I am reprogramming fibroblasts to iPSCs. I see small iPSC colonies around D21 and want to isolate them. I have two questions here.
1. Many iPSC colonies are already yellow from the center. Does this mean that the iPSCs are dying already? (Example one at Day 24 in the picture)
2. The fibroblasts are so dense that when I scratch the colonies they float away alongwith the fibroblast layer. Is there a good way to help detaching the ipsc from the fibros?
Thanks in advance!
Dear Kalaivani, happy to bring you into contact with our Field Application Sientists for iPSCs. Please write me at [email protected]
Best Alex
From the image, it looks like the cells are aggregating themselves and growing in a multilayer to form something that resembles embryoid bodies. This usually happens in iPSC cultures that are over confluent.
I would recommend using versene solution or 0.5mM EDTA in DPBS (without calcium and magnesium) to dissociate iPSCs. This non-enzymatic method prevents fibroblasts from detaching.
Kalaivani Manibarathi You are welcome.
Were you able to passage iPSCs without dissociating fibroblasts?
Yes Muhammad Nihad A S ...I just plated them directly on Matrigel 24well plates then passaged them when they were confluent with PBS/EDTA !!
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