Hello! I'm a beginner to hiPSC culture, and while my cultures look acceptably good, I'd be grateful for any advice on how I can revive my slightly stressed culture to an un-stressed, healthy state before I can start differentiating them.
The biggest source of possible stresses to my iPSCs so far has been imperfect ReLeSR splitting.
I have had to pipette cells through a p1000 multiple (6-8 times) to get an acceptable clump size, resulting in some clumps that are in an acceptable size range - 50 - 250 uM, but some consist of just 3-4 cells. The cells survive, but I must assume they must be stressed.
How do you all make your iPSC cultures great again? :P
Use laminin 511 culture surface to make them tolerant to passage as single cells. Then accutase for passaging as single cells. Freeze them as single cells in something like mFreSR or Cryo-Brew. Optionally use CloneR2 for thawing, but you don't need to. Really the laminin surface takes care of most of the problem. 5% O2 is really valuable too.
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