无血清无饲养层条件下培养小鼠胚胎干细胞

Serum-and Feeder-Free Culture of Mouse Embryonic Stem Cells

目的研究在无血清无饲养层条件下小鼠胚胎干细胞的培养方法,为最终建立无血清无饲养层培养系统打下基础。方法比较小鼠胚胎干细胞ES-S8株在无血清培养体系和有血清培养体系中的生长情况,分析ES-S8细胞克隆形成效率,测定其生长速度;然后在撤去血清和饲养层的条件下培养ES-S8细胞,进行AKP染色和表面标记物SSEA-1免疫荧光检测。结果ES-S8细胞在无血清培养条件下细胞生长速度减缓,克隆形成率降低,但AKP染色、SSEA-1免疫荧光均显阳性;在无血清无饲养层条件下ES-S8细胞培养仍能形成克隆,且AKP染色、SSEA-1免疫荧光均显阳性。结论研究表明ES-S8细胞能够在无血清无饲养层的培养条件下生长,保持其良好的未分化特性。

Objective The aim of this study was to establish a serum- and feeder-free culture method of mouse embryonic stem cells. Methods Mouse stem cell line ES-S8 cells were cultured in serum and serum-free culture medium, respectively, and their colony formation efficiency and growth rate were compared. Then ES-S8 cells were cultured without both serum and feeder layer cells. The cell growth status was identified by expression of alkaline phosphatase and SSEA-1. Results ES-S8 cells cultured without serum were positive for alkaline phosphatase activity and SSEA-1 staining, but the colony formation efficiency and growth rate were reduced. Upon withdrawal of both feeder layer cells and serum, ES-S8 cells still formed colonies and expressed pluripotent markers （AKP and SSEA-1 ）. Conclusion ES-S8 cells can grow in serum- and feeder-free culture medium, and maintain their undifferentiated characteristics.