提高人胚胎干细胞建系率的优选培养体系与方法

目的:建立一种从废弃胚胎中提高囊胚形成率和质量的培养体系,寻找多种促进内细胞团(ICM)数目增多、贴壁、增值的方法,提高人胚胎干细胞(human embryonic stem cell,hESC)建系效率,建立人胚胎干细胞库。方法:将179枚IVFDay3废弃的胚胎放入优选培养体系中培养(G2.5培养液中添加10%人血清蛋白,人白细胞抑制生长因子(hLIF),碱性成纤维细胞生长因子(bFGF))。到Day7将形成的囊胚全部用机械法分离ICM,接种于丝裂霉素C灭活处理的原代小鼠胚胎成纤维细胞(MEF)上,培养8-9天,每4-5天传代1次。结果:优选培养体系的囊胚形成率为29.1%(52/179),其中A级囊胚形成率为11.2%(20/179),50个ICM贴壁生长,20个出现克隆形态,成功建立11株hESC(FY-hES-11至FY-hES-21)。11株hESC均具有共同的多能性生物学特性。结论:优选培养体系可以明显提高囊胚形成的质量,促进ICM的增值,纯熟的机械切割法可以避免损伤ICM并提高其贴壁率,原代灭活的MEF饲养层可以明显促进细胞增殖。

The Modified Culture Medium and Special Method to Increase the Derivation of Human Embryonic Stem Cells

Objective: To establish a modified culture medium to improve the blastocyst formation rate and quality from the discard-ed embryos,and find good methods to promote the number of inner cell mass(ICM) increase,adherence and growth,so as to increase the derivation of human embryonic stem cells(human embryonic stem cell,hESC) and build hESC bank.Methods: We collected 179 Day3 discarded embryos into the modified culture medium(G2.5 medium supplemented with 10% human serum albumin,human leukocyte in-hibiting growth factor(hLIF),basic fibroblast growth factor(bFGF)).At Day7 separated ICM from the blastocyst and seeded on the mouse embryonic fibroblasts(MEF) of passage 0 which have been inactivated by mitomycin C.The cells were cultured 8 to 9 days with hESC medium and passaged every 4 to 5 days.Results: The data showed that the formation of blastocyst in the modified culture medium was 29.1%(52/179),and the blastocyst formation rate with grate A was 11.2%(20/179).The number of ICM adherence were 50,and 20 clones appeared,finally established of 11 hESC which name were(FY-hES-11 to FY-hES-21).All of the hESC lines with common pluripotent biological characteristic.Conclusions: Our Studies have shown that the modified culture medium can improve the blastocyst formation rate and increase the number of the ICM.The proficiency skill of mechanical cutting method can avoid damage the ICM and improve the rate of ICM adherence.MEF of passage 0 inactivated as feeder layer can promote cell proliferation significantly.