小鼠胚胎成纤维细胞的原代培养及饲养层制备

Preparation of Feeder Layer and Mouse Embryonic Fibroblast Cells in Primary Culture

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。

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 （10μg/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.