**3. 3D culturing systems based on natural components of the ECM**

Gels present the easiest, most affordable and quickest way to provide cells with a threedimensional environment that can be further manipulated by variety of methods in order to modulate their properties. Different types of gels have been utilized during the past few decades with collagen, fibrin, hyaluronan and basement membrane extract (BME) gels being the most frequently employed.

#### **3.1. Collagen gels**

Collagens represent the most abundant type of proteins in mammals making up to 35% of the whole body protein content. They are found in fibrillar and non-fibrillar forms and are widely distributed among tissues with a distinct structural, organizational and density variance among tissues and organs. Currently, 28 types of collagen have been described that are known to be products of 49 different α-chain gene products [37]. Collagen gels are one of the first employed 3D culturing systems used to study cellular adhesion and migration in threedimensional environment [17, 38]. Currently collagen gels have found application in a wide variety of studies in the field of cellular motility [11] and the importance of physical parameters such as density, stiffness and elasticity for cellular adhesion, proliferation, migration and contraction [39]. Fibroblast cells, seeded in collagen gels, have been shown to acquire different morphology when compared to fibroblasts grown on two-dimensional surfaces and appear similar to their counterparts in normal connective tissue. The three-dimensional environment of the gel mimics the in vivo conditions and also causes normal polarization and differentiation of epithelial cells [16]. It has also been developed as a model for studying the progression of prostate and breast cancer [40, 41].

Few types of collagen gels are most widely employed as three-dimensional culturing systems. Pure gels that remain attached to the dish after polymerization are considered as "stressed". The cells plated within this 3D environment usually exert isometric tension. If the gels are released, detached from the bottom of the Petri dish, they contract and represent the so called "relaxed and loaded" gels. If the gels are detached from the dish before cells are being seeded, they represent "relaxed and unloaded" gels. The ease of manipulation of the collagen gel's stiffness provides a useful tool for the evaluation of the role of isometric tension for cell survival and differentiation as well as for the investigation of mechanical features of the environment and it's reorganization during wound healing processes or various pathological conditions [39]. Some interesting features of cell migration in 3D have been established using those different types of collagen gels. Migration in softer substrates, or compliant matrices, results in migratory activity that is independent of the small GTPase Rho [39], thus, the cell acquires an amoeboid phenotype. As tension increases and the extracellular matrix becomes more rigid, the migratory mode of fibroblasts switches over to Rho dependent migration [42] with a mesenchymal phenotype. Moreover, in order for fibroblasts to differentiate to myofibroblasts upon TGFβ stimulation, a typical process during wound healing, increased tension in the extracellular matrix is required [43]. Reorganization of the extracellular matrix also appears to be dependent on the mechanical properties of the substrate as incorporation of fibronectin in the extracellular matrix requires internal tension of the ECM [44].

Collagen gel-based dressings were also used in some of the first attempts to create in vitro equivalents of full thickness skin for regenerative medicine and tissue engineering applications [45]. Currently there are a few temporary and semi-permanent dressings available as off-theshelf products, intended for use as bioconstructs for skin reconstruction [46].

#### **3.2. Fibrin gels**

modulate their properties. Different types of gels have been utilized during the past few decades with collagen, fibrin, hyaluronan and basement membrane extract (BME) gels being

Collagens represent the most abundant type of proteins in mammals making up to 35% of the whole body protein content. They are found in fibrillar and non-fibrillar forms and are widely distributed among tissues with a distinct structural, organizational and density variance among tissues and organs. Currently, 28 types of collagen have been described that are known to be products of 49 different α-chain gene products [37]. Collagen gels are one of the first employed 3D culturing systems used to study cellular adhesion and migration in threedimensional environment [17, 38]. Currently collagen gels have found application in a wide variety of studies in the field of cellular motility [11] and the importance of physical parameters such as density, stiffness and elasticity for cellular adhesion, proliferation, migration and contraction [39]. Fibroblast cells, seeded in collagen gels, have been shown to acquire different morphology when compared to fibroblasts grown on two-dimensional surfaces and appear similar to their counterparts in normal connective tissue. The three-dimensional environment of the gel mimics the in vivo conditions and also causes normal polarization and differentiation of epithelial cells [16]. It has also been developed as a model for studying the progression of

