Perspectives for Clinical Translation of Adipose Stromal/Stem Cells

Adipose stromal/stem cells (ASCs) are an ideal cell type for regenerative medicine applications, as they can easily be harvested from adipose tissue in large quantities. ASCs have excellent proliferation, differentiation, and immunoregulatory capacities that have been demonstrated in numerous studies. Great interest and investment have been placed in efforts to exploit the allogeneic use and immunomodulatory and anti-inflammatory effects of ASCs. However, bridging the gap between in vitro and in vivo studies and moving into clinical practice remain a challenge. For the clinical translation of ASCs, several issues must be considered, including how to characterise such a heterogenic cell population and how to ensure their safety and efficacy. This review explores the different phases of in vitro and preclinical ASC characterisation and describes the development of appropriate potency assays. In addition, good manufacturing practice requirements are discussed, and cell-based medicinal products holding marketing authorisation in the European Union are reviewed. Moreover, the current status of clinical trials applying ASCs and the patent landscape in the field of ASC research are presented. Overall, this review highlights the applicability of ASCs for clinical cell therapies and discusses their potential.Peer reviewe

Effects of macromolecular crowding on human adipose stem cell culture in fetal bovine serum, human serum and defined xeno-free/serum-free conditions

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.Peer reviewe

Evaluation of the effect of donor weight on adipose stromal/stem cell characteristics by using weight-discordant monozygotic twin pairs

Background Adipose stromal/stem cells (ASCs) are promising candidates for future clinical applications. ASCs have regenerative capacity, low immunogenicity, and immunomodulatory ability. The success of future cell-based therapies depends on the appropriate selection of donors. Several factors, including age, sex, and body mass index (BMI), may influence ASC characteristics. Our aim was to investigate the effect of acquired weight on ASC characteristics under the same genetic background using ASCs derived from monozygotic (MZ) twin pairs. Methods ASCs were isolated from subcutaneous adipose tissue from five weight-discordant (WD, within-pair difference in BMI > 3 kg/m(2)) MZ twin pairs, with measured BMI and metabolic status. The ASC immunophenotype, proliferation and osteogenic and adipogenic differentiation capacity were studied. ASC immunogenicity, immunosuppression capacity and the expression of inflammation markers were investigated. ASC angiogenic potential was assessed in cocultures with endothelial cells. Results ASCs showed low immunogenicity, proliferation, and osteogenic differentiation capacity independent of weight among all donors. ASCs showed a mesenchymal stem cell-like immunophenotype; however, the expression of CD146 was significantly higher in leaner WD twins than in heavier cotwins. ASCs from heavier twins from WD pairs showed significantly greater adipogenic differentiation capacity and higher expression of TNF and lower angiogenic potential compared with their leaner cotwins. ASCs showed immunosuppressive capacity in direct cocultures; however, heavier WD twins showed stronger immunosuppressive capacity than leaner cotwins. Conclusions Our genetically matched data suggest that a higher weight of the donor may have some effect on ASC characteristics, especially on angiogenic and adipogenic potential, which should be considered when ASCs are used clinically.Peer reviewe

Preventing White Adipocyte Browning during Differentiation In Vitro : The Effect of Differentiation Protocols on Metabolic and Mitochondrial Phenotypes

Mitochondrial dysfunction in white adipose tissue is strongly associated with obesity and its metabolic complications, which are important health challenges worldwide. Human adipose-derived stromal/stem cells (hASCs) are a promising tool to investigate the underlying mechanisms of such mitochondrial dysfunction and to subsequently provide knowledge for the development of treatments for obesity-related pathologies. A substantial obstacle in using hASCs is that the key compounds for adipogenic differentiation in vitro increase mitochondrial uncoupling, biogenesis, and activity, which are the signature features of brown adipocytes, thus altering the white adipocyte phenotype towards brown-like cells. Additionally, commonly used protocols for hASC adipogenic differentiation exhibit high variation in their composition of media, and a systematic comparison of their effect on mitochondria is missing. Here, we compared the five widely used adipogenic differentiation protocols for their effect on metabolic and mitochondrial phenotypes to identify a protocol that enables in vitro differentiation of white adipocytes and can more faithfully recapitulate the white adipocyte phenotype observed in human adipose tissue. We developed a workflow that included functional assays and morphological analysis of mitochondria and lipid droplets. We observed that triiodothyronine- or indomethacin-containing media and commercially available adipogenic media induced browning during in vitro differentiation of white adipocytes. However, the differentiation protocol containing 1 mu M of the peroxisome proliferator-activated receptor gamma (PPAR gamma) agonist rosiglitazone prevented the browning effect and would be proposed for adipogenic differentiation protocol for hASCs to induce a white adipocyte phenotype. Preserving the white adipocyte phenotype in vitro is a crucial step for the study of obesity and associated metabolic diseases, adipose tissue pathologies, such as lipodystrophies, possible therapeutic compounds, and basic adipose tissue physiology.Peer reviewe

