Improvement Of Vitro Culture Methods Of The Bone Marrow Mesenchymal Stem Cells (BMSCs)

Objecyive:Through separation of bone marrow mesenchymal stem cells (BMSCs) extraction method, the medium was changed for the first time time improvement SD rat bone marrow mesenchymal stem cells (BMSCs) cultured in vitro, and in order to explore more effective and rational BMSCs cultured in vitro method for further experimental studies provide sufficient quantities of stem cells.Methods:Through bone marrow adherent method, the modified total bone marrow adherent method, density gradient centrifugation, three different methods to extract the SD rat bone marrow mesenchymal stem cells (BMSCs), and through8h,24h2different kind combination of the first time of changing medium after inoculation, observation and comparison the morphological features,proliferation in vitro culture passaging efficiency,the cell viability of passaged BMSCs, the expression of cell surface markers of the passaged BMSCs of bone marrow mesenchymal stem cells (BMSCs) in each portfolio, in order to filter more reasonable BMSCs in vitro amplification mode.Result:a small amount of spindle-shaped、triangular or polygonal cells adherent can be found when8h medium was changed for the first time in bone marrow adherent method and the modified total bone marrow adherent method, but only a part of the culture flask is visible to a very small amount of adherent cells in the density gradient centrifugation, affixed wall cells gradually increased with the first exchange of medium time pushed back, more spindle-shaped, triangular or polygonal cells were seen adherenting when the first time of changing medium changes to24h。 The first passaging time of the bone marrow adherent method and the modified total bone marrow adherent method is7-8days, the first passaging time of density gradient centrifugation is12-14days, the first two cell extracts than the latter had statistically significant; the each cell generation from the third to the eighth generation by the3cell extraction method is about7-8days, there was no significant difference among the three groups. MTT assay showed cells growth curve was basically the same in each group, when the cells extracted by the density gradient centrifugation spread to the ninth generation, cell morphology Changed, cell proliferation significantly decreased or even stagnation compared with the previous, there was no significant changes in the bone marrow adherent method and the modified total bone marrow adherent method,each group of third-generation rat BMSCs underwent trypan blue and CD34、 CD44detection, the results shown thatthere was no statistical significance in the proportion of living cells between each group, surface antigen test results shown that density gradient centrifugation group、improved bone marrow adherent the first time of changing medium after inoculation group performed well,there was statistically significant compared with the other three groups.Conclusion:The cells were extracted by bone marrow adherent method、the modified total bone marrow adherent method and density gradient centrifugation can all show the morphology of of BMSCs specific cells, and can stable passaging at a certain extent.The BMSCs extracted by the bone marrow adherent method and the modified total bone marrow adherent method had higher Adherent efficiency, shorter first passaging time, better continuing proliferation ability and cell viability; Density gradient centrifugation and the improved whole bone marrow adherent method would get higher purity of rat BMSCs.Through using the full amount of replacing liquid way and properly in advance changed the first time for the medium, the modified total bone marrow adherent method can improve the purity of rat BMSCs,and does not affect the culture efficiency activity of the BMSCs,so the method is a more suitable rat BMSCs training mode.