cells are well accepted extrahepatic SSCs [67–69], while oval cells, especially neuro-glial antigen 2 (Ng2) expressing cells (Ng2<sup>+</sup> HSP), are currently identified as intrahepatic stem/progenitor cells. Isolation of the Ng2+ HSP should be completed by using a specific protocol [70]. Other sources of SSC behaviors seeded in the DLS have also influenced liver tissue engineering. Several studies have demonstrated that liver-derived mesenchymal stem cell (MSC)-like cells can differentiate into hepatocytes and cholangiocytes in nDLS and that the functional differentiation of MSCs in certain situations could be an alternative approach for an engineered liver organ transplantation in the treatment or replacement of ESLD [35, 71]. Our recently studied animal models have revealed that intrahepatic MSC-like SSCs repaired injured livers better than extrahepatic MSCs [unpublished]. Contrary to past hypotheses, extrahepatic bone marrow-derived MSCs do not seem to directly differentiate themselves into hepatocytes, in particularly in vivo, compared to local (liver) MSC-like cells, such as above mentioned the Ng2+ HSP. As the Ng2<sup>+</sup> HSP has been demonstrated to have a role in tissue repair [70] and failed liver replacement [35] in liver cirrhosis murine model, we recently further demonstrated that the intrahepatic Ng2+ HSP cells are capable of more efficiency than extrahepatic BM-MSCs in self-renewal and hepatocyte and cholangiocyte differentiations (unpublished) (**Figure 3**). Interestingly, by using the Ng2+ HSP, Zhang et al. have successfully reconstituted a liver construct in vitro

#### **Figure 3.**

*Murine intrahepatic and extrahepatic mesenchymal stem cells (MSCs). (A) Cultured and immunofluorescently stained of intrahepatic neuro-glial antigen 2 (Ng2)-expressing mesenchymal stem cell (MSC)-like stem/progenitor cells (Ng2+ HSP). (B) Cultured and immunofluorescently stained identical bone marrow (BM)-derived-MSCs (BM-MSCs), as visualized by optical microscopy, scale bar = 100 μM.*

**101**

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered...*

that is very similar to a naïve liver organ [35]. In addition, the immuno-modulatory, anti-inflammatory, antiapoptotic, and angiogenic properties of the intrahepatic

**4. Decellularization/recellularization strategy-based liver construction**

With the development of decellularization approaches, such as the detergent perfusion technique, whole decellularized scaffolds from liver organs have been produced DLS with an ECM structure and bioactive components being used fabricate bioengineered liver tissues, thus serving as a platform for liver organ bioengineering. Within the past several decades, numerous accomplishments have been driven by the development of these construction strategies. To date, decellularization-based liver construction strategies are constantly advancing such

Despite the well-conserved macroscopic structure of a liver organ obtained by using decellularization, it is still difficult to avoid some disruption to the ECM composition and ultrastructure through decellularization, which leads to impairment of the natural 3D microenvironment, for example, an impairment of glycosaminoglycans within the ECM by enzymes [73] can cause altered stiffness. Therefore, improved measures for preserving the integrity of the ECM during the decellularization process are required [74, 75]. An functional engineered liver tissue usually uses stem cells or progenitor cells that need to differentiate into multiple kinds of repair cells, which is a challenge to directly seed cells to colonize in relevant sites of DLS to induce their differentiation into specific cell types. Whether an engineering formed liver organ can successfully fulfill its functions depends not only on its physically decellularized scaffold structure but also on an effective recellularization. Therefore, how to populate seed cells like differentiated hepatocytes from different kinds of seed cells or stem cells themselves onto the DLS needs to be carefully considered. In particular, how to manipulate the DLS to enhance the targeted specific colonization of cells to specific areas of DLS such as perfused endothelial cells [76–78], has drawn much attention. To ensure the long-term survival of an engineered liver by allowing exchanges of oxygen, nutrients, and disposal of metabolic waste [79], a functional vascular network and thrombosis after transplantation also needs to be considered. Despite the conservation of the general vascular structure by DLS, the formation of a functional vascular network remains a challenge for liver organ construction. The mainstream strategy to fabricate an engineered liver organ with a functional vascular network includes also the procedure of prevascularization. The initial approaches have been successfully used in spontaneous lineage of endothelial cells in DLS vascular networks after recellularization with stem cells [80–82] to challenge thrombosis after transplantation when exposed to blood, thus leading to localized organ failures [83]. There are two nice approaches showed that endothelialization of vasculature and immobilization of heparin on nDLS could reduce its incidence of thrombosis [84]. More recently, from a pretreated naïve liver obtained rDLS, exhibited except for strong promoting primary hepatocyte survival but also antithrombosis more effect [biomaterials 2018]. Notably, after transplantation guiding the rDLS/cells complex forms complex liver-like tissues (geometries) more effective on rDLS than on nDLS (**Figure 4**), meanwhile combined with better organization of endothelial lineage in rDLS than in nDLS [42]. This suggests that rDLS possesses an advanced "bioactive natural regeneration state niche" relative to the nDLS, which preserves a "still state niche." Therefore, the spontaneous manipulation of the ECM on DLS is a more promising strategy for decellularization-based

HSP in the liver still need to be further investigated for liver tissue

*DOI: http://dx.doi.org/10.5772/intechopen.89493*

as maintaining complete hepatic vessel networks [72].

MSC-like Ng2+

engineering.

that is very similar to a naïve liver organ [35]. In addition, the immuno-modulatory, anti-inflammatory, antiapoptotic, and angiogenic properties of the intrahepatic MSC-like Ng2+ HSP in the liver still need to be further investigated for liver tissue engineering.

#### **4. Decellularization/recellularization strategy-based liver construction**

With the development of decellularization approaches, such as the detergent perfusion technique, whole decellularized scaffolds from liver organs have been produced DLS with an ECM structure and bioactive components being used fabricate bioengineered liver tissues, thus serving as a platform for liver organ bioengineering. Within the past several decades, numerous accomplishments have been driven by the development of these construction strategies. To date, decellularization-based liver construction strategies are constantly advancing such as maintaining complete hepatic vessel networks [72].

Despite the well-conserved macroscopic structure of a liver organ obtained by using decellularization, it is still difficult to avoid some disruption to the ECM composition and ultrastructure through decellularization, which leads to impairment of the natural 3D microenvironment, for example, an impairment of glycosaminoglycans within the ECM by enzymes [73] can cause altered stiffness. Therefore, improved measures for preserving the integrity of the ECM during the decellularization process are required [74, 75]. An functional engineered liver tissue usually uses stem cells or progenitor cells that need to differentiate into multiple kinds of repair cells, which is a challenge to directly seed cells to colonize in relevant sites of DLS to induce their differentiation into specific cell types. Whether an engineering formed liver organ can successfully fulfill its functions depends not only on its physically decellularized scaffold structure but also on an effective recellularization. Therefore, how to populate seed cells like differentiated hepatocytes from different kinds of seed cells or stem cells themselves onto the DLS needs to be carefully considered. In particular, how to manipulate the DLS to enhance the targeted specific colonization of cells to specific areas of DLS such as perfused endothelial cells [76–78], has drawn much attention. To ensure the long-term survival of an engineered liver by allowing exchanges of oxygen, nutrients, and disposal of metabolic waste [79], a functional vascular network and thrombosis after transplantation also needs to be considered. Despite the conservation of the general vascular structure by DLS, the formation of a functional vascular network remains a challenge for liver organ construction. The mainstream strategy to fabricate an engineered liver organ with a functional vascular network includes also the procedure of prevascularization. The initial approaches have been successfully used in spontaneous lineage of endothelial cells in DLS vascular networks after recellularization with stem cells [80–82] to challenge thrombosis after transplantation when exposed to blood, thus leading to localized organ failures [83]. There are two nice approaches showed that endothelialization of vasculature and immobilization of heparin on nDLS could reduce its incidence of thrombosis [84]. More recently, from a pretreated naïve liver obtained rDLS, exhibited except for strong promoting primary hepatocyte survival but also antithrombosis more effect [biomaterials 2018]. Notably, after transplantation guiding the rDLS/cells complex forms complex liver-like tissues (geometries) more effective on rDLS than on nDLS (**Figure 4**), meanwhile combined with better organization of endothelial lineage in rDLS than in nDLS [42]. This suggests that rDLS possesses an advanced "bioactive natural regeneration state niche" relative to the nDLS, which preserves a "still state niche." Therefore, the spontaneous manipulation of the ECM on DLS is a more promising strategy for decellularization-based

*Xenotransplantation - Comprehensive Study*

engineering [65, 66].

expressing cells (Ng2<sup>+</sup>

mentioned the Ng2+

the Ng2+

tor cells. Isolation of the Ng2+

cases, the characterization reported is not sufficient to determine the fetal versus mature nature of the resulting differentiated hepatic cells. Given the seemingly fetal nature of iPS-derived hepatic cells produced to date, it is apparent that additional, careful modification of differentiation protocols is still required for further investigation before clinical implementation. Somatic stem cells could overcome the obstacles caused by ECSs, thereby making them more appropriate for liver tissue

SSCs are composed of intrahepatic SSCs and extrahepatic SSCs. Bone marrow-, umbilical-, and fat tissue-derived mesenchymal stem cells are well accepted extrahepatic SSCs [67–69], while oval cells, especially neuro-glial antigen 2 (Ng2)-

[70]. Other sources of SSC behaviors seeded in the DLS have also influenced liver tissue engineering. Several studies have demonstrated that liver-derived mesenchymal stem cell (MSC)-like cells can differentiate into hepatocytes and cholangiocytes in nDLS and that the functional differentiation of MSCs in certain situations could be an alternative approach for an engineered liver organ transplantation in the treatment or replacement of ESLD [35, 71]. Our recently studied animal models have revealed that intrahepatic MSC-like SSCs repaired injured livers better than extrahepatic MSCs [unpublished]. Contrary to past hypotheses, extrahepatic bone marrow-derived MSCs do not seem to directly differentiate themselves into hepatocytes, in particularly in vivo, compared to local (liver) MSC-like cells, such as above

tissue repair [70] and failed liver replacement [35] in liver cirrhosis murine model,

of more efficiency than extrahepatic BM-MSCs in self-renewal and hepatocyte and cholangiocyte differentiations (unpublished) (**Figure 3**). Interestingly, by using

HSP, Zhang et al. have successfully reconstituted a liver construct in vitro

HSP. As the Ng2<sup>+</sup>

we recently further demonstrated that the intrahepatic Ng2+

*Murine intrahepatic and extrahepatic mesenchymal stem cells (MSCs). (A) Cultured and* 

*immunofluorescently stained of intrahepatic neuro-glial antigen 2 (Ng2)-expressing mesenchymal stem cell* 

*marrow (BM)-derived-MSCs (BM-MSCs), as visualized by optical microscopy, scale bar = 100 μM.*

*HSP). (B) Cultured and immunofluorescently stained identical bone* 

HSP), are currently identified as intrahepatic stem/progeni-

HSP should be completed by using a specific protocol

HSP has been demonstrated to have a role in

HSP cells are capable

**100**

**Figure 3.**

*(MSC)-like stem/progenitor cells (Ng2+*

#### **Figure 4.**

*Comparison of the murine liver-lobule-like tissue construction formation between rDLS and nDLS after portal-renal arterialized auxiliary heterotopic liver transplantation. (A) Schematic of the procedure. The left green cycle indicates the DLS, and the right green cycle indicates the end-to-end anastomosis of the PV (scaffold)-L-RA (recipient). The green arrows in the panels indicate the right-RA. The right bottom cartoon shows the end-to-side anastomosis of the IVC (scaffold)-IVC (recipient). (Ba–d) Exposure of the right-side kidney (the square indicates the kidney) (a). Nephrectomy of the right-side kidney (the square indicates the lack of kidney) (b). The cell-loaded DLS where the kidney was removed (the bold arrow indicates the PV, thin arrow indicates the IVC, and the green arrow indicates a right renal artery (right-RA)). The left-side renal artery (L-RA) was connected to the PV with cross-clamping of the PV and the IVC of the recellularized scaffold (c). The noncell loaded DLS was connected to the recipient by the same procedure as the cell-loaded DLS where the kidney was removed (d). (Ca–c) DLS seeded with Ng2+ HSP cells formed a liver-lobule-like construct in rDLS (a and b) after approximately 20–40 days (a, indicated as a cycle), for two lobes with better blood patency (b), represented with a white arrow; there was no visible blood flow in the nDLS loaded with Ng2<sup>+</sup> HSP cells for the same time (c). (D) Blood flow velocity (flow, arbitrary unit, AU) was measured in rDLS and nDLS at 45 min within 100 s after the operation by a near-infrared-LDF system, scale bar = 50 μM.*

liver tissue. In the future, the objective of a decellularization-based liver construction strategy could be based on generating a 3D decellularized biomaterial scaffold with natural "regenerative bioactive niche" for the seed cell attachment, proliferation and differentiation of cells, and developing a transplantable "new" liver in vitro that maintains the structures and functions of a naïve liver.

In summary, compared with other strategies that can only fabricate partial structures, a decellularization/recellularization-based liver tissue engineering strategy enables the construction of the liver structures with complete blood vessel network at a clinically relevant scale, thus becoming a more promising approach for liver tissue engineering. However, in order to provide a promising route for developing a functional bioartificial liver with potential applications for humans by

**103**

Ng2+

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered...*

such strategy, several questions must be answered: (1) Is the use of a decellularized liver matrix the only possible solution? (2) What kinds of cells need to be chosen for recellularization? Extrahepatic cells? or possibly resident stem/progenitors cells? (3) What is the optimal decellularized liver scaffold (DLS)? (4) What is the length of time for incubation in a bioreactor? (5) Would the technique be applicable to a

Clearly, decellularization/recellularization through the development of in vitro and in vivo tissue and organ models for liver bioengineering are advancing strategies. This, combined with multidisciplinary team-workers performing focused, systematic studies to address critical questions, is essential for the success of this strategy. The following critical issues might need to be addressed before clinical applications: (1) preservation and modification of a functional ECM structure to better mimic the regenerative niche; (2) selection of effective seed cell sources for recellularization; (3) modification of blood-vessel networks for "endothelialized DLS"; (4) long-term survival by preventing from thrombosis and functions after transplantation; and (5) immune rejection. In the coming years, many new techniques will be explored, which are expected to have the potential to address these

This work was funded by the National Natural Science Foundation of China (NNSFC), Grant/Award Numbers: 81570573 and 81873586; the Army Medical University, Grant/Award Numbers: 2018XLC2009; and the Army Medical University-affiliated Southwest Hospital, Grant/Award Numbers:

nDLS the decellularized scaffold that generated from naïve liver rDLS the decellularized scaffold that generated from pretreated liver

*DOI: http://dx.doi.org/10.5772/intechopen.89493*

**5. Conclusion and challenges**

challenges.

**Funding information**

SWH2017ZYLX-03.

**Abbreviations**

ESLD end-stage liver disease ECM extracellular matrix 3D three-dimensional

HGF hepatic growth factor

ESCs embryonic stem cells SSCs somatic stem cells

MSC mesenchymal stem cell

IL-6 interleukin 6

OLT orthotopic liver transplant DLS decellularized liver scaffold

TGF-α transforming growth factor-alpha

b-FGF fibroblast *growth* factor-beta VEGF vascular endothelial growth factor

iPS inducible pluripotent stem cells

HSP neuro-glial antigen 2 (Ng2)-expressing cells

human liver with its extensively sinusoidal surface?

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered... DOI: http://dx.doi.org/10.5772/intechopen.89493*

such strategy, several questions must be answered: (1) Is the use of a decellularized liver matrix the only possible solution? (2) What kinds of cells need to be chosen for recellularization? Extrahepatic cells? or possibly resident stem/progenitors cells? (3) What is the optimal decellularized liver scaffold (DLS)? (4) What is the length of time for incubation in a bioreactor? (5) Would the technique be applicable to a human liver with its extensively sinusoidal surface?

#### **5. Conclusion and challenges**

*Xenotransplantation - Comprehensive Study*

**102**

**Figure 4.**

*Ng2<sup>+</sup>*

liver tissue. In the future, the objective of a decellularization-based liver construction strategy could be based on generating a 3D decellularized biomaterial scaffold with natural "regenerative bioactive niche" for the seed cell attachment, proliferation and differentiation of cells, and developing a transplantable "new" liver in vitro

*and nDLS at 45 min within 100 s after the operation by a near-infrared-LDF system, scale bar = 50 μM.*

*construct in rDLS (a and b) after approximately 20–40 days (a, indicated as a cycle), for two lobes with better blood patency (b), represented with a white arrow; there was no visible blood flow in the nDLS loaded with* 

*HSP cells for the same time (c). (D) Blood flow velocity (flow, arbitrary unit, AU) was measured in rDLS* 

*HSP cells formed a liver-lobule-like* 

*Comparison of the murine liver-lobule-like tissue construction formation between rDLS and nDLS after portal-renal arterialized auxiliary heterotopic liver transplantation. (A) Schematic of the procedure. The left green cycle indicates the DLS, and the right green cycle indicates the end-to-end anastomosis of the PV (scaffold)-L-RA (recipient). The green arrows in the panels indicate the right-RA. The right bottom cartoon shows the end-to-side anastomosis of the IVC (scaffold)-IVC (recipient). (Ba–d) Exposure of the right-side kidney (the square indicates the kidney) (a). Nephrectomy of the right-side kidney (the square indicates the lack of kidney) (b). The cell-loaded DLS where the kidney was removed (the bold arrow indicates the PV, thin arrow indicates the IVC, and the green arrow indicates a right renal artery (right-RA)). The left-side renal artery (L-RA) was connected to the PV with cross-clamping of the PV and the IVC of the recellularized scaffold (c). The noncell loaded DLS was connected to the recipient by the same procedure as the cell-loaded* 

In summary, compared with other strategies that can only fabricate partial structures, a decellularization/recellularization-based liver tissue engineering strategy enables the construction of the liver structures with complete blood vessel network at a clinically relevant scale, thus becoming a more promising approach for liver tissue engineering. However, in order to provide a promising route for developing a functional bioartificial liver with potential applications for humans by

that maintains the structures and functions of a naïve liver.

*DLS where the kidney was removed (d). (Ca–c) DLS seeded with Ng2+*

Clearly, decellularization/recellularization through the development of in vitro and in vivo tissue and organ models for liver bioengineering are advancing strategies. This, combined with multidisciplinary team-workers performing focused, systematic studies to address critical questions, is essential for the success of this strategy. The following critical issues might need to be addressed before clinical applications: (1) preservation and modification of a functional ECM structure to better mimic the regenerative niche; (2) selection of effective seed cell sources for recellularization; (3) modification of blood-vessel networks for "endothelialized DLS"; (4) long-term survival by preventing from thrombosis and functions after transplantation; and (5) immune rejection. In the coming years, many new techniques will be explored, which are expected to have the potential to address these challenges.

#### **Funding information**

This work was funded by the National Natural Science Foundation of China (NNSFC), Grant/Award Numbers: 81570573 and 81873586; the Army Medical University, Grant/Award Numbers: 2018XLC2009; and the Army Medical University-affiliated Southwest Hospital, Grant/Award Numbers: SWH2017ZYLX-03.

#### **Abbreviations**


*Xenotransplantation - Comprehensive Study*

#### **Author details**

Quanyu Chen1,2, Xiaolin You2 , Jiejuan Lai<sup>2</sup> , Shifang Jiang2 , Hongyu Zhang2 and Lianhua Bai1,2\*

1 Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Chongqing, China

2 Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing, China

\*Address all correspondence to: qqg63@outlook.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**105**

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered...*

Biomedical Materials Research. Part B, Applied Biomaterials. 2019;**107**:19-28

[10] Brown JH, Das P, DiVito MD, Ivancic D, Tan LP, Wertheim JA. Nanofibrous PLGA electrospun scaffolds modified with type I collagen influence hepatocyte function and support viability in vitro. Acta Biomaterialia. 2018;**73**:217-227

[11] Tandon V, Zhang B, Radisic M, Murthy SK. Generation of tissue constructs for cardiovascular regenerative medicine: From cell procurement to scaffold design. Biotechnology Advances. 2013;**31**:

[12] Rowley AT, Nagalla RR, Wang SW, Liu WF. Extracellular matrix-based strategies for immunomodulatory biomaterials engineering. Advanced Healthcare Materials. 2019;**8**:e1801578

[13] Perez RA, Won JE, Knowles JC, Kim HW. Naturally and synthetic smart composite biomaterials for tissue regeneration. Advanced Drug Delivery

[14] Atala A, Kasper FK, Mikos AG. Engineering complex tissues. Science Translational Medicine.

[15] Lakshmanan R, Kumaraswamy P,

Engineering a growth factor embedded nanofiber matrix niche to promote vascularization for functional cardiac regeneration. Biomaterials.

