Dear Esra Bedir

do you refer to Expi293F or HEK293F cells?

Since the protocol that you mentioned its work well for Expi293, but Expi293 is a special cell lines that is able to growth up to high cell density (in my past experience they growth linearly up to about 6milion/ml and they can reach a maximun of about 8 milion/ml. However you results with HEK293 do not sound soo strange to me since i suspect that HEK293F has same ability to growth to soo high cell density even if you are using the Expi293 expression ,medium. I suggest to you to use the Expi293, since even this strain is expensive, the most of the costs are represented from the Expi293 Transfection kit, otherwise you have to determine the growth profile of the HEK293F and perform trasfection at lower cell density (about 50% of the maxium that can be reached from the cells).

If you are interested at the following link of my blog

https://www.blogger.com/video.g?token=AD6v5dyTuEwDXD2ztCnLIFHkG06cCp1jaRuUHOGFtHdiTFnj-yS_TyCyPqzvi6RogJaSP_sXqbJnMDIYplkXODpfOc21BETPPh74J7FJuwMPxHW4X79mURcAl7-V0ApA8BQrGUAgcSc

you can find some example about the results that i obtained with the Expi293 in the past.

best

Manuele

Dear Manuele Martinelli,

Thank you very much for your answer. I'm studying with Expi293F cells. I think the problem is the medium as you say. That's why I'll definitely take your suggestions into consideration in my new study. It's a really difficult cell line, I hope I can do it.

Hello Esra Bedir ,

We had a similar problem earlier this year when we tried to do some new stocks of Expi293F cells : they stopped growing and were not in good shape. It appeared that they don't like being cultured in a large volume, ie when we reached a volume of more than 1/3 of the maximum volume of our cell culture bottle, they started to die.

I think they are sensitive to gas exchanges and they also need to be agitated just the right way, so when your volume is too large compared to the maximum volume that you can put in your culture bottle (or erlen, or anyhting you are using), they don't like it.

For example, if you cultivate the cells in a 100mL bottle, don't put a higher volume than ~35mL, and see how they behave then ? In our case it fixed everything, and we could freeze some huge stocks !

Also, they are quite resilient cells so they get better very quickly and easily if you put them back in the right conditions.

I hope it will help a bit, good luck with your cells !

Dear Bénédicte Giroudot,

Thank you very much for your answer and suggestions, it was very helpful. You also said that you freeze stock of your cells. How did you achieve this? Because I can't grow the suspended cells again when I could freeze with DMSO. I think, I need to read a lot more about this cell line.

Dear Esra,

I'm glad I could help you !

For freezing I use the same procedure as every other cell line that we have in my facility : for suspensions I count the cells and calculate how many cryotubes I can prepare with 10^6 cells/tube.

For the freezing medium, I calculate 1mL of culture medium per tube + 10% Fœtal Calf Serum (any serum would work I guess, you can try 20% also, as the Expi medium doesn't contain any) + 10% final of DMSO.

Once the tubes are ready we use Cryofreeze or Coolcell containers to freeze them at least 4h at -80°C (to reach the temperature gradually), and after that transfer them in liquid nitrogen for longer and better conservation. You can keep your cells at -80°C for a month if it is easier, after that it is better to keep them in liquid nitrogen.

I hope it will help again, good luck with everything !

Thank you very much again. You've been very helpful, I'll definitely try it. Have a nice day :)