Isolation,Culture And Multi-Directional Differentiation Potential Of Human Adipose-derived Stem Cells(ADSCs)

This study aims to standardize the production of adipose-derived stem cells(ADSCs)from human adipose tissue under cGMP conditions,and to expand and culture adipose-derived stem cells(ADSCs)under serum-free culture conditions to obtain higher yields while ensuring quality.Standardized identification and functional verification of the obtained cell quality.Establish a non-differentiated,large-scale expansion culture,and it is expected to form an international standard culture system.The lipoaspirate was transported to the laboratory in a closed manner.The Stromal Vascular Fraction(SVF)was separated by enzyme digestion and differential centrifugation.The ADSCs were purified by differential adherence method.Amplification culture in serum medium.Thethird-generation ADSCs were cultured in a 6-well plate,and induced differentiation,adipogenic differentiation,osteogenic differentiation and chondrogenic differentiation were performed with the induced adipogenic differentiation inducer,osteogenic differentiation inducer and chondrogenic differentiation inducer,respectively.After 2-3 weeks,the cell differentiation ability was identified by staining with 2% oil red O stain,alizarin red staining agent and Alisin blue glacial acetic acid staining agent respectively.The cells obtained by separation and purification showed no differentiation when cultured stably for10 generations or more under serum-free culture conditions.The cell morphology was fine,short,small spindle-shaped,the cells were full,the size was uniform,and the growth was spiral.When the cell culture density is 5000/cm2,the doubling time is 20-30 h,and the cell growth vigor is strong when the cell diameter is in the range of 18-20 μm,and there is no aging phenomenon.Flow cytometry results show that the phenotypic characteristics of ADSCs cells are very consistent.More than 90% of ADSCs have D90,CD105,CD44,CD73,CD146,CD166 and CD29 expressed by mesenchymal stem cells.ADSCs also share cell surface antigens with fibroblasts and pericytes.ADSCs have low expression of hematopoietic hepatocyte markers(CD45,HLA-DR)and endothelialmarkers(CD31).The immunophenotype of adipose stem cells is uniform,indicating that high-purity adipose stem cells are obtained and have phenotypic characteristics of MSC.The results of directed differentiation assay of ADSCs showed that the cells were successfully differentiated into the desired cells in a medium containing adipocytes,osteoblasts and chondrocyte differentiation inducers.In this study we successfully isolated and purified ADSCs and established a primary culture system.ADSCs cells have good morphological characteristics and excellent immunophenotype.ADSCs are differentiated into differentiated potentials for adipogenic,osteogenic and chondrogenic cells.