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目的从人胚胎躯干中分离、培养人胚胎成纤维细胞,建立人胚胎成纤维细胞饲养层用于人精原干细胞的培养。方法利用酶消化法从孕5～9周龄人胚胎躯干中分离培养人胚胎成纤维细胞,制作饲养层,使用不同浓度丝裂霉素C处理,以细胞形态、生长曲线作为胚胎成纤维细胞和饲养层的评价指标。结果从人胚胎中成功分离培养出人胚胎成纤维细胞,该细胞可传代15代以上,且经过传代及冷冻复苏后生物学特性无改变。12.5mg/L丝裂霉素C作用2h可达到较好处理效果。结论成功分离和培养人胚胎来源的成纤维细胞,用于人精原干细胞饲养层的制作。
OBJECTIVE: To isolate and culture human embryonic fibroblasts from the human embryo trunk and to establish a human embryonic fibroblast feeder layer for the culture of human spermatogonial stem cells. Methods Human embryonic fibroblasts were isolated and cultured from human embryos of 5 ~ 9 weeks of gestation by enzyme digestion method. The feeder layer was made by using different concentrations of mitomycin C, and the cell morphology and growth curve were taken as embryonic fibroblasts and Feeding layer evaluation index. Results Human embryonic fibroblasts were successfully isolated and cultured from human embryos. The cells were passaged for more than 15 passages and their biological characteristics were unchanged after passage and cryopreservation. 12.5mg / L mitomycin C role 2h to achieve a better treatment effect. Conclusion Human embryo-derived fibroblasts were successfully isolated and cultured for the production of human spermatogonial feeder cells.