We have had a lot of success over the past few years growing and differentiating these cells from nasal and bronchial brushings, but since August, we can not even get them to stick to the plate overnight. Things we have done:
Replaced ALL media etc with brand new stock. (also tried different suitable media from different supplier)
Replaced ALL tips/cultureware etc with brand new stock
Incubator tested and CO2 and temperature perfect.
MBSC airflow tested AND peroxide fumigation.
Tried culturing in different incubators - no difference.
Different collagens and concentrations - no difference
NO appreciable infection problem - antibiotics are in the media as they are patient derived cells.
The cells look absolutely fine once taken, then they are put into the plate overnight at 37oC as we have done for years, but by morning, they are apoptotic and there are rounded up cells everywhere. The very few that have stuck down look granular and different to what we have seen previously. We have also tracked back and nothing has changed in August to make us think there was a trigger. If anybody has any suggestions I would appreciate it. Thanks in advance.
Just to add : We are also growing another epithelial cell line (Detroit 562) and they are growing fine under the same conditions.
Hi
Is there any problem with collagen that you are using? I mean pH of the media in collagen is stable ?
If that is also all right then try increasing the concentration of FBS .
Good Luck
Thanks. Yes we have checked pH all through the process and it is within culture parameters. I am a bit hesitant to add anything, as this process has been validated numerous times, and we havent changed protocol or anything, it has just stopped working, but thank you so much for your reply,
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