Few types of collagen gels are most widely employed as three-dimensional culturing systems. Pure gels that remain attached to the dish after polymerization are considered as "stressed". The cells plated within this 3D environment usually exert isometric tension. If the gels are released, detached from the bottom of the Petri dish, they contract and represent the so called "relaxed and loaded" gels. If the gels are detached from the dish before cells are being seeded, they represent "relaxed and unloaded" gels. The ease of manipulation of the collagen gel's stiffness provides a useful tool for the evaluation of the role of isometric tension for cell survival and differentiation as well as for the investigation of mechanical features of the environment and it's reorganization during wound healing processes or various pathological conditions [39]. Some interesting features of cell migration in 3D have been established using those different types of collagen gels. Migration in softer substrates, or compliant matrices, results in migratory activity that is independent of the small GTPase Rho [39], thus, the cell acquires an amoeboid phenotype. As tension increases and the extracellular matrix becomes more rigid, the migratory mode of fibroblasts switches over to Rho dependent migration [42] with a mesenchymal phenotype. Moreover, in order for fibroblasts to differentiate to myofibroblasts upon TGFβ stimulation, a typical process during wound healing, increased tension in the extracellular matrix is required [43]. Reorganization of the extracellular matrix also appears to be dependent on the mechanical properties of the substrate as incorporation of fibronectin in

Collagen gel-based dressings were also used in some of the first attempts to create in vitro equivalents of full thickness skin for regenerative medicine and tissue engineering applications

the extracellular matrix requires internal tension of the ECM [44].

the most frequently employed.

224 Regenerative Medicine and Tissue Engineering

prostate and breast cancer [40, 41].

**3.1. Collagen gels**

Fibrin gels are another type of easily manufactured 3D culturing systems. They are formed as a result of thrombin cleavage of fibrinogen, resulting in a mesh of fibrin fibers. Stressed, relaxed and loaded and relaxed and unloaded types of fibrin gels are also frequently used model systems. In normal physiological conditions fibrin represents the provisional matrix of the clot, formed after wounding. Thus, the defined use of fibrin gels to study cellular invasion and contraction during the processes of wound healing [47] has emerged. Historically, polymer‐ ized fibrin gels were one of the first scaffolds used for skin tissue engineering after severe burns or for treatment of chronic wounds [48]. Currently fibrin gels have found further applications in the field of vascular tissue engineering and are being extensively studied especially as a possible resolution of a number of rapidly growing problems related to arterial occlusive diseases [49]. Studies in cardiac tissue engineering and cartilage regeneration and reconstruc‐ tion have also benefited of the use of this natural, biodegradable scaffold [50, 51].

#### **3.3. Hyaluronan gels**

Hyaluronan or hyaluronic acid is widely distributed throughout all tissues glycosaminogly‐ can. It is an anionic, nonsulfated polysaccharide, formed on the plasma membrane instead of in the Golgi apparatus, with molecular weight varying between 5 kDa and 2,000 kDa. Hyaluronan is a major component of the cartilage, synovial fluid, the extracellular matrix of the skin and has a major role during the developmental processes [52]. Presumably, due to the high abundance of the molecule and its role during development, wound healing and migration, hyaluronan gels became another suitable three-dimensional model for studies in numerous fields.

As a natural component of the cartilage, hyaluronan is being used as a milieu for culturing chondrocytes in vitro. Current research indicates that culturing in tissue-like hyaluronic 3D environment sustains chondrocytes phenotype, leading to increased proliferation, sulphated glycosaminoglycans production as well as collagen type II and aggrecan synthesis and indeed supports chondrogenic differentiation [53]. Additional studies have shown promising results concerning the use of hyaluronan for cartilage repair and as scaffold for regenerative medicine [54-56]. Another intriguing direction of studies is the utilization of three-dimensional hyalur‐ onic acid scaffolds for culturing mesenchymal stem cells. Promising results from culturing stem cells in three-dimensional hyaluronan-based scaffolds have been obtained in regard to the generation of cartilage-like tissue for the use of regenerative medicine [57]. Hyaluronic acid gels have been also investigated as possible 3D scaffolds for culturing cardiomyocytes and hepatocytes in vitro [58, 59]. Therapeutical use of hyaluronan gels has been reported also in the field of adipose tissue engineering [60] as well as in neuromedicine. The use of this biodegradable material has revealed promising results in cell and drug delivery to the central nervous system[61].

