**4. Manipulation of adipose tissue and ADSCs**

#### **4.1. Introduction**

adiponectin, angiotensin, cathepsin D, penetraxin, pregnancy zone protein and retinol binding protein, as well as stromal cell-derived growth factor (CXCL12) have been found in the conditioned media of ADSCs differentiating towards the adipocyte lineage. ADSCs secrete also oher different well characterized cytokines (GM-CSF, TGF-β, PGE2, IGF-1) and their release can be modulated by exposure to different agents, such as b-FGF and EGF or inflam‐ matory stimula, like lipopolysaccharide (LPS). The role of these and other factors has been investigated by multiple studies regarding one or more possible applications of ADSCs in the field of regenerative medicine. Brain Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Glial Derived Neurotrophic Factor (GDNF) are thought to be important molecules secreted by ADSCs mediating neurotrophic effects and modulating in animal models of Parkinson Disease the recovery after hypoxic-ischemic injuries. Hepatocyte Growth Factor (HGF) and Vascular Endothelial Growth factor (VEGF) are the most important factors capable of inducing angiogenesis in areas that have undergone ischemic episodes and their importance is particularly relevant in wound healing. In cardiac regeneration, IGF-1 and VEGF mediate respectively an anti-apoptotic and angiogenic action, to which is attributed the capacity of ADSCs to have beneficial effects when transplanted/injected in different animal models of myocardial infarction/failure. In conclusion, most of ADSCs secreted factors act through mechanisms that mediate protection against cell death or, alternatively, induce cell migration and proliferation. Alternatively, they can indirectly act on the targeted cell popula‐ tions: by promoting vascularization they can be indirectly linked to an increase of oxygen and nutrients in the affected areas, which may in turn promote local regenerative processes. Indeed, up to now most reports have focused on a limited set of known factors but it is expected that

other molecules are responsible for the regenerative effects of ADSCs.

The regenerative potential of ADSCs has been related also to their immune-modulatory abilities. ADSCs have been shown to be an immune-privileged site, preventing severe graftversus-host response after transplantation procedures in vitro and in vivo. A concern of fundamental importance is the interplay between ADSCs and the host tissue, with particular focus on the immune system. Several studies have shown that ADSCs can be used either for autologous or allogenic cell transplants: this feature would be a major advantage for the employment of adipose tissue as a source for cell-based therapies. Furthermore ADSCs seem to act also as modulators of the immune system. The allogenic potential of these cells could be explained by the property of ADSCs to decrease the expression of hematopoietic markers and HLA-DR after subsequent passages. In addition, it has been observed that ADSCs only express HLA class I, but not HLA class II molecules: the latter can only be induced in ADSCs after incubation with IFN-γ. Furthermore, several experiments have proved that ADSCs do not stimulate lymphocyte proliferation and they do not elicit a response by Mixed Lymphocyte Reaction (MLR): in addition, they can also inhibit phyohemagglutinin (PHA)-stimulated lymphocyte proliferation. These immune-suppressive effects are likely mediated by soluble factors, among which PGE-2 seems to be the most important. Notably, the secretion of cytokines by ADSCs can be modulated not only by the inflammatory stimulus but also by the

*3.2.3. Immunomodulatory properties of ADSCs*

186 Regenerative Medicine and Tissue Engineering

Human subcutaneous adipose tissue provides an ideal alternative source of autologous pluripotent stem cells showing several advantages compared with other sources. As a matter of fact it is ubiquitous and commonly easily obtainable in large quantity with minimal invasive harvesting procedures or methods (either liposuction aspirates or subcutaneous adipose tissue fragments), limited patient discomfort and minimal ethical considerations: it may be trans‐ planted safely and efficaciously. The abundance of stem cells available enables the direct therapeutic adoption of primary cells without any need for culture expansion. Moreover, adipose tissue is also uniquely expandable: currently available procedures for cell isolation yield a high amount of stem cells with remarkable properties of stable proliferation and potential differentiation in vitro, being attractive candidates for clinical applications offering protocols that may provide alternative therapeutic solutions in cell-based therapies and tissue engineering to repair or regenerate damaged tissues and organs. The technologies for adipose tissue harvesting, processing, and transplantation have substantially evolved in recent years together with appropriate commercial development and with updated refinements and information regarding extraction, isolation, storage, options for cultures, growth and differ‐ entiation, cryopreservation and its effect on survival and proliferation of isolated ADSCs, also related to their adoption in tissue-engineered constructs involving biomaterials and scaffolds. Inconsistencies in literature regarding the handling of ADSCs require more extensive inves‐ tigations and controls, in particular in the in vitro processing and differences between the regenerative properties of freshly-processed heterogeneous stromal vascular fraction cells and of culture-expanded relatively homogeneous ADSCs, or the related risk of complications and possible adverse events. There is a need for stronger evidence of the safety, reproducibility and quality of the ADSCs prior to a more extensive use in clinical applications. As a matter of fact, despite the clinical use of adipose tissue grafts and ADSCs worldwide has dramatically increased, questions concerning the safety and efficacy of these treatments are still opened and currently the use of isolated ADSCs for medical indications in a clinical setting has been approved only in selected cases and few countries.

