ICR小鼠胚胎干细胞的分离与培养

Isolation and cultivation of ICR mouse embryonic stem cells(ESCs)

为了研究不同条件对ICR小鼠ES细胞的影响，试验以12．5～13．5dICR小鼠胚胎成纤维细胞（MEF）为饲养层，以3．5dICR小鼠胚胎为试验材料，探讨了血清、生长因子及传代方法对ICR小鼠Es细胞分离培养的影响。结果表明：采用含15％FBS的Es细胞培养液的囊胚贴壁率及ICM增殖率（79．3％，69．0％）均比含15％KSR、5％FBS＋10％KSR的细胞培养液高（42．9％，28．6％；75．0％，54．2％），Es细胞最高传至6代；培养液中添加10．g／m LLIF＋10ng／mLSCF的效果比单独添加1种因子的效果好，最高传至6代，高于单独添加1种因子的传代数（4代，2代）；用3种传代方法进行传代时，采用差异贴壁法传代效果最佳，最高传至8代，酶消化法传至4代，机械加酶消化法传至6代。

To study the different conditions on ICR mouse ESCs, the mouse embryonic flbroblasts（MEFs） were made by 12.5 - to 13.5 - day mouse fetus and 3.5 - day mouse embryos. The results suggested that the adherence rate of blastocysts and proliferation rate of ICM for the me- dium contained with 15% FBS（79.3% ,69.0% ） were both larger than the mediums contained with 15% KSR or 5% FBS ad 10% KSR （42.9% ,28.6% ;75.0% ,54.2% ）, and the ESC clones were cultured for six passages. The medium added 10 ng/mL LIF and 10 ng/mL SCF was better than the one added only one factor, and the ESC clones were cultured for six passages which was higher than the passage number of the medium added one kind of factors （ four passages, two passages）. Comparing the three kinds of passage ways, the diversity adherence way was the best way of passage and the ESC clones were cultured for eight passages, but the ESC clones were cultured for four passages by the en- zyme digestion and cultured for six passages by the mechanic and enzyme digestion.