It is a bit unclear from your question if your cells do not grow by itself in serum-free or after you initiated ExpiCHO if your cells fail with ExpiCHO system protocol that it might be the protein that you are producing to be the culprit. Try to run ExpiCHO protocol with something nondisruptive and straightforward, i.e., GFP to see if it is change anything you can also quickly visualize the success of your transfection. If your cells are dying even before you started the protein production try to change serum-free growth supplement. Most cells are not particularly happy in serum-free media, try to change the media bottle maybe something not right with it as well.

Hi Yogesh,

Could you please attach pictures of your cultured ExpiCHO cells at time zero and after 36 hours?

HI Yogesh,

I also experienced similar problem with my HEK293 cells when using Freestyle 293 expression medium, which is a serum free medium that allows suspension cell growth.

So when I tried to expand the cells in this serum free media, the cell die within two generation. Apparently, the gas exchange is not very good in suspension culture.

Later on I reasoned that cells should be grown in enough numbers using the serum containing medium and then resuspended in the serum free medium for transfection and recombinant protein production.

This will allow exposure of your cells for minimal period in the serum free medium while allowing a serum free condition for recombinant protein production.

Best,

Sudeep

Anton Lennikov

The cells were bought from Thermo, which was frozen in a serum-free condition.

I have worked with them before in serum free conditions and did not face such issue in a different lab.

I tried another experiment where in, I just passaged the cells.

But they also died after the density crossed 5-6 million per mL.

Therefore, I doubt there is a contaminant (Other than e.coli and fungal as I did not observe them under the microscope)

Radhwan Al-Zidan

Hi, I do not have an image but then the cells are stressed after 36 hours. (upon microscopic observation)

I am unable to find out what it is, and as they grow and reach higher cell densities, they die.

Hi,

Did you ever solve the problem? I’ve been having similar problems lately.

Hi, I am having the same issues these days.

When thawing ExpiCHO cells, the density usually is really slow to increase (compared to the data in the guidelines).

But lately all of the aliquots I thaw can't survive past 2 to 3 days. Should I check for any form of contamination? I have already verified the culture medium, without any visible contaminant.

Thank you.

Deeann Martinez-Guzman I am sorry for the late reply. Was not online.

Yes, I did solve the problem. There was a problem with gas exchange as the incubator CO2 pressure was not what was shown on the monitor.

And we also changed the media which increased the density of the cells.

Hi Yogesh,

I am experiencing the same problem. Which medium you are using now?

Hi, I am facing the same trouble with ExpiCHO. Can you please tell which media you have used? did you use serum?