Preventing White Adipocyte Browning during Differentiation In Vitro : The Effect of Differentiation Protocols on Metabolic and Mitochondrial Phenotypes

Mitochondrial dysfunction in white adipose tissue is strongly associated with obesity and its metabolic complications, which are important health challenges worldwide. Human adipose-derived stromal/stem cells (hASCs) are a promising tool to investigate the underlying mechanisms of such mitochondrial dysfunction and to subsequently provide knowledge for the development of treatments for obesity-related pathologies. A substantial obstacle in using hASCs is that the key compounds for adipogenic differentiation in vitro increase mitochondrial uncoupling, biogenesis, and activity, which are the signature features of brown adipocytes, thus altering the white adipocyte phenotype towards brown-like cells. Additionally, commonly used protocols for hASC adipogenic differentiation exhibit high variation in their composition of media, and a systematic comparison of their effect on mitochondria is missing. Here, we compared the five widely used adipogenic differentiation protocols for their effect on metabolic and mitochondrial phenotypes to identify a protocol that enables in vitro differentiation of white adipocytes and can more faithfully recapitulate the white adipocyte phenotype observed in human adipose tissue. We developed a workflow that included functional assays and morphological analysis of mitochondria and lipid droplets. We observed that triiodothyronine- or indomethacin-containing media and commercially available adipogenic media induced browning during in vitro differentiation of white adipocytes. However, the differentiation protocol containing 1 mu M of the peroxisome proliferator-activated receptor gamma (PPAR gamma) agonist rosiglitazone prevented the browning effect and would be proposed for adipogenic differentiation protocol for hASCs to induce a white adipocyte phenotype. Preserving the white adipocyte phenotype in vitro is a crucial step for the study of obesity and associated metabolic diseases, adipose tissue pathologies, such as lipodystrophies, possible therapeutic compounds, and basic adipose tissue physiology.Peer reviewe

Development of fully defined xeno-free culture system for the preparation and propagation of cell therapy-compliant human adipose stem cells.

Introduction Adipose tissue is an attractive and abundant source of multipotent stem cells. Human adipose stem cells (ASCs) have shown to have therapeutic relevancy in diverse clinical applications. Nevertheless, expansion of ASCs is often necessary before performing clinical studies. Standard in vitro cell-culture techniques use animal-derived reagents that should be avoided in clinical use because of safety issues. Therefore, xeno- and serum-free (XF/SF) reagents are highly desirable for enhancing the safety and quality of the transplanted ASCs. Methods In the current study, animal component-free isolation and cell-expansion protocols were developed for ASCs. StemPro MSC SFM XF medium with either CELLstart™ CTS™ coating or Coating Matrix Kit were tested for their ability to support XF/SF growth. Basic stem-cell characteristics such as immunophenotype (CD3, CD11a, CD14, CD19, CD34, CD45RO, CD54, CD73, CD80, CD86, CD90, CD105, HLA-DR), proliferation, and differentiation potential were assessed in XF/SF conditions and compared with human serum (HS) or traditionally used fetal bovine serum (FBS) cultures. Results ASCs cultured in XF/SF conditions had significantly higher proliferation rates compared with HS/FBS cultures. Characteristic immunophenotypes of ASCs were maintained in every condition; however, cells expanded in XF/SF conditions showed significantly lower expression of CD54 (intercellular adhesion molecule 1, ICAM-1) at low passage number. Further, multilineage differentiation potential of ASCs was maintained in every culture condition. Conclusions Our findings demonstrated that the novel XF/SF conditions maintained the basic stem cell features of ASCs and the animal-free workflow followed in this study has great potential in clinical cell therapies.BioMed Central open acces

Näkemyksiä rasvakudoksen kantasolujen käytöstä kliinisissä soluterapioissa: uudet viljelyolosuhteet sekä solujen monikykyisyyden ja immunogeenisten ominaisuuksien karakterisointi

