Before passaging, and freezing, this ipsc passage's sum was 63
after culturing, I passaged and froze.
First, I found that the transferred cell was not attached on matrigel plates (0.5mg/plate)
I thought that may be related to plate coating or my thawing mistakes.
Then thawed them to another person's matrigel by themself and my matrigel by myself. But it didn't attach. But colony size was great, and colony density was also high.
Moreover, cells are taken away changing the medium ( two days after subculture. )
I am still trying to figure out the reason.
I used gibco's Verene, Wicell E8.
Those pictures are captured after 24h subcultured.
In the picture, non-attach colonies are soooo weird!
Could you find some reasons?
Thank you
it looks like the frozen cells are not happy for some reason. following could be the reasons:
1. Is there any possibility that the frozen vials were not stored at proper temperature / had been too long in Mr. Frosty before being transferred to liquid Nitrogen or -150C freezer?
2. Is the composition of the freezing and thawing medium the same (as in were these cells also frozen down in Wicell E8 media and on the same matrigel concentration) ?
3. Also you can double check if there is Rock-inhibitor included in your thawing medium
good luck !
Is it possible that there's some kind of medium contamination going on (micoplasma, bacteria or fungus)?
At this point, after confirming everything thing else is ok, I'd suggest you go back to basics, thaw a batch onto a feederlayer and give them time to grow into iPS colonies with decent morphology.
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