#### **3.4. Basement membrane extract gels**

Almost 30 years ago the Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell line was found to secrete vast amount of unknown protein mixture. Later it was determined that this mixture was composed of the typical for the basement membrane proteins collagen IV, laminin, entactin and the heparansulfate proteoglycan [62]. Further components like matrix metallo‐ proteinases as well as number of growth factors were also identified in this complex mixture. This protein composite is marketed by BD Biosciences under the trade name Matrigel®, but similar products are available from other sources. Matrigel resembles the complex extracellular matrix found in many tissues and is thus used by cell biologists as a substrate for cell culturing.

Utilization of the basement membrane matrix led to numerous scientific discoveries. For the first time the EHS matrix was used as a substrate to cultivate Sertoli cells, which led to their survival and the differentiation of the accompanying germ cells[63]. The first in vitro myeli‐ nation was also observed in cultures based on the basement membrane matrix. Carey et al. demonstrated in 1986 that rat Schwann cells cultured in 3D conditions based on basement membrane matrix show increased dendrite outgrowth and myelination [64]. Thus a reliable in vitro model for investigation of nerve regeneration was established.

Another interesting observation was made when epithelial and endothelial cells were cultured on basement membrane matrix. Both cell types show different morphology compared to flat 2D surfaces, but also form specific structures depending on whether cells are cultured on top or within the matrix. Ducts were formed when epithelial cells were cultured on top of the matrix, and acinar-like structures appeared when cells were embedded within the BME gel [65, 66]. Different types of acinar epithelial cells (breast epithelial cells, salivary gland cells, pancreatic and prostate cells) form distinct structures in 3D indeed clearly supporting the importance of the extracellular matrix in cellular differentiation and proliferation. Endothelial cells form capillary-like structures when seeded in lower counts, or monolayers when seeded in higher counts. These observations made possible the development of in vitro models for studying angiogenesis. Tumor-induced angiogenesis is a key prerequisite for neoplastic progression thus angiogenesis suppression is one of the major directions investigated as possible cancer treatment. Vascular endothelial cells form capillary-like structures when plated on 3D basement membrane gels and have provided a suitable model for testing pharmacological substances and screening of chemical agents as angiogenic inhibitors [67].

Basement membrane extract gels provided also another important model system aimed at investigation of cancer invasion and metastasis. Kramer et al. noticed that normal, nonmalignant cells polarize on top of the matrix whereas malignant cells invaded the 3D basement membrane [68]. While normal cells attached, polarized and differentiated on top of the matrix and showed almost no migratory tendency, malignant cells exhibited increased invasive and migratory phenotype within the matrix. Formation of long protrusions in the direction of migration and channels of degraded matrix behind the cells were observed. Evidently, malignant cells mimicked their in vivo invasive behaviour since synthesis of proteolitic enzymes and degradation of the basement membrane are key events during tumor metastasis. Continuous investigation led to the development and improvement of invasion and metastasis assays [69, 70] providing identical conditions and criteria to measure the invasiveness of tumors and the efficiency of treatment.

Maybe one of the most significant discoveries related to the progress of regenerative medicine and tissue engineering is the possibility to culture stem cells on basement membrane matrix [71]. Normally the basement membrane is the first ECM to be synthesized in the developing embryo [72] hence the logical use for culturing stem cells. Feeder layers of irradiated mouse embryonic fibroblasts were required for long term culturing and limited the large scale production of human embryonic stem cells. Moreover, they present the possibility of viral cross contaminations. Utilization of other extracellular matrix components like fibronectin, collagen I, collagen IV or artificial substrates has failed to support undifferentiated stem cells growth [73], whereas coating of the dish with BME extract retained the undifferentiated state of human embryonic stem cells for up to 30 passages, maintaining their proliferation rate, high telomerase activity, normal karyotype and the expression of the pluripotency markers.

#### **3.5. Cell-derived matrices**

**3.4. Basement membrane extract gels**

226 Regenerative Medicine and Tissue Engineering

Almost 30 years ago the Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell line was found to secrete vast amount of unknown protein mixture. Later it was determined that this mixture was composed of the typical for the basement membrane proteins collagen IV, laminin, entactin and the heparansulfate proteoglycan [62]. Further components like matrix metallo‐ proteinases as well as number of growth factors were also identified in this complex mixture. This protein composite is marketed by BD Biosciences under the trade name Matrigel®, but similar products are available from other sources. Matrigel resembles the complex extracellular matrix found in many tissues and is thus used by cell biologists as a substrate for cell culturing.

