人脐血间充质干细胞分离培养方法的优化

目的探索脐血来源的间充质干细胞体外分离培养的更优方法。方法无菌条件下抽取正常足月剖宫产胎儿的脐带血,肝素抗凝,用淋巴细胞分离液分离脐血单个核细胞。50份脐血分为对照组和改良组,每组25份,分别以1.5×10^7/ml的细胞密度接种于25 cm^2培养瓶中。对照组贴壁3 d后弃上清,去除未贴壁细胞,更换新鲜培养液;改良组贴壁0.5 h后弃上清,去除未贴壁细胞,更换新鲜培养液。结果对照组杂细胞较多,最终成功培养出8份脐血间充质干细胞;改良组杂细胞较少,最终成功培养出18份脐血间充质干细胞。经流式细胞仪检测脐血间充质干细胞的免疫表型,结果显示,所培养出的脐血间充质干细胞不表达或极弱表达CD34、CD106等造血细胞标志,稳定地高表达CD29、CD44、CD105等间充质细胞相关的表面抗原标记物。结论采取脐血单个核细胞贴壁0.5 h弃上清更换新鲜培养液可以明显提高脐血间充质干细胞的培养成功率,其结果优于脐血单个核细胞贴壁3 d弃上清更换新鲜培养液。

Optimization of Isolation and Cultivation Method of Human Umbilical Cord Blood- derived Metsenchymal Stem Cells

Objective To explore an optimum method of isolation and cultivation of mesenchymal stem cells(MSCs) derived from human umbilical cord blood. Methods Umbilical cord blood(UCB) was collected from full term normal delivery or cesarean section delivery infants.Totally 50 samples of UCB were collected,with 50～60ml per sample.The umbilical cord blood mononuclear cells were isolated by density gradient centrifugation with a lymphocyte separation medium.50 samples of UCB were divided into control group and experimental group(each n=25),and the mononuclear cells were inoculated into culture flask with the final density of 1.5×107/ml.The control group discarded supernatant after adhering for 3 days and then the culture solution was changed.The experimental group discarded supernatant after adhering for 0.5 hour and then the culture solution was changed. Results There were many parenchyma cells in control group,and 8 samples of MSCs were finally cultured.There were fewer parenchyma cells in experimental group,and 18 samples of MSCs were finally cultured.The flow cytometer was used to detect immune phenotype of MSCs,and the results indicated that MSCs derived from human umbilical cord blood hardly expressed or expressed few haematopoietic cells antigens(CD34 and CD106),but stably expressed MSCs-related antigens(CD29,CD44,and CD105). Conclusions The method of discarding supernatant after umbilical blood mononuclear cell adhering for 0.5 h and then changing the culture solution can significantly improve the success rate of culturing MSCs derived from human umbilical cord blood,and the result of the above method is superior to that of the method of discarding supernatant after umbilical blood mononuclear cell adhering for 3 days and then changing the culture solution.