title: Characterization of human adipose-derived stem cells cultured in autologous serum after subsequent passaging and long term cryopreservation.
abstract: The aim of this study was to evaluate human adipose-derived stem cells (ASCs) from passage 2 (P2) to P8 cultured in medium containing 5% autologous serum (AS) after a long-term cryopreservation with regards to their surface marker expression, differentiation potential, and immunosuppressive effect in vitro. 8-color flow cytometry and real time PCR were used to determine mesenchymal stem cell (MSC) surface marker expression on ASCs from various passages. In vitro differentiation ability and immunomodulatory properties of ASCs were also tested. Flow cytometry showed that all ASCs express typical MSC markers CD29, CD44, CD73, CD90, CD105 simultaneously, but do not express such markers as HLA-DR, CD34, CD14, CD19, and CD45. Furthermore, median fluorescence intensity of positive cell surface markers increased with each subsequent passage indicating the accumulation of protein expression. The multilineage differentiation demonstrated the ability of ASCs from P6 to efficiently differentiate into adipocytes and chondrocytes, but their potential of osteogenic differentiation was diminished. Data from co-culture of ASCs and autologous peripheral blood mononuclear cells (PBMNCs) indicated that ASCs from P3, P6, and P9 significantly reduce the proliferation of PBMNCs at ASCs:PBMNCs ratio 1:1 and this suppression is dose dependent. This study demonstrated that ASCs from P2 to P8, cultured in the presence of AS, represent a highly homogeneous cell population with a peak accumulation of MSC surface proteins at P5 possessing multilineage differentiation ability and significant immunosuppressive properties after double freezing and more than 4 years of cryopreservation.
authors.affiliation: Latvian Biomedical Research and Study Centre, Ratsupites Street 1, Riga, LV-1067, Latvia.
authors.forename: Ance
authors.initials: A
authors.lastname: Bogdanova
authors.affiliation: Latvian Biomedical Research and Study Centre, Ratsupites Street 1, Riga, LV-1067, Latvia.
authors.forename: Uldis
authors.initials: U
authors.lastname: Berzins
authors.affiliation: University Children’s Hospital, Clinical Laboratory, Vienibas Gatve 45, Riga, LV-1004, Latvia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Nikulshin
authors.affiliation: University Children's Hospital Clinical Laboratory Vienibas Gatve 45 Riga LV-1004 Latvia.
authors.forename: Dace
authors.initials: D
authors.lastname: Skrastina
authors.affiliation: University Children's Hospital Clinical Laboratory Vienibas Gatve 45 Riga LV-1004 Latvia.
authors.forename: Agnese
authors.initials: A
authors.lastname: Ezerta
authors.affiliation: University Children's Hospital Clinical Laboratory Vienibas Gatve 45 Riga LV-1004 Latvia.
authors.forename: Diana
authors.initials: D
authors.lastname: Legzdina
authors.affiliation: University Children's Hospital Clinical Laboratory Vienibas Gatve 45 Riga LV-1004 Latvia.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Kozlovska
mesh_terms: D017667:Adipocytes / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology*; D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D002450:Cell Communication; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002469:Cell Separation; D048430:Cell Shape; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism; D018920:Coculture Techniques; D015925:Cryopreservation; D005434:Flow Cytometry; D006801:Humans; D007108:Immune Tolerance; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008297:Male; D059630:Mesenchymal Stem Cells / Q000276:immunology / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction; D044968:Regenerative Medicine / Q000379:methods*; D044967:Serum / Q000378:metabolism*; D013997:Time Factors
chemical_list: D015415:Biomarkers