Utilization of the basement membrane matrix led to numerous scientific discoveries. For the first time the EHS matrix was used as a substrate to cultivate Sertoli cells, which led to their survival and the differentiation of the accompanying germ cells[63]. The first in vitro myeli‐ nation was also observed in cultures based on the basement membrane matrix. Carey et al. demonstrated in 1986 that rat Schwann cells cultured in 3D conditions based on basement membrane matrix show increased dendrite outgrowth and myelination [64]. Thus a reliable

Another interesting observation was made when epithelial and endothelial cells were cultured on basement membrane matrix. Both cell types show different morphology compared to flat 2D surfaces, but also form specific structures depending on whether cells are cultured on top or within the matrix. Ducts were formed when epithelial cells were cultured on top of the matrix, and acinar-like structures appeared when cells were embedded within the BME gel [65, 66]. Different types of acinar epithelial cells (breast epithelial cells, salivary gland cells, pancreatic and prostate cells) form distinct structures in 3D indeed clearly supporting the importance of the extracellular matrix in cellular differentiation and proliferation. Endothelial cells form capillary-like structures when seeded in lower counts, or monolayers when seeded in higher counts. These observations made possible the development of in vitro models for studying angiogenesis. Tumor-induced angiogenesis is a key prerequisite for neoplastic progression thus angiogenesis suppression is one of the major directions investigated as possible cancer treatment. Vascular endothelial cells form capillary-like structures when plated on 3D basement membrane gels and have provided a suitable model for testing pharmacological substances and screening of chemical agents as angiogenic inhibitors [67].

Basement membrane extract gels provided also another important model system aimed at investigation of cancer invasion and metastasis. Kramer et al. noticed that normal, nonmalignant cells polarize on top of the matrix whereas malignant cells invaded the 3D basement membrane [68]. While normal cells attached, polarized and differentiated on top of the matrix and showed almost no migratory tendency, malignant cells exhibited increased invasive and migratory phenotype within the matrix. Formation of long protrusions in the direction of migration and channels of degraded matrix behind the cells were observed. Evidently, malignant cells mimicked their in vivo invasive behaviour since synthesis of proteolitic enzymes and degradation of the basement membrane are key events during tumor metastasis. Continuous investigation led to the development and improvement of invasion and metastasis

in vitro model for investigation of nerve regeneration was established.

The use of three-dimensional gels of collagen, fibrin, basement membrane matrix or glycosa‐ minoglycans represents a significant advance in cell culturing. Those gels provide the much needed dimensionality to bring the environmental conditions closer to the in vivo settings. A major disadvantage of those models though is the fact that they still lack the chemical com‐ plexity and spatial organization of the ECM, characteristic for tissues (Figure 1). Indeed cells plated in three-dimensional gels degrade and reorganize to some extent the constituents of their surroundings and also secrete and integrate new components within the existing extracellular matrix [74]. Presumably cells are trying to shape their environment by their liking, but all those processes are believed to keep cells in an "activated" state that is unnatural for healthy tissue. In the quest for creating better model systems that approximate closer to different healthy tissues a few groups have developed during the last few decades affordable methods for creating cell derived three-dimensional cultures and matrices [9, 75-79]. Those methods are based on in vitro preparation of three-dimensional extracellular matrices made by the cells themselves. The result is a naturally synthesized and organized extracellular matrix, providing in vivo-like conditions.

Conducted research, based on these tissue-like cultures has already demonstrated significant differences between cells grown in three-dimensional cell derived matrices and other 3D culturing methods. Ahlfors and Billiar [79] demonstrated that culturing fibroblasts in appro‐ priate conditions induces synthesis of extracellular matrix components, thus resulting in a multilayer culture, with mechanical properties approximating normal tissue. Investigation of the mechanical properties of these cell derived 3D cultures revealed that the resulting matrices are stronger than collagen or fibrin 3D cultures. Moreover cells in naturally synthesized cultures had higher protein synthesis rate than fibroblasts cultured in collagen or fibrin gels, and more importantly, inhibition of collagen synthesis was not observed at later stages of cultivation. The production of matrices was achieved in serum supplemented media as well as in chemically defined media. The second option makes exploitation of this method relatively

