Optimising growth of thawed mammalian cells - - is it OK to use a T25 flask? (Jul/04/2005 )
HI
I'm trying to optimise conditions for mammalian cells to revive after they are thawed from liquid nitrogen storage. I understand some cell type especially require to be in close contact with fellow cells in order to start growing optimally. So I thought of putting my thawed 1mL of cells into a T25 flask instead of the regular T75 flask. Anyone who has done this before? Is it OK to use a T25 flask for the typical 1mL of frozen cells- or is the surface area too small? Thanks.
Its impossible to say without knowing how many cells were frozen. I often thaw into T25's but that is when I know only a small number of cells were frozen.
Hi, thanks for your note. Well, the 1mL of frozen cells represent one fifth of all the cells in a T75 flask grown to confluency. Usually I have no problems thawing into a T75. But recently, the next day after I remove media to get rid of DMSO, I see only a handful of cells are attached, and they were rounded, not the usual shape. Instead, I observed good numbers of cells were floating, and these got dumped when I changed media. So if I now use T25, I wonder if that would help in getting recovery faster?
If you move to the t25, unless your cells are pretty fragile, I would take your thawed cells into media, then spin them down and resuspend in fresh media so that the DMSO is removed right away since you will be in a smaller volume for ON culture.
If you haven't had a problem with this before it is also possible that there were problems during the freezing of this batch of cells and that is causing the low survival.
If you haven't had a problem with this before it is also possible that there were problems during the freezing of this batch of cells and that is causing the low survival.
Hi
Thanks for your advice. I'm in a dilemma to decide if these cells are too fragile for spinning or not. These are CHO cells, which normally will recover fine without spinning. But they were shipped to me from my collaborators, such that they were in dry ice for 7 days. This shipping condition may have may have damaged it somewhat, as the first tube didn't give me cells. Under normal conditions, dilution into normal media for overnight is fine, the diluted DMSO can be tolerated by the cells. The question is this time to spin or not. But certainly I think I will increase the FBS, from 5 to 10-20%, and omit antibiotics until the first passage.