Rasvakudos on monikykyisten kantasolujen ehtymätön lähde. Rasvakudoksen kantasoluilla on erinomainen kyky jakautua ja erilaistua usean eri solutyypin suuntaan. Ne eivät myöskään aiheuta voimakasta immunologista vastetta, vaan niillä on kyky säädellä elimistön immunologisia reaktioita. Edellä mainitut ominaisuudet tekevät rasvakudoksen kantasoluista lupaavan vaihtoehdon erilaisiin kliinisiin sovelluksiin uudistavan lääketieteen alalla sekä immuunipuolustuksen säätelyä vaativiin hoitoihin. Kantasoluhoitoihin tarvittavat solumäärät ovat kuitenkin usein suuria, joten solujen määrää tulee lisätä laboratorio-olosuhteissa tehokkaan hoitovasteen aikaansaamiseksi. Perinteiset laboratoriossa käytettävät soluviljelytekniikat hyväksikäyttävät eläinperäisiä ainesosia, joita ei suositella kliiniseen käyttöön mahdollisten turvallisuusriskien, kuten allergisten reaktioiden tai hyljinnän takia. Tästä syystä täysin seerumittomat ja eläinperäisistä aineista vapaat reagenssit ovat toivottuja ja niillä voidaan parantaa rasvakudoksen kantasolujen turvallisuutta ja laatua kliinisiä soluhoitoja ajatellen. Tässä väitöskirjatyössä kehitettiin seerumittomat ja eläinperäisistä aineista vapaat soluviljelymenetelmät rasvakudoksen kantasolujen laboratoriossa tapahtuvaan eristykseen ja kasvatukseen. Solujen perusominaisuudet, kuten immunofenotyyppi, jakautumiskyky ja erilaistuspotentiaali arvioitiin seerumittomissa ja eläinperäisistä aineista vapaissa kasvatusolosuhteissa ja niitä verrattiin ihmisen seerumia sekä naudan seerumia sisältäviin olosuhteisiin. Lisäksi tutkittiin viljelymediumin makromolekulaarisen täytön eli Ficollin lisäyksen vaikutusta soluihin ja kykyä tukea solujen luonnollista mikrotason elinympäristön tuottoa laboratorio-olosuhteissa. Ficollin lisäyksen vaikutusta rasvakudoksen kantasolujen ominaisuuksiin tutkittiin kolmessa yllämainitussa kasvatusolosuhteessa. Lisäksi rasvakudoksen kantasolujen immunogeenisyys, kyky estää immuunivasteen muodostusta sekä signaaliproteiini-eritys määritettiin käyttäen lymfosyyttien yhteisviljelmiä kolmessa yllämainitussa laboratorio-olosuhteessa. Lopuksi tutkittiin bioaktiivisen lasin sekä β-trikalsiumfosfaatin sekä luun morfogeneettisten proteiinien (BMP) vaikutusta rasvakudoksen kantasolujen luuerilaistuskykyyn ihmisen seerumia sisältävässä kasvatusliuoksessa. Tulokset osoittivat, että seerumittomissa ja eläinperäisitä aineista vapaissa viljelyolosuhteissa rasvakudoksen kantasolut jakautuivat merkittävästi nopeammin verrattuna viljelyliuoksiin, jotka sisälsivät ihmisen tai naudan seerumia. Soluille ominainen immunofenotyyppi sekä solujen monikykyinen erilaistumispotentiaali säilyivät kaikissa kolmessa viljelyolosuhteessa. Ficollin lisääminen kasvatusliuokseen ei tukenut solujen jakautumista, mutta solujen erilaistuskyky rasva- ja luusolujen suuntaan oli tehokasta seerumia sisältävissä viljelyolosuhteissa Ficollin lisäyksen jälkeen. Menetelmä ei kuitenkaan soveltunut seerumittomiin olosuhteisiin, sillä rasvakudoksen kantasolujen elinkyky heikentyi merkittävästi Ficollin vaikutuksesta. Rasvakudoksen kantasolut eivät aiheuttaneet voimakasta immunologista vastetta missään tutkitussa olosuhteessa. Solujen kyky estää immuunivasteen muodostumista oli voimakkain naudan seerumia sisältävässä olosuhteessa, mutta suuremmilla kantasolumäärillä estävä vaikutus voitiin nähdä myös muissa tutkituissa olosuhteissa. Luun morfogeneettiset proteiinit vähensivät rasvakudoksen kantasolujen määrää ja luuerilaistuskykyä molempien biomateriaalien kanssa. Rasvakudoksen kantasolujen luuerilaistuskyky oli tehokkainta, kun niitä viljeltiin bioaktiivisella lasilla ilman osteogeenisiä suplementteja. Tämä väitöstutkimus osoitti, että uudet seerumittomat ja eläinperäisistä aineista vapaat viljelyolosuhteet pitävät yllä rasvakudoksen kantasolujen perusominaisuuksia ja kyseisillä olosuhteissa voi olla edellytyksiä myös kliiniseen käyttöön. Rasvakudoksen kantasolujen huolellinen karakterisointi erilaisissa viljelyolosuhteissa, immunologisten omaisuuksien arviointi sekä solujen käyttäytymisen tutkiminen