#### **4.2. Origins and delivery of adipose tissue grafts**

Adipose tissue have been used for long time for reconstructive purposes through fat grafting or autologous fat transfer, a method according to which fat from the patient is removed from one area of the body ad reinserted into the desired recipient location. [9] Fat grafting has shown to be beneficial as a reconstructive and cosmetic procedure for patients with volume losses to soft tissues due to disease, trauma, congenital defects or aging. Even so, outcomes of these techniques are often unpredictable and rates of graft reabsorption may be disappointing. As a matter of fact, fat tissue is re-vascularized at the transplantation site within 48 hours from the surgical procedure, in the meantime being fed by diffused materials from surrounding free plasma. The survival rates of the graft are dependent on size of transplanted fat particles and on surface area from which these cells could re-establish their blood supply. In order to minimize reabsorption, studies have demonstrated the efficacy of less traumatic methods of harvesting, processing and injecting. Microinjection of fat by means of the "lipostructure technique" known also as Coleman's technique has been adopted by many plastic surgeons. [10] This technique distributes fat grafts in small aliquots by meticulous injection through multiple access sites, from which the graft fans out into various subcutaneous layers. The abundance of stem cells obtaina‐ ble in many common procedures, such as liposuction and liposculture, enables their direct therapeutic adoption without any need for culture expansion. [11] Even so, precursor cells can be purified by a variety of processes and enzymatic techniques may be adopted to obtain an ADSC-rich stromal vascular fraction (SVF). This issues are currently investigat‐ ed as adjuvants to free fat transfer in order to increase yield of graft retention (cellassisted lipotransfer). The ADSCs contained in the stromal vascular fraction have been applied clinically as early as 2004 for the treatment of perianal fistulas in Crohn's dis‐ ease. [12] However, it is worth pointing out that, even though harvesting and first processing steps overlap, fat grafts SVF cells and ADSCs represent three different therapeu‐ tic options. Fat grafts are obtained directly after centrifugation of lipoaspirates. They contain predominantly mature adipocytes and are poor in ADSCs. The stromal vascular fraction, as mentioned above, is obtained by digestion with collagenase of the lipoaspi‐ rate sample and a subsequent centrifugation step: its cellular composition is heterogene‐ ous, being rich in ADSCs but containing also circulating blood cells, fibroblasts, pericytes and endothelial cells. The adoption of a pure ADSC population requires plating of the SVF and expansion of the stem cell population and thus, differently from the previous two options, ADSCs cannot be harvested and implanted in a one step-procedure. Even if all these approaches exploit to some extent the regenerative potential of adipose tissue they

are quite different procedures having also different therapeutic indications. Thus, atten‐ tion has to be paid in order to avoid confusion. As for harvesting of ADSCs, several factors related to the patient, such as Body Mass Index (BMI) and age, have been analyzed for their impact on cell viability and number. Results are controversial, there is no evidence of a strong correlation of BMI with stem cells viability, number or size. Instead, there seems to be a negative correlation between age and rates of pre-adipocytes proliferation or differentiation, with higher lipolitic activity in the younger population and lower levels of apoptosis. [13] The body region of the donor site is another important variable patientdependent. The abdomen, according to some studies, seems to be the best harvest site, while medial thigh and knee seem to have the lowest levels of viability of ASDCs. [14] These differences have not been proved in other studies. Effects of infiltration of local anesthetics during harvesting have also been investigated: lidocaine and adrenaline seem to have no effects on adipocyte viability. The method of harvest can affect not only viability of ADSCs but also their level of adhesiveness to extracellular matrix proteins. Standard liposuction allows the harvest of larger volumes of adipose tissue but it might result in up to 90% rate of adipocyte rupture. For this reason this technique is not ideal for fat grafting, while it could be more appropriate for ADSCs harvesting. An equivalent damage to preadipocytes has been measured comparing syringe aspiration with fat surgical excision. It is accepted that a larger cannula diameter at harvest correlates with improved cell viability. Partial purification of lipoaspirate can be carried out in the operatory room. The first step is centrifugation, which separates harvested fat into three layers: infra-natant (lowest layer composed of blood, tissue fluid and local anesthetics), middle portion (mostly composed by fatty tissue) and supra-natant (least dense upper layer including lipids). Infra-natant components can be ejected from the base of the syringe, while supra-natant can be poured off and soaked up using absorbent materials. While this technique is the most practical and today commonly used for fat grafting, it may not produce the best fraction of ADSCs possible. Several studies have been conducted on this issue, revealing that gentle centrifu‐ gation produces the highest cell viability, while long periods of centrifugation lead to isolation of the most proliferative cell type. When comparing decantation, washing and centrifugation, stem cells concentration results greater in washed lipoaspirates and pellets contained at the bottom of the centrifuged samples contain the highest concentration of stem cells.

