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目的探讨能够达到临床细胞治疗标准的人脐血间充质干细胞的体外培养与纯化方法。方法以15%FBS/DMEM作为培养基,采用淋巴细胞分离液间接分离法,分离MSCs,以贴壁培养法进行原代培养和纯化。倒置显微镜下观察生长状态和形态学变化,共聚焦显微镜下观察细胞表面抗原CD34,CD44免疫组化染色的鉴定。结果细胞计数显示人脐血间充质干细胞产品的细胞成活率大于95%,其纯度大于85%,符合卫生部第三类医疗技术的有关标准。结论脐带血内含MSCs,可在体外分离培养纯化增殖,获得大量相对纯化的MSCs,达到临床应用水平。
Objective To investigate the in vitro culture and purification of human umbilical cord blood-derived mesenchymal stem cells that can reach the standard of clinical cell therapy. Methods 15% FBS / DMEM was used as the culture medium, and the MSCs were separated by indirect method of lymphocyte separation. The primary culture and purification were performed by adherent culture method. Under inverted microscope, the growth and morphological changes were observed. Confocal microscopy was used to identify the cell surface antigens CD34 and CD44. Results The cell count showed that the cell survival rate of human umbilical cord blood mesenchymal stem cells was more than 95% and the purity was more than 85%, which accorded with the third standard of medical technology of Ministry of Health. Conclusion MSCs contained in cord blood can be isolated, cultured, purified and proliferated in vitro, and a large number of relatively purified MSCs can be obtained, reaching the clinical application level.