Isolation,culture and identification of rat adipose derived stem cells

AIM: To obtain and identify a large number of adipose derived stem cells(ADSCs) by monoclonal culture,and to investigate the characteristics and differentiation potential of ADSCs derived from the monoclone in vitro.METHODS: Adipose tissue was isolated from 4-day-old rat.After sufficient mixing and filtering,the rat adipose cells were seeded onto culture plates at a low density.Monoclonal cells were indentified through microscope and were picked out with micromanipulative technique when these monoclones were obvious.The ADSCs were identified by colony-forming assay and membrane antigens expression.Expression of Stro-1,CD90,CD105,CD34 were detected with flowcytometry(FCM).Multipotential differentiations of ADSCs into adipocyte,bone and neuron were identified by oil red O staining,alizarin red staining and morphological observation.RESULTS: The obtained cells had high colony-forming efficiency.FCM results indicated that the positive rate of Stro-1,CD90,CD105 and CD34 were 8.1%,78.2%,96.9% and 1.2%,respectively.Under specific conditions,they could differentiate into adipocytes,osteoblasts and nerve cells in vitro.CONCLUSION: Limiting dilution assay is an effective method to isolate ADSCs and the single-cell-derived clonal cell populations demonstrate the properties of stem cells in vitro.