#### **4.3. Origins and delivery of ADSCs**

of culture-expanded relatively homogeneous ADSCs, or the related risk of complications and possible adverse events. There is a need for stronger evidence of the safety, reproducibility and quality of the ADSCs prior to a more extensive use in clinical applications. As a matter of fact, despite the clinical use of adipose tissue grafts and ADSCs worldwide has dramatically increased, questions concerning the safety and efficacy of these treatments are still opened and currently the use of isolated ADSCs for medical indications in a clinical setting has been

Adipose tissue have been used for long time for reconstructive purposes through fat grafting or autologous fat transfer, a method according to which fat from the patient is removed from one area of the body ad reinserted into the desired recipient location. [9] Fat grafting has shown to be beneficial as a reconstructive and cosmetic procedure for patients with volume losses to soft tissues due to disease, trauma, congenital defects or aging. Even so, outcomes of these techniques are often unpredictable and rates of graft reabsorption may be disappointing. As a matter of fact, fat tissue is re-vascularized at the transplantation site within 48 hours from the surgical procedure, in the meantime being fed by diffused materials from surrounding free plasma. The survival rates of the graft are dependent on size of transplanted fat particles and on surface area from which these cells could re-establish their blood supply. In order to minimize reabsorption, studies have demonstrated the efficacy of less traumatic methods of harvesting, processing and injecting. Microinjection of fat by means of the "lipostructure technique" known also as Coleman's technique has been adopted by many plastic surgeons. [10] This technique distributes fat grafts in small aliquots by meticulous injection through multiple access sites, from which the graft fans out into various subcutaneous layers. The abundance of stem cells obtaina‐ ble in many common procedures, such as liposuction and liposculture, enables their direct therapeutic adoption without any need for culture expansion. [11] Even so, precursor cells can be purified by a variety of processes and enzymatic techniques may be adopted to obtain an ADSC-rich stromal vascular fraction (SVF). This issues are currently investigat‐ ed as adjuvants to free fat transfer in order to increase yield of graft retention (cellassisted lipotransfer). The ADSCs contained in the stromal vascular fraction have been applied clinically as early as 2004 for the treatment of perianal fistulas in Crohn's dis‐ ease. [12] However, it is worth pointing out that, even though harvesting and first processing steps overlap, fat grafts SVF cells and ADSCs represent three different therapeu‐ tic options. Fat grafts are obtained directly after centrifugation of lipoaspirates. They contain predominantly mature adipocytes and are poor in ADSCs. The stromal vascular fraction, as mentioned above, is obtained by digestion with collagenase of the lipoaspi‐ rate sample and a subsequent centrifugation step: its cellular composition is heterogene‐ ous, being rich in ADSCs but containing also circulating blood cells, fibroblasts, pericytes and endothelial cells. The adoption of a pure ADSC population requires plating of the SVF and expansion of the stem cell population and thus, differently from the previous two options, ADSCs cannot be harvested and implanted in a one step-procedure. Even if all these approaches exploit to some extent the regenerative potential of adipose tissue they

approved only in selected cases and few countries.

188 Regenerative Medicine and Tissue Engineering

**4.2. Origins and delivery of adipose tissue grafts**

Embryonic stem cells have an enormous multilineage potential but many ethical and political issues accompany their use. Therefore researchers have directed their attention on pluripotent adult stem cells. Adult stem cells were initially thought to have the differentiation capacity limited to their tissue of origin, however, as already mentioned above, many studies have now demonstrated that stem cells have the capacity to differentiate into cells of mesodermal, endodermal and ectodermal origin. MSCs from the bone marrow show extensive proliferative capacity and a multilineage differentiation potential into several lineages, including osteo‐ blasts, chondrocytes, adipocytes and myoblasts. However, pain, morbidity and low cell numbers upon harvest represent an obstacle to their extensive clinical application. The harvesting of adipose tissue, in comparison, is much less expensive than bone marrow. ADSCs can be isolated both from tissue samples and from lipoaspirate with less invasive procedures and are available in greater quantities (5 x 105 stem cells from 400 to 600 mg tissue). [15] ADSCs can be easily cultured and expanded, retaining their stem cell phenotypes and mesenchymal pluripotency still after several passages, features that make them an ideal source of stem cells for clinical applications.

