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目的建立分离培养小鼠胚胎成纤维细胞(Mouse embryonic fibroblast MEF)的体系,探讨诱导多能干细胞(induced pluripotent stem cell,iPSC)培养所需饲养层的最佳制备方法,制备高质量的IPSC饲养层细胞。方法采用胰蛋白酶消化法,将昆明小鼠胚胎制成细胞悬液接种培养。用不同浓度的丝裂霉素C处理MEF细胞制备饲养层,观察处理后的细胞活力及有无再增殖现象,在制备的饲养层细胞上接种IPS细胞,观察各组IPS细胞生长状态。结果采用0.0625%胰蛋白酶消化孕12.5-14.5d雌鼠胚胎组织获得高活力的MEF细胞,用15μg/ml的丝裂霉素C处理后MEF细胞无明显增值,细胞活力在90%以上,其IPS细胞生长状况最好,效果最佳。结论该方法可获得高活力的MEF细胞。15μg/ml的丝裂霉素C处理1.5h后的MEF细胞制成的饲养层最适于IPS细胞生长。
OBJECTIVE: To establish a system for isolation and culture of mouse embryonic fibroblast MEF, and to explore the best preparation method for feeder layer of induced pluripotent stem cell (iPSC) and to prepare high quality IPSC feeder layer cell. Methods The trypsin digestion method was used to inoculate Kunming mouse embryo into cell suspension. MEF cells were treated with different concentrations of mitomycin C to prepare feeder layer. The viability of cells after treatment and the phenomenon of repopulation were observed. IPS cells were inoculated on feeder cells and the growth of IPS cells in each group was observed. Results MEF cells with high viability were obtained by digestion with 0.0625% trypsin in embryos of 12.5-14.5 d pregnant women. MEF cells treated with 15 μg / ml mitomycin C showed no significant proliferation and cell viability was over 90% The best cell growth, the best. Conclusion This method can obtain high-activity MEF cells. Feeder layers made from MEF cells treated with 15 μg / ml mitomycin C for 1.5 h were the most suitable for IPS cell growth.