Hello Everyone,
I am having some issues with regards to some iPSC stocks I have been trying to revive for a while. The initial lines that we ordered thawed fine and grew fine, so we froze down a lot of stocks using cs-10 as per the Stemcell manual (we cultured the cells in mTESR and followed guidelines from said manual + what is recommended by the company we purchased these cells from). I thawed some of the stocks I made for some staining + imaging and they were also growing fine (+ ROCK inhibitor the first 24h). But all of a sudden, when thawing more stocks for further experiments, they just stopped recovering and cannot seem to stay attached. I have tried a new bottle of media, tried thawing with Stemflex, tried new matrigel coating, and even decontaminated the incubator prior, but I am still coming across the same issue. If there is anyone that has had the same issue that has some feedback, this would be highly appreciated.
Sounds like most likely your frozen stocks got killed. How often do people rummage through your boxes, leaving them on the counter while all the cells warm up? Can you rule out that somebody ran out of ln2 altogether, quietly filled it back up and didn't tell you? These things happen all the time...
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