kliinisessä käytössä olevien biomateriaalien kanssa ovat ensiarvoisen tärkeitä soluterapioiden positiivisen kehityksen kannalta. Uudet tutkimustulokset rasvakudoksen kantasolujen ominaisuuksista tarjoavat työkaluja myös kantasoluhoitojen regulaatioon. Lisää solujen turvallisuutta ja tehokkuutta arvioivia prekliinisiä tutkimuksia kuitenkin tarvitaan ennen siirtymää kliinisiin soluhoitoihin.Adipose tissue is an abundant source of multipotent stem cells, known as adipose stromal/stem cells (ASCs). The excellent proliferation capacity, multilineage differentiation potential, low immunogenicity and ability for immunomodulation make ASCs promising candidates for diverse clinical applications in regenerative medicine and immunomodulation therapies. Nevertheless, expansion of ASCs is often necessary to obtain a clinically sufficient cell number for effective cell treatments. Standard cell-culture techniques use animal-derived reagents that are not recommended in clinical use because of safety concerns with respect to allergic reactions, rejection and zoonoses. Therefore, xeno- and serum-free (XF/SF) reagents are highly desirable for enhanced safety and quality of transplanted ASCs. In this thesis, animal-component-free isolation and cell-expansion protocols were developed for ASCs. Basic stem cell characteristics such as immunophenotype, proliferation, and differentiation potential were assessed in XF/SF conditions and compared with those in allogeneic human serum (HS) or the traditionally used fetal bovine serum (FBS) cultures. Additionally, macromolecular crowding (MMC) was used to support cells in re-creating their own microenvironments in vitro. The effects of MMC on ASC characteristics were analyzed under three culture conditions, i.e., in FBS, HS and XF/SF. The immunogenicity and immunosuppression of ASCs as well as the secretion of signaling proteins were also determined through mixed lymphocyte reactions after cell isolation and expansion in FBS- and HS-supplemented medium and in XF/SF conditions. Finally, the effects of bioactive glass (BAG) S53P4 or β- tricalcium phosphate (β-TCP) and bone morphogenetic protein (BMP) -2 and -7 on the osteogenic differentiation of ASCs were investigated in HS-supplemented medium. The results showed that ASCs cultured in XF/SF conditions had significantly higher proliferation rates compared with those in HS and FBS cultures. The characteristic immunophenotype and multilineage differentiation potential of ASCs were maintained in all conditions. Although MMC did not support ASC proliferation under any of the studied conditions, adipogenic and osteogenic differentiation was efficient under MMC in FBS and HS media. However, the MMC method was not suitable for the XF/SF cultures studied because the ASCs did not remain viable in long-term exposure to MMC. Furthermore, our immunology studies showed that ASCs were weakly immunogenic when expanded under the three conditions. The significantly strongest suppression was observed with cells expanded in FBS conditions, whereas higher ASC numbers were required to display suppression in HS or XF/SF conditions. The results of the biomaterial studies showed that BMP supplementation decreased the cell number and osteogenic differentiation of ASCs with both the BAG and β-TCP materials. The most efficient osteogenic differentiation of ASCs was observed with BAG cultured without osteogenic supplements. Our findings demonstrated that the novel XF and XF/SF conditions maintained the basic stem cell features of ASCs, and thus, these animal-free workflows may have potential in clinical cell therapies. Careful characterization of ASCs with respect to in vitro culture conditions, immunological properties and cell behavior with clinically approved biomaterials is highly important for positive development of cell-based therapies. Thus, these novel findings of ASC characteristics will provide valuable tools for regulatory authorities. Nevertheless, additional preclinical safety and efficacy studies are required prior to clinical translation