**Figure 1.** Schematic presentation of the main characteristics of 3D cell culture systems. While artificially fabricated 3D scaffolds can be designed with specific three dimensional organization and desired mechanical properties, they, how‐ ever, generally lack the chemical complexity of the natural ECM. This limitation can be compensated to a certain ex‐ tent by incorporation of natural ECM proteins or their peptides (indicated by additional small arrow). 3D gels, made out of natural ECM components have also restricted chemical complexity and in addition their structural organization is a result of spontaneous, rather than cell-directed polymerization. Cell-derived 3D matrices and decellularized tissues and organs meet the general requirements for in vivo-like 3D environment. However, they often pose difficulties in preparation and restrictions in their mass production (see text for details).

expensive, but increases the chances of utilization of such cultures for the needs of tissue engineering by greatly diminishing the possibilities of species or viral cross contamination.

As we have previously discussed, cellular morphology of fibroblasts differs between 2D and 3D conditions. Even though fibroblasts appear to acquire the same morphology when plated on collagen gels and cell derived matrices, there are a number of issues to be considered – fibroblasts attach, proliferate and migrate at much higher rate in cell derived matrices compared to collagen gels [9]. Though dimensionality influences fibroblast morphology one must also take into account the heterogeneity and specific organization of the extracellular matrix which are of significant importance as well. The fact that 3D matrix adhesions were established in cell derived extracellular matrices, but not in fibrin or collagen gels, or 2D surfaces coated with ECM components [9] supports the notion of the importance of structural organization of the environment. Both, the components and their colocalization in these adhesion structures differ from focal and fibrillar adhesions. Formation of such in vivo-like adhesions appears to depend on the heterogeneity and organization of the ECM and their formation does not require de novo protein synthesis during cellular adhesion. Major differ‐ ences in signal transduction of fibroblasts in cell derived matrices were also discovered. The focal adhesion kinase (FAK), a molecule with exceptionally important role for cellular adhesion in conventional monolayer cultures, appears to be bypassed in in vivo-like conditions [8, 9]. In spite of this, downstream molecules maintain or even augment their activities, resulting in increased proliferation for example. Furthermore culturing of fibroblasts as a cell derived, three-dimensional multilayer culture leads to altered distribution of plasma membrane lipids [80-82]. Changes of the localization and properties of plasma membrane cholesterol and sphingomyelin in 3D are a probable cause for lowered oxidative stress, thus supporting the growing number of studies indicating that 2D cultures are an inappropriate, stressful method for cultivation of cells. Moreover the differences in the structural organization of the membrane coupled with the higher content of cholesterol and sphingomyelin, the major lipid components of the lipid rafts, in the plasma membranes of cells in 3D tissue-like environment, probably contribute to the differences in cell signalling.

The major difference between other 3D culturing systems and the cell derived in vivo-like three-dimensional matrices is the fact that their ECM is synthesized and organized naturally by the cells. In vivo, the extracellular matrix main functions are to present proper and specific 3D environment to cells and thus to define boundaries between different tissues. It provides the required elasticity and integrity during tissue and organ development, but is also being degraded and remodelled during both developmental processes and disease. Serving as an adhesive substrate, the ECM directs migratory cells and variations in its components concen‐ trations may act as chemotactic gradient as well as differences in its mechanical properties can serve as durotactic gradient. The extracellular matrix also participates in the accumulation, storage, release and presentation of growth factors to the cells. The synthesis and immobili‐ zation of growth factors is spatially and temporally regulated by the ECM and the release of the incorporated ligands is also dependent upon appropriate cell-mediated forces, proteolitic degradation and proper presentation to cells. As a supportive structure, the ECM also participates in the reciprocal mechanical signalling. The transmittion of forces to and by the cells is regulated by their mechanical receptors – the integrins – and is manifested by changes in the intracellular signalling resulting in activation of the cytoskeleton machinery, growth factor production, proliferation rate alteration, etc. [83]. Among all these specific characteristic of the extracellular matrix probably the most important one is the fact that it is a dynamic structure, being constantly remodelled by cells in response to intrinsic signals, depending on the specific periods of organism's development or due to occurring diseases.

