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诱导多能干细胞(IPS)是近年来研究的热门,而IPS细胞体外培养需要饲养层细胞。小鼠胚胎成纤维细胞(MEF)可分泌白血病抑制因子,作为IPS细胞的饲养层细胞。目的建立分离培养小鼠胚胎成纤维细胞的体系,制作高质量的IPS饲养层细胞。方法选妊娠11.5~15.5d的昆明小鼠制作MEF细胞,选择第三代MEF细胞用不同浓度丝裂霉素C处理,制作饲养层细胞,在饲养层细胞上接种IPS细胞,观察各组IPS细胞生长状态并计算克隆数目。结果妊娠第13.5天的胚胎制作的MEF,经10μg/ml的丝裂霉素C处理3h的饲养层细胞状态较好,其IPS生长状况最好,克隆数目和质量最佳。结论不同胎龄胚胎分离的MEF可用做IPS饲养层,第13.5d的胚胎分离的MEF细胞经10μg/ml的丝裂霉素C处理3h制成的饲养层细胞最适于IPS细胞生长。
Induction of pluripotent stem cells (IPS) is a hot topic in recent years, whereas in vitro culture of IPS cells requires feeder cells. Mouse embryonic fibroblasts (MEFs) secrete leukemia inhibitory factor as feeder cells for IPS cells. OBJECTIVE To establish a system to isolate and culture mouse embryonic fibroblasts and make high quality IPS feeder cells. Methods MEF cells were selected from Kunming mice of 11.5 ~ 15.5 days of gestation. MEF cells of the third generation were treated with mitomycin C at different concentrations to produce feeder cells. IPS cells were inoculated on feeder cells, and the number of IPS cells Growth status and calculate the number of clones. Results MEF embryo produced on the 13.5th day of gestation had a good state of feeder layer when treated with 10μg / ml mitomycin C for 3 hours. The IPS grew best and the number and quality of clones were the best. Conclusions MEF isolated from embryos of different gestational ages can be used as IPS feeder layer. Feeder cells made from 13.5th embryo isolated MEF cells treated with 10μg / ml mitomycin C for 3h are most suitable for IPS cell growth.