Näkemyksiä rasvakudoksen kantasolujen käytöstä kliinisissä soluterapioissa: uudet viljelyolosuhteet sekä solujen monikykyisyyden ja immunogeenisten ominaisuuksien karakterisointi

Rasvakudos on monikykyisten kantasolujen ehtymätön lähde. Rasvakudoksen kantasoluilla on erinomainen kyky jakautua ja erilaistua usean eri solutyypin suuntaan. Ne eivät myöskään aiheuta voimakasta immunologista vastetta, vaan niillä on kyky säädellä elimistön immunologisia reaktioita. Edellä mainitut ominaisuudet tekevät rasvakudoksen kantasoluista lupaavan vaihtoehdon erilaisiin kliinisiin sovelluksiin uudistavan lääketieteen alalla sekä immuunipuolustuksen säätelyä vaativiin hoitoihin. Kantasoluhoitoihin tarvittavat solumäärät ovat kuitenkin usein suuria, joten solujen määrää tulee lisätä laboratorio-olosuhteissa tehokkaan hoitovasteen aikaansaamiseksi. Perinteiset laboratoriossa käytettävät soluviljelytekniikat hyväksikäyttävät eläinperäisiä ainesosia, joita ei suositella kliiniseen käyttöön mahdollisten turvallisuusriskien, kuten allergisten reaktioiden tai hyljinnän takia. Tästä syystä täysin seerumittomat ja eläinperäisistä aineista vapaat reagenssit ovat toivottuja ja niillä voidaan parantaa rasvakudoksen kantasolujen turvallisuutta ja laatua kliinisiä soluhoitoja ajatellen. Tässä väitöskirjatyössä kehitettiin seerumittomat ja eläinperäisistä aineista vapaat soluviljelymenetelmät rasvakudoksen kantasolujen laboratoriossa tapahtuvaan eristykseen ja kasvatukseen. Solujen perusominaisuudet, kuten immunofenotyyppi, jakautumiskyky ja erilaistuspotentiaali arvioitiin seerumittomissa ja eläinperäisistä aineista vapaissa kasvatusolosuhteissa ja niitä verrattiin ihmisen seerumia sekä naudan seerumia sisältäviin olosuhteisiin. Lisäksi tutkittiin viljelymediumin makromolekulaarisen täytön eli Ficollin lisäyksen vaikutusta soluihin ja kykyä tukea solujen luonnollista mikrotason elinympäristön tuottoa laboratorio-olosuhteissa. Ficollin lisäyksen vaikutusta rasvakudoksen kantasolujen ominaisuuksiin tutkittiin kolmessa yllämainitussa kasvatusolosuhteessa. Lisäksi rasvakudoksen kantasolujen immunogeenisyys, kyky estää immuunivasteen muodostusta sekä signaaliproteiini-eritys määritettiin käyttäen lymfosyyttien yhteisviljelmiä kolmessa yllämainitussa laboratorio-olosuhteessa. Lopuksi tutkittiin bioaktiivisen lasin sekä β-trikalsiumfosfaatin sekä luun morfogeneettisten proteiinien (BMP) vaikutusta rasvakudoksen kantasolujen luuerilaistuskykyyn ihmisen seerumia sisältävässä kasvatusliuoksessa. Tulokset osoittivat, että seerumittomissa ja eläinperäisitä aineista vapaissa viljelyolosuhteissa rasvakudoksen kantasolut jakautuivat merkittävästi nopeammin verrattuna viljelyliuoksiin, jotka sisälsivät ihmisen tai naudan seerumia. Soluille ominainen immunofenotyyppi sekä solujen monikykyinen erilaistumispotentiaali säilyivät kaikissa kolmessa viljelyolosuhteessa. Ficollin lisääminen kasvatusliuokseen ei tukenut solujen jakautumista, mutta solujen erilaistuskyky rasva- ja luusolujen suuntaan oli tehokasta seerumia sisältävissä viljelyolosuhteissa Ficollin lisäyksen jälkeen. Menetelmä ei kuitenkaan soveltunut seerumittomiin olosuhteisiin, sillä rasvakudoksen kantasolujen elinkyky heikentyi merkittävästi Ficollin vaikutuksesta. Rasvakudoksen kantasolut eivät aiheuttaneet voimakasta immunologista vastetta missään tutkitussa olosuhteessa. Solujen kyky estää immuunivasteen muodostumista oli voimakkain naudan seerumia sisältävässä olosuhteessa, mutta suuremmilla kantasolumäärillä estävä vaikutus voitiin nähdä myös muissa tutkituissa olosuhteissa. Luun morfogeneettiset proteiinit vähensivät rasvakudoksen kantasolujen määrää ja luuerilaistuskykyä molempien biomateriaalien kanssa. Rasvakudoksen kantasolujen luuerilaistuskyky oli tehokkainta, kun niitä viljeltiin bioaktiivisella lasilla ilman osteogeenisiä suplementteja. Tämä väitöstutkimus osoitti, että uudet seerumittomat ja eläinperäisistä aineista vapaat viljelyolosuhteet pitävät yllä rasvakudoksen kantasolujen perusominaisuuksia ja kyseisillä olosuhteissa voi olla edellytyksiä myös kliiniseen käyttöön. Rasvakudoksen kantasolujen huolellinen karakterisointi erilaisissa