Currently cell-derived extracellular matrices are being applied with great success for in vitro investigation of developmental processes [84], tumor cell invasion and the role of the accom‐ panying stroma cells [85], the mechanisms of fibrosis [86], studies of cellular migration [12, 87], as drug screening systems [88], for exploring the processes of wound healing [78] and numerous other applications. Despite the advantages of this model system, there are still a lot of considerable hindrances for its application in regenerative medicine and tissue engineering. Therefore numerous groups have undertaken a "reverse" approach towards meeting the requirements of modern medicine (see below).

#### **3.6. Decellularized tissues and organs as scaffolds for tissue engineering**

expensive, but increases the chances of utilization of such cultures for the needs of tissue engineering by greatly diminishing the possibilities of species or viral cross contamination.

preparation and restrictions in their mass production (see text for details).

228 Regenerative Medicine and Tissue Engineering

**Figure 1.** Schematic presentation of the main characteristics of 3D cell culture systems. While artificially fabricated 3D scaffolds can be designed with specific three dimensional organization and desired mechanical properties, they, how‐ ever, generally lack the chemical complexity of the natural ECM. This limitation can be compensated to a certain ex‐ tent by incorporation of natural ECM proteins or their peptides (indicated by additional small arrow). 3D gels, made out of natural ECM components have also restricted chemical complexity and in addition their structural organization is a result of spontaneous, rather than cell-directed polymerization. Cell-derived 3D matrices and decellularized tissues and organs meet the general requirements for in vivo-like 3D environment. However, they often pose difficulties in

As we have previously discussed, cellular morphology of fibroblasts differs between 2D and 3D conditions. Even though fibroblasts appear to acquire the same morphology when plated on collagen gels and cell derived matrices, there are a number of issues to be considered – fibroblasts attach, proliferate and migrate at much higher rate in cell derived matrices compared to collagen gels [9]. Though dimensionality influences fibroblast morphology one must also take into account the heterogeneity and specific organization of the extracellular matrix which are of significant importance as well. The fact that 3D matrix adhesions were established in cell derived extracellular matrices, but not in fibrin or collagen gels, or 2D surfaces coated with ECM components [9] supports the notion of the importance of structural organization of the environment. Both, the components and their colocalization in these adhesion structures differ from focal and fibrillar adhesions. Formation of such in vivo-like adhesions appears to depend on the heterogeneity and organization of the ECM and their formation does not require de novo protein synthesis during cellular adhesion. Major differ‐ ences in signal transduction of fibroblasts in cell derived matrices were also discovered. The focal adhesion kinase (FAK), a molecule with exceptionally important role for cellular adhesion in conventional monolayer cultures, appears to be bypassed in in vivo-like conditions [8, 9]. In spite of this, downstream molecules maintain or even augment their activities, resulting in increased proliferation for example. Furthermore culturing of fibroblasts as a cell derived, three-dimensional multilayer culture leads to altered distribution of plasma membrane lipids

The need for organ transplantation in modern society far much exceeds the donor availability. Moreover the immunological incompatibility limits further the patient's possibilities for finding a matching donor for the subsequent transplantation. Even if all those criteria are met more often than not, patients are treated with powerful immunosupressants to reduce the chances of transplant rejection.

The aspiring role of the ECM in governing the appropriate behaviour of a number of cell types led to the investigation of the properties and possible use in regenerative medicine of decel‐ lularized organs. The diversity of the extracellular matrix, its structure and micro-patterns suggest that specific cell types would overall "feel and perform better" if embedded in their corresponding natural matrix. There is a growing number of research to confirm this view. Culturing of hepatocytes as conventional monolayers, outside of their natural environment, results in loss of specific hepatocyte functions thus limiting the possibilities for their use in regenerative medicine. However, culturing of human hepatocytes in porcine liver-derived extracellular matrix supports albumin secretion and ammonia metabolism as well as restora‐ tion of hepatic transport activity [89]. Characteristic extracellular matrix is even required for the differentiation of human or murine embryonic stem cells to pneumocytes [90]. To date a number of ECM derived scaffolds have been described and investigated for use as organ reproducing systems, including heart-derived ECM [91], liver-derived ECM [92] and lungderived ECM [93].

