I have a problem growing Human mammary epithelial cells. I have ordered twice now from different suppliers, so it's getting to be an expensive problem.
I thaw the vials and seed into a flask - they attach fine and grow to confluency. Then I trypsinize and wash, and reseed. They will not reattach!
I have tried coating plates in gelatin, different brands of flasks, plates, dishes. No good.
I'm using the proper media, everything added. I've also tried added 10% FCS in case that helps, it doesn't.
Can anyone help?
Could be a trypsin inhibitor problem. What is your protocol for neutralizing the trypsin?
Trypsin inhibitors are often necessary for serum free culture. You may want to reduce the contact time or the concentration of the trypsin to see if that helps.
You may be interested in this paper Article Trypsin-induced proteome alteration during cell subculture i...
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