人类原始生殖细胞的体外培养

为了探讨人原始生殖细胞(primordial germ cells,PGCs)的体外分离培养条件及相关影响因素,以小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEF)作为饲养层,室温下用1．25 g／L胰蛋白酶+0．2 g／L EDTA消化人胚胎组织5～9 min,或用1 g／L胶原酶(IV型)消化20～40 min,分离得到PGCs,然后用不同培养液培养,从培养液角度较为系统的研究原代培养人原始生殖细胞的条件。结果表明,在KSR培养液(体积分数15％ Knockout serum replacement+高糖DMEM+10 ng／mL人重组白血病抑制因子(LIF)+4 ng／mL人重组碱性成纤维细胞生长因子+10 ng／mL Forskolin)和STO(建系的小鼠胚胎成纤维细胞)条件培养液中培养的原代人原始生殖细胞,有较高的克隆形成率;培养液中添加不同质量浓度的LIF对克隆形成率影响差异不大。

Culture of human primordial germ cells in vitro

The study aims to investigate the proper conditions and affecting factors in culturing human primordial germ cells (PGCs) in vitro. By digesting the early embryonic tissue in 1. 25 g/L trypsin + 0. 2 g/L EDTA or 1 g/L Collagenase IV at the room temperature,PGCs were separated and cultured on the mouse embryonic fibroblasts cells. The proliferation and colony forming efficiency of the PGCs were investigated and the effects of the different concentrations of LIF (leukemia inhibitory factor) and the conditions medium (CM) on PGCs were studied. PGCs were identified by alkaline phosphatase activity,and staining was also investigated. The proliferation and the colony- forming efficiency of the PGCs were increased when PGCs were cultured by KSR (Knockout serum replacement+ 10 ng/mL LIF +4 ng/mL bFGF+10 ng/mL Forskolin) or STO CM. The proliferation and the colony- forming efficiency of the PGCs was not different when the concentration of LIF was between 5 - 15 ng/mL. KSR or STO CM was best to isolate and culture human primordial germ cells in vitro. The effects of the different concentrations of LIF (leukemia inhibitory factor) on PGCs were little.