I have worked with HEK cells before and have done freezing and thawing with no problems. I am currently working with HEK 293 T cells to make lentivirus and after thawing them, I can grow them with no trouble in T75 flasks to approximately 80% confluency. However, the problem right now is that after I split the cells in the T75 flasks into 10 cm dishes, I observe a lot of dead cells floating in the media and only a minimum number of cells attached to the surface. The cells that have attached are still clumped together and don't seem to be growing. I have been very gentle with my cells and don't think that it's my protocol that's causing the issue but I could be wrong. I would greatly appreciate any advice.
In addition, I use DMEM 4.5 g/L D Glucose with 10% heat inactivated FBS and 4 mM Glutamax and 0.05% Trypsin with EDTA
I had similar problem few years ago with HEK293T cells.
1. Try to thaw the cells from the lower passage
2. or try to use the Tryple or accutase to split the cells
293T are very easy to remove from the substrate, use very low amount of detache agent.
Does 100mm culture dish are from the same manufacturer? If not, perhaps, the cells do not "like" the surface. Try new one
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