Hello, I have A549 cells in culture. Normally they attached really nice on normal flask, T25 or T75 without coating the flask. Now I freeze some of these cells and after thawing they don't attached. They are not dead, they grow in the media.
Any idea?
Hi,
although I find it quite unusual, that the cells do not exhibit their characteristical adherent growth, I have seen several cell lines behaving in an unexpected manner after being frozen, stored in about 10% DMSO at -80°C or below and then being thawed and reactivated.
In the simpelest form, they do not grow or express very well.
For experimental usage, it is recommended or even necessary for many cell lines to passage them several times after cryo storage in the desired media unless they regain all their normal properties prior to application.
Hello Christoph,
maybe they have this problems and I have to passage them several times now, but I thaw this kind of cells before also and it works. Maybe the problem is the freeze aid? Because cells I frozen without works, with freeze aid it doesn't work. I just thaw again cells which I frozen without freeze aid.
In that case, I would also think of some material problem, most probably the freeze protection. The dishes / flasks cannot be the problem, since adherent cells attach to every PS surface, unless the flasks were coated with something unfitting / attachment-preventing or have been rotated during cell growth.
I hope, you are more lucky with the fresh thawed cells w/o freeze aid, otherwise I would try recultivation of cells frozen with dilution series of your preferred and maybe some other freeze aid, e.g. pure DMSO 5-10%, to see if I could avoid that effect under certain conditions while regaining vital cells, cause for freezing and thawing them w/o freeze protection, you'll surely need some fortune.
Best regards,
I think something go definetly wrong in the freezing step. Because now I thaw an older stock and everything works fine. In the older freezing steps I don't use a freezing aid, these cells works. The last time I use a freezing aid and this cells don't attach. Maybe the freezing with the freezing aid is not fast enough?
Thank a lot for your comments!!! and viele Grüße nach Hamburg ;-)
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