Problem with Passaging MEFs - (Sep/02/2010 )

thx for ur advice. I agree with you regarding it being too confluent. But for the occasions when i passage them at 80% confluency, they don't grow. I don't understand. That's the reason i wait it until they're fully confluent before passaging. Is my problem due to the quality of my cells? Btw, i only manage to passage up to P7. And most of the cells that i freeze-thaw barely survive. Any advice? thx

I'm not surprised that you can't passage further than P7, I never passage my MEF further than P4. With primary cells, you should limit the number of passages.
After that, they start to change morphology, grow slower, quite often not reaching 100% confluent, and they also don't support our virus growth as well as for earlier passages.

Also, our MEF are fine being at 100% confluent (and even left that way for a day) before splitting. I doubt that is your problem (would be, of course, if you were working with an immortal cell line with no contact inhibition).