小鼠胚胎成纤维细胞的原代培养及饲养层制备

摘要

Objective To establish a stable and effective system of mouse embryonic fibroblasts ( MEFs) feeder layer in order to culture mouse embryonic stem cells (ESC) in vitro. Methods Embryonic fibroblast cells for primary culture were derived from mouse embryos (pregnant 13-15 days) through trypsin digestion. Results After being treated with mitomycin C (10( μg/mL) , MEFs proliferation could be efficiently repressed and be made into the feeder layer for ESC Undifferentiated growth. Conclusion MEFs feeder layer could effectively support mouse embryonic stem cells to grow.%目的 建立小鼠胚胎成纤维细胞(MEF)饲养层,用于胚胎干细胞的培养.方法 取孕13～ 15d的昆明种小鼠,分离原代成纤维细胞,在37℃时,用0.25％胰蛋白酶(含0.04％ EDTA)消化组织块5min,重复消化多次,24h首次换液,培养3d后传代.采用10μg/mL丝裂霉素C处理胚胎成纤维细胞3h,制备出的饲养层细胞能有效地抑制胚胎干细胞的分裂,且不影响其活力.结果 胎鼠分离原代成纤维细胞经10μg/mL丝裂霉素C处理后,细胞仍保持分泌多种生长因子的能力,在10d内既不增殖,也不死亡,能够很好的维持胚胎干细胞克隆的生长.结论 该方法制备的饲养细胞层适用于胚胎干细胞的培养.