**•** Isolation and culture of ADSCs

Since Rodbell's description of isolated pre-adipocytes from adipose tissue, a variety of methods have been developed. [16] Today, most laboratories use several common steps to process cells from adipose tissue. These methods include: washing, enzymatic digestion/mechanical disruption, centrifugal separation for isolation of cells which can be used directly, after cryopreservation, or after culture expansion for the generation of ADSCs. Still, despite the extensive use of ADSCs for research purposes, there is no any widely-accepted unique standard protocol for isolating and culturing these cells. For enzymatic digestion most laboratories use collagenases of different subtypes, trypsin, or a mixture of both, at various concentrations with an average incubation time of one hour, at 37°C, in constant shaking. The optimal centrifugation speed is considered to be around 1200g for 5 to 10 minutes. Some additional purification procedures can include filtration through nylon meshes and incubation with an erytrocyte-lysing buffer, usually Krebs Ringer Buffer (KRB) or NH4Cl. This procedure, however, seems to have a negative influence on the growth of ADSCs. Some investigators, after the identification of ADSCs surface immunophenotype, use immune-magnetic beads or flow cytometry to purify the stem cell population directly from the heterogeneous sample, using the CD34+ antigen. The most used culture medium are α-Modified Eagle's Medium (α-MEM), or Dulbecco's Modified Eagle's Medium (DMEM), after addition of fetal bovine/calf serum, (FBS/FCS), L-glutamine, penicillin and streptomycin.

**•** Cryopreservation of ADSCs

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. [17] Cryopreservation is regarded as a promising technique and many studies have focused on this procedure. Other protocols investigated drying (anhydrobiosis) and freeze drying (lyophilization). The majority of in vitro studies agree that cryopreservation of adipocytes in liquid nitrogen, preferably using a set cooling and re-warming protocol, provides the lowest damage to cell viability. These results have been replicated in vivo (murine models) showing that grafts frozen in liquid nitrogen and stored at -35°C had a similar viability and histology compared to fresh tissue: in addition, this method obtained better results than freeze drying and immersion in glycerol. Recently, in order to increase the yield of adipose-derived stem cells post-thawing, the use of cryoprotective agents, such as dimethyl sulphoxide (DMSO) has been examined: samples frozen with DMSO achieved better outcomes than unprotected ones. Thus, cryoprotective agents are now considered as an essential part of any cryopreservation protocol aiming to provide appropriate conditions for the survival of ADSCs and adipocytes.

#### **4.4. Safety concerns**

harvesting of adipose tissue, in comparison, is much less expensive than bone marrow. ADSCs can be isolated both from tissue samples and from lipoaspirate with less invasive procedures and are available in greater quantities (5 x 105 stem cells from 400 to 600 mg tissue). [15] ADSCs can be easily cultured and expanded, retaining their stem cell phenotypes and mesenchymal pluripotency still after several passages, features that make them an ideal source of stem cells

Since Rodbell's description of isolated pre-adipocytes from adipose tissue, a variety of methods have been developed. [16] Today, most laboratories use several common steps to process cells from adipose tissue. These methods include: washing, enzymatic digestion/mechanical disruption, centrifugal separation for isolation of cells which can be used directly, after cryopreservation, or after culture expansion for the generation of ADSCs. Still, despite the extensive use of ADSCs for research purposes, there is no any widely-accepted unique standard protocol for isolating and culturing these cells. For enzymatic digestion most laboratories use collagenases of different subtypes, trypsin, or a mixture of both, at various concentrations with an average incubation time of one hour, at 37°C, in constant shaking. The optimal centrifugation speed is considered to be around 1200g for 5 to 10 minutes. Some additional purification procedures can include filtration through nylon meshes and incubation with an erytrocyte-lysing buffer, usually Krebs Ringer Buffer (KRB) or NH4Cl. This procedure, however, seems to have a negative influence on the growth of ADSCs. Some investigators, after the identification of ADSCs surface immunophenotype, use immune-magnetic beads or flow cytometry to purify the stem cell population directly from the heterogeneous sample, using the CD34+ antigen. The most used culture medium are α-Modified Eagle's Medium (α-MEM), or Dulbecco's Modified Eagle's Medium (DMEM), after addition of fetal bovine/calf

