小鼠胚胎干细胞成纤维细胞液养层的制备

采用受孕12.5 d、13.5 d、14.5 d的ICR品系小鼠的胚胎制备原代成纤维细胞,在相同条件下观察不同胚龄胚胎成纤维细胞的增殖情况。结果表明受孕13.5 d胚胎为分离小鼠胚胎原代成纤维细胞的最适胚龄。分离出的原代成纤维细胞在37 ℃时,用0.25%胰蛋白酶0.02%EDTA消化组织的时间为3 min,可在培养3 d后传代。F1代和F2代较F3代成纤维细胞分泌的抑制干细胞分化因子LIF的量要多。小鼠胚胎成纤维细胞冻存于-135 ℃~-150 ℃10 个月,复苏后能正常传代。丝裂霉素C抑制胚胎成纤维细胞增殖的最适浓度为10μg/105细胞,作用时间为2.5 h,丝裂霉素C处理过的胚胎成纤维细胞可以在12 d内即不增殖也不死亡,但在6 d内使用效果最佳。

Preparation of the feeder layer of mouse embryonic stem cells

The mouse primary embryonic fibroblst were isolated from ICR strain mouse that the embryo phases of mouse were 12.5d, 13.5d and 14.5d. In the same condition, the proliferation of fibroblasts and inhibition of mytomycin-C were observed. The results showed that the optimal embeyonic age for isolation of mouse embryonic fibroblasts was from 13.5d gestational mouse. At the temperature of 37 ℃, the optimal time for digestion of fetus tissue to obtain fibroblasts was 3min with digestion 0.25% trypsin-0.02%EDTA solution. The optimal concentration of mytomycin-C to inhibit proliferation of fibroblasts was 10μg/10~5 for 2.5h, at that condition of treatment of mytomycin-C the fibroblasts were alive for 12d without proliferation.