Isolation,cultivation and biological characterisitics of human adipose derived mesenchymal stem cells in vitro

Objective To explore a method for effective isolation and multiplication of human adipose derived mesenchymal stem cells ( ADSCs ),observe its biological characteristics and verification by morphology and multi-potential differentiation. Methods The subcutaneous adipose tissue was collected from the operation. Primary ADSCs were isolated subsequently by the methods of stirring digestion within collagenase type I. The morphological changes of the ADSCs were observed under the microscope. Growth curve of cells was measured by MTT method,and the membrane antigens expression of cells,such as CD29,CD31,CD34,CD45,CD90,CD105 were detected through flow cytometry ( FCM ). Adipocyte differentiation was induced by DMEM supplemented with dexamethasone,3-isobutyl-1-methylxanthine,indomethacin and fetal bovine serum. Osteocyte differentiation was induced by DMEM supplemented with dexamethasone,β-glycerophosphte,antiscorbic acid,insulin and fetal bovine serum. Results The ADSCs in both primary and passage culture were fusiform shapes,and stable in growth with active proliferation. ADSCs were in latency for 2 days,converted into growth period on the 3rd day and entered the stationary phase on the 5th day. FCM results indicated that the positive rate of CD29,CD31,CD34,CD45,CD90,CD105 were respectively 73.4%,3.6%,4.5%,2.0%,97.3%,80.4%. After purposively induced,ADSCs had the phenotype characterisitics of adipocyte or osteocyte. Conclution The methods of stirring digestion within collagenase can be effectively used to isolate and purify human ADSCs. The cultured ADSCs are stable in growth with active proliferation and share the general biological characterisitics of ADSCs,which will further make them ideal seed cells for tissue engineering.