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. [17] Cryopreservation is regarded as a promising technique and many studies have focused on this procedure. Other protocols investigated drying (anhydrobiosis) and freeze drying (lyophilization). The majority of in vitro studies agree that cryopreservation of adipocytes in liquid nitrogen, preferably using a set cooling and re-warming protocol, provides the lowest damage to cell viability. These results have been replicated in vivo (murine models) showing that grafts frozen in liquid nitrogen and stored at -35°C had a similar viability and histology compared to fresh tissue: in addition, this method obtained better results than freeze drying and immersion in glycerol. Recently, in order to increase the yield of adipose-derived stem cells post-thawing, the use of cryoprotective agents, such as dimethyl sulphoxide (DMSO) has been examined: samples frozen with DMSO achieved better outcomes than unprotected ones. Thus, cryoprotective agents are now considered as an essential part of any cryopreservation protocol aiming to

for clinical applications.

**•** Isolation and culture of ADSCs

190 Regenerative Medicine and Tissue Engineering

**•** Cryopreservation of ADSCs

serum, (FBS/FCS), L-glutamine, penicillin and streptomycin.

provide appropriate conditions for the survival of ADSCs and adipocytes.

Inconsistencies in literature regarding the handling of ADSCs require more extensive inves‐ tigations and controls. In particular, a focus should be placed on in vitro processing as well as differences between the regenerative properties of freshly-processed heterogeneous adipose cells and those of culture-expanded relatively homogeneous ADSCs. Related risks of compli‐ cations and possible adverse events like fat necrosis, seromas, oncological recurrences, should be accurately considered. In addition, adiponectin is implicated in the pathogenesis of insulinresistant states, such as obesity and diabetes type 2. In particular, several studies reported that differentiated WAT cells and WAT resident progenitors may promote cancer growth and metastasis by means of a variety of different mechanisms (endocrine, paracrine, autocrine interactions). The main cellular component of WAT are adipocytes, the large cells accumulat‐ ing tryglicerides in lipid droplets. In particular, in conditions like obesity, adipocytes in WAT may eventually became under oxygenated, leading to hypoxia, increased oxidative stress, recruitment of inflammatory leukocytes and eventually fibrosis. In recent experimental models, some adipokines showed to be able to promote tumor growth along with fatty acids released by adipocytes. High levels of adiponectin have been associated with the development of endometrial carcinoma and breast cancer. Leptin has been identified in regulation of cell proliferation and neo-vascularization in malignant and normal cells of different origins, including lung, gastric, colonic, kidney, leukemic, hematopoietic and epithelial cells. Notably, these molecules can enhance proliferation and survival of malignant cells and/or of tumor vasculature. So far, studies investigating the role of WAT in cancer have predominantly focused on pro-tumorigenic effect of ADSCs. In fact the increased proliferation and survivor of malignant cells may result from the engagement of perivascular ADSCs into angiogenesis and vascular maturation, resulting in improved tumor blood perfusion. Cytokines such as adiponectin, leptin, interleukin-6, and TNF alfa seem to be responsible for a chronic low-grade inflammation. Furthermore, mesenchymal cells are known to suppress the activation of Tkiller cells: this finding suggests that also ADSCs may help tumors to evade the host immune response. Thus, adipocytes may be able to produce adipokines and several secretions which could potentially induce cancer reappearance by "fueling" dormant breast cancer cells in tumor bed true "tumor-stroma interaction": even so, up to now, especially for grafting of adipose tissue after breast cancer treatment, there is no strong clinical evidence or international agreement on this topic. [18-19] Depending on country, the safety of adipose tissue grafting is still a controversial issue. In 2009, the American Society of Plastic surgeons Fat Graft task Force concluded that no reliable studies could confirm definitely the oncologic safety of lipofilling in breast cancer patients. A more accurate point of view is provided by a large multicentric observational study on adipose tissue grafting in patients previously affected by breast cancer: considered parameters included the complication rate of the technique, the risk of modification of mammography and a rigorous long-term clinical/instrumental follow-up. [20] At the moment no studies on the effects of lipotransfer on human cancer breast cells in vivo are available. We cannot provide the definitive proof of the safety of lipofilling in terms of cancer recurrence or distant metastasis, but until then, should be performed in experienced hands, and a cautious oncologic follow-up protocol is advised.
