I am currently culturing primary human keratinocytes. Until last week it always worked rather well. But since last monday, the cells in the culture flasks started to spantaneously detach. There are still lots of cells attached and prloiferating, but this slows everything down enormeously.
After splitting and passaging only about half of the cells actually seem to attach at all. This is especially annoying for my assays, as it takes much much longer for cells to get confluent and to get valuable results. Another phenomenon is, that in some flasks all of the keratinocytes seem to differentiate.
All my cells are in first to 4th sub-culture, so not too old.
The problem is that it all started so abruptly. Before cell attachment was just fine.
I am using Serum Free Keratinocyte Medium 2 from PromoCell (with Supplement, Streptomycine+Penicilline and CaCl2), and Falcon T75 Flasks (we never coated them). Medium is changed twice a week. No bacterial contamination is visible. I also tried a new bottle of medium, but I cant really say if things are getting better now. I know that calcium induces differentiation, but I am using this CaCl2 supplement since november (the producer implies that the addition to the serum is required for effective cell growth).
Has anyone else ever experienced this? Any ideas whats happening?
Thanks a lot!