临床应用型人脐血间充质干细胞分离培养与鉴定

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。

Culture and purification of mesenchymal stem cells of human cord blood

Objective To explore a practical method of culture and purification of mesenchymal stem cells （MSCs） from human cord blood in vitro in order to satisfy the clinical application. Methods The MSCs from human cord blood were cultured in 15% FBS/DMEM and separated indirectly by lymphocyte separating medium. The primary culture and purification were performed by adherent method. Growth condition and morphology change of MSCs were observed under the inverted microscope, and the cell surface antigen was identified by CD34 and CD44 immunohistochemistry and observed by laser scanning confocal microscope. Results The obtained human MSCs showed CD44 positve and CD34 negative. The purity of human MSCs was more than 85% and about 95% of them were viable, which was in line with the standard of third category of national medical technology issued by Ministry of Health of China. Conclusion The MSCs from human cord blood were separated, cultured and purified successfully, which meets the need of clinical treatment by MSCs transplantation.