Padera RF, Henderson PW, Spector JA, Putnam D. Design of an injectable synthetic and biodegradable surgical biomaterial. Proceedings of the National Academy of Sciences

Krishnan UM, Sethuraman S.

[16] Zawaneh PN, Singh SP,

Reviews. 2013;**65**:471-496

2012;**4**:112r-160r

2016;**97**:176-195

722-735

*DOI: http://dx.doi.org/10.5772/intechopen.89493*

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**Author details**

and Lianhua Bai1,2\*

Chongqing, China

Quanyu Chen1,2, Xiaolin You2

, Jiejuan Lai<sup>2</sup>

Sciences, Southwest University, Chongqing, China

\*Address all correspondence to: qqg63@outlook.com

provided the original work is properly cited.

1 Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life

2 Hepatobiliary Institute, Southwest Hospital, The Army Medical University,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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[73] He M, Callanan A. Comparison of methods for whole-organ

[74] Struecker B, Hillebrandt KH, Voitl R, Butter A, Schmuck RB, Reutzel-Selke A, et al. Porcine liver decellularization under oscillating pressure conditions: A technical

of the decellularization process. Tissue Engineering. Part C, Methods.

[75] Manso AM, Okada H,

[76] Shirakigawa N, Ijima H, Takei T. Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering. Journal of Bioscience and Bioengineering. 2012;**114**:546-551

[77] Devalliere J, Chen Y, Dooley K, Yarmush ML, Uygun BE. Improving functional re-endothelialization of

2015;**21**:303-313

decellularization in tissue engineering of bioartificial organs. Tissue Engineering. Part B, Reviews. 2013;**19**:194-208

refinement to improve the homogeneity

Sakamoto FM, Moreno E, Monkley SJ, Li R, et al. Loss of mouse cardiomyocyte talin-1 and talin-2 leads to beta-1 integrin reduction, costameric

instability, and dilated cardiomyopathy. Proceedings of the National Academy of Sciences of the United States of America. 2017;**114**:E6250-E6259

*DOI: http://dx.doi.org/10.5772/intechopen.89493*

for liver regeneration in mice. The Journal of Clinical Investigation.

[63] Hirata M, Yamaoka T. Hepatocytic

differentiation of iPS cells on decellularized liver tissue. Journal of Artificial Organs. 2017;**20**:318-325

[64] Schepers A, Li C, Chhabra A, Seney BT, Bhatia S. Engineering a perfusable 3D human liver platform from iPS cells. Lab on a Chip. 2016;

[65] Grath A, Dai G. Direct cell

[67] Klimczak A, Kozlowska U. Mesenchymal stromal cells and tissuespecific progenitor cells: Their role in tissue homeostasis. Stem Cells International. 2016;**2016**:4285215

[69] Coronado RE, Somaraki-Cormier M, Ong JL, Halff GA. Hepatocyte-like cells derived from human amniotic epithelial, bone marrow, and adipose stromal cells display enhanced functionality when cultured on decellularized liver substrate. Stem Cell Research.

[70] Zhang H, Siegel CT, Shuai L, Lai J, Zeng L, Zhang Y, et al. Repair of liver mediated by adult mouse liver

[68] Verstegen M, Willemse J, van den Hoek S, Kremers GJ, Luider TM, van Huizen NA, et al. Decellularization of whole human liver grafts using controlled perfusion for transplantable organ bioscaffolds. Stem Cells and Development. 2017;**26**:1304-1315

reprogramming for tissue engineering and regenerative medicine. Journal of Biological Engineering. 2019;**13**:14

[66] Kumar SA, Delgado M, Mendez VE, Joddar B. Applications of stem cells and bioprinting for potential treatment of diabetes. World Journal of Stem Cells.

2010;**120**:3120-3126

**16**:2644-2653

2019;**11**:13-32

*Optimization of a Decellularization/Recellularization Strategy for Transplantable Bioengineered... DOI: http://dx.doi.org/10.5772/intechopen.89493*

for liver regeneration in mice. The Journal of Clinical Investigation. 2010;**120**:3120-3126

*Xenotransplantation - Comprehensive Study*

Archivos de Bronconeumología.

[55] Lorzadeh N, Kazemirad N. Embryonic stem cells and infertility. American Journal of Perinatology.

[56] Yu Y, Fisher JE, Lillegard JB, Rodysill B, Amiot B, Nyberg SL. Cell therapies for liver diseases. Liver Transplantation. 2012;**18**:9-21

[57] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell.

[58] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;**131**:861-872

Corbineau S, Martinez A, Martinet C, Branchereau S, et al. Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology.

[59] Touboul T, Hannan NR,

[60] Sharma NS, Wallenstein EJ, Novik E, Maguire T, Schloss R, Yarmush ML. Enrichment of

hepatocyte-like cells with upregulated metabolic and differentiated function derived from embryonic stem cells using S-NitrosoAcetylPenicillamine. Tissue Engineering. Part C, Methods.

[61] Moore RN, Dasgupta A, Rajaei N, Yarmush ML, Toner M, Larue L, et al. Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytes. Biotechnology and Bioengineering. 2008;**101**:1332-1343

[62] Espejel S, Roll GR, McLaughlin KJ, Lee AY, Zhang JY, Laird DJ, et al. Induced pluripotent stem cell-derived hepatocytes have the functional and proliferative capabilities needed

2018;**35**:925-930

2006;**126**:663-676

2010;**51**:1754-1765

2009;**15**:297-306

[47] Murphy SV, Atala A. Organ engineering—Combining stem cells, biomaterials, and bioreactors to produce bioengineered organs for transplantation. BioEssays.

[48] Uygun BE, Yarmush ML.

Engineered liver for transplantation. Current Opinion in Biotechnology.

[49] Jain E, Damania A, Kumar A. Biomaterials for liver tissue engineering.

[50] Kaserman JE, Wilson AA. Patientderived induced pluripotent stem cells for alpha-1 antitrypsin deficiency disease modeling and therapeutic discovery. Chronic Obstructive Pulmonary Disease. 2018;**5**:258-266

[51] Zhang L, Ye JS, Decot V, Stoltz JF, de Isla N. Research on stem cells as candidates to be differentiated into hepatocytes. Bio-medical Materials and

[52] Zhang L, Zhao YH, Guan Z, Ye JS, de Isla N, Stoltz JF. Application potential of mesenchymal stem cells derived from Wharton's jelly in liver tissue engineering. Bio-medical Materials and

Engineering. 2012;**22**:105-111

Engineering. 2015;**25**:137-143

2014;**466**:1847-1857

2019;**20**:813-829

Martin CA, Reddy MS,

[53] Ren X, Ott HC. On the road to bioartificial organs. Pflügers Archiv.

[54] Radhakrishnan S, Trentz OA,

Rela M, Chinnarasu M, et al. Effect of passaging on the stemness of infrapatellar fat padderived stem cells and potential role of nucleostemin as a prognostic marker of impaired stemness. Molecular Medicine Reports.

Hepatology International.

2018;**54**:31-38

2013;**35**:163-172

2013;**24**:893-899

2014;**8**:185-197

**108**

[63] Hirata M, Yamaoka T. Hepatocytic differentiation of iPS cells on decellularized liver tissue. Journal of Artificial Organs. 2017;**20**:318-325

[64] Schepers A, Li C, Chhabra A, Seney BT, Bhatia S. Engineering a perfusable 3D human liver platform from iPS cells. Lab on a Chip. 2016; **16**:2644-2653

[65] Grath A, Dai G. Direct cell reprogramming for tissue engineering and regenerative medicine. Journal of Biological Engineering. 2019;**13**:14

[66] Kumar SA, Delgado M, Mendez VE, Joddar B. Applications of stem cells and bioprinting for potential treatment of diabetes. World Journal of Stem Cells. 2019;**11**:13-32

[67] Klimczak A, Kozlowska U. Mesenchymal stromal cells and tissuespecific progenitor cells: Their role in tissue homeostasis. Stem Cells International. 2016;**2016**:4285215

[68] Verstegen M, Willemse J, van den Hoek S, Kremers GJ, Luider TM, van Huizen NA, et al. Decellularization of whole human liver grafts using controlled perfusion for transplantable organ bioscaffolds. Stem Cells and Development. 2017;**26**:1304-1315

[69] Coronado RE, Somaraki-Cormier M, Ong JL, Halff GA. Hepatocyte-like cells derived from human amniotic epithelial, bone marrow, and adipose stromal cells display enhanced functionality when cultured on decellularized liver substrate. Stem Cell Research. 2019;**38**:101471

[70] Zhang H, Siegel CT, Shuai L, Lai J, Zeng L, Zhang Y, et al. Repair of liver mediated by adult mouse liver neuro-glia antigen 2-positive progenitor cell transplantation in a mouse model of cirrhosis. Scientific Reports. 2016;**6**:21783

[71] Martin C, Olmos É, Collignon M, De Isla N, Blanchard F, Chevalot I, et al. Revisiting MSC expansion from critical quality attributes to critical culture process parameters. Process Biochemistry. 2017;**59**:231-243

[72] Shirakigawa N, Ijima H. Decellularization of liver and organogenesis in rats. Methods in Molecular Biology. 2018;**1577**:271-281

[73] He M, Callanan A. Comparison of methods for whole-organ decellularization in tissue engineering of bioartificial organs. Tissue Engineering. Part B, Reviews. 2013;**19**:194-208

[74] Struecker B, Hillebrandt KH, Voitl R, Butter A, Schmuck RB, Reutzel-Selke A, et al. Porcine liver decellularization under oscillating pressure conditions: A technical refinement to improve the homogeneity of the decellularization process. Tissue Engineering. Part C, Methods. 2015;**21**:303-313

[75] Manso AM, Okada H, Sakamoto FM, Moreno E, Monkley SJ, Li R, et al. Loss of mouse cardiomyocyte talin-1 and talin-2 leads to beta-1 integrin reduction, costameric instability, and dilated cardiomyopathy. Proceedings of the National Academy of Sciences of the United States of America. 2017;**114**:E6250-E6259

[76] Shirakigawa N, Ijima H, Takei T. Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering. Journal of Bioscience and Bioengineering. 2012;**114**:546-551

[77] Devalliere J, Chen Y, Dooley K, Yarmush ML, Uygun BE. Improving functional re-endothelialization of

acellular liver scaffold using REDV cellbinding domain. Acta Biomaterialia. 2018;**78**:151-164

[78] Agarwal T, Maiti TK, Ghosh SK. Decellularized caprine liver-derived biomimetic and pro-angiogenic scaffolds for liver tissue engineering. Materials Science & Engineering. C, Materials for Biological Applications. 2019;**98**:939-948

[79] Sun Y, Liu Y, Li S, Liu C, Hu Q. Novel compound-forming technology using bioprinting and electrospinning for patterning a 3D scaffold construct with multiscale channels. Micromachines (Basel). 2016;**7**:238-256

[80] Unger RE, Sartoris A, Peters K, Motta A, Migliaresi C, Kunkel M, et al. Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillarylike structures on three-dimensional porous biomaterials. Biomaterials. 2007;**28**:3965-3976

[81] Unger RE, Dohle E, Kirkpatrick CJ. Improving vascularization of engineered bone through the generation of proangiogenic effects in co-culture systems. Advanced Drug Delivery Reviews. 2015;**94**:116-125

[82] Ko IK, Peng L, Peloso A, Smith CJ, Dhal A, Deegan DB, et al. Bioengineered transplantable porcine livers with re-endothelialized vasculature. Biomaterials. 2015;**40**:72-79

[83] Watanabe M, Yano K, Okawa K, Yamashita T, Tajima K, Sawada K, et al. Construction of sinusoid-scale microvessels in perfusion culture of a decellularized liver. Acta Biomaterialia. 2019;**95**:307-318

[84] Hussein KH, Park KM, Kang KS, Woo HM. Heparin-gelatin mixture improves vascular reconstruction efficiency and hepatic function in bioengineered livers. Acta Biomaterialia. 2016;**38**:82-93

**111**

Section 3

Regeneration

Section 3

## Regeneration

*Xenotransplantation - Comprehensive Study*

acellular liver scaffold using REDV cellbinding domain. Acta Biomaterialia.

[78] Agarwal T, Maiti TK, Ghosh SK. Decellularized caprine liver-derived biomimetic and pro-angiogenic scaffolds for liver tissue engineering. Materials Science & Engineering. C, Materials for Biological Applications. 2019;**98**:939-948

[79] Sun Y, Liu Y, Li S, Liu C, Hu Q. Novel compound-forming technology using bioprinting and electrospinning

construct with multiscale channels. Micromachines (Basel). 2016;**7**:238-256

[80] Unger RE, Sartoris A, Peters K, Motta A, Migliaresi C, Kunkel M, et al. Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillarylike structures on three-dimensional porous biomaterials. Biomaterials.

[81] Unger RE, Dohle E, Kirkpatrick CJ. Improving vascularization of engineered bone through the generation of proangiogenic effects in co-culture systems. Advanced Drug Delivery Reviews.

[82] Ko IK, Peng L, Peloso A, Smith CJ, Dhal A, Deegan DB, et al. Bioengineered transplantable porcine livers with re-endothelialized vasculature. Biomaterials. 2015;**40**:72-79

[83] Watanabe M, Yano K, Okawa K, Yamashita T, Tajima K, Sawada K, et al. Construction of sinusoid-scale microvessels in perfusion culture of a decellularized liver. Acta Biomaterialia.

[84] Hussein KH, Park KM, Kang KS, Woo HM. Heparin-gelatin mixture improves vascular reconstruction efficiency and hepatic function in bioengineered livers. Acta Biomaterialia.

for patterning a 3D scaffold

2007;**28**:3965-3976

2015;**94**:116-125

2019;**95**:307-318

2016;**38**:82-93

2018;**78**:151-164

**110**

**113**

**Chapter 7**

**Abstract**

Controllable Immunosuppression

Along with a growing interest in regenerative medicine, pigs are becoming a popular model for preclinical studies on human cell therapy. Due to pharmaceutical species difference and inability to self-medicate, specific modification and care are necessary in immunosuppressive regimen for pigs. Here, we summarize recent literature on immunosuppression in pigs for experimental transplantation. Based on literature and our own experiences, a practical protocol has been proposed in this report. In early studies of allogeneic organ transplantation, recipient pigs were administered cyclosporine or tacrolimus, and mycophenolate mofetil at slightly higher dose than that in human cases, because of relatively poor effectiveness of the drugs in pigs. Steroids may be effective but sometimes can cause debilitating side effects. Cell transplantation studies follow the basic protocol, but it remains to be clarified whether the smaller graft mass, even if it is xenogeneic, requires the same scale of immunosuppression as organ transplantation. To obtain reliable results, the use of gastrostomy tube and blood trough level monitoring are highly recommended. Nonpharmaceutical immunosuppression such as thymic intervention and

the use of severe combined immunodeficient pigs have also been discussed.

**Keywords:** pig, experimental transplantation, immunosuppression, human cell

The number of preclinical studies conducted using pigs has been increasing, especially in the field of cell therapy [1]. The merits of using pigs include (1) size advantages that enable to mimic clinical procedures; (2) availability of various experimental pigs such as miniature, microminiature, and gene-engineered strains; and (3) worldwide trend of discouragement of using dogs as research models.

However, immunosuppressive treatment has not been established well in pigs. Initially, pigs were used as models for performing allogeneic organ transplantation; now, the hope is to use them as xenogeneic organ donors. In the latter case, immunosuppressive protocols have been designed for primate recipients, while the number of reports mentioning immunosuppressive protocols for xenogeneic transplantation in pigs is unexpectedly few. In addition, insufficient medications are occasionally found in studies conducted by researchers who are not accustomed

in Pigs as a Basis for Preclinical

Studies on Human Cell Therapy

*Shin Enosawa and Eiji Kobayashi*

therapy, regenerative medicine

**1. Introduction**

to organ transplantation.

#### **Chapter 7**

## Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy

*Shin Enosawa and Eiji Kobayashi*

#### **Abstract**

Along with a growing interest in regenerative medicine, pigs are becoming a popular model for preclinical studies on human cell therapy. Due to pharmaceutical species difference and inability to self-medicate, specific modification and care are necessary in immunosuppressive regimen for pigs. Here, we summarize recent literature on immunosuppression in pigs for experimental transplantation. Based on literature and our own experiences, a practical protocol has been proposed in this report. In early studies of allogeneic organ transplantation, recipient pigs were administered cyclosporine or tacrolimus, and mycophenolate mofetil at slightly higher dose than that in human cases, because of relatively poor effectiveness of the drugs in pigs. Steroids may be effective but sometimes can cause debilitating side effects. Cell transplantation studies follow the basic protocol, but it remains to be clarified whether the smaller graft mass, even if it is xenogeneic, requires the same scale of immunosuppression as organ transplantation. To obtain reliable results, the use of gastrostomy tube and blood trough level monitoring are highly recommended. Nonpharmaceutical immunosuppression such as thymic intervention and the use of severe combined immunodeficient pigs have also been discussed.

**Keywords:** pig, experimental transplantation, immunosuppression, human cell therapy, regenerative medicine

#### **1. Introduction**

The number of preclinical studies conducted using pigs has been increasing, especially in the field of cell therapy [1]. The merits of using pigs include (1) size advantages that enable to mimic clinical procedures; (2) availability of various experimental pigs such as miniature, microminiature, and gene-engineered strains; and (3) worldwide trend of discouragement of using dogs as research models.

However, immunosuppressive treatment has not been established well in pigs. Initially, pigs were used as models for performing allogeneic organ transplantation; now, the hope is to use them as xenogeneic organ donors. In the latter case, immunosuppressive protocols have been designed for primate recipients, while the number of reports mentioning immunosuppressive protocols for xenogeneic transplantation in pigs is unexpectedly few. In addition, insufficient medications are occasionally found in studies conducted by researchers who are not accustomed to organ transplantation.

We summarize here the pharmaceutical immunosuppressive regimens in experimental transplantation of organs, tissues, and cells in pigs (**Table 1**), as well as highlight the usefulness of thymic intervention and severe combined immunodeficient (SCID) pigs. Finally, we propose an appropriate introductory protocol that will fit human cell and tissue transplantation.


*Abbreviations: half maximal inhibitory concentration (IC50); major histocompatibility complex (MHC); cyclosporine (Cys); tacrolimus (Tac); azathioprine (Aza); rapamycin (rap); mycophenolate mofetil (MMF); methylprednisolone (MP); prednisone (P); prednisolone (Pl); per os (po); intravenous injection (iv); intramuscular injection (im).*

**115**

*Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy*

The half maximal inhibitory concentration (IC50) of major immunosuppressants was reported to be higher in mitogen response of pig lymphocytes than that in human lymphocytes [2]. The IC50 of cyclosporine, tacrolimus, and azathioprine was 19.1 times [1.72 μg/ml (pig) vs. 0.09 μg/ml (human)], 13.0 times [2.99 ng/ml (pig) vs. 0.23 ng/ml (human)], and 11.0 times [1.43 μg/ml [pig] vs. 0.13 μg/ml (human)] higher in pigs than in human lymphocytes, respectively. The species differences decreased in case of rapamycin and mycophenolate mofetil (MMF); IC50 values of rapamycin and MMF were 2.28 times [2.05 ng/ml (pig) vs. 0.90 ng/ml (human)] and 1.45 times [10.75 ng/ml (pig) vs. 7.42 ng/ml (human)] higher in pigs than in humans, respectively. In contrast, the IC50 value of methylprednisolone in pigs was only 0.41 times [0.11 μg/ml (pig) vs. 0.27 μg/ml (human)] the value in humans. These results suggest that the blood concentration of calcineurin inhibitors should be kept higher in pigs than in humans to suppress blast formation of lymphocytes in

**3. Immunosuppressive medications in allogeneic transplantation**

effective immunosuppression with low side effects.

tion period (90 days) [6] or for the first 30 days [7].

of tacrolimus was kept at 5–15 ng/ml.

