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In this experiment, human adipose-derived stem cells (human adipose-derived stem cells, hASCs) study, in vitro isolation, culture, amplification, and identification of the adipogenic, osteogenic into cartilage induced differentiation and self-renewal capacity of its in vitro evaluation and multi-differentiation potential, seed cells for tissue engineering and clinical soft tissue organ reconstruction. In this study, using collagenase digestion and adherent screening combining adipose-derived stem cells extracted from the surgical resection of adipose tissue (adipose-derived stem cells, ASCs) inverted cell morphology was observed under a microscope, MTS law growth curve required to freeze deposit recovery experiments, the use of flow cytometry to detect cell surface markers on the extraction of cultured cells were identified. Induced hASCs in vitro to adipogenic, osteogenic and chondrogenic differentiation. Results isolated from human adipose tissue extracted hASCs in vitro amplification. hASCs showed a fibroblast-like growth, the growth curve of cell proliferation undergoes growth stagnant period, the logarithmic growth phase and growth plateau, very active in their proliferative ability to divide. hASCs after cryopreservation, higher survival, proliferation and differentiation capacity did not change significantly. Flow cytometry hASCs surface markers, expression of CD29, CD44, CD73, CD90, CD105 and CD166, expression of CD31, CD34, CD45 and HLA-DR, in line with the characteristics of the the hASCs surface flag. After adipogenic 14d, cell volume increases, intracytoplasmic full round lipid droplets, was a bunch of grapes like some lipid droplets integration presents large lipid droplets. Oil Red O staining showed bright red particles proved indeed fat droplets. The osteogenic induction 28d, Alizarin Red S (red pigment sulfate Qian S) staining: cells were stratified growth, enrichment extracellular matrix extracellular matrix presented dyed orange-red, and number, as nodular or tablets like existence, to say explicitly calcium deposition and calcium salts nodules. Alcian Blue / Sirius Red (Alcian blue / picrosirius) stained hASCs into the of cartilage induction 42d, rearrange, cell arrangement rules, Chuo-like hyaline cartilage-like structure, rich blue dye cartilage matrix cells ball appear the similar cartilage lacunar structure, the center of the pit like chondrocytes, and around, compared dyed red collagen ingredients in the middle. In summary, successfully established a simple and effective method of in vitro isolation and culture of hASCs. Get the growth of hASCs proliferation, save with multipotent differentiation capacity. hASCs can be used as an ideal seed cells for cell transplantation therapy and tissue engineering.
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