6 replies to this topic

[cellthetruth]

Hi,

I have the problem that over the weekend my cells did not grow. We started to use Mouse Embryonic Fibroblasts (MEF) and observed last week they grow  nice in DMEM + FCS. Now after only two passages they stopped growing. They are not dead but won't proliferate anymore. How to explain? Is there a medium other than DMEM suitable for MEF? Usually I work with HEK and NIH3T3 cells which both like DMEM.

Thank you, cheers

[bob1]

What passage are they?  Have a google for Hayflick limit.

[cellthetruth]

Hi Bob,

cells were in passage 2, after splitting they are now in passage 5. The Hayflick limit is interesting but should not be the problem here. Some of the cells don't even have a fibroblast like phenotype anymore but others do. Next steps I plan to do is defreeze new aliquots and repeat the procedure. Maybe this helps.

[leelee]

Yes, the Hayflick limit will be the problem you are having.

We do not use our MEFs past passage 4 (5 at the most, if they are a particularly good batch) but after that they are no good.

MEF are primary cells, so cannot be passaged indefinitely, unlike your 3T3 or HEK.

[cellthetruth]

Ok, I did not know that they encounter already in that low passage numbers these hayflick limit. Our cells were immortalized by a company therefore I thought this should not be an issue because then they aren't anymore primary? Or am I wrong here?

[bob1]

Hmmm, if they were immortalized - how did they do it?   It is quite likely that they used an insert like large T antigen, in which case the MEFs may have lost it (and they certainly won't be passage 2, more like passage 30 since the initial isolation and having to get the gene stably expressed).   You may need to find out how they did the immortalization and either re-do it or try adding some of the selective antibiotic to the medium to ensure that the immortalizing agent's expression is maintained.

[cellthetruth]

Thank you both for your advice.

I think best is to get in touch with the company and discuss the matter with them. I just thought that there maybe a simple solution to my problem.

cheers