想问下大家，用no feeder体系诱导ips.细胞大量死亡

2374691002

最近用no feeder方法诱导ips，先复苏原代成纤维细胞（胎儿P1），长满后分盘，每盘10万个细胞，18小时后加入慢病毒，连续诱导2次，polybrene浓度为5ug／ul，诱导4天后，等细胞长满，将其转移至用matrigel处理的6孔板里，每个孔20万个细胞，转后第一天用成纤维细胞培养基和tesr1（1:1），第二天1:3.第三天换成全tesr1.结果第四天细胞形态突然变差，细胞大量死亡，想问各位大虾，是什么原因？是不是tesr1不适合养成纤维细胞

lchanlon

I think what happened in your iPSc generation experiment is normal. This is happened in my experiment. But I am a little bit confused about 100, 000 cells per plate/dish. What kind of dish are you using for lentivirus transduction? if 100, 000 fibroblast cells was in one well of six-well plate, this might be good. It is terrible in a 100-mm dish. Based on my experience on human primary fibroblast culture, cell density is a factor to keep primary cells healthy. Are you sure the polybrene working concentration is 5 ug / ul? I remember I was using 6 ug / ml for virus transduction. After cells reseeding, what i was doing is culture the cells for 2-3 days in fibroblast medium, then replace medium with tesr1 or E8. Cells is not happy after media changing and many cells are dying. The lentivirus for transduction is concentrated virus or just cell supernatant. is it commercial virus? if not, what kind of method did you use for virus packaging? Using lipofectamin or calcium?

wangxiang

E8或是tesr1不适合重编程导入因子后的培养吧，现在有专门的重编程培养基，你可以进行尝试，E8和tes1里面的某些成分不适合于重编程。