Identification of cultured human adipose-derived stem cells in vitro

Objective Subcutaneous adipose-derived cell populations would be isolated and cultured in vitro.In accordance with international standard,we will confirm that cell populations obtained in vitro are adipose-derived mesenchymal stem cells by morphology,immunophenotyping and identifying of multiple differentiation potential.Methods Using dualdigestion method for separation of cells in vitro with 0.1% collagenase I and 0.25% trypsin to get it,adding LG-DMEM medium containing the volume fraction of 10% fetal bovine serum,culturing at 37℃ and 5% of CO 2 saturated humidity conditions,then amplifying of digestion and passage until the cell growth to 70% to 80%.Cell morphology would be observed through inverted microscope.Flow cytometry detect cell phenotype expression.The third passage cells would be directed induction of differentiation by added osteogenic and adipogenic induction agents into the culture medium,and then induced cells would be staining.Results Isloated cell populations are long fusiform and whirlpool-like adherent growth.P2 cells detected by Flow cytometry,which of CD73 and CD105,C90,CD44,CD49d and CD29 are positive expression,but of CD14 and CD34,CD45,CD106and HLA-DR are negative expression.Alizarin red staining,alkaline phosphatase staining and VON-KOSSA stain tested positive 3 weeks after osteogenic induction.2 weeks after the adipogenic induction,there are small lipid droplets appearing,and oil red O staining is positive.Conclusion The Cells that using collagenase and trypsin digestion method isolated from adipose tissue can be attached to the wall growth.Flow cytometry confirme that isolated cells can express the mesenchyme stem cells consistent phenotype and have osteogenic and adipogenic differentiation ability after directed induced,the cultured cells are consistent with the international standard about the characteristics of mesenchyme stem cells.