In allogeneic orthotopic small bowel transplantation, high-dose tacrolimus monotherapy and low-dose tacrolimus-MMF combination therapy were compared using Large White-Landrace pig strain as donors and recipients (both weighing approximately 26 kg) [3, 4]. Tacrolimus dose was controlled to keep trough at 15–25 ng/ml (high-dose group) or 5–15 ng/ml (low-dose group). The average dosage amount of tacrolimus in the high single dose group was 0.3 mg/kg/day intramuscularly from the day of operation to day 6 and 0.61 ± 0.26 mg/kg/day via gastrostomy after day 7. In the low-dose combination group, recipients were administered 0.1 mg/kg of tacrolimus intramuscularly on the day of operation and 0.43 ± 0.14 mg/kg/day (average) of tacrolimus and 10 mg/kg twice a day of MMF via gastrostomy. All recipients in the high single dose group died within 46 days, while 7 out of the 10 recipients in the low-dose combination group survived for more than 60 days; the nontreated controls died within 15 days [3]. The subgroup study of tacrolimus-MMF combined group revealed that the recipients with low trough level of tacrolimus showed better survival, suggesting that higher trough level increases side effects of infection [4]. In general, the protocol consisting of calcineurin inhibitors (cyclosporine or tacrolimus) and MMF suppresses the immune responses of T and B cells, respectively, and the combination treatment leads to

A pharmacokinetic study recommended the oral administration of 0.25 mg/kg of tacrolimus and 500 mg of MMF at 12 h intervals to pigs weighing 22–30 kg [5]. MMF dose was calculated to be around 20 mg/kg at each administration. The trough level

When orthotopic forelimb transplantation was performed between outbred pigs weighing 13–24 kg, recipients were administered cyclosporine or tacrolimus and MMF orally once a day [6, 7]. The desired trough levels were 100–300 ng/ml in case of cyclosporine and 3–8 ng/ml for tacrolimus. A total of 500 mg of MMF was administered, i.e., 21–38 mg/kg. They also used steroids; 500 mg of methylprednisolone was injected intravenously during the operation, and 2.0 mg/kg/day of prednisone was given on the first postoperative day and then tapered by 0.5 mg/kg/ day every 3 days to a maintenance dose of 0.1 mg/kg/day until the end of observa-

*DOI: http://dx.doi.org/10.5772/intechopen.89521*

**immunosuppressants in vitro**

transplantation studies.

**2. Role of species differences in the effectiveness of** 

#### **Table 1.**

*Summary of immunosuppressive protocols.*

*Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy DOI: http://dx.doi.org/10.5772/intechopen.89521*

#### **2. Role of species differences in the effectiveness of immunosuppressants in vitro**

*Xenotransplantation - Comprehensive Study*

In vitro culture, IC50 estimation [2]

[5]

[6, 7]

Orthotopic allogeneic small bowel transplantation [3, 4]

Trough level determination

Orthotopic allogeneic forelimb transplantation

Orthotopic allogeneic (Class I disparate) kidney transplantation [8–10]

Heterotopic allogeneic (Class I disparate) heart transplantation [11]

Immortalized human hepatocytes transplantation

Human bone marrow mesenchymal stem cell transplantation to myocardium [14]

Human umbilical mesenchymal stem cell transplantation to cardiac

muscle [15, 16]

Human iPS cell-derived cardiomyocyte sheet to endocardium [18]

Human ES cell-derived retinal sheet to retina [19]

Orthotopic allogeneic (Class I disparate) kidney transplantation [20]

Human hepatocyte transplantation to spleen

Human artificial vascular tubes for neck arteriovenous shunt [22]

*Summary of immunosuppressive protocols.*

[21]

to liver [13]

will fit human cell and tissue transplantation.

We summarize here the pharmaceutical immunosuppressive regimens in experimental transplantation of organs, tissues, and cells in pigs (**Table 1**), as well as highlight the usefulness of thymic intervention and severe combined immunodeficient (SCID) pigs. Finally, we propose an appropriate introductory protocol that

Unknown Cys, Tac, Aza, Rap, MMF, MP

kg × 2/day, po

P (tapered)

ml) [10]

Landrace, 6 kg Tac 0.5 mg/kg/day, im

Tac 0.43 ± 0.14 mg/kg/day, po (keep trough 5–15 ng/ml), MMF 10 mg/kg × 2/day, po

Tac 0.25 mg/kg × 2/day, po, MMF 20 mg/

100–300 ng/ml) or Tac 1.5 mg/kg/day, po (keep trough 4–8 ng/ml), MMF 500 mg/day, MP and

MMF 1.5 g × 2 /day, iv or Cys 10–13 mg/kg/day,

Cys 4–10 mg/kg/day, po, 1–2/day, some cases

Cys 40 mg/kg/day, po (keep trough

Cys 10–13 mg/kg/day, iv (keep trough 400–800 ng/ml) [8, 9] or Tac 0.15 or 0.30 mg/ kg/day, iv (keep trough 20–40 or 45–80 ng/

iv (keep trough 400–800 ng/ml)

Cys 5 mg/kg/day, route unknown

Thymectomy and donor thymus

Thymectomy and splenectomy, and Tac 0.5 mg/ kg/day, MMF 60 mg/kg/day, prednisolone

treated with steroids

Minipig, 20–25 kg Tac 0.75 mg/kg/day, MMF 500 mg/day, Pl 20 mg/day

transplantation

20 mg/day, po

Neonatal thymectomy

Yucatan minipig Cys, po, details unknown

**Study design Pigs Immunosuppression**

Large White × Landrace, 26.4 ± 4.7 kg

Outbred farm pig, 13–24 kg (6–8-week-old)

MHC-defined miniature swine, 5–7-month-old

MHC-defined miniature swine, 3–6-month-old

Domestic pig, 35–40 kg

unknown

MHC-defined miniature swine, 3–6-month-old

Microminiature pig, 13–16-month-old

Göttingen minipigs, 6–7-month-old, ≥15 kg

*Abbreviations: half maximal inhibitory concentration (IC50); major histocompatibility complex (MHC); cyclosporine (Cys); tacrolimus (Tac); azathioprine (Aza); rapamycin (rap); mycophenolate mofetil (MMF); methylprednisolone (MP); prednisone (P); prednisolone (Pl); per os (po); intravenous injection (iv); intramuscular injection (im).*

Yorkshire, size and age

22–30 kg

Yorkshire × Landrace,

**114**

**Table 1.**

The half maximal inhibitory concentration (IC50) of major immunosuppressants was reported to be higher in mitogen response of pig lymphocytes than that in human lymphocytes [2]. The IC50 of cyclosporine, tacrolimus, and azathioprine was 19.1 times [1.72 μg/ml (pig) vs. 0.09 μg/ml (human)], 13.0 times [2.99 ng/ml (pig) vs. 0.23 ng/ml (human)], and 11.0 times [1.43 μg/ml [pig] vs. 0.13 μg/ml (human)] higher in pigs than in human lymphocytes, respectively. The species differences decreased in case of rapamycin and mycophenolate mofetil (MMF); IC50 values of rapamycin and MMF were 2.28 times [2.05 ng/ml (pig) vs. 0.90 ng/ml (human)] and 1.45 times [10.75 ng/ml (pig) vs. 7.42 ng/ml (human)] higher in pigs than in humans, respectively. In contrast, the IC50 value of methylprednisolone in pigs was only 0.41 times [0.11 μg/ml (pig) vs. 0.27 μg/ml (human)] the value in humans. These results suggest that the blood concentration of calcineurin inhibitors should be kept higher in pigs than in humans to suppress blast formation of lymphocytes in transplantation studies.

#### **3. Immunosuppressive medications in allogeneic transplantation**

In allogeneic orthotopic small bowel transplantation, high-dose tacrolimus monotherapy and low-dose tacrolimus-MMF combination therapy were compared using Large White-Landrace pig strain as donors and recipients (both weighing approximately 26 kg) [3, 4]. Tacrolimus dose was controlled to keep trough at 15–25 ng/ml (high-dose group) or 5–15 ng/ml (low-dose group). The average dosage amount of tacrolimus in the high single dose group was 0.3 mg/kg/day intramuscularly from the day of operation to day 6 and 0.61 ± 0.26 mg/kg/day via gastrostomy after day 7. In the low-dose combination group, recipients were administered 0.1 mg/kg of tacrolimus intramuscularly on the day of operation and 0.43 ± 0.14 mg/kg/day (average) of tacrolimus and 10 mg/kg twice a day of MMF via gastrostomy. All recipients in the high single dose group died within 46 days, while 7 out of the 10 recipients in the low-dose combination group survived for more than 60 days; the nontreated controls died within 15 days [3]. The subgroup study of tacrolimus-MMF combined group revealed that the recipients with low trough level of tacrolimus showed better survival, suggesting that higher trough level increases side effects of infection [4]. In general, the protocol consisting of calcineurin inhibitors (cyclosporine or tacrolimus) and MMF suppresses the immune responses of T and B cells, respectively, and the combination treatment leads to effective immunosuppression with low side effects.

A pharmacokinetic study recommended the oral administration of 0.25 mg/kg of tacrolimus and 500 mg of MMF at 12 h intervals to pigs weighing 22–30 kg [5]. MMF dose was calculated to be around 20 mg/kg at each administration. The trough level of tacrolimus was kept at 5–15 ng/ml.

When orthotopic forelimb transplantation was performed between outbred pigs weighing 13–24 kg, recipients were administered cyclosporine or tacrolimus and MMF orally once a day [6, 7]. The desired trough levels were 100–300 ng/ml in case of cyclosporine and 3–8 ng/ml for tacrolimus. A total of 500 mg of MMF was administered, i.e., 21–38 mg/kg. They also used steroids; 500 mg of methylprednisolone was injected intravenously during the operation, and 2.0 mg/kg/day of prednisone was given on the first postoperative day and then tapered by 0.5 mg/kg/ day every 3 days to a maintenance dose of 0.1 mg/kg/day until the end of observation period (90 days) [6] or for the first 30 days [7].

The Massachusetts General Hospital group uses genetically defined mini pigs, swine leukocyte antigen (SLA)gg (class Ic /class IId ) donors, and SLAdd (class Id /class IId ) recipients [8–11]. In orthotopic kidney transplantation, 10–13 mg/kg of cyclosporine once a day, administered with a catheter to the external jugular vein, kept the trough level at 400–800 ng/ml [8, 9]. The first 12-day administration resulted in the survival of well-functioning major histocompatibility complex (MHC) class I-disparate kidney grafts for over 90 days regardless of use of steroids. In another study [10], continuous intravenous injection of 0.15 or 0.30 mg/kg/day of tacrolimus treatment kept the drug level at 20–40 or 45–80 ng/ml, respectively, and the first 12-day administration resulted in well-functioning kidney grafts that survived for over 5 months. In addition, the high-dose regimen achieved successful engraftment of MHC class Ic /class IIc -mismatched kidney. The same group also compared the separate effect of cyclosporine and MMF on the survival of class I-disparate heterotopic heart graft [11]. The treatment protocol of cyclosporine was the same as above [8] and MMF was administered at 1.5 g twice a day through a catheter into the external jugular vein to keep the trough level at 3–5 μg/ml. The survival days of the test heart grafts were 53 ± 7.5 days (mean ± SD) and over 124 days in cyclosporine and MMF groups, respectively. The graft vascular changes were also mild in the MMF group.

#### **4. Immunosuppressive medications in xenogeneic transplantation**

Because pigs are hoped to be a xenogeneic donor, the major objectives in pig experiments include establishing xenogeneic antigen-free pigs and developing strategies for long-term survival in nonhuman primates [12]. In such studies, immunosuppression is almost equivalent to human clinics using tacrolimus, MMF, and antibody remedies. Pig recipients in xenotransplantation appear in the preclinical studies of human cell and tissue therapy.

In a short-term experiment of intrahepatic transplantation of human hepatocyte cell line, 0.5 mg/kg of tacrolimus was injected intramuscularly for 7 days [13]. When pigs received xenogeneic human-lined hepatocytes, the recipients survived D-galactosamine-induced hepatic injury. Human albumin appeared in the recipient serum 2 days after transplantation but disappeared at day 7, suggesting that the cells survived only for a few days.

Intramyocardial transplantation of mesenchymal stem cells had been actively investigated in not only basic research but also clinical practice. Because of the size advantage, pigs were used for preclinical studies to explore proof of concept by mimicking clinical procedure. Human bone marrow-derived mesenchymal stem cells labeled with radioactive indium (111In) were transplanted in porcine myocardium via catheter inserted from a femoral artery and traced by whole body scanning for 6 days [14]. Recipient pigs were orally treated with 5 mg/kg of cyclosporine from 3 days before to 6 days after cell transplantation. Immunosuppressed pigs retained the radioactivity far longer than nontreated controls. In another study, pigs received human umbilical mesenchymal stem cells in artificial cardiac infarct area and were administered 5 mg/kg of cyclosporine orally twice a day, from the day before to 8 weeks after cell transplantation [15]. In a similar study, 10 mg/kg of cyclosporine was administered orally, twice a day from 3 days before to 8 weeks after the cell transplantation [16]. As a more sophisticated approach, cardiomyocyte sheet transplantation is being undertaken [17, 18]. When cell sheets consisting of human induced pluripotent stem (iPS) cell-derived myocytes were transplanted onto the epicardium of minipigs (weighing 20–25 kg) mimicking clinical trial, 0.75 mg/kg/day of tacrolimus, 500 mg/day of MMF, and 20 mg/day of prednisolone were administered from 5 days before to 8 weeks after the transplantation [18].

**117**

*Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy*

Transplantation of human embryonic stem cell-derived retinal pigmented epithelium was also performed in pigs [19]. Although immunosuppression was precarious, cyclosporine was added in the feed and their blood level 2 h after administration was only 1 pg/ml, and the graft tissue was detectable after 3 months. Because retina is known as an immune-privileged site, additional immunosuppres-

**5. Nonpharmaceutical immunosuppression: thymic intervention and** 

Studies on genetically defined minipigs by Massachusetts General Hospital group emphasize the role of thymus in the establishment and maintenance of immunological tolerance. As quoted above [8], the 12-day administration of cyclosporine induced the long-term engraftment of MHC class I-disparate kidneys, but not if the thymus was removed. They also stated that old recipients tend to be difficult to establish tolerance [9]. These observations lead to the concept of tolerance induction by thymic transplantation. In this, unlike conventional thymic tissue transplantation, the donor thymus is transplanted by vascular anastomosis that assures immediate and perfect function of the thymus. Three weeks after the complete removal of thymus, the recipient was transplanted with MHC fully-disparate donor thymus in the neck region and infused continuously with 0.15 mg/kg/day of tacrolimus (trough level, 30–40 ng/ml) for 12 days. After 3–4 months, the recipient accepted a kidney from the thymus donor without

Previously, we reported the effectiveness of thymectomy on the acceptance of xenogeneic human hepatocytes and artificial vascular tubes [21, 22]. Upon hepatocyte transplantation, the blood human albumin levels were higher in neonatallythymectomized microminiature pigs than in nonthymectomized controls [21]. In another study, thymus and spleen were removed from Göttingen minipigs aged 6–7 months (≥ 15 kg), followed by the administration of 0.5 mg/kg/day of tacrolimus, 60 mg/kg/day of MMF, and 20 mg/day of prednisolone [22]. Seven days after the removal, an artificial vascular tube made from human fibroblasts was transplanted in between a carotid artery and a jugular vein to form an arteriovenous shunt. While the shunt was obstructed completely by thrombus 2 weeks after the operation in pigs without the removal of thymus and spleen, the shunt was functional in pigs with thymectomy and splenectomy, even though the immunosuppres-

Finally, we would like to refer briefly to the availability of SCID pigs in preclinical study on human cell therapy. According to a well-constructed review [23], there are 11 SCID pig strains so far; one was naturally found and others were genetically modified. The mutated genes in these strains are *ARTEMIS* (a gene encoding a nuclear protein that is involved in V(D)J recombination and DNA repair), *interleukin 2 receptor gamma chain (IL2RG)*, *recombination-activating genes (RAG)1*, and *RAG2*. Three strains have double mutations, namely *RAG1* and *2*, *RAG2* and *IL2RG*, and *ARTEMIS* and *IL2RG*. In accordance with gene function, each strain lacks specific immune-competent cell lineages such as T, B, and natural killer (NK) cells. Human cell transplant experiments were reported as iPS cell teratoma formation [24] and ovarian cancer engraftment [25], both of which did not focus on preclinical study on human cell therapy. SCID pigs need the highest antibacterial care because of their vulnerability to infection. Indeed, they have been reported to survive for only 6 months at the longest [23]. In addition, as general features of mutant pigs, there are diversities in phenotypic severity and small litter size. If

*DOI: http://dx.doi.org/10.5772/intechopen.89521*

sion may not be necessary.

immunosuppression [20].

sive treatment administered was equal.

**SCID pigs**

*Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy DOI: http://dx.doi.org/10.5772/intechopen.89521*

Transplantation of human embryonic stem cell-derived retinal pigmented epithelium was also performed in pigs [19]. Although immunosuppression was precarious, cyclosporine was added in the feed and their blood level 2 h after administration was only 1 pg/ml, and the graft tissue was detectable after 3 months. Because retina is known as an immune-privileged site, additional immunosuppression may not be necessary.

#### **5. Nonpharmaceutical immunosuppression: thymic intervention and SCID pigs**

Studies on genetically defined minipigs by Massachusetts General Hospital group emphasize the role of thymus in the establishment and maintenance of immunological tolerance. As quoted above [8], the 12-day administration of cyclosporine induced the long-term engraftment of MHC class I-disparate kidneys, but not if the thymus was removed. They also stated that old recipients tend to be difficult to establish tolerance [9]. These observations lead to the concept of tolerance induction by thymic transplantation. In this, unlike conventional thymic tissue transplantation, the donor thymus is transplanted by vascular anastomosis that assures immediate and perfect function of the thymus. Three weeks after the complete removal of thymus, the recipient was transplanted with MHC fully-disparate donor thymus in the neck region and infused continuously with 0.15 mg/kg/day of tacrolimus (trough level, 30–40 ng/ml) for 12 days. After 3–4 months, the recipient accepted a kidney from the thymus donor without immunosuppression [20].

Previously, we reported the effectiveness of thymectomy on the acceptance of xenogeneic human hepatocytes and artificial vascular tubes [21, 22]. Upon hepatocyte transplantation, the blood human albumin levels were higher in neonatallythymectomized microminiature pigs than in nonthymectomized controls [21]. In another study, thymus and spleen were removed from Göttingen minipigs aged 6–7 months (≥ 15 kg), followed by the administration of 0.5 mg/kg/day of tacrolimus, 60 mg/kg/day of MMF, and 20 mg/day of prednisolone [22]. Seven days after the removal, an artificial vascular tube made from human fibroblasts was transplanted in between a carotid artery and a jugular vein to form an arteriovenous shunt. While the shunt was obstructed completely by thrombus 2 weeks after the operation in pigs without the removal of thymus and spleen, the shunt was functional in pigs with thymectomy and splenectomy, even though the immunosuppressive treatment administered was equal.

Finally, we would like to refer briefly to the availability of SCID pigs in preclinical study on human cell therapy. According to a well-constructed review [23], there are 11 SCID pig strains so far; one was naturally found and others were genetically modified. The mutated genes in these strains are *ARTEMIS* (a gene encoding a nuclear protein that is involved in V(D)J recombination and DNA repair), *interleukin 2 receptor gamma chain (IL2RG)*, *recombination-activating genes (RAG)1*, and *RAG2*. Three strains have double mutations, namely *RAG1* and *2*, *RAG2* and *IL2RG*, and *ARTEMIS* and *IL2RG*. In accordance with gene function, each strain lacks specific immune-competent cell lineages such as T, B, and natural killer (NK) cells. Human cell transplant experiments were reported as iPS cell teratoma formation [24] and ovarian cancer engraftment [25], both of which did not focus on preclinical study on human cell therapy. SCID pigs need the highest antibacterial care because of their vulnerability to infection. Indeed, they have been reported to survive for only 6 months at the longest [23]. In addition, as general features of mutant pigs, there are diversities in phenotypic severity and small litter size. If

*Xenotransplantation - Comprehensive Study*

swine leukocyte antigen (SLA)gg (class Ic

/class IIc

cal studies of human cell and tissue therapy.

survived only for a few days.

IId

MHC class Ic

The Massachusetts General Hospital group uses genetically defined mini pigs,

) recipients [8–11]. In orthotopic kidney transplantation, 10–13 mg/kg of cyclosporine once a day, administered with a catheter to the external jugular vein, kept the trough level at 400–800 ng/ml [8, 9]. The first 12-day administration resulted in the survival of well-functioning major histocompatibility complex (MHC) class I-disparate kidney grafts for over 90 days regardless of use of steroids. In another study [10], continuous intravenous injection of 0.15 or 0.30 mg/kg/day of tacrolimus treatment kept the drug level at 20–40 or 45–80 ng/ml, respectively, and the first 12-day administration resulted in well-functioning kidney grafts that survived for over 5 months. In addition, the high-dose regimen achieved successful engraftment of

rate effect of cyclosporine and MMF on the survival of class I-disparate heterotopic heart graft [11]. The treatment protocol of cyclosporine was the same as above [8] and MMF was administered at 1.5 g twice a day through a catheter into the external jugular vein to keep the trough level at 3–5 μg/ml. The survival days of the test heart grafts were 53 ± 7.5 days (mean ± SD) and over 124 days in cyclosporine and MMF groups, respectively. The graft vascular changes were also mild in the MMF group.

