CF-1小鼠胚胎成纤维细胞饲养层的制备**

背景：小鼠胚胎成纤维细胞作为干细胞生长用饲养层,优于无饲养层,它能够分泌一些既促进干细胞生长又能抑制干细胞分化的因子。目的：建立CF-1小鼠胚胎成纤维细胞饲养层最佳的分离培养方法,分析丝裂霉素C抑制成纤维细胞增殖的最佳浓度与时间,用于培养诱导多能性干细胞。方法：取孕10~15 d CF-1小鼠,分离胎鼠原代成纤维细胞,经不同质量浓度(5,10,15,20 mg/L)丝裂霉素C处理不同时间(1,1.5,2,2.5,3 h)制备饲养层,并观察其增殖情况。将人诱导多潜能干细胞或胚胎干细胞在丝裂霉素C处理过的饲养层上培养,观察细胞集落生长情况。结果与结论：制备CF-1小鼠胚胎成纤维细胞饲养层的最佳胎龄为13.0~14.0 d。丝裂霉素C抑制CF-1小鼠胚胎成纤维细胞增殖的最佳浓度及作用时间为10 mg/L,2.5 h,成纤维细胞饲养层可以维持5~7 d。诱导多能性干细胞与胚胎干细胞在经10 mg/L丝裂霉素C处理2.5 h后饲养层上能发育成典型的“鸟巢”状干细胞集落。关键词：胚胎成纤维细胞;诱导多潜能分化干细胞;饲养层;丝裂霉素C;细胞集落doi:10.3969/j.issn.1673-8225.2012.10.017

Preparation of CF-1 mouse embryonic fibroblast feeder layers

BACKGROUND: Mouse embryo fibroblasts as the feeder layer for growth of stem cells exhibit better effects than the circumstance without feeder layer because they can secrete some factors which can promote the growth of stem cells and inhibit the differentiation of stem cells. OBJECTIVE: To establish the optimal method for isolation and culture of CF-1 mouse embryo fibroblast feeder layer and investigate the optimal concentration of mitomycin C in inhibiting the proliferation of fibroblasts and the processing time.METHODS: Fetal mice were taken from CF-1 mice at pregnancy days 10-15. Primary fibroblasts were isolated and treated with different concentrations (5, 10, 15, 20 mg/L) of mitomycin C for different time periods (1, 1.5, 2, 2.5, 3 hours) to prepare feeder layers. Cell proliferation was observed. Human induced pluripotent stem cells or embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed.RESULTS AND CONCLUSION: The optimal fetal age for preparing CF-1 mouse embryo fibroblast feeder layer was 13.0-14.0 days. Treatment with 10 mg mitomycin C for 2.5 hours showed optimal effects on inhibiting the proliferation of CF-1 mouse embryo fibroblasts and fibroblast feeder layer could maintain 5-7 days. Induced pluripotent stem cells and embryonic stem cells can develop into typical “bird nest”-shaped colony of stem cells after treatment with 10 mg/L mitomycin C for 2.5 hours.