昆明小鼠胚胎干细胞最佳分离方法及培养液选择

Optimized isolation method and medium choice of embryonic stem cells from Kunming mice

背景：到目前为止已经建立数百个小鼠胚胎干细胞系,昆明小鼠是中国应用最多的实验小鼠,其建系成功报道极少。目的：建立一简单有效的昆明小鼠胚胎干细胞分离培养体系,以提高昆明鼠胚胎干细胞建系成功率。方法：用添加血清替代物或胎牛血清培养液的小鼠胚胎成纤维细胞制备饲养层。昆明小鼠经孕马血清促性腺激素和人绒毛膜促性腺激素促排卵,交配后3.5d冲洗子宫取胚胎,将胚胎接种在饲养层中培养,观察胚胎贴壁、内细胞团集落形成率。4~6d后在0.05%trypsin-0.02%EDTA消化液中用切割法处理增殖的内细胞团块,再接种到新的饲养层上。倒置显微镜下观察形成的胚胎干细胞样集落形态。结果与结论：妊娠3.5d孕鼠适合胚胎干细胞的分离培养;在0.05%trypsin-0.02%EDTA消化液中采用切割法进行内细胞团集落的分离,集落增殖快、分化低;在含胎牛血清培养液中进行干细胞培养较血清替代物培养液中传代次数多,此培养液更适宜进行分离培养昆明鼠胚胎干细胞。

BACKGROUND：Up to date,hundreds of mouse embryonic stem cell（ESC） line has been established.Mouse of Kunming line is commonly used in experiment,and rare reports have addressed the success establishment.OBJECTIVE：To establish simple,efficient protocol for the derivation of ESCs from Kunming mice,and to elevate the success rate of establishment.METHODS：Mouse ESCs were incubated with the media containing serum surrogate or fetal bovine serum.Females of Kunming mice were superovulated with an injection of priganant mare＇s serum gonadotropin and human chorionic gonadotropin.Blastocysts were collected by flushing the uterus at 3.5 days after copulation.Embryos were transferred in dish with fresh mouse embryonic fibroblasts feeder layer.Blastocysts detachment and colony forming efficiency of inner cell mass were observed.4-6 days after culture,inner cell masses were digested mechanically in 0.05% trypsin-0.02%ethylenediamine tetraacetic acid.The disaggregated inner cell masses were transferred into a new feeder layer.Typical ESCs morphology was observed through an inverted microscope.RESULTS AND CONCLUSION：Pregnant mice of 3.5 days were the best choice to derive ESCs.Inner cell masses were separated through cutting in drops of 0.05% trypsin-0.02% ethylenediamine tetraacetic acid.The disaggregated inner cell masses grew more rapidly and maintained less differentiation.The ESCs were passaged many times in fetal bovine serum medium.This medium is more suitable to separate ESCs from Kunming mouse.