Hi, everybody. I'm coming today with a technical question. I'm having problems culturing HEK293 cells. Under the same culturing conditions, my HEK293T cells survive after splitting while the HEK293 cells don't. I searched for differences in culturing requirements or any specific condition they need but I didn't find anything relevant. Have you ever experienced this problem? Do you have any idea to solve it? Thanks so much in advance.
Hello, there is a difference between the cultivation of HEK293 and HEK293T cell lines. HEK293T cells are derived from HEK293 cells and have been engineered to express the SV40 large T antigen, which allows for higher transfection efficiency. As a result, HEK293T cells are typically more commonly used for transient transfection experiments, while HEK293 cells are often used for stable transfection experiments. Additionally, HEK293T cells tend to grow more rapidly than HEK293 cells.
Maybe contact Sigma? They seem to sell both lines so they should have specific culture conditions for them. Good luck!! Ellen
https://www.sigmaaldrich.com/US/en/search/hek293?facet=facet_related_category%3AMammalian%20Cell%20Lines&focus=products&page=1&perpage=30&sort=relevance&term=hek293&type=product
It's not uncommon to experience differences in cell behavior even when using closely related cell lines like HEK293 and HEK293T. Here are a few troubleshooting steps you can consider.
Confirm that you are indeed working with authentic HEK293 cells. Cell lines can change or become contaminated over time, leading to unexpected behavior.
Check the passage number of both HEK293 and HEK293T cells. Some cell lines, including HEK293, can exhibit changes in behavior as they are passaged.
Ensure that you are using the appropriate culture medium and supplements for both HEK293 and HEK293T cells. Different cell lines can have variations in nutrient requirements.
Try adjusting the subculture ratio for HEK293 cells. Some cell lines might require a different ratio of cells to fresh medium during passaging.
Experiment with the trypsinization time. Some cell lines are more sensitive to trypsin exposure than others. Try reducing the exposure time and monitor the cells closely under the microscope.
Hello, perhaps you could share your culturing conditions and splitting ratio or cell concentration. Did you buy it or was some vial given to you? Sometimes when cells weren't properly stored or frozen, you can have viability issues. Are they growing normally or do you some changes in growth rate or morphology? Did you try with different vials/stocks?
Hello, everybody
Thank you so much for the suggestions. This is a modified HEK293 cell line provided by a colleague. After being unfrozen, cells behave normally. They grow excellently. The problem came after splitting them. I usually trypsinize cells for 5' at 37° C, which works well for HeLa, HEK293T, and SY5Y, for example. But HEK293 cells almost completely died after that. And not only with this special clone but also with another HEK293 cell line in the lab. Finally, I tried diminishing the trypsinization time to 3' and it works way better. This cell line seems to be more sensitive to trypsinization, evidently. I don't know if somebody else has had this same issue.
Once again, thank you so much for the comments. Have a nice week.
Favio
Please reduce the trypsinization time to maximum 2 mins. or 1 min 30 secs. you will thank me later Favio Pesaola
I reduced the time to 2 minutes yesterday and the cells look like very well so far. Thanks Bbumba Patrick .
HEK293 is readily detach from the surface and start dying, you can use poly L-Lysine coated culture plates?
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