**4. Immunosuppressive medications in xenogeneic transplantation**

Because pigs are hoped to be a xenogeneic donor, the major objectives in pig experiments include establishing xenogeneic antigen-free pigs and developing strategies for long-term survival in nonhuman primates [12]. In such studies, immunosuppression is almost equivalent to human clinics using tacrolimus, MMF, and antibody remedies. Pig recipients in xenotransplantation appear in the preclini-

In a short-term experiment of intrahepatic transplantation of human hepatocyte

Intramyocardial transplantation of mesenchymal stem cells had been actively investigated in not only basic research but also clinical practice. Because of the size advantage, pigs were used for preclinical studies to explore proof of concept by mimicking clinical procedure. Human bone marrow-derived mesenchymal stem cells labeled with radioactive indium (111In) were transplanted in porcine myocardium via catheter inserted from a femoral artery and traced by whole body scanning for 6 days [14]. Recipient pigs were orally treated with 5 mg/kg of cyclosporine from 3 days before to 6 days after cell transplantation. Immunosuppressed pigs retained the radioactivity far longer than nontreated controls. In another study, pigs received human umbilical mesenchymal stem cells in artificial cardiac infarct area and were administered 5 mg/kg of cyclosporine orally twice a day, from the day before to 8 weeks after cell transplantation [15]. In a similar study, 10 mg/kg of cyclosporine was administered orally, twice a day from 3 days before to 8 weeks after the cell transplantation [16]. As a more sophisticated approach, cardiomyocyte sheet transplantation is being undertaken [17, 18]. When cell sheets consisting of human induced pluripotent stem (iPS) cell-derived myocytes were transplanted onto the epicardium of minipigs (weighing 20–25 kg) mimicking clinical trial, 0.75 mg/kg/day of tacrolimus, 500 mg/day of MMF, and 20 mg/day of prednisolone were administered from 5 days before to 8 weeks after the transplantation [18].

cell line, 0.5 mg/kg of tacrolimus was injected intramuscularly for 7 days [13]. When pigs received xenogeneic human-lined hepatocytes, the recipients survived D-galactosamine-induced hepatic injury. Human albumin appeared in the recipient serum 2 days after transplantation but disappeared at day 7, suggesting that the cells

/class IId


) donors, and SLAdd (class Id

/class

**116**

these difficulties can be overcome, SCID pigs will be useful experimental animals for preclinical study on human cell therapy.

#### **6. Conclusion**

Due to the lack of identifiable sign of rejection, immunosuppression in cell transplant experiment is hard to control. Successful protocol is established only based on case-by-case experiences. Here, we suggest an introductory regimen of immunosuppression in human cell and tissue transplantation into pigs using tacrolimus and MMF. Preliminary doses are 0.5 mg/kg of tacrolimus orally and 40 mg/kg of MMF orally, and the administration should start 3 and 5 days before transplantation, respectively. Drugs can be administered by mixing in the powdered feed before transplantation; however, after transplantation, it should be given through a gastrostomy tube to assure the dosage in order to not be affected by appetite. Periodical examination of drug trough levels is indispensable and should be reflected in subsequent dose. Steroids should be carefully tested because their immunosuppressive dose has a risk of side effects such as gastrointestinal ulcer and systemic over immunosuppression. In addition, unavailability of exogenous steroid monitoring makes dosage control difficult. If surgical skill is available, the combination of thymectomy and splenectomy is recommended. Since the graft mass of cell and tissue transplantation is far smaller and not fully vascularized than organs, the recipients may need less immunosuppression. Data accumulation and optimization are desired in this field.

#### **Acknowledgements**

The authors acknowledge Dr. Kazuaki Nakano of Meiji University School of Agriculture for providing current information about genetically engineered SCID pigs.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Shin Enosawa\* and Eiji Kobayashi Department of Organ Fabrication, Keio School of Medicine, Tokyo, Japan

\*Address all correspondence to: enosawa-s@ncchd.go.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy*

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[5] Jensen-Waern M, Kruse R,

Lundgren T. Oral immunosuppressive medication for growing pigs in transplantation studies. Laboratory Animals. 2012;**46**:148-151. DOI:

[6] Ustüner ET, Zdichavsky M, Ren X, Edelstein J, Maldonado C, Ray M, et al. Long-term composite tissue allograft survival in a porcine model with cyclosporine/mycophenolate mofetil therapy. Transplantation.

expanim.17-0086

**References**

*Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy DOI: http://dx.doi.org/10.5772/intechopen.89521*

#### **References**

*Xenotransplantation - Comprehensive Study*

**6. Conclusion**

tion are desired in this field.

**Acknowledgements**

**Conflict of interest**

**Author details**

Shin Enosawa\* and Eiji Kobayashi

provided the original work is properly cited.

The authors declare no conflict of interest.

\*Address all correspondence to: enosawa-s@ncchd.go.jp

SCID pigs.

for preclinical study on human cell therapy.

these difficulties can be overcome, SCID pigs will be useful experimental animals

Due to the lack of identifiable sign of rejection, immunosuppression in cell transplant experiment is hard to control. Successful protocol is established only based on case-by-case experiences. Here, we suggest an introductory regimen of immunosuppression in human cell and tissue transplantation into pigs using tacrolimus and MMF. Preliminary doses are 0.5 mg/kg of tacrolimus orally and 40 mg/kg of MMF orally, and the administration should start 3 and 5 days before transplantation, respectively. Drugs can be administered by mixing in the powdered feed before transplantation; however, after transplantation, it should be given through a gastrostomy tube to assure the dosage in order to not be affected by appetite. Periodical examination of drug trough levels is indispensable and should be reflected in subsequent dose. Steroids should be carefully tested because their immunosuppressive dose has a risk of side effects such as gastrointestinal ulcer and systemic over immunosuppression. In addition, unavailability of exogenous steroid monitoring makes dosage control difficult. If surgical skill is available, the combination of thymectomy and splenectomy is recommended. Since the graft mass of cell and tissue transplantation is far smaller and not fully vascularized than organs, the recipients may need less immunosuppression. Data accumulation and optimiza-

The authors acknowledge Dr. Kazuaki Nakano of Meiji University School of Agriculture for providing current information about genetically engineered

Department of Organ Fabrication, Keio School of Medicine, Tokyo, Japan

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

**118**

[1] Kobayashi E, Hanazono Y, Kunita S. Swine used in the medical university: Overview of 20 years of experience. Experimental Animals. 2018;**67**:7-13. DOI: 10.1538/ expanim.17-0086

[2] Wright DC, Deol HS, Tuch BE. A comparison of the sensitivity of pig and human peripheral blood mononuclear cells to the antiproliferative effects of traditional and newer immunosuppressive agents. Transplant Immunology. 1999;**7**:141-147

[3] Alessiani M, Spada M, Dionigi P, Arbustini E, Regazzi M, Fossati GS, et al. Combined immunosuppressive therapy with tacrolimus and mycophenolate mofetil for small bowel transplantation in pigs. Transplantation. 1996;**62**:563-567

[4] Spada M, Alessiani M, Ferrari P, Iacona I, Abbiati F, Viezzoli A, et al. Tacrolimus and mycophenolate mofetil in pig small bowel transplantation: Different protocols and their outcome. Transplantation Proceedings. 1997;**29**:1819-1820

[5] Jensen-Waern M, Kruse R, Lundgren T. Oral immunosuppressive medication for growing pigs in transplantation studies. Laboratory Animals. 2012;**46**:148-151. DOI: 10.1258/la.2012.011152

[6] Ustüner ET, Zdichavsky M, Ren X, Edelstein J, Maldonado C, Ray M, et al. Long-term composite tissue allograft survival in a porcine model with cyclosporine/mycophenolate mofetil therapy. Transplantation. 1998;**66**:1581-1587

[7] Jones JW Jr, Ustüner ET, Zdichavsky M, Edelstein J, Ren X, Maldonado C, et al. Long-term survival of an extremity composite tissue allograft with FK506-mycophenolate

mofetil therapy. Surgery. 1999;**126**:384-388

[8] Yamada K, Gianello PR, Ierino FL, Lorf T, Shimizu A, Meehan S, et al. Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts. The Journal of Experimental Medicine. 1997;**186**:497-506

[9] Yamada K, Gianello PR, Ierino FL, Fishbein J, Lorf T, Shimizu A, et al. Role of the thymus in transplantation tolerance in miniature swine: II. Effect of steroids and age on the induction of tolerance to class I mismatched renal allografts. Transplantation. 1999;**67**:458-467

[10] Utsugi R, Barth RN, Lee RS, Kitamura H, LaMattina JC, Ambroz J, et al. Induction of transplantation tolerance with a short course of tacrolimus (FK506): I. rapid and stable tolerance to two-haplotype fully mhc-mismatched kidney allografts in miniature swine. Transplantation. 2001;**71**:1368-1379

[11] Schwarze ML, Houser SL, Muniappan A, Allan JS, Menard MT, McMorrow I, et al. Effects of mycophenolate mofetil on cardiac allograft survival and cardiac allograft vasculopathy in miniature swine. The Annals of Thoracic Surgery. 2005;**80**:1787-1793

[12] Sekijima M, Waki S, Sahara H, Tasaki M, Wilkinson RA, Villani V, et al. Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout swine. Transplantation. 2014;**98**:419-426. DOI: 10.1097/TP.0000000000000314

[13] Totsugawa T, Yong C, Rivas-Carrillo JD, Soto-Gutierrez A, Navarro-Alvarez N, Noguchi H, et al. Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with tamoxifenmediated self-recombination. Journal of Hepatology. 2007;**47**:74-82

[14] Lyngbaek S, Ripa RS, Haack-Sørensen M, Cortsen A, Kragh L, Andersen CB, et al. Serial in vivo imaging of the porcine heart after percutaneous, intramyocardially injected 111In-labeled human mesenchymal stromal cells. The International Journal of Cardiovascular Imaging. 2010;**26**:273-284. DOI: 10.1007/s10554-009-9532-4

[15] Ghodsizad A, Niehaus M, Kögler G, Martin U, Wernet P, Bara C, et al. Transplanted human cord bloodderived unrestricted somatic stem cells improve left-ventricular function and prevent left-ventricular dilation and scar formation after acute myocardial infarction. Heart. 2009;**95**:27-35. DOI: 10.1136/hrt.2007.139329

[16] Gahremanpour A, Vela D, Zheng Y, Silva GV, Fodor W, Cardoso CO, et al. Xenotransplantation of human unrestricted somatic stem cells in a pig model of acute myocardial infarction. Xenotransplantation. 2013;**20**:110-122. DOI: 10.1111/xen.12026

[17] Kawamura M, Miyagawa S, Miki K, Saito A, Fukushima S, Higuchi T, et al. Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a porcine ischemic cardiomyopathy model. Circulation. 2012;**126**:S29-S37

[18] Kawamura M, Miyagawa S, Fukushima S, Saito A, Miki K, Ito E, et al. Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart. Circulation. 2013;**128**:S87-S94. DOI: 10.1161/ CIRCULATIONAHA.112.000366

[19] Brant Fernandes RA, Koss MJ, Falabella P, Stefanini FR, Maia M, Diniz B, et al. An innovative surgical technique for subretinal transplantation of human embryonic stem cellderived retinal pigmented epithelium in Yucatan mini pigs: Preliminary results. Ophthalmic Surgery, Lasers & Imaging Retina. 2016;**47**:342-351. DOI: 10.3928/23258160-20160324-07

[20] Kamano C, Vagefi PA, Kumagai N, Yamamoto S, Barth RN, LaMattina JC, et al. Vascularized thymic lobe transplantation in miniature swine: Thymopoiesis and tolerance induction across fully MHC-mismatched barriers. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**:3827-3832

[21] Hsu HC, Enosawa S, Yamazaki T, Tohyama S, Fujita J, Fukuda K, et al. Enhancing survival of human hepatocytes by neonatal thymectomy and partial hepatectomy in microminiature pigs. Transplantation Proceedings. 2017;**49**:153-158. DOI: 10.1016/j.transproceed.2016.11.023

[22] Itoh M, Mukae Y, Kitsuka T, Arai K, Nakamura A, Uchihashi K, et al. Development of an immunodeficient pig model allowing long-term accommodation of artificial human vascular tubes. Nature Communications. 2019;**10**:2244. DOI: 10.1038/s41467-019-10107-1

[23] Boettcher AN, Loving CL, Cunnick JE, Tuggle CK. Development of severe combined immunodeficient (SCID) pig models for translational cancer modeling: Future insights on how humanized SCID pigs can improve preclinical cancer research. Frontiers in Oncology. 2018;**8**:559. DOI: 10.3389/ fonc.2018.00559

[24] Lee K, Kwon DN, Ezashi T, Choi YJ, Park C, Ericsson AC, et al. Engraftment of human iPS cells and allogeneic porcine cells into pigs with

**121**

*Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy*

*DOI: http://dx.doi.org/10.5772/intechopen.89521*

inactivated RAG2 and accompanying severe combined immunodeficiency. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**:7260-7265. DOI:

[25] Boettcher AN, Kiupel M, Adur MK, Cocco E, Santin AD, Bellone S, et al. Human ovarian cancer tumor formation in severe combined immunodeficient (SCID) pigs. Frontiers in Oncology. 2019;**9**:9. DOI: 10.3389/fonc.2019.00009

10.1073/pnas.1406376111

*Controllable Immunosuppression in Pigs as a Basis for Preclinical Studies on Human Cell Therapy DOI: http://dx.doi.org/10.5772/intechopen.89521*

inactivated RAG2 and accompanying severe combined immunodeficiency. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**:7260-7265. DOI: 10.1073/pnas.1406376111

*Xenotransplantation - Comprehensive Study*

[19] Brant Fernandes RA, Koss MJ, Falabella P, Stefanini FR, Maia M, Diniz B, et al. An innovative surgical technique for subretinal transplantation

of human embryonic stem cellderived retinal pigmented epithelium in Yucatan mini pigs: Preliminary results. Ophthalmic Surgery, Lasers & Imaging Retina. 2016;**47**:342-351. DOI:

10.3928/23258160-20160324-07

Kumagai N, Yamamoto S, Barth RN, LaMattina JC, et al. Vascularized thymic lobe transplantation in miniature swine: Thymopoiesis and tolerance induction across fully MHC-mismatched barriers. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**:3827-3832

[21] Hsu HC, Enosawa S, Yamazaki T, Tohyama S, Fujita J, Fukuda K, et al. Enhancing survival of human

hepatocytes by neonatal thymectomy and partial hepatectomy in microminiature pigs. Transplantation Proceedings. 2017;**49**:153-158. DOI: 10.1016/j.transproceed.2016.11.023

[22] Itoh M, Mukae Y, Kitsuka T,

Arai K, Nakamura A, Uchihashi K, et al. Development of an immunodeficient pig model allowing long-term accommodation of artificial human vascular tubes. Nature

Communications. 2019;**10**:2244. DOI:

Cunnick JE, Tuggle CK. Development of severe combined immunodeficient (SCID) pig models for translational cancer modeling: Future insights on how humanized SCID pigs can improve preclinical cancer research. Frontiers in Oncology. 2018;**8**:559. DOI: 10.3389/

10.1038/s41467-019-10107-1

fonc.2018.00559

[23] Boettcher AN, Loving CL,

[24] Lee K, Kwon DN, Ezashi T, Choi YJ, Park C, Ericsson AC, et al. Engraftment of human iPS cells and allogeneic porcine cells into pigs with

[20] Kamano C, Vagefi PA,

Navarro-Alvarez N, Noguchi H, et al. Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with tamoxifenmediated self-recombination. Journal of

[14] Lyngbaek S, Ripa RS, Haack-Sørensen M, Cortsen A, Kragh L, Andersen CB, et al. Serial in vivo imaging of the porcine heart after percutaneous, intramyocardially injected 111In-labeled human mesenchymal stromal cells. The

International Journal of Cardiovascular

Kögler G, Martin U, Wernet P, Bara C, et al. Transplanted human cord bloodderived unrestricted somatic stem cells improve left-ventricular function and prevent left-ventricular dilation and scar formation after acute myocardial infarction. Heart. 2009;**95**:27-35. DOI:

[16] Gahremanpour A, Vela D, Zheng Y, Silva GV, Fodor W, Cardoso CO, et al. Xenotransplantation of human

unrestricted somatic stem cells in a pig model of acute myocardial infarction. Xenotransplantation. 2013;**20**:110-122.

[17] Kawamura M, Miyagawa S, Miki K, Saito A, Fukushima S, Higuchi T, et al. Feasibility, safety, and therapeutic efficacy of human induced pluripotent stem cell-derived cardiomyocyte sheets in a porcine ischemic cardiomyopathy model. Circulation. 2012;**126**:S29-S37

[18] Kawamura M, Miyagawa S, Fukushima S, Saito A, Miki K, Ito E, et al. Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart. Circulation. 2013;**128**:S87-S94. DOI: 10.1161/ CIRCULATIONAHA.112.000366

Imaging. 2010;**26**:273-284. DOI: 10.1007/s10554-009-9532-4

[15] Ghodsizad A, Niehaus M,

10.1136/hrt.2007.139329

DOI: 10.1111/xen.12026

Hepatology. 2007;**47**:74-82

**120**

[25] Boettcher AN, Kiupel M, Adur MK, Cocco E, Santin AD, Bellone S, et al. Human ovarian cancer tumor formation in severe combined immunodeficient (SCID) pigs. Frontiers in Oncology. 2019;**9**:9. DOI: 10.3389/fonc.2019.00009

**123**

**Chapter 8**

**Abstract**

pluripotent stem cells

scaffold for the regeneration of human organs.

**1. Introduction**

A Novel Strategy for

Xeno-Regenerative Therapy

*Toshinari Fujimoto, Takashi Yokoo and Eiji Kobayashi*

**Keywords:** kidney regeneration, xeno-embryonic kidney transplantation, organogenic niche method, nephron progenitor cell replacement system, induced

Currently, the only definitive treatment for end-stage organ failure is transplantation. However, the global scarcity of organs is a critical challenge, necessitating that novel alternatives be developed. Xenotransplantation is a revolutionary therapy that can supply organs stably. In recent years, gene editing techniques, such as CRISPR/Cas9, have been developed to produce animals that generate organs at low risk of rejection and infection. Given that our understanding of xenogenic immune barriers has expanded, xenotransplantation may have a promising outlook. Presently, the strong antigenicity of xenogenic organs is the main barrier to xenotransplantation and has resulted in the development of methods of xeno-embryonic transplantation that use less antigenic organs. Although embryonic organs are prematurely transplanted, they can mature in vivo in a self-sustaining manner to perform their function. Xeno-embryonic organs, therefore, have some utility as a

Regenerative medicine is anticipated to be a promising alternative when tackling the problem of a shortfall in organ availability. Recent advances in stem cell research have enabled the reproduction of miniature organs called organoids, which are derived in vitro from human induced pluripotent stem cells (iPSCs). However, we

The shortage of organs for transplantation is of critical importance worldwide. Xenotransplantation or xeno-embryonic organ transplantation can stably supply organs and is considered to be an established alternative treatment. Regenerative medicine is another option, and recent advances in stem cell research have enabled the reproduction of miniature organs, called organoids, derived in vitro from human induced pluripotent stem cells. However, the in vitro production of large and complex organs that can efficiently function in vivo is not yet accomplished. We proposed a novel strategy for xenotransplantation in which a chimeric kidney is constructed by injecting human nephron progenitor cells into a porcine embryonic kidney, thereby eliminating pig nephron progenitor cells and allowing transplantation into a human and long-term survival. In this chapter, we discussed advantages and pitfalls of xenotransplantation and xeno-embryonic kidney transplantation. Recent attempts of human organoids and blastocyst complementation were reviewed. Finally, we proposed our novel xeno-regenerative therapeutic strategy.

#### **Chapter 8**

## A Novel Strategy for Xeno-Regenerative Therapy

*Toshinari Fujimoto, Takashi Yokoo and Eiji Kobayashi*

#### **Abstract**

The shortage of organs for transplantation is of critical importance worldwide. Xenotransplantation or xeno-embryonic organ transplantation can stably supply organs and is considered to be an established alternative treatment. Regenerative medicine is another option, and recent advances in stem cell research have enabled the reproduction of miniature organs, called organoids, derived in vitro from human induced pluripotent stem cells. However, the in vitro production of large and complex organs that can efficiently function in vivo is not yet accomplished. We proposed a novel strategy for xenotransplantation in which a chimeric kidney is constructed by injecting human nephron progenitor cells into a porcine embryonic kidney, thereby eliminating pig nephron progenitor cells and allowing transplantation into a human and long-term survival. In this chapter, we discussed advantages and pitfalls of xenotransplantation and xeno-embryonic kidney transplantation. Recent attempts of human organoids and blastocyst complementation were reviewed. Finally, we proposed our novel xeno-regenerative therapeutic strategy.

