不同分离方法及培养条件对人脐带间充质干细胞生物活性的影响

Effects of Separation Methods and Culture Conditions on Biological Characteristics of Human Umbilical Cord-mesenchymal Stem Cells

目的:观察不同分离方法及培养条件对人脐带间充质干细胞(Human umbilical cord-mesenchymal stem cells,hUCMSCs)生物活性的影响。方法:分别用组织块贴壁法、酶消化法获取hUCMSCs,进行原代培养,记录各组首次传代时间,相差显微镜观察细胞的形态,流式细胞仪鉴定其CD29、CD44、HLA-ABC、CD34、CD45、HLA-DR抗原表达。分别用DMEM-LG、DMEM-HG和DMEM-F123种培养基培养第3代、第7代细胞,MTT法比较不同时间点3种培养基培养细胞的生长情况。结果:两种分离方法均能得到较均一的梭形或者多角形贴壁细胞,胞核大胞浆丰富,CD29、CD44、HLA-ABC抗原表达阳性,CD34、CD45、HLA-DR抗原表达阴性。用DMEM-F12培养的细胞活力更好,细胞生长更迅速。结论:两种分离方法均能得到hUCMSCs,DMEM-F12培养基更能够促进细胞的增殖,较适合于培养hUCMSCs。

Objective To observe the effects of different separation methods and culture conditions on biological characteristics of hUCMSCs.Methods The hUCMSCs were isolated form human umbilical cord by tissue adherence and digested with collagenase,the time of passage was recorded,in addition,the cell morphology was observed by phase contrast microscope,and the CD29、CD44、HLA-ABC、CD34、CD45、HLA-DR antigen expression was identifi ed using fl ow cytometry.The 3rd and the 7th generation cells were cultured with DMEM-LG,MEM-HG,and DMEM-F12 culture medium.MTT was used to evaluate the growth of hUCMSCs.Phase contrast microscope was used to observe the morphological changes of aging cells.Results hUCMSCs could be separated by each method.The adherent cells showed shuttle or multiple angle shapes,with rich cytoplasm,and positive for CD29、CD44、HLA-ABC antigen,negative for CD34、CD45 and HLA-DR.DMEM-F12 could promote the proliferation of quiescent cells.And the cells presented the better viability.Conclusion Both of the two methods can produce hUCMSCs,DMEM-F12 medium is more suitable for the culture of hUCMSCs.