Objective   To establish a three-dimensional culture method for the culture of SD rat bone marrow mesenchymal stem cells (BMSCs) in vitro.     Methods   BMSCs were obtained from femurs and tibiae of SD rats and cultured by the way of density gradient centrifugation combined with attachment culture method. The cell cycles were detected by flow cytometry and cell internal structure by electronic microscopy. BMSCs membrane antigen phenotypes were identified with an immunocytochemical method. BMSCs were suspended in the extracellular matrix (ECM) gel and cultured at the interface of air and medium.    

Results   The cells showed shuttle shape with single nuclear and strong proliferative ability in vitro. The cell cycle analysis showed that 93.10% of BMSCs were at G

0

/G

1

phase. Transmission electron microscopy revealed that the ratio of nucleus/cytoplasm of the cells was larger. Immunochemistry staining revealed significant expressions of vimentin and fibronectin. BMSCs grew well in ECM gel.     Conclusion   Highly purified BMSCs obtained from SD rats can grow well in ECM gel and are of highly proliferative ability and stability.