**Keywords:** kidney regeneration, xeno-embryonic kidney transplantation, organogenic niche method, nephron progenitor cell replacement system, induced pluripotent stem cells

#### **1. Introduction**

Currently, the only definitive treatment for end-stage organ failure is transplantation. However, the global scarcity of organs is a critical challenge, necessitating that novel alternatives be developed. Xenotransplantation is a revolutionary therapy that can supply organs stably. In recent years, gene editing techniques, such as CRISPR/Cas9, have been developed to produce animals that generate organs at low risk of rejection and infection. Given that our understanding of xenogenic immune barriers has expanded, xenotransplantation may have a promising outlook. Presently, the strong antigenicity of xenogenic organs is the main barrier to xenotransplantation and has resulted in the development of methods of xeno-embryonic transplantation that use less antigenic organs. Although embryonic organs are prematurely transplanted, they can mature in vivo in a self-sustaining manner to perform their function. Xeno-embryonic organs, therefore, have some utility as a scaffold for the regeneration of human organs.

Regenerative medicine is anticipated to be a promising alternative when tackling the problem of a shortfall in organ availability. Recent advances in stem cell research have enabled the reproduction of miniature organs called organoids, which are derived in vitro from human induced pluripotent stem cells (iPSCs). However, we

have not yet achieved in vitro reproduction of large and complex organs that function efficiently in vivo. Because kidneys comprise a three-dimensional and complex combination of various cell types that must perform homeostatic and endocrine functions, in vitro regeneration of the kidneys is particularly challenging compared with other organs. To overcome this challenge, we sought to use xeno-embryonic kidneys as a scaffold for development of human progenitor cells. By transplanting exogenous nephron progenitor cells (NPCs) into the metanephric mesenchyme of a xenogenic fetus, we aimed to regenerate whole neo-kidneys from the transplanted NPCs via their xenogenic development program. Specifically, we propose a novel xenotransplantation strategy wherein a chimeric kidney is constructed by injecting human NPCs into a porcine embryonic kidney and transplanted into a human after eliminating pig NPCs.

In this chapter, we discussed advantages and pitfalls of xenotransplantation and xeno-embryonic kidney transplantation. Recent attempts of human organoids and blastocyst complementation were reviewed. Finally, we proposed our novel xenoregenerative therapeutic strategy.

#### **2. Kidney xenotransplantation**

Xenotransplantation is a revolutionary therapy used to solve the problem of organ shortage. The concept has existed for more than 100 years, with the first kidney xenotransplantation performed in 1906. In this procedure, a pig kidney was heterotopically transplanted into a patient with renal failure but had to be removed after 3 days because of vessel thrombosis [1]. Subsequent attempts at renal xenografting failed, and the practice was abandoned. However, xenotransplantation reemerged as an option following the development of powerful immunosuppressive agents. In 1964, the kidney of a chimpanzee was successfully transplanted into a patient with renal failure and functioned for 9 months before the patient ultimately died of pneumonia [2]. Nonhuman primates were often used as a xenograft source at this time because the similarities between species produced good outcomes. However, this practice was abandoned because of the relative scarcity of nonhuman primate sources, concerns about disease transmission, and ethical issues. By contrast, pigs are almost limitlessly available as a transplant source, and their kidneys are similar in size and physiological function to those of humans. At present, porcine kidneys are therefore considered a suitable xenotransplantation source [3].

Nevertheless, using porcine kidneys in xenotransplantation presents some problems that must be addressed. The most critical problem is the presence of α-galactose-1,3-galactose (Gal). This galactose moiety is added to cell surface sugars in swine by α-1,3-galactosyltransferase (GalT), whereas primates, including humans, do not inherit GalT and possess anti-Gal antibodies as natural antibodies [4]. When a pig kidney is transplanted into a primate, anti-Gal antibodies bind to the Gal antigen expressed on porcine vascular endothelial cells, activate human complement, and cause hyperacute vascular rejection that immunosuppressants alone cannot prevent. Recently, genetically modified pigs with low risk of rejection potential were created with gene editing technology; a representative example is the GalT-knockout (KO) pig [5]. Given that these transgenic pigs do not express Gal, the anti-Gal antibody in primates does not react with them. Pigs expressing human complement regulatory proteins (e.g., human CD55 and CD46) that suppress human complement activation have also been reported [6, 7]. Other attempts have been made to overcome the risk of coagulation dysfunction by introducing human coagulation-regulatory genes, such as thrombomodulin, into pigs [8]. Moreover, pigs are now available that have various combinations of these genetic

**125**

compared to an adult graft [23].

*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

from infection due to strong immunosuppression [12].

**3. Xeno-embryonic kidney transplantation**

a scaffold for the regeneration of human organs.

be complete [10].

was produced [16].

modifications, and research is ongoing as to the optimal combination for transplantation. Actually, it is technically possible to produce pigs with multiple KO genes and multiple transgenes simultaneously [9, 10]. One such example is the double gene KO pig including GalT-KO that expresses three human complement regulatory genes and two anti-inflammatory genes; however, this combination might not yet

Improved immunosuppressive regimens are also contributing to the progress seen in xenotransplantation of pig kidneys. There has been particular interest in blockade inhibiting the CD40/CD154 pathway, with anti-CD40 or anti-CD154 antibody therapy contributing significantly to a prolongation of renal xenograft survival [11, 12]. In 2019, a GalT-KO kidney expressing CD55 was transplanted from pig to rhesus macaque and had the longest survival of life-sustaining xenograft to date (499 days) [13]. However, although hyperacute rejection by Gal antibodies has been largely overcome, late antibody-mediated injury by non-Gal antibodies remains a problem. Another problem that needs to be resolved is recipient death

The possibility of zoonotic infection cannot be ignored in xenotransplantation. Given that pigs can be bred in pathogen-free environments, the risk of acquiring zoonotic infections is lower than that of primates. However, the risk of porcine endogenous retrovirus (PERV) that integrates along chromosomes cannot be removed by this approach. In previous research, it was reported that PERV can infect human cells in vitro [14]. In 2015, CRISPR/Cas9 succeeded in knocking out 62 copies of the PERV pol gene in porcine cells [15], and in 2017, a PERV-free pig

As shown in this section, the measures taken against rejection and infection mean that kidney xenotransplantation is rapidly approaching clinical reality.

The use of xeno-embryonic transplantation may broaden the organ pool. This approach seems to benefit from a lower risk of rejection compared with adult organ transplantation, making a potentially invaluable therapeutic resource. Although an embryonic organ is transplanted prematurely, it can mature in a self-sustaining way in vivo to become functional. Xeno-embryonic organs may be particularly useful as

Metanephroi have generally been used for embryonic kidney transplantation [17] because this form is already committed to becoming a kidney. When transplanted into a recipient, the metanephroi is free to differentiate and mature into a whole kidney. The transplanted metanephroi promotes angiogenesis, encouraging host blood vessel infiltrating, thus resulting in glomeruli that are composed of host-derived vasculature [18]. The developed metanephroi produces urine, and anastomosing the ureter of the metanephroi and the ureter of the host has been shown to prolong the survival time of host anephric rats [19]. Moreover, the developed metanephroi acquires endocrine function, producing both renin and erythropoietin [20, 21]. Conveniently, the metanephroi is a fetal organ that may have low immunogenicity, potentially making it especially suitable for transplantation. Contrasting with adult grafts that already have the donor vessels, the avascular metanephroi is only vascularized by host vessels after it is transplanted. Thus, humoral immunity to donor endothelial cells is less likely to occur when using the metanephroi for transplantation [22]. Additionally, we can expect a reduced expression of donor antigens, such as HLA class I and II, on a developing metanephroi graft when

#### *A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

*Xenotransplantation - Comprehensive Study*

eliminating pig NPCs.

regenerative therapeutic strategy.

**2. Kidney xenotransplantation**

have not yet achieved in vitro reproduction of large and complex organs that function efficiently in vivo. Because kidneys comprise a three-dimensional and complex combination of various cell types that must perform homeostatic and endocrine functions, in vitro regeneration of the kidneys is particularly challenging compared with other organs. To overcome this challenge, we sought to use xeno-embryonic kidneys as a scaffold for development of human progenitor cells. By transplanting exogenous nephron progenitor cells (NPCs) into the metanephric mesenchyme of a xenogenic fetus, we aimed to regenerate whole neo-kidneys from the transplanted NPCs via their xenogenic development program. Specifically, we propose a novel xenotransplantation strategy wherein a chimeric kidney is constructed by injecting human NPCs into a porcine embryonic kidney and transplanted into a human after

In this chapter, we discussed advantages and pitfalls of xenotransplantation and xeno-embryonic kidney transplantation. Recent attempts of human organoids and blastocyst complementation were reviewed. Finally, we proposed our novel xeno-

Xenotransplantation is a revolutionary therapy used to solve the problem of organ shortage. The concept has existed for more than 100 years, with the first kidney xenotransplantation performed in 1906. In this procedure, a pig kidney was heterotopically transplanted into a patient with renal failure but had to be removed after 3 days because of vessel thrombosis [1]. Subsequent attempts at renal xenografting failed, and the practice was abandoned. However, xenotransplantation reemerged as an option following the development of powerful immunosuppressive agents. In 1964, the kidney of a chimpanzee was successfully transplanted into a patient with renal failure and functioned for 9 months before the patient ultimately died of pneumonia [2]. Nonhuman primates were often used as a xenograft source at this time because the similarities between species produced good outcomes. However, this practice was abandoned because of the relative scarcity of nonhuman primate sources, concerns about disease transmission, and ethical issues. By contrast, pigs are almost limitlessly available as a transplant source, and their kidneys are similar in size and physiological function to those of humans. At present, porcine kidneys are therefore considered a suitable xenotransplantation source [3]. Nevertheless, using porcine kidneys in xenotransplantation presents some problems that must be addressed. The most critical problem is the presence of α-galactose-1,3-galactose (Gal). This galactose moiety is added to cell surface sugars in swine by α-1,3-galactosyltransferase (GalT), whereas primates, including humans, do not inherit GalT and possess anti-Gal antibodies as natural antibodies [4]. When a pig kidney is transplanted into a primate, anti-Gal antibodies bind to the Gal antigen expressed on porcine vascular endothelial cells, activate human complement, and cause hyperacute vascular rejection that immunosuppressants alone cannot prevent. Recently, genetically modified pigs with low risk of rejection potential were created with gene editing technology; a representative example is the GalT-knockout (KO) pig [5]. Given that these transgenic pigs do not express Gal, the anti-Gal antibody in primates does not react with them. Pigs expressing human complement regulatory proteins (e.g., human CD55 and CD46) that suppress human complement activation have also been reported [6, 7]. Other attempts have been made to overcome the risk of coagulation dysfunction by introducing human coagulation-regulatory genes, such as thrombomodulin, into pigs [8]. Moreover, pigs are now available that have various combinations of these genetic

**124**

modifications, and research is ongoing as to the optimal combination for transplantation. Actually, it is technically possible to produce pigs with multiple KO genes and multiple transgenes simultaneously [9, 10]. One such example is the double gene KO pig including GalT-KO that expresses three human complement regulatory genes and two anti-inflammatory genes; however, this combination might not yet be complete [10].

Improved immunosuppressive regimens are also contributing to the progress seen in xenotransplantation of pig kidneys. There has been particular interest in blockade inhibiting the CD40/CD154 pathway, with anti-CD40 or anti-CD154 antibody therapy contributing significantly to a prolongation of renal xenograft survival [11, 12]. In 2019, a GalT-KO kidney expressing CD55 was transplanted from pig to rhesus macaque and had the longest survival of life-sustaining xenograft to date (499 days) [13]. However, although hyperacute rejection by Gal antibodies has been largely overcome, late antibody-mediated injury by non-Gal antibodies remains a problem. Another problem that needs to be resolved is recipient death from infection due to strong immunosuppression [12].

The possibility of zoonotic infection cannot be ignored in xenotransplantation. Given that pigs can be bred in pathogen-free environments, the risk of acquiring zoonotic infections is lower than that of primates. However, the risk of porcine endogenous retrovirus (PERV) that integrates along chromosomes cannot be removed by this approach. In previous research, it was reported that PERV can infect human cells in vitro [14]. In 2015, CRISPR/Cas9 succeeded in knocking out 62 copies of the PERV pol gene in porcine cells [15], and in 2017, a PERV-free pig was produced [16].

As shown in this section, the measures taken against rejection and infection mean that kidney xenotransplantation is rapidly approaching clinical reality.

#### **3. Xeno-embryonic kidney transplantation**

The use of xeno-embryonic transplantation may broaden the organ pool. This approach seems to benefit from a lower risk of rejection compared with adult organ transplantation, making a potentially invaluable therapeutic resource. Although an embryonic organ is transplanted prematurely, it can mature in a self-sustaining way in vivo to become functional. Xeno-embryonic organs may be particularly useful as a scaffold for the regeneration of human organs.

Metanephroi have generally been used for embryonic kidney transplantation [17] because this form is already committed to becoming a kidney. When transplanted into a recipient, the metanephroi is free to differentiate and mature into a whole kidney. The transplanted metanephroi promotes angiogenesis, encouraging host blood vessel infiltrating, thus resulting in glomeruli that are composed of host-derived vasculature [18]. The developed metanephroi produces urine, and anastomosing the ureter of the metanephroi and the ureter of the host has been shown to prolong the survival time of host anephric rats [19]. Moreover, the developed metanephroi acquires endocrine function, producing both renin and erythropoietin [20, 21].

Conveniently, the metanephroi is a fetal organ that may have low immunogenicity, potentially making it especially suitable for transplantation. Contrasting with adult grafts that already have the donor vessels, the avascular metanephroi is only vascularized by host vessels after it is transplanted. Thus, humoral immunity to donor endothelial cells is less likely to occur when using the metanephroi for transplantation [22]. Additionally, we can expect a reduced expression of donor antigens, such as HLA class I and II, on a developing metanephroi graft when compared to an adult graft [23].

The ultimate size of the developed metanephroi appears to be imprinted during the early stages of embryonic development. Considering human clinical application, pigs are an ideal resource for metanephric transplantation as with adult kidney xenotransplantation. In the case of allogenic porcine transplantation, the metanephroi on embryonic day 28 (E28) has been successfully transplanted into a nonimmunosuppressed recipient pig and shown to differentiate into a mature kidney without rejection [24]. Allogenic adult kidney grafts are easily rejected without immunosuppression. Transplants originating from pig embryos on E27 to E28 all exhibited significant growth and full differentiation, while those harvested on E20 and E25 failed to develop and only differentiated into few glomeruli and tubules, together with other derivatives, such as blood vessels, cartilage, and bone [25]. This indicates that metanephroi that are too immature may be incompletely pre-programmed and may differentiate into nonrenal structures. However, agedependent graft growth and survival in allogenic rats was shown to be optimal from E15 and worsened progressively for metanephroi obtained on E16 to E21. The developed metanephroi obtained on E15 showed maturation of renal elements and no sign of rejection, whereas those obtained on E20 had a poor renal architecture and a dense lymphocytic infiltrate [26]. Importantly, there appears to be an optimal window for harvesting metanephroi to obtain good transplantation outcomes.

Successful xeno-metanephric transplantation has been reported previously. In an important study, E28 pig metanephroi or adult kidneys were transplanted into recipient rats with and without immunosuppression. Those transplanted into nonimmunosuppressed rats showed tissue rejection, whereas those transplanted into hosts treated with CTLA-4-Ig underwent growth and differentiation. By contrast, adult kidney grafts showed disturbed morphology, necrotic tissue, and a high degree of lymphocyte infiltration, even when hosts were treated with CTLA-4-Ig [25].

The immune advantage of metanephroi over developed adult kidneys has been demonstrated by direct comparison of xenotransplantation into host animals treated with immunosuppressants. Next, it will be necessary to study the xenotransplantation of pig metanephroi into nonhuman primates.

In the case of xenotransplantation of pig islets, embryonic islet tissues are regarded as a choice for xenotransplantation with several advantages including reduced immunogenicity, long-term proliferative potential, and revascularization by host endothelium. However, the embryonic implants exhibit a delayed insulin response to glucose in vivo (>3 months) and limited effect on improvement of blood glucose level [27]. Fetal and neonatal pig islets have the higher expression of GAL and will be more susceptible to xenorejection than adult pig islets [28, 29]. Therefore, adult pig is regarded as the primary donor source of islet xenografts, which can supply an adequate amount of viable islet cells and start functioning immediately after implantation.

#### **4. Kidney organoids derived from pluripotent stem cells (PSCs)**

The field of stem cell research is growing at a rapid pace. The reproduction of organoids derived from human iPSCs in vitro is already possible in several organs, including the optic cup, intestines, and liver [30–32]. Although embryologic kidney development is complicated, the reproduction of kidney organoids has been reported. Kidneys arise from metanephroi, which develop via the reciprocal interaction between the metanephric mesenchyme, containing NPCs and stromal progenitor cells, and the ureteric bud. Takasato et al. reported simultaneously inducing metanephric mesenchyme and ureteral buds from human iPSCs to

**127**

*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

**5. Blastocyst complementation**

organs in pancreas-deficient pigs [39].

a challenge before this research has translational potential.

produce kidney organoids. The generated organoids contained nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells [33]. Taguchi et al. also reported the successful differentiation of human iPSCs into NPCs and ureteric buds in vitro, by repeating the development of the metanephric kidney [34, 35]. Additionally, they reconstructed kidney organoids with higher-order structures, containing embryonic branching morphogenesis, by reaggregating NPCs and ureteric buds derived from mouse PSCs and stromal progenitor cells from mouse embryos. However, a method for differentiating human iPSCs into stromal progenitor cells is yet to be established. Furthermore, neither of the developed kidney organoids have a urine drainage system, and both are too small to function in vivo. Therefore, generating functional kidneys in vitro remains

As an alternative to in vitro directed differentiation of iPSCs, previous studies have considered methods of regenerating solid organs from transplanted exogenous cells to function in vivo by borrowing a xenogenic development program. One such method is blastocyst complementation. When PSCs are transplanted into blastocysts, which are early animal embryos, chimeras containing blastocysts and PSCs are formed. When PSCs are injected into blastocysts that have undergone genetic manipulation not to generate a target organ, the missing organ is formed from the injected PSCs by systemic chimera formation. Using the method of blastocyst complementation, kidneys derived from mouse iPSCs have been regenerated in sall1 knockout mice that lack kidneys [36]. Successful kidney regeneration has also been derived from mouse iPSCs in sall1 knockout rats [37]. Therefore, this generation mechanism appears to have interspecies compatibility. The renal lineage cells were derived from the injected PSCs, whereas nonrenal lineages such as blood vessels and stromal cells in kidneys were chimeric for both blastocyst cells and PSCs. Recently, mouse PSC-derived vascular endothelial cells were regenerated into *Flk-1* knockout mice, lacking a key gene for vascular endothelial development [38]. By simultaneously disrupting Flk-1 and genes required for genesis of the target organ, rejection-free organs could be generated from patient-specific iPSCs. The size of the regenerated organ will be affected by the size of the host animal blastocyst. Successful allogenic blastocyst complementation has been shown to regenerate large

Given that human iPSCs fundamentally lack the ability to form chimeras, blastocyst complementation cannot be applied directly to humans. Inducing the expression of anti-apoptotic genes could give some chimera-forming ability to human iPSCs [40, 41], but the long-term safety would require clarification because these are also recognized oncogenes. Another issue is that basing this method on systemic chimera formation leads to the serious ethical concern of chimera formation in host gametes or neural tissue other than the target organs. The introduction of the heterologous cells during insemination must be thoroughly considered for the loss of the personal identity of a living being [42]. If these problems can be resolved, such a method that can produce organs that function in vivo would be highly significant.

We have developed an organogenic niche method that utilizes a xenogenic development program. In this method, exogenous organ progenitor cells are transplanted

**6. Organogenic niche method and NPC replacement system**

*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

*Xenotransplantation - Comprehensive Study*

The ultimate size of the developed metanephroi appears to be imprinted during the early stages of embryonic development. Considering human clinical application, pigs are an ideal resource for metanephric transplantation as with adult kidney xenotransplantation. In the case of allogenic porcine transplantation, the metanephroi on embryonic day 28 (E28) has been successfully transplanted into a nonimmunosuppressed recipient pig and shown to differentiate into a mature kidney without rejection [24]. Allogenic adult kidney grafts are easily rejected without immunosuppression. Transplants originating from pig embryos on E27 to E28 all exhibited significant growth and full differentiation, while those harvested on E20 and E25 failed to develop and only differentiated into few glomeruli and tubules, together with other derivatives, such as blood vessels, cartilage, and bone [25]. This indicates that metanephroi that are too immature may be incompletely pre-programmed and may differentiate into nonrenal structures. However, agedependent graft growth and survival in allogenic rats was shown to be optimal from E15 and worsened progressively for metanephroi obtained on E16 to E21. The developed metanephroi obtained on E15 showed maturation of renal elements and no sign of rejection, whereas those obtained on E20 had a poor renal architecture and a dense lymphocytic infiltrate [26]. Importantly, there appears to be an optimal window for harvesting metanephroi to obtain good transplantation outcomes.

