Dear scientist,
We are currently working with iPSCs and their differentiation into pancreatic B-cells. We have noticed that after thawing, the growth and expansion rate of iPSCs has not been ideal.
We are using mTeSR1 for the expansion and maintenance of iPSCs before their differentiation.
Has anyone had similar problems in expansion and maintenance processes?
How could the growth and expansion rate be improved?
Thank you very much for your help and support
Dear Felipe.
As I know, there are four critical points that could improve the iPSCs expansion.
1) The pre-coated plate, which coating did you use for iPSCs adhesion?
2) ROCK inhibitor, the ROCK inhibitor should be added on the first day of seeding and removed after 24h.
3) Free of contamination, please check out the Mycoplasma contamination
4) NANOG and OCT3/4, please check out the pluripotency of iPSCs that you used, maybe they started to differentiate.
Best regards,
Fatemeh.
Dear Fatemeh Etezadi
Thank you very much for your answer.
We are currently using vitronectin XF to pre-coat the wells as the manufacturer recommends, but we are not using ROCK inhibitor in the moment, I already read about the potential benefits that it has in the first stages of the cells growth.
It is possitive that we do not have Mycoplasma contamination, and I will follow your advise to check the pluripotency markers.
Thank you very much again
Great
I recommend you to use ROCK inhibitor (Y-27632; Wako) at the first day of seeding then should be removed after 24h (more than 24h has negative effect on cell growth)
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