I tried to revive a2780 cells cryopreserved in -80 degrees. Its been more than 12 hours and its still not attaching. What could possibly be happening? What should I do?
Hi Manita, it depends how cells were frozen. if proper protocol was followed (e.g., cells were not left too log in 10% DMSO), and how they were thawed like at 37C, and immediately placed in excessive amount of medium and place for culture without centrifuging (Avoid immediately centrifugation, that breaks cell walls, Next day you can change medium once cells attach
Hello Manita Raina
First and foremost, mammalian cells are not recommended to be stored at -80 degree C for long-term storage. For long-term storage, you need to store A2780 cells in liquid nitrogen. For how long have these cells been stored at -80 degree C? You may store mammalian cells at -80 degree C for a maximum of 4-6 months.
Have you taken the viability count after thawing? This will give you an idea about the viability percent. If you record a good viability of about 85-95%, then you may have to consider environmental stress as a possibility. What could cause environmental stress?
Watch out for the following:
1. Contamination,
2. Inappropriate cell surface for attachment,
3. Temperature fluctuation in the incubator,
4. Improper gas mixture in the environment,
5. Inadequate or altered nutrients in the culture media. You may have to change the FBS which you are presently using.
Check for each of the above. If you still do not succeed, then as Dr. Subhash C. Juneja mentioned, you may have to revisit your freezing and thawing protocols. In short, you will have to perform gradual freezing at -1 degree C /min of cells whenever you have to prepare stock, and quick thawing whenever you have to revive these cells.
Best.
This could stem from the process when you cryo-preserve the cells, and/or the process when you revive the cells. Cells usually need to be cryopreserved in liquid nitrogen instead of -80C. For checking your cryopreservation process, you can check this: https://lab.plygenind.com/how-to-cryopreserve-mammalian-cell-lines
For checking your revival process, read this: https://lab.plygenind.com/considerations-revive-cell-cryogenic-preservation
Liquid nitrogen storage is recommended for long term storage and not -80C.
Freeze media formulation could be another issue. Appropriate amount of DMSO, FBS, and media. Check for the cell line you work with.
How long did you took to thaw the frozen vial of cells, recommendation is rapid thaw.
What to do now!
Add more 20-30% FBS in the cell culture flask, this might have been happening due to heat shock. Adding more FBS before you pour thawed cell into the flask has worked for me. Even though this should have been in your Freeze media recipe to begin with.
Avoid centrifugation of the frozen cells, just pour the content directly into the flask and replace the media next day once they are attached.
if nothing works, try adding a tiny layer of matrigel, might help with attachment.
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