Successful xeno-metanephric transplantation has been reported previously. In an important study, E28 pig metanephroi or adult kidneys were transplanted into recipient rats with and without immunosuppression. Those transplanted into nonimmunosuppressed rats showed tissue rejection, whereas those transplanted into hosts treated with CTLA-4-Ig underwent growth and differentiation. By contrast, adult kidney grafts showed disturbed morphology, necrotic tissue, and a high degree of lymphocyte infiltration, even when hosts were treated with

The immune advantage of metanephroi over developed adult kidneys has been

demonstrated by direct comparison of xenotransplantation into host animals treated with immunosuppressants. Next, it will be necessary to study the xeno-

In the case of xenotransplantation of pig islets, embryonic islet tissues are regarded as a choice for xenotransplantation with several advantages including reduced immunogenicity, long-term proliferative potential, and revascularization by host endothelium. However, the embryonic implants exhibit a delayed insulin response to glucose in vivo (>3 months) and limited effect on improvement of blood glucose level [27]. Fetal and neonatal pig islets have the higher expression of GAL and will be more susceptible to xenorejection than adult pig islets [28, 29]. Therefore, adult pig is regarded as the primary donor source of islet xenografts, which can supply an adequate amount of viable islet cells and start functioning

**4. Kidney organoids derived from pluripotent stem cells (PSCs)**

The field of stem cell research is growing at a rapid pace. The reproduction of organoids derived from human iPSCs in vitro is already possible in several organs, including the optic cup, intestines, and liver [30–32]. Although embryologic kidney development is complicated, the reproduction of kidney organoids has been reported. Kidneys arise from metanephroi, which develop via the reciprocal interaction between the metanephric mesenchyme, containing NPCs and stromal progenitor cells, and the ureteric bud. Takasato et al. reported simultaneously inducing metanephric mesenchyme and ureteral buds from human iPSCs to

transplantation of pig metanephroi into nonhuman primates.

**126**

CTLA-4-Ig [25].

immediately after implantation.

produce kidney organoids. The generated organoids contained nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells [33]. Taguchi et al. also reported the successful differentiation of human iPSCs into NPCs and ureteric buds in vitro, by repeating the development of the metanephric kidney [34, 35]. Additionally, they reconstructed kidney organoids with higher-order structures, containing embryonic branching morphogenesis, by reaggregating NPCs and ureteric buds derived from mouse PSCs and stromal progenitor cells from mouse embryos. However, a method for differentiating human iPSCs into stromal progenitor cells is yet to be established. Furthermore, neither of the developed kidney organoids have a urine drainage system, and both are too small to function in vivo. Therefore, generating functional kidneys in vitro remains a challenge before this research has translational potential.

#### **5. Blastocyst complementation**

As an alternative to in vitro directed differentiation of iPSCs, previous studies have considered methods of regenerating solid organs from transplanted exogenous cells to function in vivo by borrowing a xenogenic development program. One such method is blastocyst complementation. When PSCs are transplanted into blastocysts, which are early animal embryos, chimeras containing blastocysts and PSCs are formed. When PSCs are injected into blastocysts that have undergone genetic manipulation not to generate a target organ, the missing organ is formed from the injected PSCs by systemic chimera formation. Using the method of blastocyst complementation, kidneys derived from mouse iPSCs have been regenerated in sall1 knockout mice that lack kidneys [36]. Successful kidney regeneration has also been derived from mouse iPSCs in sall1 knockout rats [37]. Therefore, this generation mechanism appears to have interspecies compatibility. The renal lineage cells were derived from the injected PSCs, whereas nonrenal lineages such as blood vessels and stromal cells in kidneys were chimeric for both blastocyst cells and PSCs. Recently, mouse PSC-derived vascular endothelial cells were regenerated into *Flk-1* knockout mice, lacking a key gene for vascular endothelial development [38]. By simultaneously disrupting Flk-1 and genes required for genesis of the target organ, rejection-free organs could be generated from patient-specific iPSCs. The size of the regenerated organ will be affected by the size of the host animal blastocyst. Successful allogenic blastocyst complementation has been shown to regenerate large organs in pancreas-deficient pigs [39].

Given that human iPSCs fundamentally lack the ability to form chimeras, blastocyst complementation cannot be applied directly to humans. Inducing the expression of anti-apoptotic genes could give some chimera-forming ability to human iPSCs [40, 41], but the long-term safety would require clarification because these are also recognized oncogenes. Another issue is that basing this method on systemic chimera formation leads to the serious ethical concern of chimera formation in host gametes or neural tissue other than the target organs. The introduction of the heterologous cells during insemination must be thoroughly considered for the loss of the personal identity of a living being [42]. If these problems can be resolved, such a method that can produce organs that function in vivo would be highly significant.

#### **6. Organogenic niche method and NPC replacement system**

We have developed an organogenic niche method that utilizes a xenogenic development program. In this method, exogenous organ progenitor cells are transplanted into the region of the xenogenic fetus where the target organ develops. The transplantation of progenitor cells into host tissue matched by developmental stage may be critical for efficient cell grafting. In our experiments, we first injected human mesenchymal stem cells (hMSC) expressing glial cell line-derived neurotrophic factor into the embryonic rat site where budding of the ureteric bud occurred. Second, the transplanted host rat embryo was grown in a whole-embryo culture system [43]. Third, the transplanted hMSCs were integrated into the metanephroi and differentiated into tubular epithelial cells, interstitial cells, and glomerular epithelial cells [44]. Fourth, we transplanted the developed metanephroi into recipient rats. Using this approach, the metanephroi integrated with the vessels of recipient rats and the vascularized nephrons (derived from hMSCs) regenerated. The neo-kidney derived from hMSCs also produced urine by filtering the host blood, and the level of urea nitrogen and creatinine in the urine was higher than that of the host serum [45], and it also secreted human erythropoietin in response to host anemia [46]. Thus, we successfully regenerated human cell-derived neo-kidneys with in vivo function. As described, instead of PSCs, we used stem cells or progenitor cells that have limited potency. These cells were only locally transplanted into embryos at mid-to-late gestational ages, thereby ensuring that chimera formation only occurs in the kidney and avoiding any potential ethical concerns.

Existing native host cells inhibit the engraftment of transplanted donor cells. We recently developed a new method combining an organogenic niche with eliminating host NPCs to increase the efficiency of donor cell engraftment [47]. In this method, we used transgenic mice in which the diphtheria toxin receptor (DTR) was specifically expressed on Six2-positive NPCs (Six2-iDTR transgenic mouse). Rodents such as mice and rats naturally lack the DTR, so Six2-positive NPCs selectively undergo apoptosis with the administration of diphtheria toxin. When donor mouse NPCs are transplanted into host mouse metanephroi, they became chimeric with the existing native host NPCs, and contribution rate of the donor cells was 30% of cap mesenchyme cells. Administering diphtheria toxin eliminated the host mouse NPCs and allowed 100% replacement with donor mouse NPCs that could generate neo-nephrons [47] (**Figure 1**). In this way, we succeeded in achieving full replacement with heterogeneous donor rat NPCs. Importantly, we revealed that nephrons derived from rat NPCs could connect to the host mouse collecting ducts, even when nephrons and collecting ducts were heterogeneous. Next, we examined the possibility of in vivo regeneration of interspecies kidneys

**Figure 1.**

*Schematic of the drug-induced cell elimination system to exchange native NPCs with exogenous NPCs.*

**129**

*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

resulting in functional neo-glomeruli filtration [48].

does not affect human cells (unpublished data).

**7. Stepwise peristaltic ureter system**

kidneys generated by stem cell methods.

urine excretion may be important for normal development.

using NPC replacement. In subsequent research, we successfully regenerated rat nephrons using the Six2-iDTR mouse metanephroi as a scaffold in recipient rats receiving immunosuppressive therapy. We showed that neo-kidneys were vascularized by blood vessels originating from the recipient rats using the species-specific antibody for detection. Furthermore, we injected fluorescent-labeled dextran into the recipient rats, and the accumulation of dextran in Bowman's space of neoglomeruli and in the lumen of neo-tubules was confirmed. Our findings confirmed that neo-kidneys were incorporated into blood circulatory system of recipients,

In the future, we aim to use this system of kidney regeneration with pig fetuses as the bioreactor and human iPSC-derived NPCs as the cell source. It is not difficult to supply a cell source because protocols for inducing NPCs from human iPSCs have already been developed [33, 34]; additionally, expansion of NPC culture is possible [49, 50]. A fundamental problem with our proposals is that human cells permanently express DTR and can undergo apoptosis when treated with diphtheria toxin. Therefore, the DTR system cannot be applied directly to humans. Recently, we developed a new transgenic model to ablate NPCs using an alternative drug that

Although transplanted metanephroi can produce urine, they lack a urine excretion pathway, gradually become hydronephrotic, and cease functioning. Neo-kidneys regenerated using metanephroi as a bioreactor may also follow the same mechanism. Recently, we developed a urine excretion strategy for embryonic

The ultimate size of a metanephric graft is determined by the size of the source animal. Pigs are therefore considered a suitable resource from this perspective. To eliminate the potential for rejection, we transplanted metanephroi from cloned pig fetuses into syngenic hosts. All transplanted metanephroi differentiated successfully into mature kidneys, growing to 5–7 mm in length by 3 weeks. After 5 weeks, metanephroi grew to more than 1 cm and retained urine in the developed ureters, and after 8 weeks, they grew to about 3 cm and started to develop hydronephrosis as urine production increased [51]. Ureteral primordia start peristalsis during the embryonic stage and normally excrete urine into the bladder, and this sustained

To delay the onset of hydronephrosis and to promote the growth of metanephric

grafts, we transplanted metanephroi with ureters and a bladder (MNB) into a recipient animal. After 4 weeks, hydronephrosis occurred in the group with normal metanephroi transplants but not in the group with MNB transplants. In the MNB group, urine retention in the bladder was observed. Histopathologic examination also showed more pronounced tubular luminal dilation and interstitial fibrosis, and greater reductions in the number of glomeruli, in the metanephroi group than with the MNB group. Moreover, urine volumes and urinary levels of urea nitrogen and

creatinine were higher in the MNB group than in the metanephroi group.

Furthermore, we demonstrated the generation of a urine excretion channel in MNB by using the stepwise peristaltic ureter (SWPU) system. Briefly, at an appropriate time, we connected the host ureter to the MNB graft containing urine produced by the metanephroi. The SWPU system allowed for continuous urine drainage from the developed bladder of the MNB into the recipient bladder via the recipient ureter. Even 8 weeks after transplantation, the MNB showed no hydronephrosis and had maintained mature renal structures, such as glomeruli and renal

#### *A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

*Xenotransplantation - Comprehensive Study*

and avoiding any potential ethical concerns.

into the region of the xenogenic fetus where the target organ develops. The transplantation of progenitor cells into host tissue matched by developmental stage may be critical for efficient cell grafting. In our experiments, we first injected human mesenchymal stem cells (hMSC) expressing glial cell line-derived neurotrophic factor into the embryonic rat site where budding of the ureteric bud occurred. Second, the transplanted host rat embryo was grown in a whole-embryo culture system [43]. Third, the transplanted hMSCs were integrated into the metanephroi and differentiated into tubular epithelial cells, interstitial cells, and glomerular epithelial cells [44]. Fourth, we transplanted the developed metanephroi into recipient rats. Using this approach, the metanephroi integrated with the vessels of recipient rats and the vascularized nephrons (derived from hMSCs) regenerated. The neo-kidney derived from hMSCs also produced urine by filtering the host blood, and the level of urea nitrogen and creatinine in the urine was higher than that of the host serum [45], and it also secreted human erythropoietin in response to host anemia [46]. Thus, we successfully regenerated human cell-derived neo-kidneys with in vivo function. As described, instead of PSCs, we used stem cells or progenitor cells that have limited potency. These cells were only locally transplanted into embryos at mid-to-late gestational ages, thereby ensuring that chimera formation only occurs in the kidney

Existing native host cells inhibit the engraftment of transplanted donor cells. We recently developed a new method combining an organogenic niche with eliminating host NPCs to increase the efficiency of donor cell engraftment [47]. In this method, we used transgenic mice in which the diphtheria toxin receptor (DTR) was specifically expressed on Six2-positive NPCs (Six2-iDTR transgenic mouse). Rodents such as mice and rats naturally lack the DTR, so Six2-positive NPCs selectively undergo apoptosis with the administration of diphtheria toxin. When donor mouse NPCs are transplanted into host mouse metanephroi, they became chimeric with the existing native host NPCs, and contribution rate of the donor cells was 30% of cap mesenchyme cells. Administering diphtheria toxin eliminated the host mouse NPCs and allowed 100% replacement with donor mouse NPCs that could generate neo-nephrons [47] (**Figure 1**). In this way, we succeeded in achieving full replacement with heterogeneous donor rat NPCs. Importantly, we revealed that nephrons derived from rat NPCs could connect to the host mouse collecting ducts, even when nephrons and collecting ducts were heterogeneous. Next, we examined the possibility of in vivo regeneration of interspecies kidneys

*Schematic of the drug-induced cell elimination system to exchange native NPCs with exogenous NPCs.*

**128**

**Figure 1.**

using NPC replacement. In subsequent research, we successfully regenerated rat nephrons using the Six2-iDTR mouse metanephroi as a scaffold in recipient rats receiving immunosuppressive therapy. We showed that neo-kidneys were vascularized by blood vessels originating from the recipient rats using the species-specific antibody for detection. Furthermore, we injected fluorescent-labeled dextran into the recipient rats, and the accumulation of dextran in Bowman's space of neoglomeruli and in the lumen of neo-tubules was confirmed. Our findings confirmed that neo-kidneys were incorporated into blood circulatory system of recipients, resulting in functional neo-glomeruli filtration [48].

In the future, we aim to use this system of kidney regeneration with pig fetuses as the bioreactor and human iPSC-derived NPCs as the cell source. It is not difficult to supply a cell source because protocols for inducing NPCs from human iPSCs have already been developed [33, 34]; additionally, expansion of NPC culture is possible [49, 50]. A fundamental problem with our proposals is that human cells permanently express DTR and can undergo apoptosis when treated with diphtheria toxin. Therefore, the DTR system cannot be applied directly to humans. Recently, we developed a new transgenic model to ablate NPCs using an alternative drug that does not affect human cells (unpublished data).

#### **7. Stepwise peristaltic ureter system**

Although transplanted metanephroi can produce urine, they lack a urine excretion pathway, gradually become hydronephrotic, and cease functioning. Neo-kidneys regenerated using metanephroi as a bioreactor may also follow the same mechanism. Recently, we developed a urine excretion strategy for embryonic kidneys generated by stem cell methods.

The ultimate size of a metanephric graft is determined by the size of the source animal. Pigs are therefore considered a suitable resource from this perspective. To eliminate the potential for rejection, we transplanted metanephroi from cloned pig fetuses into syngenic hosts. All transplanted metanephroi differentiated successfully into mature kidneys, growing to 5–7 mm in length by 3 weeks. After 5 weeks, metanephroi grew to more than 1 cm and retained urine in the developed ureters, and after 8 weeks, they grew to about 3 cm and started to develop hydronephrosis as urine production increased [51]. Ureteral primordia start peristalsis during the embryonic stage and normally excrete urine into the bladder, and this sustained urine excretion may be important for normal development.

To delay the onset of hydronephrosis and to promote the growth of metanephric grafts, we transplanted metanephroi with ureters and a bladder (MNB) into a recipient animal. After 4 weeks, hydronephrosis occurred in the group with normal metanephroi transplants but not in the group with MNB transplants. In the MNB group, urine retention in the bladder was observed. Histopathologic examination also showed more pronounced tubular luminal dilation and interstitial fibrosis, and greater reductions in the number of glomeruli, in the metanephroi group than with the MNB group. Moreover, urine volumes and urinary levels of urea nitrogen and creatinine were higher in the MNB group than in the metanephroi group.

Furthermore, we demonstrated the generation of a urine excretion channel in MNB by using the stepwise peristaltic ureter (SWPU) system. Briefly, at an appropriate time, we connected the host ureter to the MNB graft containing urine produced by the metanephroi. The SWPU system allowed for continuous urine drainage from the developed bladder of the MNB into the recipient bladder via the recipient ureter. Even 8 weeks after transplantation, the MNB showed no hydronephrosis and had maintained mature renal structures, such as glomeruli and renal

tubules. The levels of urea nitrogen and creatinine were much higher in the urine from the MNB than in the sera of recipients. Finally, the SWPU system significantly prolonged the lifespan of anephric rats in the MNB group compared with the nontransplanted group.

In a previous study, researchers demonstrated that they could create a urinary pathway by directly connecting the ureter of the transplanted metanephroi to the ureter of the host (ureteroureterostomy) to prolong the short-term survival of anephric rats [19]. However, the SWPU system is more efficient than ureteroureterostomy in terms of preventing hydronephrosis and allowing maturation of the metanephroi. Surgery for the SWPU system is also easier than that for ureteroureterostomy because the bladder of the MNB expands with urinary retention. Furthermore, we can join two metanephroi to a host ureter using the SWPU system, whereas it is difficult to connect two metanephroi to the host ureter. In a previous study, it was reported that the survival time in anephric rats correlated with the total volume of the grown metanephroi [52]. It is possible that the SWPU system is more effective than the conventional method in prolonging survival time for this reason.

Assuming that MNBs can be used as a scaffold for kidney regeneration before transplantation into patients with renal failure, we investigated the effects of host renal failure on the structure and activity of the transplanted MNB. Uremic conditions were reproduced using a 5/6 renal infarction rat model, and 4 weeks after transplantation, the developed bladder was successfully anastomosed to the host ureter. At 8 weeks after transplantation, histological analysis showed the presence of mature glomeruli and tubules in the groups with and without renal failure. There were also no differences between these groups in terms of survival in anephric host rats, indicating that the grafts were responsible for prolonging host survival, even under renal failure conditions [53]. The results of this study demonstrate that a transplanted MNB can grow and function effectively, even under uremic conditions.

The use of MNB as a kidney regeneration scaffold can provide new treatment for patients with renal failure. We assume that the SWPU system will be applicable to human neo-kidneys regenerated via the NPC replacement system, using a pig MNB as a scaffold to establish the urinary excretion pathway. In brief, human iPSC-derived NPCs may be injected into the metanephroi of porcine fetuses that

**131**

**Conflict of interest**

*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

**failure**

**9. Conclusions**

main targets to rejection, could decrease antigenicity.

are genetically manipulated to have an NPC elimination system. Human nephrons may then regenerate in porcine metanephroi by eliminating the porcine NPCs and replacing them with human NPCs. The MNB that has human kidneys will be transplanted into patients with end-stage renal disease, and an excretion pathway will be constructed. In this case, the regenerated kidneys will be of human origin, but the ureters and bladder of MNB will be of porcine origin (**Figure 2**). Although further investigation is required, we assume that replacing nephrons, which are the

**8. Regenerative potential of iPSCs derived from patients with renal** 

with ESRD may still be a useful cell source for kidney regeneration.

the translation of our strategies to clinical settings.

The authors declare no conflict of interest.

