利用冷冻胚胎建立人胚胎干细胞系

目的利用辅助生殖技术中废弃的冷冻胚胎建立人胚胎干细胞系。方法将体外受精一胚胎移植周期中废弃的冷冻胚胎解冻，序贯培养至囊胚，分离出内细胞团，接种至混合饲养层，进行传代培养，对可连续传代的细胞系进行鉴定。结果共收集废弃冷冻胚胎44枚，形成囊胚15枚，原代接种内细胞团10枚，其中一枚已稳定传至25代，目前生长状态良好，经鉴定证实为胚胎干细胞。结论利用辅助生殖技术中废弃的冷冻胚胎，可成功建立人胚胎干细胞系。

Establishment of human embryonic stem cell line from frozen embryos discarded in assistant reproductive technology

Objective： To establish embryonic stem cell line from frozen embryos discarded in assisted reproduc rive technology. Methods： The frozen embryos were cultured to develop to blastocysts. Inner cell masses （ICM） were isolated, and cultured and subcultured on the feeder layer that is a mixture of mouse embryonic fibroblast and human foreskin fibroblast. The derived cell line was identified by the expression of markers, and in vivo and in vitro development characteristics. Results： A_ total of 44 embryos were collected in the experiment, 15 of which developed to blastocytes. 10 ICMs were cultured and one has been in continuous eulture and is now at passage level 25. The cell line expresses the markers of Oct-4, SSEA and alkaline phosphatase, and shows the pluripotency （forming embryoid bodies in culture and teratomas in severe combined immunodeficient mice）. The karyotype of the cell line is normal 46, XX. Conclusions： Human embryonic stem cell line can be established with frozen embyos.