饲养层细胞的接种密度对分离培养胚胎干细胞的影响

Effect of densities of feeder layer for isolation and culturing of Kunming white mouse embryonic stem cells

建立有效的昆明白小鼠胎儿成纤维细胞(Mouse embryonic fibroblast,MEF)饲养层培养体系,用于分离和培养小鼠胚胎干细胞(Embryonic stem cell,ES细胞)的研究。(1)取怀孕14.5 d昆明白小鼠胎儿分离成纤维细胞,利用体外培养体系分离传代,选取生长旺盛并且已纯化的P3代的MEF,经丝裂霉素处理后,用细胞计数板计算活细胞数,分别按1×104、1×106、1×108/mL密度接种,制备饲养层,观察不同密度饲养层的生长状况。(2)取怀孕4 d的昆明白小鼠囊胚,接种在不同密度饲养层上。观察不同密度饲养层上囊胚、ICM及ES细胞的克隆生长情况。结果显示:囊胚在密度为1×106/mL的饲养层上,贴壁率和ICM孵出率分别为(97.0±3.606)%和(96.3±2.887)%,显著高于其他2组;密度为1×106/mL饲养层上的ES细胞克隆形成率高于密度为1×104/mL(差异极显著,P<0.01)和1×108/mL饲养层上的ES细胞克隆形成率(差异显著,P<0.05);而1×104/mL饲养层和1×108/mL饲养层上的ES细胞克隆形成率差异不显著(P>0.05)。结果表明:以1×106/mL密度接种的MEF作为饲养层,最适合用于分离培养昆明白小鼠ES细胞,有利于囊胚的发育,ICM的增殖,促进ES细胞的增殖,并起到抑制其分化的作用。

To estahlish feeder layer culture system of Kunming mouse embryonic fibroblast（MEF）cells for isolation and culturing of embryonic stem（ES）cells in vitro. （1）MEF were isolated from the 14.5 d fetus mouse. By in vitro culture system of MEF, after isolated and subcuhured, using Generation 3 MEF were selected. After treated with Mitomycin C,living cell were inoculated at concentrations of 1 × 10^4 , 1 × 10^6 and 1 × 10^8/mL after their number counted as the feeder layers of ES cells. The growth behavior of different density of MEF was observed. （2）The blastulas of 4 d from Kunming mouse were collected,cultured on medium with different density of MEF. The growth of the blastulas, ICM and ES cells were observed. The blastulas cultured on MEF with a density of 1 × 10^6/mL showed that attachment rate and ICM growing rate were （97. 0 ± 3. 606）% and （96. 3 ± 2. 887）% respectively, which expressed more than those cultured on the other two kinds of feeders. The clone forming efficiency of ES cells on 1 × 10^6/mL MEF were higher than those of on 1 × 10^4/mL MEF（P（0.01） and on 1 × 10^8/mL MEF（P（0.05）. While the clone forming efficiency of ES cells on 1 × 10^4/mL MEF and 1 × 10^8/mL MEF were not significant（P〉0. 05）. As feeder cell of MEF with a density of 1 × 10^6/mL is the best for isolation and culturing of embryonic stem （ES）cells in vitro ,which can be beneficial for the development of blastulas,the proliferation of ICM, inducing the proliferation of ES cells,and inhibiting their diferentiation.