The use of iPSCs generated from patients holds promise for tailored therapy that uses patient-derived cells, tissues, or organs. In clinical settings, it is desirable to use patient-derived iPSCs as the cell source for neo-kidneys to circumvent immune rejection. However, because uremia can reduce the function of stem cells, it may be problematic to use stem cells derived from patients in renal failure. Previous studies have shown that uremia causes many toxic effects, including reduced proliferation capacity, abnormalities of differentiation, and angiogenic dysfunction in stem cells [54, 55]. We previously reported that gene and protein expression of p300-/CBPassociated factor was significantly suppressed and that in vivo angiogenesis activation was decreased in hMSCs derived from patients with end-stage renal disease (ESRD) [56]. However, there have been no reports about the biological properties of iPSCs derived from patients with ESRD. In our recent study, iPSCs derived from patients with ESRD could differentiate into NPCs as efficiently as iPSCs derived from healthy controls. Moreover, NPCs derived from patients with ESRD showed the potential to become mature and vascularized nephrons in vivo, similar to the process in healthy control [57]. These findings suggest that iPSCs from patients

In this chapter, we have described several potential alternatives to allotransplantation, focusing on our novel xeno-regenerative therapeutic strategy for kidney regeneration. Although there are issues to be overcome with the treatment alternatives that are being developed, recent advances in genetic recombination technology and stem cell research may make them available in clinical practice. We have addressed the development of genetically modified pigs that possess an NPC elimination system and have performed experiments with NPCs derived from human iPSCs. To date, each step of our proposed strategy for kidney regeneration has been accomplished successfully in rodent models. This includes the regeneration of kidneys derived from transplanted NPCs via NPC replacement, the transplantation of regenerated kidneys into host animals, and the construction of a urine excretion pathway (i.e., the SWPU system). Looking to the future, we aim to complete a series of studies to allow transplantation from pig to human, which should facilitate

**Figure 2.**

*Schematic of our novel xeno-regenerative therapeutic strategy for kidney regeneration.*

*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

*Xenotransplantation - Comprehensive Study*

nontransplanted group.

tubules. The levels of urea nitrogen and creatinine were much higher in the urine from the MNB than in the sera of recipients. Finally, the SWPU system significantly prolonged the lifespan of anephric rats in the MNB group compared with the

In a previous study, researchers demonstrated that they could create a urinary pathway by directly connecting the ureter of the transplanted metanephroi to the ureter of the host (ureteroureterostomy) to prolong the short-term survival of anephric rats [19]. However, the SWPU system is more efficient than ureteroureterostomy in terms of preventing hydronephrosis and allowing maturation of the metanephroi. Surgery for the SWPU system is also easier than that for ureteroureterostomy because the bladder of the MNB expands with urinary retention. Furthermore, we can join two metanephroi to a host ureter using the SWPU system, whereas it is difficult to connect two metanephroi to the host ureter. In a previous study, it was reported that the survival time in anephric rats correlated with the total volume of the grown metanephroi [52]. It is possible that the SWPU system is more effective than the conventional method in prolonging survival time for this reason. Assuming that MNBs can be used as a scaffold for kidney regeneration before transplantation into patients with renal failure, we investigated the effects of host renal failure on the structure and activity of the transplanted MNB. Uremic conditions were reproduced using a 5/6 renal infarction rat model, and 4 weeks after transplantation, the developed bladder was successfully anastomosed to the host ureter. At 8 weeks after transplantation, histological analysis showed the presence of mature glomeruli and tubules in the groups with and without renal failure. There were also no differences between these groups in terms of survival in anephric host rats, indicating that the grafts were responsible for prolonging host survival, even under renal failure conditions [53]. The results of this study demonstrate that a transplanted MNB can grow and function effectively, even under uremic

The use of MNB as a kidney regeneration scaffold can provide new treatment for patients with renal failure. We assume that the SWPU system will be applicable to human neo-kidneys regenerated via the NPC replacement system, using a pig MNB as a scaffold to establish the urinary excretion pathway. In brief, human iPSC-derived NPCs may be injected into the metanephroi of porcine fetuses that

**130**

**Figure 2.**

conditions.

*Schematic of our novel xeno-regenerative therapeutic strategy for kidney regeneration.*

are genetically manipulated to have an NPC elimination system. Human nephrons may then regenerate in porcine metanephroi by eliminating the porcine NPCs and replacing them with human NPCs. The MNB that has human kidneys will be transplanted into patients with end-stage renal disease, and an excretion pathway will be constructed. In this case, the regenerated kidneys will be of human origin, but the ureters and bladder of MNB will be of porcine origin (**Figure 2**). Although further investigation is required, we assume that replacing nephrons, which are the main targets to rejection, could decrease antigenicity.

#### **8. Regenerative potential of iPSCs derived from patients with renal failure**

The use of iPSCs generated from patients holds promise for tailored therapy that uses patient-derived cells, tissues, or organs. In clinical settings, it is desirable to use patient-derived iPSCs as the cell source for neo-kidneys to circumvent immune rejection. However, because uremia can reduce the function of stem cells, it may be problematic to use stem cells derived from patients in renal failure. Previous studies have shown that uremia causes many toxic effects, including reduced proliferation capacity, abnormalities of differentiation, and angiogenic dysfunction in stem cells [54, 55]. We previously reported that gene and protein expression of p300-/CBPassociated factor was significantly suppressed and that in vivo angiogenesis activation was decreased in hMSCs derived from patients with end-stage renal disease (ESRD) [56]. However, there have been no reports about the biological properties of iPSCs derived from patients with ESRD. In our recent study, iPSCs derived from patients with ESRD could differentiate into NPCs as efficiently as iPSCs derived from healthy controls. Moreover, NPCs derived from patients with ESRD showed the potential to become mature and vascularized nephrons in vivo, similar to the process in healthy control [57]. These findings suggest that iPSCs from patients with ESRD may still be a useful cell source for kidney regeneration.

#### **9. Conclusions**

In this chapter, we have described several potential alternatives to allotransplantation, focusing on our novel xeno-regenerative therapeutic strategy for kidney regeneration. Although there are issues to be overcome with the treatment alternatives that are being developed, recent advances in genetic recombination technology and stem cell research may make them available in clinical practice. We have addressed the development of genetically modified pigs that possess an NPC elimination system and have performed experiments with NPCs derived from human iPSCs. To date, each step of our proposed strategy for kidney regeneration has been accomplished successfully in rodent models. This includes the regeneration of kidneys derived from transplanted NPCs via NPC replacement, the transplantation of regenerated kidneys into host animals, and the construction of a urine excretion pathway (i.e., the SWPU system). Looking to the future, we aim to complete a series of studies to allow transplantation from pig to human, which should facilitate the translation of our strategies to clinical settings.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Xenotransplantation - Comprehensive Study*

#### **Author details**

Toshinari Fujimoto1 , Takashi Yokoo1 \* and Eiji Kobayashi<sup>2</sup>

1 Division of Nephrology and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan

2 Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan

\*Address all correspondence to: tyokoo@jikei.ac.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**133**

*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

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anti-gal antibody with alpha-

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10.1002/mrd.20305

10.1038/srep29081

xen.12166

ajt.15329

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gene stacking and gene editing. Scientific Reports. 2016;**6**:29081. DOI:

Pre-transplant antibody screening and anti-CD154 costimulation

2015;**22**:221-230. DOI: 10.1111/

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#### **References**

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**132**

Japan

**Author details**

Toshinari Fujimoto1

, Takashi Yokoo1

The Jikei University School of Medicine, Tokyo, Japan

\*Address all correspondence to: tyokoo@jikei.ac.jp

provided the original work is properly cited.

\* and Eiji Kobayashi<sup>2</sup>

1 Division of Nephrology and Hypertension, Department of Internal Medicine,

2 Department of Organ Fabrication, Keio University School of Medicine, Tokyo,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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[3] Yamada K, Griesemer A, Okumi M. Pigs as xenogeneic donors. Transplantation Reviews. 2005;**19**:164- 177. DOI: 10.1016/j.trre.2005.10.004

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[7] Diamond LE et al. A human CD46 transgenic pig model system for the study of discordant xenotransplantation. Transplantation. 2001;**71**:132-142. DOI: 10.1097/00007890-200101150-00021

[8] Miwa Y et al. Potential value of human thrombomodulin and DAF expression for coagulation control in pig-to-human xenotransplantation. Xenotransplantation. 2010;**17**:26-37. DOI: 10.1111/j.1399-3089.2009.00555.x

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[12] Iwase H et al. Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation. 2017;**24**:e12293. DOI: 10.1111/xen.12293

[13] Kim SC et al. Long-term survival of pig-to-rhesus macaque renal xenografts is dependent on CD4 T cell depletion. American Journal of Transplantation. 2019;**19**(8):2174-2185. DOI: 10.1111/ ajt.15329

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[25] Dekel B et al. Human and porcine early kidney precursors as a new source for transplantation. Nature Medicine. 2003;**9**:53-60. DOI: 10.1038/nm812

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[28] Bennet W et al. A comparison of fetal and adult porcine islets with regard to gal alpha (1,3)gal expression and the role of human immunoglobulins and complement in islet cell cytotoxicity. Transplantation. 2000;**69**:1711-1717. DOI: 10.1097/00007890-200004270-00030

[29] Omori T et al. A study of the xenoantigenicity of neonatal porcine islet-like cell clusters (NPCC) and the efficiency of adenovirus-mediated DAF (CD55) expression. Xenotransplantation. 2006;**13**:455-464. DOI: 10.1111/j.1399-3089.2006.00335.x

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*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

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[41] Huang K et al. BMI1 enables interspecies chimerism with human pluripotent stem cells. Nature Communications. 2018;**9**:4649. DOI: 10.1038/s41467-018-07098-w

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DOI: 10.1006/meth.2001.1154

10.1073/pnas.0406878102

ASN.2005101043

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[46] Yokoo T et al. Generation of a transplantable erythropoietin-producer derived from human mesenchymal

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stem cells. Transplantation. 2008;**85**:1654-1658. DOI: 10.1097/

TP.0b013e318173a35d

electroporation. Methods. 2001;**24**:35-42.

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stem.2016.10.013

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DOI: 10.1073/pnas.1222902110

from human iPS cells contain multiple lineages and model human nephrogenesis. Nature. 2015;**526**:564-568.

DOI: 10.1038/nature15695

stem.2013.11.010

stem.2017.10.011

ajpath.2012.03.007

stemcr.2018.08.015

nature12271

*A Novel Strategy for Xeno-Regenerative Therapy DOI: http://dx.doi.org/10.5772/intechopen.89275*

*Xenotransplantation - Comprehensive Study*

2017;**357**:1303-1307. DOI: 10.1126/

of pig metanephroi. ASAIO Journal. 2003;**49**:48-52. DOI: 10.1097/01. MAT.0000044737.04648.F5

[25] Dekel B et al. Human and porcine early kidney precursors as a new source for transplantation. Nature Medicine. 2003;**9**:53-60. DOI: 10.1038/nm812

[26] Foglia RP et al. Fetal allograft survival in immunocompetent recipients is age dependent and organ specific. Annals of Surgery. 1986;**204**:402-410. DOI: 10.1097/00000658-198610000-00008

[27] Hecht G et al. Embryonic pig pancreatic tissue for the treatment of diabetes in a nonhuman primate model. Proceedings of the National Academy of Sciences of the United States of America. 2009;**106**:8659-8664. DOI:

[28] Bennet W et al. A comparison of fetal and adult porcine islets with regard to gal alpha (1,3)gal expression and the role of human immunoglobulins and complement in islet cell cytotoxicity. Transplantation. 2000;**69**:1711-1717. DOI: 10.1097/00007890-200004270-00030

[29] Omori T et al. A study of the xenoantigenicity of neonatal porcine islet-like cell clusters (NPCC) and the efficiency of adenovirus-mediated DAF (CD55) expression. Xenotransplantation.

2006;**13**:455-464. DOI:

nature09941

nature07935

10.1111/j.1399-3089.2006.00335.x

[30] Eiraku M et al. Self-organizing optic-cup morphogenesis in threedimensional culture. Nature. 2011;**472**:51-56. DOI: 10.1038/

[31] Sato T et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;**459**:262-265. DOI: 10.1038/

10.1073/pnas.0812253106

[17] Hammerman MR. Renal organogenesis from transplanted metanephric primordia. Journal of the American Society of Nephrology. 2004;**15**:1126-1132. DOI: 10.1097/01.

asn.0000106020.64930.64

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[19] Rogers SA, Hammerman MR. Prolongation of life in anephric rats following de novo renal organogenesis. Organogenesis. 2004;**1**:22-25. DOI:

[20] Yokote S et al. The effect of metanephros transplantation on blood pressure in anephric rats with induced acute hypotension. Nephrology, Dialysis, Transplantation. 2012;**27**:3449-3455.

Xenotransplanted embryonic kidney provides a niche for endogenous mesenchymal stem cell differentiation into erythropoietin-producing tissue. Stem Cells. 2012;**30**:1228-1235. DOI: 10.1002/stem.1101

Hammerman MR. Differential origin for endothelial and mesangial cells after transplantation of pig fetal renal primordia into rats. Transplant Immunology. 2006;**15**:211-215. DOI:

DOI: 10.1093/ndt/gfs006

[21] Matsumoto K et al.

[22] Takeda S, Rogers SA,

10.1016/j.trim.2005.10.003

[24] Rogers SA, Talcott M,

Hammerman MR. Transplantation

[23] Statter MB et al. Correlation of fetal kidney and testis congenic graft survival with reduced major histocompatibility complex burden. Transplantation. 1989;**47**:651-660. DOI: 10.1097/00007890-198904000-00017

10.4161/org.1.1.1009

science.aan4187

**134**

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[36] Usui J et al. Generation of kidney from pluripotent stem cells via blastocyst complementation. The American Journal of Pathology. 2012;**180**:2417-2426. DOI: 10.1016/j. ajpath.2012.03.007

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[41] Huang K et al. BMI1 enables interspecies chimerism with human pluripotent stem cells. Nature Communications. 2018;**9**:4649. DOI: 10.1038/s41467-018-07098-w

[42] Kobayashi E et al. Organ fabrication using pigs as an in vivo bioreactor. The Keio Journal of Medicine. [published online ahead of print August 6, 2019]. DOI: 10.2302/kjm.2019-0006-OA

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**137**

**Chapter 9**

**Abstract**

**1. Introduction**

wide problem.

Pigs as Models of Preclinical

Generation of Human Organs

*Yujiro Kawai, Shugo Tohyama, Hideyuki Shimizu,* 

*Keiichi Fukuda and Eiji Kobayashi*

animal models that accept human cells, tissues, or organs.

**Keywords:** regenerative therapy, transplant, bioreactor, immune tolerance

Organ transplantation is often the only possible treatment for a patient with organ failure. The organs are donated from either living or deceased donors, and thus the number of transplantable organs is limited and insufficient to meet the clinical demand. Consequently, some illegal or unethical transplantations along with transplant commercialism and tourism have emerged, representing a world-

The discovery of the potential of pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), to regenerate tissues or organs offers new hope to overcome this situation. Human ESCs [1] and iPSCs [2] are now widely used to generate tissues or organs, and techniques for the in vitro production of specific cell types have been developed [3]. However, these strategies still have several limitations for clinical application, including the size, maturity,

To solve these problems, large animal models for the transplantation of human PSC-derived cells, tissues, or organs are required. In this review, we summarize the animal models currently used in the development for xenotransplantation and highlight the efficacy and prospects of pig models to accept human tissues or organs.

function, and risk of tumor formation after transplantation [4].

Studies and In Vivo Bioreactors for

Pigs are valuable and essential large animal models for human medical applications, including for stem cell therapy. Moreover, substantial effort has been made to directly engraft genetically engineered pig organs in the human body and to use pigs as in vivo bioreactors for the growth and development of human cells, tissue, or organs. However, engraftment of human cells in pigs has not yet been achieved. Although severe combined immunodeficient pigs have been developed, which can accept human biological materials, these pigs do not have practical value at present owing to difficulty in their care. To overcome these current limitations, we have proposed the generation of operational immunodeficient pig models by simply removing the thymus and spleen, enabling the long-term accommodation of human tissue. In this review, we summarize research progress on xenotransplantation

#### **Chapter 9**

*Xenotransplantation - Comprehensive Study*

regeneration of interspecies chimeric kidneys using a nephron progenitor cell replacement system. Scientific Reports. 2019;**9**:6965. DOI: 10.1038/

[56] Yamanaka S et al. Adipose tissuederived mesenchymal stem cells in long-term dialysis patients display downregulation of PCAF expression and poor angiogenesis activation. PLoS One. 2014;**9**:e102311. DOI: 10.1371/

[57] Tajiri S et al. Regenerative potential of induced pluripotent stem cells derived from patients undergoing haemodialysis in kidney regeneration. Scientific Reports. 2018;**8**:14919. DOI: 10.1038/s41598-018-33256-7

journal.pone.0102311

[49] Li Z et al. 3D culture supports longterm expansion of mouse and human nephrogenic progenitors. Cell Stem Cell. 2016;**19**:516-529. DOI: 10.1016/j.

[50] Tanigawa S et al. Selective In vitro propagation of nephron progenitors derived from embryos and pluripotent stem cells. Cell Reports. 2016;**15**:801- 813. DOI: 10.1016/j.celrep.2016.03.07

[51] Yokote S et al. Urine excretion strategy

for stem cell-generated embryonic kidneys. Proceedings of the National Academy of Sciences of the United States of America. 2015;**112**:12980-12985. DOI: 10.1073/pnas.1507803112

[52] Marshall D et al. Increasing renal mass improves survival in anephric rats following metanephros transplantation. Experimental Physiology. 2007;**92**:263-271. DOI: 10.1113/expphysiol.2006.036319

10.1007/s10157-016-1337-x

[55] Klinkhammer BM et al. Mesenchymal stem cells from rats with chronic kidney disease exhibit premature senescence and loss of regenerative potential. PLoS One. 2014;**9**:e92115. DOI: 10.1371/journal.

[54] Noh H et al. Uremia induces functional incompetence of bone marrow-derived stromal cells.

Nephrology, Dialysis, Transplantation. 2012;**27**:218-225. DOI: 10.1093/ndt/

[53] Fujimoto E et al. Embryonic kidney function in a chronic renal failure model in rodents. Clinical and Experimental Nephrology. 2017;**21**:579-588. DOI:

[48] Fujimoto T et al. In vivo

s41598-019-43482-2

stem.2016.07.016

**136**

pone.0092115

gfr267

## Pigs as Models of Preclinical Studies and In Vivo Bioreactors for Generation of Human Organs

*Yujiro Kawai, Shugo Tohyama, Hideyuki Shimizu, Keiichi Fukuda and Eiji Kobayashi*

#### **Abstract**

Pigs are valuable and essential large animal models for human medical applications, including for stem cell therapy. Moreover, substantial effort has been made to directly engraft genetically engineered pig organs in the human body and to use pigs as in vivo bioreactors for the growth and development of human cells, tissue, or organs. However, engraftment of human cells in pigs has not yet been achieved. Although severe combined immunodeficient pigs have been developed, which can accept human biological materials, these pigs do not have practical value at present owing to difficulty in their care. To overcome these current limitations, we have proposed the generation of operational immunodeficient pig models by simply removing the thymus and spleen, enabling the long-term accommodation of human tissue. In this review, we summarize research progress on xenotransplantation animal models that accept human cells, tissues, or organs.

**Keywords:** regenerative therapy, transplant, bioreactor, immune tolerance

#### **1. Introduction**

Organ transplantation is often the only possible treatment for a patient with organ failure. The organs are donated from either living or deceased donors, and thus the number of transplantable organs is limited and insufficient to meet the clinical demand. Consequently, some illegal or unethical transplantations along with transplant commercialism and tourism have emerged, representing a worldwide problem.

The discovery of the potential of pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), to regenerate tissues or organs offers new hope to overcome this situation. Human ESCs [1] and iPSCs [2] are now widely used to generate tissues or organs, and techniques for the in vitro production of specific cell types have been developed [3]. However, these strategies still have several limitations for clinical application, including the size, maturity, function, and risk of tumor formation after transplantation [4].

To solve these problems, large animal models for the transplantation of human PSC-derived cells, tissues, or organs are required. In this review, we summarize the animal models currently used in the development for xenotransplantation and highlight the efficacy and prospects of pig models to accept human tissues or organs.

#### **2. Xenotransplantation in small animals (mouse and rat)**

Immunosuppression is a key requirement for an animal to accept human tissues or organs, as a functional immune system will result in the host animals rejecting the human grafts. The nude mouse was the first immunosuppressed animal model developed in 1962 [5]. Nude mice lack T cells and can therefore accept human tumor cells. Subsequently, severe combined immunodeficient (SCID) mice were developed in 1983, which lack both B cells and T cells [6]. McCune et al. [7] successfully transplanted a human fetal thymus, liver cells, and lymph node into SCID mice, resulting in the differentiation of human T cells and B cells. However, the rate of engraftment of human cells in these mice was low due to maintenance of their natural killer (NK)-T cell activity. Gerling et al. [8] developed NOD/SCID mice by crossbreeding SCID mice with NOD mice, a diabetes model due to autoimmunity in the pancreas, which also show low NK-T cell activity and macrophage function [9]. Combining the low activity of NK-T cells and macrophages in NOD mice with the lack of B cells and T cells in SCID mice, the use of NOD/SCID mice improved the engraftment rate of hematopoietic stem cells [10]. Ito et al. [11] produced NOG mice as a crossbreed of NOD/SCID mice and gamma(c)(null) mice, which completely lack NK-T cells, and achieved a dramatically improved engraftment rate of human hematopoietic cells.

