I've been working with HEK293T cell line for the last 3 months and over the last month, they seem to keep dying after a week. I restore the cell line from stocks frozen in liquid nitrogen and leave for 1/2 days in a T25 flask with DMEM-high glucose (with 10% FBS, 5% L-glutamate and 5% penicillin and streptomycin) until confluent and then split 1:2 into a T75 flask where after 2/3 days the cells are confluent again. However, every time I split/seed from this point into a T75 flask or 24 well plate, my cells just don't seem to grow and are beginning to die within 3/4 days. This problem has only arisen in the last 3/4 weeks and was wondering if anyone has had a similar experience. (Note: I use PBS for washes and detach using trypsin ~5 minutes).
HEK293 cells are not very sticky at all and don't need to be trypsinised. I just pipette wash the cells to detach with fresh DMEM, perhaps that's the issue.
Hi, I worked with HEK cells. I used DMEM+10%FBS+P/S but not L-glutamate. They grew fine in that growth medium mix. You shouldn't grow your cells so they reach full flask confluency. HEK cells consume growth medium very rapidly and then they detach from the surface of your flask. If you are working every day with them seed them at 40% confluency (not more) so they never reach more then 90% confluency. Doubling time is around 24 hours so you should have enough cells to work with. I used trypsin well and never had problem with cells dying from it so I do not think that is the problem in your case.
Hello
Have you started using a new batch of FBS recently? Cells in culture come under stress when there are changes in the composition of the medium, presence of contamination especially mycoplasma, pH changes. You need to check on these. Cells under stress in culture fail to attach to the substratum and eventually die.
I also agree with @ Mateo Glumac. Avoid subculturing cells when they are over confluent.
Best Wishes.
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