crazy ATCC lady eh? - (Jun/17/2011 )

Problem: Working w/ CHO K-1 cell lines. Thawed frozen aliquots of cells and dumped them into Ham's F-12 complete media. Cells take 36hrs just to adhere and then even after 3 days, some cells have gotten back their shape and remaining are still rounded up.

ATCC lady says: I dumped these cells into T-150cm flasks with 30ml media. She thinks its a huge flask to start off with AND its a lot of media so the cells have gotten diluted. She says start off with a small flask, like T-75 maybe with 15ml media. It should become 70% confluent within 2 days she says. Is she making sense?

Confused: I used to thaw fresh vials of cells before and dump them into T-150cm flasks and they would still be OK. They would become confluent in 2 days. Why are my cells behaving differently all of a sudden if they belong to the same batch? Is the ATCC lady crazy?

I'm confused about your question-
Firstly, the frozen aliquots you thawed, are they straight from the ATCC or did you expand them up and then freeze aliquots yourself?
Secondly, when you say its worked before with the same batch, do you meant cells from the same origin (as in all expanded from the same original vial) or are they multiple vials that were frozen down at the same time with the same number of cells?
Thirdly, do you know how many cells are in each vial?

In short, no she is not crazy and yes she is making sense- cells grow better when there are other cells close by, by thawing straight into a P150 you may well have indeed diluted the cells so much that they will take some time to recover. (I don't agree with her about the media volume though, its only surface area to grow on that I would think makes a difference).

Volume can make a difference too - the cell:cell signalling factors are all passed between the cells through the medium. Too big a volume will cause these factors to be diluted.

Seconding the ATCC lady being correct.

Ah,now that makes sense bob, I hadn't thought of that (obviously!!)

Yep. All you guys R awesome-ly right.

I re-tried thawing cells again, removing DMSO by centrifugation and dumped them in t-75 with 12ml media. By 2 days the cells were confluent and happy.

Thanks again for all your time... it really means a lot!

suhas