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目的:探讨人脐带血中的间充质干细胞(umibilicalcordbloodmesenchymalstemcells,UBCMSCs)的分离培养方法。方法:采用密度梯度离心法结合直接贴壁法分离UBCMSCs,MDEM(低糖)培养基加20%新生小牛血清培养并传代,进行形态观察并应用FACScan流式细胞仪检测细胞表面抗原表达情况。结果:将密度梯度离心收获的单个核细胞接种培养后,24h后即可见有少量细胞贴壁,呈圆形;48h后贴壁细胞增多,少量贴壁细胞形态呈单极梭形突出;反复换液后梭形细胞明显增多,最后获得均一的贴壁细胞。培养6天后细胞开始呈对数生长。在单个核细胞中,CD44+细胞的比例占16.8%,CD34+为29.6%;经培养14天后,CD44+细胞显著增多,占54.4%,而CD34+细胞的比例降至6.0%。结论:密度梯度离心结合直接贴壁法可在体外分离培养UBCMSCs,可望用于大量制备人源性MSCs。
Objective: To investigate the isolation and culture of umbilical cord blood mesenchymal stem cells (UBCMSCs) in human umbilical cord blood. Methods: UBC MSCs were isolated by density gradient centrifugation combined with direct adherent method. Cultured and passaged MDEM (low glucose) medium supplemented with 20% newborn calf serum for morphological observation and cell surface antigen expression detection by FACScan flow cytometry. Results: After mononuclear cells harvested by density gradient centrifugation, a small number of adherent cells were observed after 24 hours of inoculation. The number of adherent cells was increased after 48h, and a small number of adherent cells appeared monopolar spindle-shaped protrusions. After fluid spindle cells increased significantly, and finally get uniform adherent cells. After 6 days of culture, the cells began to grow logarithmically. In mononuclear cells, CD44 + cells accounted for 16.8%, CD34 + 29.6%; after 14 days of culture, CD44 + cells increased significantly, accounting for 54.4%, while the proportion of CD34 + cells decreased to 6.0%. Conclusion: Density gradient centrifugation combined with direct adherent method can be used to isolate and culture UBCMSCs in vitro, which is expected to be used for mass production of human MSCs.