两株人胚胎干细胞的无饲养层扩增

目的:建立两株中国人胚胎干细胞(hESCs)的无饲养层的培养体系,观察条件培养基所需的饲养层密度,并探索最佳的传代方式。方法:两株人胚胎干细胞hES-846XX和hES-1846XY分别使用不同密度(3×105/mL、6×105/mL和1.2×106/mL)获得的条件培养基(CM)在无饲养层培养体系培养12代以上,用机械分离法和Ⅳ型胶原酶法进行传代,观察比较结果并对所得hESCs鉴定。结果:6×105/mL～1.2×106/mL之间的密度都是合适密度;Ⅳ型胶原酶在长期传代中优于机械法传代;所得细胞维持干细胞未分化状态和全能性。结论:无饲养层的培养体系可以用于中国胚胎干细胞培养,两株中国人胚胎干细胞(hESCs)的倍增周期、所需饲养层密度及分化率等方面与国外hESCs有明显差别。

Propagation of two human embryonic stem cell lines on feeder - free culture system

Objective:To establish the feeder-free culture system on two chinese human embryonic stem cell lines,observe the optimal density of feeder-layer,make condition medium and the proper way for dissociation.Methods:Two human embryonic stem cell lines hES-8 and hES-18 were cultured in feeder-free culture system with condition medium which was collected from mouse embryonic fibroblasts feeder-layer in the density of 3×105/mL,6×105/mL and 1.2×106/mL respectively for 12 passages.Collagenase Ⅳ and mechanical method were used in the experiments for cell passage.The growth information and efficiency of two dissociated methods were detected and the undifferentiated state and pluripotency of cells obtained from such culture system were estimated.Results:Densities from 6×105/mL to 1.2×106/mL were suitable for collecting condition medium which could maintain human embryonic stem cells undifferentiated state sufficiently.Collagenase Ⅳ was better than mechanical way for dissociation in long term culture.Both two lines maintained undifferentiated state and pluripotency in feeder-free culture system by estimate.Conclusion:Feeder-free culture system can be used on chinese human embryonic stem cell lines successfully and there are obvious differences between two chinese lines with others reported abroad in the double time,rate of differerentiated cells and density of feeder-layer.