小鼠胚胎成纤维细胞饲养层制备条件的研究

Conditions for establishing mouse embryonic fibroblast feeder layers

目的:建立小鼠胚胎成纤维细胞饲养层最佳分离培养及丝裂霉素C(MMC)处理的方法,用于培养胚胎干细胞。方法:取不同胎龄的胎鼠分离原代成纤维细胞,观察不同胎龄和培养、分离条件对细胞增殖的影响;观察不同浓度和不同作用时间下MMC对细胞增殖的抑制作用。用MTT法测定细胞增殖的数量。取妊娠3.5d的囊胚在经MMC处理的饲养层上培养,观察胚胎干细胞集落生成情况。结果:制备小鼠胚胎成纤维细胞饲养层的最佳胎龄是12.5～14.5d;室温条件下,0.25%胰蛋白酶消化时间最好在3～5min之内;原代细胞培养2～4d后即可传代,MMC能抑制胚胎成纤维细胞的增殖,最佳的作用浓度和时间是20μg/ml,2～4h,成纤维细胞饲养层可以维持10d。囊胚在饲养层上能发育成典型的“鸟巢”状干细胞集落。结论:从12.5～14.5d胎龄小鼠胚胎分离培养的胚胎成纤维细胞质量最佳,经20μg/mlMMC处理2～4h后,可以作为饲养层用于胚胎干细胞的分离克隆。

Objective: To establish mouse fibroblast cell feeder layers to support embryonic stem cells in vitro. Methods: Under different culture and separation conditions, the mouse primary embryonic fibroblast cells(PMEFs) were isolated from mouse fetuses of different gestational ages. The inhibitory effect of mytomycin C (MMC) on fibroblasts was observed by measuring cell numbers through MTT assay. The 3.5-day blastocysts from Kunming species mice were collected and cultured on the fibroblast cell feeder layers treated with MMC. Results: The optimal gestational age for isolation of mouse embryonic fibroblasts(MEFs) was 12.5 to 14.5 days. At room temperature, the optimal time for digestion of fetal tissue was 3 to 5 minutes with 0.25% trypsin. When MMC concentrated on 20 μg/ml and acted for 2～4 hours, it worked best to inhibit proliferation of fibroblasts. MEFs kept alive for 10 days after being treated. The stem cells grew with characterized 'nest' morphology. Conclusion: MEFs isolated from 12.5～14.5-day gestational mouse fetuses have the best state. Mouse embryonic stem cells grow well on feeder layers when MEFs are inhibited by 20 μg/ml MMC for 2～4 hours.