viljelyolosuhteissa, immunologisten omaisuuksien arviointi sekä solujen käyttäytymisen tutkiminen kliinisessä käytössä olevien biomateriaalien kanssa ovat ensiarvoisen tärkeitä soluterapioiden positiivisen kehityksen kannalta. Uudet tutkimustulokset rasvakudoksen kantasolujen ominaisuuksista tarjoavat työkaluja myös kantasoluhoitojen regulaatioon. Lisää solujen turvallisuutta ja tehokkuutta arvioivia prekliinisiä tutkimuksia kuitenkin tarvitaan ennen siirtymää kliinisiin soluhoitoihin.Adipose tissue is an abundant source of multipotent stem cells, known as adipose stromal/stem cells (ASCs). The excellent proliferation capacity, multilineage differentiation potential, low immunogenicity and ability for immunomodulation make ASCs promising candidates for diverse clinical applications in regenerative medicine and immunomodulation therapies. Nevertheless, expansion of ASCs is often necessary to obtain a clinically sufficient cell number for effective cell treatments. Standard cell-culture techniques use animal-derived reagents that are not recommended in clinical use because of safety concerns with respect to allergic reactions, rejection and zoonoses. Therefore, xeno- and serum-free (XF/SF) reagents are highly desirable for enhanced safety and quality of transplanted ASCs. In this thesis, animal-component-free isolation and cell-expansion protocols were developed for ASCs. Basic stem cell characteristics such as immunophenotype, proliferation, and differentiation potential were assessed in XF/SF conditions and compared with those in allogeneic human serum (HS) or the traditionally used fetal bovine serum (FBS) cultures. Additionally, macromolecular crowding (MMC) was used to support cells in re-creating their own microenvironments in vitro. The effects of MMC on ASC characteristics were analyzed under three culture conditions, i.e., in FBS, HS and XF/SF. The immunogenicity and immunosuppression of ASCs as well as the secretion of signaling proteins were also determined through mixed lymphocyte reactions after cell isolation and expansion in FBS- and HS-supplemented medium and in XF/SF conditions. Finally, the effects of bioactive glass (BAG) S53P4 or β- tricalcium phosphate (β-TCP) and bone morphogenetic protein (BMP) -2 and -7 on the osteogenic differentiation of ASCs were investigated in HS-supplemented medium. The results showed that ASCs cultured in XF/SF conditions had significantly higher proliferation rates compared with those in HS and FBS cultures. The characteristic immunophenotype and multilineage differentiation potential of ASCs were maintained in all conditions. Although MMC did not support ASC proliferation under any of the studied conditions, adipogenic and osteogenic differentiation was efficient under MMC in FBS and HS media. However, the MMC method was not suitable for the XF/SF cultures studied because the ASCs did not remain viable in long-term exposure to MMC. Furthermore, our immunology studies showed that ASCs were weakly immunogenic when expanded under the three conditions. The significantly strongest suppression was observed with cells expanded in FBS conditions, whereas higher ASC numbers were required to display suppression in HS or XF/SF conditions. The results of the biomaterial studies showed that BMP supplementation decreased the cell number and osteogenic differentiation of ASCs with both the BAG and β-TCP materials. The most efficient osteogenic differentiation of ASCs was observed with BAG cultured without osteogenic supplements. Our findings demonstrated that the novel XF and XF/SF conditions maintained the basic stem cell features of ASCs, and thus, these animal-free workflows may have potential in clinical cell therapies. Careful characterization of ASCs with respect to in vitro culture conditions, immunological properties and cell behavior with clinically approved biomaterials is highly important for positive development of cell-based therapies. Thus, these novel findings of ASC characteristics will provide valuable tools for regulatory authorities. Nevertheless, additional preclinical safety and efficacy studies are required prior to clinical translation