Isolation,cultivation,purification and identification of mice bone marrow mesenchymal stem cells in vitro

Objective To explore a new method for the isolation,cultivaton,purification and identification of MSCs and observe the biological features of mice MSCs in vitro.Methods Bone marrow was extracted from the tibia and thighbone of mice.The marrow liquid were isolated with 1.073 g/ml oercoll.MSCs were obtained by removing the non-adherent cells.Then the MSCs were purified and expanded through passage in time.The growth curve was drawn and the morphology was observed.Cell cycle and the antigen expression of P3 MSCs were measured with FACS.Results The MSCs exhibited a small round shape after fresh separation.After cultivated and passaged,the MSCs were homogenenously fusiform shaped.The growth curves of P1,P2 and P3 MSCs were S shape.The cells of G0-G1 stage account for 75.27%.The expression of CD 44 was positive,while the expression of CD34 was negative.Conclusion The method of density gradient centrifugation combined with adherent culture could isolate MSCs from bone marrow simplely.DMEM-LG medium supplemented with 15%fetal bovine serum is suitable for the culture of MSCs.The cultured MSCs lineage is stable and can be used for further research.