The process of removal of cells from the organ and obtaining a cell-free scaffold is crucial for preserving the scaffold's qualities and usually includes several stages. Physical methods, involving freezing and thawing, mechanical agitation and sonication, could be considered non-harmful to the remaining biological scaffold and are usually combined with enzymatic and/or chemical methods, depending on the organ that is being decellularized. Treatment with exo-and endonucleases yields better results than physical methods, but is likely to affect the extracellular matrix as well. Harsh chemical treatment with acid or alkaline solutions, espe‐ cially detergents, either ionic or non-ionic, is known to extract cells from the extracellular matrix, but also severely damages the remaining scaffold. Depending on the organ, its cellular content, overall lipid content, ECM biochemical composition, structure and complexity an appropriate method for decellularization has to be selected. More often than not a combination of methods is used to achieve better results. Successful removal of cells would ideally yield DNA- and cell debris-free extracellular matrix that is not affected or altered by the applied treatments, thus resulting in a minimal or even absent immunological reaction towards the allogenic or xenogenic extracellular matrix. Some protocols have already been developed and successfully used for producing biological scaffolds for heart, liver and lung [91-93].

Decellularization of organs would ultimately provide the most suitable scaffold for recon‐ struction of an organ – a natural extracellular matrix. Repopulation with cells of the obtained scaffold is a process which also poses some difficulties in organ reconstruction. Reintroduction of cells to such scaffold requires organ-specific types of cells as well as endothelial and epithelial cells for rebuilding of blood vessels, stem or progenitor type of cells to support future cell renewal in the organ and most of all, distinct methods for introduction of the appropriate cell types to their targeted environment. When possible, autologous cells are used since they are less likely to provoke immune response and be rejected. Such cells also present lesser chance of inducing cancer or the possible non-immune toxic reactions caused by immunosu‐ pressants [94]. Allogenic cells from matching donors could also be used for regenerative purposes when autologus cells cannot be harvested, or are terminally damaged. Although these cells are not derived from the patient, they have some advantages too. The required cell types can be derived from healthy individuals, characterized and maintained until needed, thereby providing the opportunity for faster therapy application. A well known example of such use of allogenic cells is provided by the mesenchymal bone marrow cells.

The aspiring role of the ECM in governing the appropriate behaviour of a number of cell types led to the investigation of the properties and possible use in regenerative medicine of decel‐ lularized organs. The diversity of the extracellular matrix, its structure and micro-patterns suggest that specific cell types would overall "feel and perform better" if embedded in their corresponding natural matrix. There is a growing number of research to confirm this view. Culturing of hepatocytes as conventional monolayers, outside of their natural environment, results in loss of specific hepatocyte functions thus limiting the possibilities for their use in regenerative medicine. However, culturing of human hepatocytes in porcine liver-derived extracellular matrix supports albumin secretion and ammonia metabolism as well as restora‐ tion of hepatic transport activity [89]. Characteristic extracellular matrix is even required for the differentiation of human or murine embryonic stem cells to pneumocytes [90]. To date a number of ECM derived scaffolds have been described and investigated for use as organ reproducing systems, including heart-derived ECM [91], liver-derived ECM [92] and lung-

The process of removal of cells from the organ and obtaining a cell-free scaffold is crucial for preserving the scaffold's qualities and usually includes several stages. Physical methods, involving freezing and thawing, mechanical agitation and sonication, could be considered non-harmful to the remaining biological scaffold and are usually combined with enzymatic and/or chemical methods, depending on the organ that is being decellularized. Treatment with exo-and endonucleases yields better results than physical methods, but is likely to affect the extracellular matrix as well. Harsh chemical treatment with acid or alkaline solutions, espe‐ cially detergents, either ionic or non-ionic, is known to extract cells from the extracellular matrix, but also severely damages the remaining scaffold. Depending on the organ, its cellular content, overall lipid content, ECM biochemical composition, structure and complexity an appropriate method for decellularization has to be selected. More often than not a combination of methods is used to achieve better results. Successful removal of cells would ideally yield DNA- and cell debris-free extracellular matrix that is not affected or altered by the applied treatments, thus resulting in a minimal or even absent immunological reaction towards the allogenic or xenogenic extracellular matrix. Some protocols have already been developed and

successfully used for producing biological scaffolds for heart, liver and lung [91-93].

Decellularization of organs would ultimately provide the most suitable scaffold for recon‐ struction of an organ – a natural extracellular matrix. Repopulation with cells of the obtained scaffold is a process which also poses some difficulties in organ reconstruction. Reintroduction of cells to such scaffold requires organ-specific types of cells as well as endothelial and epithelial cells for rebuilding of blood vessels, stem or progenitor type of cells to support future cell renewal in the organ and most of all, distinct methods for introduction of the appropriate cell types to their targeted environment. When possible, autologous cells are used since they are less likely to provoke immune response and be rejected. Such cells also present lesser chance of inducing cancer or the possible non-immune toxic reactions caused by immunosu‐ pressants [94]. Allogenic cells from matching donors could also be used for regenerative purposes when autologus cells cannot be harvested, or are terminally damaged. Although these cells are not derived from the patient, they have some advantages too. The required cell

derived ECM [93].

