How do you make your media? Do they contain glutamine?
In case your basic media contains GlutaMAX you can stop reading this article.
GlutaMAX is a temperature instable dipeptide of glutamine and was specifically developed for cell culture purposes. Glutamine is unstable and breaks down at higher temperatures.
All those scientists who come in the morning, put their media in the water bath and then take care of their cell culture in the afternoon continously reduce glutamine levels in their media.
If you make large medium bottles because you are too lazy to make fresh medium every week you might end up with half of your glutamine concentration after 5 days! And your cell count drastically reduces after 4 days.
Ever wondered why your cells are not growing properly? Maybe you just found one reason ...
Glutamine is important for cell growth and viability. It is an essential amino acid and cells can use it as an energy source if glucose levels are low. This is especially important for cells with high energy demands like rapidly dividing cells.
Experiments often challenge your cells, think about transfections or drug tests. In critical conditions you dont want to additionally limit essential nutrients. And most importantly, you want to keep your cells continously under the same conditions.
I actually dont know why people keep using glutamine instead of GlutaMAX but in both my PostDoc labs my bosses kept holding on to glutamine. Because thats how things worked till then and they didnt want to change anything. Not a really smart argument. So, I assume that there are still some folks out there who do use glutamine ...
I also loved to put media in the water bath for hours. But after working in a stem cell lab I completely changed that habit. Another temperature-sensitive media supplement is FGF (fibroblast growth factor) which is used to grow human iPSC.
This is actually the main reason why iPSC have to be fed every day. Their maintenance of pluripotency depends on FGF levels. After 24h in the incubator most of the FGF is degraded and has to be replenished by fresh medium.
I usually did not put my stem cell media in the water bath at all. Just put it in the morning under the hood and after 1-2h its at room temperature and thats good enough.
You see, cell culture looks easy but there are some little details that do make the difference between culturing cells and a successful cell culture.