人胚胎干细胞饲养层的制备及生物学活性

背景：建立一种既可以大量制备,又能保存并保持较高活性的饲养层是胚胎干细胞培养研究不可缺少的环节。目的：体外分离培养、冻存复苏ICR小鼠胚胎成纤维细胞,观察其生物学特性。方法：取ICR小鼠13.5d胚胎,用胰蛋白酶分步消化法分离培养小鼠成纤维细胞,对冻存复苏后的小鼠胚胎成纤维细胞形态、生长曲线、贴壁率、细胞化学染色及支持人胚胎干细胞生长特性等进行观察。结果与结论：复苏后的小鼠胚胎成纤维细胞在体外传代30min时80%以上细胞贴壁,生长曲线显示细胞增殖活跃,细胞化学染色AKP、PAS、POX阴性,能长期支持人胚胎干细胞传代生长。提示此方法所获得的小鼠胚胎成纤维细胞复苏后有较高的生物学活性,可为人胚胎干细胞扩增提供稳定、优质的饲养层细胞。

Preparation and biological characteristics of feeder cells for human embryonic stem cells

BACKGROUND：It is important to establish and save a large number of high-activity feeder cells for the culture of human embryonic stem cells（hESCs） . OBJECTIVE：To separate,culture,freeze and thaw embryonic fibroblasts of ICR mice in vitro,and to explore the biological characteristics of mouse embryonic fibroblast. METHODS：ICR mouse fetuses of 13.5 days were chosen to isolate mouse embryonic fibroblasts by trypsin stepwise. We observed the cell morphology,growth curves,cells adhesive rate,and cytochemistry staining of mouse embryonic fibroblasts after cryopreservation. The effect of mouse embryonic fibroblasts was assessed by the ability to support the growth of hESCs. RESULTS AND CONCLUSION：Thirty minutes after passage,the adhesive rate of mouse embryonic fibroblasts was above 80%. Mouse embryonic fibroblasts proliferated quickly. AKP,POX and PAS cytochemistry staining of mouse embryonic fibroblasts were all negative,indicating that it could support hESCs in a long period. The thawed mouse embryonic fibroblasts have high biological activity,and can be used as the stable and high quality feeder cells for hESCs culture.