230 Regenerative Medicine and Tissue Engineering

Often isolation of highly proliferating autologous cells from most organs is an almost impos‐ sible task. The use of embryonic stem cells could help solve this problem but the employment of human embryonic stem cells is still bounded up with heated moral debates. In addition it is a quite expensive technology, demanding proven methods for directed differentiation and extensive clinical trials. Therefore the use of adult cells with similar properties emerged as possible solution. Adult stem cells represent autologus cells which are multipotent and capable of self-renew. They have been known to exist in number of tissues like the gonads, intestine, skin and blood, and further data indicates their presence in adipose tissue [95], kidney [96], lung [97] and muscle [98]. In recent years, scientists have tried to identify specific markers to ease the isolation of adult stem cells, but such insignia are yet to be defined for most of them. Furthermore, it is currently evident that adult stem cells are localized in a specific extracellular environment – their niche. Signalling to and regulation of the self-renewal or differentiation processes appears to be tightly linked to the stem cell's niche, suggesting that micro-environ‐ mental cues may also be regulating cellular "stemness" [99]. A possible localization of different types of adult stem cells in niches in close proximity also exists [100, 101]. Thus, even though the specific localization of diverse types of adult stem cells have been identified, their isolation and further characterisation has proven to be an extremely difficult task. Label retention techniques and in vivo linage tracing as well as in vitro culturing and transplantation have yielded promising results and have aided the significant advancement in the field, but isolation of adult stem cells is still a major difficulty [99]. Despite the obstacles though, different types of adult stem cells have already found application in regenerative medicine [57, 102-104].

As pointed above, harvesting highly proliferating cells from healthy tissues has rendered a difficult task. Therefore researchers have sought to develop novel technologies that would aid obtaining and multiplication of such cells for further use in regenerative medicine. A promis‐ ing technique developed just a few years ago is the induction of pluripotency in differentiated somatic cells. In 2006 Takahashi and Yamanaka introduced a method for inducing pluripo‐ tency in fibroblast cells by overexpression of four transcript factors – Oct-4, c-Myc, Sox2 and Klf4 [105]. Their results showed that the timed overexpression of these factors is sufficient to convert fibroblasts to embryonic stem cell-like cells, termed induced pluripotent stem cells (iPSC), with many subsequent articles confirming their observations. The iPS cells are able to differentiate to any type of cell, just like embryonic stem cells, but since the progenitor cells are derived from the adult organism their use is liberated from the moral burdens concerning the use of embryonic stem cells. Research indicates that the genetic profile of good quality iPSC and embryonic stem cells is nearly identical, although some articles suggest that there may be some differences, probably attributed to different laboratory practices [106-108]. Further analysis of the whole genome have indeed found 71 differently methylated regions between iPSC and embryonic stem cells and 2,179 between iPSC and fibroblast [109], supporting the hypothesis that probably even though somatic cells are converted to pluripotent cells, there is still a "memory" preserved of the type of the donor cell.

Even though there are a lot of difficulties with the production of iPS cells, already a plethora of articles has demonstrated the ability of iPS cells to differentiate to numerous types of cells, thus providing the ability to direct the in vitro differentiation of iPS cells to the required cell type for the specific therapy. Moreover significant results have been accomplished, namely the generation of adult mice from iPS cells [110] thus confirming the vast capabilities of such cells. Therapeutically, iPS cells have been used together with gene therapy to correct genetic defects in mice, with two studies already showing promising results in the treatment of different types of anaemia [111, 112]. Future efforts in the field of iPS cells though have to be made before a successful iPSC therapy for human patients becomes a reality – research is currently targeted towards the development of methods of pluripotency induction, not relying on viral transduction, therefore lowering the possibility of cancer induction by the transplanted iPS cells as well as improvement of the efficacy of reprogramming. Furthermore the remaining questions of whether the donor cells are completely reprogrammed or retain a "memory" of their differentiated state are still to be answered.