We previously reported the successful transplantation of rat cells into SCID mice [12]. Isolated hepatocytes obtained from the rat liver were injected into urokinase-type plasminogen activator (uPA)/SCID mice, in which urokinase-type plasminogen accumulates specifically in the native liver causing the damaged liver. The mice served as bioreactors to allow the transplanted rat hepatocytes to proliferate in the mouse host, resulting in more than 95% of cells in the mouse liver being of rat origin. Oldani et al. [13] successfully developed a mouse-rat chimeric liver, which was transplanted in rats. They injected hepatocytes isolated from Lewis rats into C57Bl/6Fah−/−Rag2−/−Il2rg−/<sup>−</sup> mice to create chimeric livers, which were transplanted into rats with or without immunosuppression. Without immunosuppression, the recipient rats died from acute rejection, whereas rats with immunosuppression survived for more than 112 days and maturation of rat bile ducts was observed 4 months after transplantation. We also demonstrated that the nude rat model could serve as an in vivo bioreactor. Liver grafts from Syrian hamsters were transplanted into nude rats that administered several immunosuppressive agents, including tacrolimus and mycophenolate mofetil (MMF). After auxiliary xenogenic partial liver transplantation, regeneration of the liver graft was observed, and its weight increased from pre-transplant to 7 days after transplantation [14].

These immunodeficient mouse models, including SCID, NOD/SCID, and NOG mice, are useful for research on regenerative medicine using human PSCs, allowing for evaluation of teratoma formation to confirm the differentiation of the cells into the three germ layers [1]. In addition, these models are widely utilized for evaluation of tumorigenicity in human PSC-derived cells after transplantation [4], since human PSC-derived cells or tissues have a risk of tumor formation from contamination of undifferentiated PSCs [15, 16]. Small animals such as mice and rats are widely applied as models in cell transplantation research owing to their ease of handling. However, small animals have limitations in terms of the number of cells that can be transplanted and evaluation of therapeutic efficacy, that is, a human clinical application might require the transplantation of several hundreds of million cells, which is impossible to accomplish in small animals. Moreover, large animal models are required for accurate evaluation of the efficacy of cell transplantation.

**139**

immune rejection.

*Pigs as Models of Preclinical Studies and In Vivo Bioreactors for Generation of Human Organs*

Furthermore, large animal models are expected to play roles as bioreactors for

**3. Xenotransplantation in middle and large animals (monkey and pig)**

Chong et al. [17] transplanted human ESC-derived cardiomyocytes into the hearts of pig-tailed macaques as a nonhuman primate model. The main advantage of this model is that the hearts are much larger (37–52 g) than those of mice (0.15 g), rats (1 g), and guinea pigs (3 g), which allowed for the transplantation

macaques were administered methylprednisolone, cyclosporine, and abatacept (a CTLA4 immunoglobulin) to prevent immune rejection. The efficacy of human ESC-derived cardiomyocytes in the infarcted hearts of pig-tailed macaques was demonstrated, and maturation of the transplanted ESC-derived cardiomyocytes was observed [18]. However, compared to an adult human, pig-tailed macaques are still relatively small (5.2–12.6 kg), and the heart is much smaller than that of a

Pigs are a suitable animal for preclinical studies and in vivo reactors in terms of their size and anatomy that correspond well to those of humans. To establish an immunosuppressed state that allows for transplantation of human PSC-derived cells or tissues into host pigs without rejection, SCID pigs were also developed [19]. Suzuki et al. [19] generated cloned pigs by serial nuclear transfer using fibroblasts with disruption of the X-linked interleukin 2 receptor subunit gamma (*IL2RG*) gene, as this mutation is known to cause X-linked SCID in humans. The SCID pigs accepted human cells, indicating their potential in preclinical studies and as in vivo reactors with human PSCs. However, raising these pigs is a technical challenge; among the 31 cloned piglets produced, only four survived for over 1 year. In addition, SCID pigs must be raised under meticulous hygiene conditions, which impose a further cost for their establishment and maintenance. Therefore, it is not practical

Total thymectomy is an alternative strategy to create immunosuppressed pigs that can accept human cells. Binns et al. [20] first proposed the concept of achieving immunosuppression by performing thymectomy in neonatal pigs in 1972. Microminiature pigs (MMPs) are smaller than domestic or ordinary miniature pigs and are thus suitable model animals for preclinical studies [21]. To develop immunodeficient MMPs, we performed thymectomy in neonatal pigs, which were transplanted with human hepatocytes that could engraft in the pig liver without any immunosuppressive agents [22]. To further improve the immunodeficient pig model, we performed splenectomy along with the thymectomy in 6–7-month-old miniature pigs and administered several immunosuppressive agents, including tacrolimus, MMF, and prednisolone, via a stomach tube [23]. This so-called operational immunodeficient miniature pig (OIDP) model allowed for the successful implantation of artificial human vascular tubes created by a three-dimensional bioprinting. Moreover, the human tube was inserted between the carotid artery and jugular vein to act as a shunt, and blood flow was observed for 3 months without

As mentioned above, establishment of a chimera is a potential strategy for growing human tissues or organs in large animals. Matsunari et al. [24] demonstrated that blastocyst complementation can be applied to large animals by creating chimeric pigs. Specifically, they generated embryos from clones of porcine somatic cells, which showed an apancreatic phenotype, and their complementation

to use SCID pigs as models in preclinical studies and in vivo reactors.

cells into the infarcted myocardium and subsequent engraftment. The

*DOI: http://dx.doi.org/10.5772/intechopen.90202*

of 1 × 109

human (300 g).

functionally mature human tissues or organs.

Furthermore, large animal models are expected to play roles as bioreactors for functionally mature human tissues or organs.

### **3. Xenotransplantation in middle and large animals (monkey and pig)**

Chong et al. [17] transplanted human ESC-derived cardiomyocytes into the hearts of pig-tailed macaques as a nonhuman primate model. The main advantage of this model is that the hearts are much larger (37–52 g) than those of mice (0.15 g), rats (1 g), and guinea pigs (3 g), which allowed for the transplantation of 1 × 109 cells into the infarcted myocardium and subsequent engraftment. The macaques were administered methylprednisolone, cyclosporine, and abatacept (a CTLA4 immunoglobulin) to prevent immune rejection. The efficacy of human ESC-derived cardiomyocytes in the infarcted hearts of pig-tailed macaques was demonstrated, and maturation of the transplanted ESC-derived cardiomyocytes was observed [18]. However, compared to an adult human, pig-tailed macaques are still relatively small (5.2–12.6 kg), and the heart is much smaller than that of a human (300 g).

Pigs are a suitable animal for preclinical studies and in vivo reactors in terms of their size and anatomy that correspond well to those of humans. To establish an immunosuppressed state that allows for transplantation of human PSC-derived cells or tissues into host pigs without rejection, SCID pigs were also developed [19]. Suzuki et al. [19] generated cloned pigs by serial nuclear transfer using fibroblasts with disruption of the X-linked interleukin 2 receptor subunit gamma (*IL2RG*) gene, as this mutation is known to cause X-linked SCID in humans. The SCID pigs accepted human cells, indicating their potential in preclinical studies and as in vivo reactors with human PSCs. However, raising these pigs is a technical challenge; among the 31 cloned piglets produced, only four survived for over 1 year. In addition, SCID pigs must be raised under meticulous hygiene conditions, which impose a further cost for their establishment and maintenance. Therefore, it is not practical to use SCID pigs as models in preclinical studies and in vivo reactors.

Total thymectomy is an alternative strategy to create immunosuppressed pigs that can accept human cells. Binns et al. [20] first proposed the concept of achieving immunosuppression by performing thymectomy in neonatal pigs in 1972. Microminiature pigs (MMPs) are smaller than domestic or ordinary miniature pigs and are thus suitable model animals for preclinical studies [21]. To develop immunodeficient MMPs, we performed thymectomy in neonatal pigs, which were transplanted with human hepatocytes that could engraft in the pig liver without any immunosuppressive agents [22]. To further improve the immunodeficient pig model, we performed splenectomy along with the thymectomy in 6–7-month-old miniature pigs and administered several immunosuppressive agents, including tacrolimus, MMF, and prednisolone, via a stomach tube [23]. This so-called operational immunodeficient miniature pig (OIDP) model allowed for the successful implantation of artificial human vascular tubes created by a three-dimensional bioprinting. Moreover, the human tube was inserted between the carotid artery and jugular vein to act as a shunt, and blood flow was observed for 3 months without immune rejection.

As mentioned above, establishment of a chimera is a potential strategy for growing human tissues or organs in large animals. Matsunari et al. [24] demonstrated that blastocyst complementation can be applied to large animals by creating chimeric pigs. Specifically, they generated embryos from clones of porcine somatic cells, which showed an apancreatic phenotype, and their complementation

*Xenotransplantation - Comprehensive Study*

human hematopoietic cells.

transplantation [14].

**2. Xenotransplantation in small animals (mouse and rat)**

Immunosuppression is a key requirement for an animal to accept human tissues or organs, as a functional immune system will result in the host animals rejecting the human grafts. The nude mouse was the first immunosuppressed animal model developed in 1962 [5]. Nude mice lack T cells and can therefore accept human tumor cells. Subsequently, severe combined immunodeficient (SCID) mice were developed in 1983, which lack both B cells and T cells [6]. McCune et al. [7] successfully transplanted a human fetal thymus, liver cells, and lymph node into SCID mice, resulting in the differentiation of human T cells and B cells. However, the rate of engraftment of human cells in these mice was low due to maintenance of their natural killer (NK)-T cell activity. Gerling et al. [8] developed NOD/SCID mice by crossbreeding SCID mice with NOD mice, a diabetes model due to autoimmunity in the pancreas, which also show low NK-T cell activity and macrophage function [9]. Combining the low activity of NK-T cells and macrophages in NOD mice with the lack of B cells and T cells in SCID mice, the use of NOD/SCID mice improved the engraftment rate of hematopoietic stem cells [10]. Ito et al. [11] produced NOG mice as a crossbreed of NOD/SCID mice and gamma(c)(null) mice, which completely lack NK-T cells, and achieved a dramatically improved engraftment rate of

We previously reported the successful transplantation of rat cells into SCID mice [12]. Isolated hepatocytes obtained from the rat liver were injected into urokinase-type plasminogen activator (uPA)/SCID mice, in which urokinase-type plasminogen accumulates specifically in the native liver causing the damaged liver. The mice served as bioreactors to allow the transplanted rat hepatocytes to proliferate in the mouse host, resulting in more than 95% of cells in the mouse liver being of rat origin. Oldani et al. [13] successfully developed a mouse-rat chimeric liver, which was transplanted in rats. They injected hepatocytes isolated from Lewis rats into C57Bl/6Fah−/−Rag2−/−Il2rg−/<sup>−</sup> mice to create chimeric livers, which were transplanted into rats with or without immunosuppression. Without immunosuppression, the recipient rats died from acute rejection, whereas rats with immunosuppression survived for more than 112 days and maturation of rat bile ducts was observed 4 months after transplantation. We also demonstrated that the nude rat model could serve as an in vivo bioreactor. Liver grafts from Syrian hamsters were transplanted into nude rats that administered several immunosuppressive agents, including tacrolimus and mycophenolate mofetil (MMF). After auxiliary xenogenic partial liver transplantation, regeneration of the liver graft was observed, and its weight increased from pre-transplant to 7 days after

These immunodeficient mouse models, including SCID, NOD/SCID, and NOG mice, are useful for research on regenerative medicine using human PSCs, allowing for evaluation of teratoma formation to confirm the differentiation of the cells into the three germ layers [1]. In addition, these models are widely utilized for evaluation of tumorigenicity in human PSC-derived cells after transplantation [4], since human PSC-derived cells or tissues have a risk of tumor formation from contamination of undifferentiated PSCs [15, 16]. Small animals such as mice and rats are widely applied as models in cell transplantation research owing to their ease of handling. However, small animals have limitations in terms of the number of cells that can be transplanted and evaluation of therapeutic efficacy, that is, a human clinical application might require the transplantation of several hundreds of million cells, which is impossible to accomplish in small animals. Moreover, large animal models are required for accurate evaluation of the efficacy of cell transplantation.

**138**

with allogenic blastomeres resulted in the development of a functional pancreas. Wu et al. [25] reported a successful pig-human chimera that was created by introducing human PSCs into fertilized pig eggs. Therefore, when combined with blastocyst complementation, human organs can be created in a human-pig chimera; however, these methods are associated with serious ethical and legal problems. Alternatively, the introduction of human-derived cells to pig fetuses can lead to immune tolerance, allowing for the acceptance of human PSC-derived tissues or organs.

#### **4. Immune tolerance induction for xenotransplantation**

Immune tolerance is defined as a lack of an immune response against particular antigens. In general, the immune system has tolerance to self-antigens and only responds to non-self-antigens, which is a challenge for transplantation, as the grafted cells or tissues are rejected and not able to survive in the host body. The phenomenon of immune tolerance was first described in 1945 in which anastomosis in the placenta was observed in twin calves, and they accepted each other's skin grafts [26]. Hasek et al. [27] subsequently confirmed this phenomenon in chicken and duck by producing parabiosis in fertilized eggs. In 1953, Medawar et al. [28] established actively acquired tolerance by implanting a live antigen in the fetuses of mice or embryonic chicks. Using this method, Binns et al. [29] also tried to create immune tolerance in pigs by implanting bone marrow cells or lymphocytes from another pig into fetal pigs, resulting in prolonged survival of skin graft in the treated pigs.

In addition to these examples, induction of immune tolerance to human cells or tissues has been attempted in other animals. Kenneth et al. [30] transplanted human mesenchymal stem cells into fetal sheep early in gestation. Despite the xenogeneic condition, the human mesenchymal stem cells engrafted and survived in multiple tissues for up to 13 months after transplantation. These strategies of injecting human cells into a fetus were proven to result in immune tolerance to human cells after birth.

As MMPs have emerged as suitable candidates for immune tolerance induction to accept human cells, tissues, and organs owing to their useful applications in preclinical studies and in vivo reactors, it may be possible to create MMPs with immune tolerance to human cells by injecting a human antigen into pig fetuses without requiring the need to create human-pig chimeras [31].

#### **5. Conclusions**

Our newly developed OIDPs can accept human cells, tissues, and organs derived from human PSCs. These models will allow for long-term observation after the transplantation of human PSC-derived cells or tissues to better evaluate the safety and efficacy of the procedure. Moreover, if human cells, tissues, and organs are transplanted into piglets, they will grow in vivo along with the growth of the host pig. These grafts will then mature and be of suitable size with appropriate function for human application. Therefore, pigs can be suitable models for preclinical studies and serve as in vivo bioreactors for developing human tissues or organs (**Figure 1**). Transplantable MMPs without immunosuppressive agents are expected to be developed in the near future as promising and valuable animal models for researchers, which can dramatically promote regenerative medicine and organ transplant therapies with human PSCs.

**141**

**Author details**

and Eiji Kobayashi<sup>2</sup>

\*

provided the original work is properly cited.

*mature along with the growth of the host.*

, Shugo Tohyama2,3\*, Hideyuki Shimizu1

1 Department of Cardiovascular Surgery, Keio University School of Medicine, Japan

*Schema of pigs as models of preclinical studies and in vivo bioreactors. (A) Adult operational immunodeficient miniature pigs (OIDPs) are useful for preclinical studies in regenerative medicine with human PSCs, enabling evaluation of the safety and efficacy of cell transplantation. In particular, after transplantation of human PSC-derived spheroids or organoids into the OIDPs, the risk of tumorigenicity can be evaluated. (B) Fetal or neonatal OIDPs are also useful as in vivo bioreactors, facilitating the efficient in vivo growth of immature human tissues. After immature human PSC-derived tissues or organs are transplanted into OIDPs, they will* 

2 Department of Organ Fabrication, Keio University School of Medicine, Japan

\*Address all correspondence to: shugotohyama@keio.jp and organfabri@keio.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

3 Department of Cardiology, Keio University School of Medicine, Japan

, Keiichi Fukuda<sup>3</sup>

Yujiro Kawai1

**Figure 1.**

*Pigs as Models of Preclinical Studies and In Vivo Bioreactors for Generation of Human Organs*

*DOI: http://dx.doi.org/10.5772/intechopen.90202*

*Pigs as Models of Preclinical Studies and In Vivo Bioreactors for Generation of Human Organs DOI: http://dx.doi.org/10.5772/intechopen.90202*

#### **Figure 1.**

*Xenotransplantation - Comprehensive Study*

tissues or organs.

treated pigs.

human cells after birth.

**5. Conclusions**

therapies with human PSCs.

with allogenic blastomeres resulted in the development of a functional pancreas. Wu et al. [25] reported a successful pig-human chimera that was created by introducing human PSCs into fertilized pig eggs. Therefore, when combined with blastocyst complementation, human organs can be created in a human-pig chimera; however, these methods are associated with serious ethical and legal problems. Alternatively, the introduction of human-derived cells to pig fetuses can lead to immune tolerance, allowing for the acceptance of human PSC-derived

Immune tolerance is defined as a lack of an immune response against particular antigens. In general, the immune system has tolerance to self-antigens and only responds to non-self-antigens, which is a challenge for transplantation, as the grafted cells or tissues are rejected and not able to survive in the host body. The phenomenon of immune tolerance was first described in 1945 in which anastomosis in the placenta was observed in twin calves, and they accepted each other's skin grafts [26]. Hasek et al. [27] subsequently confirmed this phenomenon in chicken and duck by producing parabiosis in fertilized eggs. In 1953, Medawar et al. [28] established actively acquired tolerance by implanting a live antigen in the fetuses of mice or embryonic chicks. Using this method, Binns et al. [29] also tried to create immune tolerance in pigs by implanting bone marrow cells or lymphocytes from another pig into fetal pigs, resulting in prolonged survival of skin graft in the

In addition to these examples, induction of immune tolerance to human cells or tissues has been attempted in other animals. Kenneth et al. [30] transplanted human mesenchymal stem cells into fetal sheep early in gestation. Despite the xenogeneic condition, the human mesenchymal stem cells engrafted and survived in multiple tissues for up to 13 months after transplantation. These strategies of injecting human cells into a fetus were proven to result in immune tolerance to

As MMPs have emerged as suitable candidates for immune tolerance induction to accept human cells, tissues, and organs owing to their useful applications in preclinical studies and in vivo reactors, it may be possible to create MMPs with immune tolerance to human cells by injecting a human antigen into pig fetuses

Our newly developed OIDPs can accept human cells, tissues, and organs derived

from human PSCs. These models will allow for long-term observation after the transplantation of human PSC-derived cells or tissues to better evaluate the safety and efficacy of the procedure. Moreover, if human cells, tissues, and organs are transplanted into piglets, they will grow in vivo along with the growth of the host pig. These grafts will then mature and be of suitable size with appropriate function for human application. Therefore, pigs can be suitable models for preclinical studies and serve as in vivo bioreactors for developing human tissues or organs (**Figure 1**). Transplantable MMPs without immunosuppressive agents are expected to be developed in the near future as promising and valuable animal models for researchers, which can dramatically promote regenerative medicine and organ transplant

without requiring the need to create human-pig chimeras [31].

**4. Immune tolerance induction for xenotransplantation**

**140**

*Schema of pigs as models of preclinical studies and in vivo bioreactors. (A) Adult operational immunodeficient miniature pigs (OIDPs) are useful for preclinical studies in regenerative medicine with human PSCs, enabling evaluation of the safety and efficacy of cell transplantation. In particular, after transplantation of human PSC-derived spheroids or organoids into the OIDPs, the risk of tumorigenicity can be evaluated. (B) Fetal or neonatal OIDPs are also useful as in vivo bioreactors, facilitating the efficient in vivo growth of immature human tissues. After immature human PSC-derived tissues or organs are transplanted into OIDPs, they will mature along with the growth of the host.*

#### **Author details**

Yujiro Kawai1 , Shugo Tohyama2,3\*, Hideyuki Shimizu1 , Keiichi Fukuda<sup>3</sup> and Eiji Kobayashi<sup>2</sup> \*

1 Department of Cardiovascular Surgery, Keio University School of Medicine, Japan

2 Department of Organ Fabrication, Keio University School of Medicine, Japan

3 Department of Cardiology, Keio University School of Medicine, Japan

\*Address all correspondence to: shugotohyama@keio.jp and organfabri@keio.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Shuji Miyagawa*

Recently, remarkable progress has been made in the area of preclinical xenotransplantation experiments. Surprisingly, a heterotopic heart from the geneediting pig continued to beat for almost 2.5 years, when implanted in the monkey abdomen, and a pig life-supporting kidney could also function for over 1.3 years in monkeys. Concerning islets, islets from gene-editing pigs could work for more than one year in monkeys. It is noteworthy that one group reported a survival of adult wildtype pig islets of over 600 days. On the other hand, the progress in these preclinical trials strongly affected not only the xenotransplantation study itself but regeneration studies to use pigs as a scaffold to foster human induced pluripotent stem cells.

Published in London, UK © 2020 IntechOpen © Reimphoto / iStock

Xenotransplantation - Comprehensive Study

Xenotransplantation

Comprehensive Study

*Edited by Shuji Miyagawa*