Isolation,Culture and Identification of Rat Mesenchymal Stem Cells In Vitro

Objective To develop the methods for isolation,culture and identification of rat mesenchymal stem cells(MSCs) in vitro and lay a foundation of a serial study on MSCs.Methods MSCs were isolated and cultured by direct wall adhesion culture of the whole bone marrow,then subcultured and observed for morphology under invert microscope.The proliferation of MSCs was determined by MTT method,based on which a growth curved was plotted.The MSCs of passage 3 were determined for cell cycle and phynotype by flow cytometry.The directional differentiation of MSCs to osteoblasts and fat-like cells were induced,and the differentiation ability was identified.Results After the whole bone marrow cells were cultured for 5 d,obvious proliferation of adherent cells were observed under microscope,and the cells were even in shape,most of which were spindle-shaped.The cells could be subcultured about 7 d after culture,and formed spindle-shaped fibroblast-like cells,i.e.MSCs after subculture for 2 ~ 3 passages.The growth curve of cells was in S shape.Flow cytometry showed that 76.01%,7.13% and 16.86% of MSCs were in G0 /G1,G2 /M and S phases respectively.No CD34 was expressed on the surface of MSCs.However,by using specified inducers,MSCs were differentiated to fibroblast-like and fat-like cells respectively.Conclusion The methods for isolation,culture and identification of rat MSCs in vitro was successfully developed,which might be used for evaluation of MSCs cultured in vitro.