Hi,
I tried several times to freeze H9c2 cells in liquid nitrogen, but as soon as I defrosted them ~6 months later, most of them were dead and I had to expand the cultur from few cells on. This happened several times with different passages.
Here the procedure how I did it:
- I resuspended (after centrifugation) in normal Dulbeco's DMEM with additives (+ 10% FCS + 1% PS + 1% L-Glut) adding 5% DMSO
- I froze the cells in 1-1,2ml medium in cryo vials (cell amount that can be cultured in T25 afterwards)
- I cooled them in a "freezy boy" first for 1 day in -80°C fridge
- then I tranfered quickly to the liquid nitrogen
Is there anyone with the same problems or somebody that has experience on that?
Thank you!!
From what you have stated, everything seems ok so it is is strange you are having these issues.
These may not even apply to you but here are some of my thoughts...
1. Ensure your frozen vials have plenty of cells in them; for instance, I normally convert 1x confluent T175 into 3 (or 4) 2 mL vials. When these are thawed out I have LOTS of cells even in a T75.
2. Ensure the thawing (at 37C) is done as fast as possible, once the frozen cells are liquid, quickly place them into a tube of media, then centrifuge this tube and resuspend the pellet with fresh warm media (so you remove the old frozen media which has dmso in it).
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