I was freezing Wharton Jelly stem cell in Mr.Frosty and forgot them in -20 degree for 48hours (due to weekend). I transferred them to -80 degree in Monday morning. Does anyone has experienced this?
In principle they should be fine. Just correct the seeding density by the amount of cells alive (as usual) and keep track of their growth curve.
Best of luck
Hello Nur Afiqah.
The cells stored at -20C would already have gone dead now without proper cryopreservation procedure. Transferring to -80C after 48h would not give any cells alive at all.
However, It would be O.K. if you would like to investigate the frozen section with stem cell markers by employing proper cryostat procedure from the beginning for immunohistochemical analysis.
Regards.
Since you said that you simply had forgotten to transfer them to the -80, I would assume that you did in fact use "proper cryopreservation procedure" other than failing to transfer them to the -80.
While I have no experience with the particular stem cells you froze, unless they are highly sensitive to cryopreservation, I believe it is quite likely that you will have viable cells as long as you have handled them properly after the 48 hours at -20.
I think that 48 hs at -20 is probably too much for cells to survive. Have you tested any of those vials yet?
Dear researchers,
Thank you for all the answer, it helps me a lot. I have thawed one vial and some of the cells are still viable.
I tried replicating this shortly after this was posted. I used human iPSCs. Survival was quite poor relative to a normal freeze, but there was some survival. I actually had expected it to be not as bad as it was.
It only was for curiosity. I used extra cells that were going to be discarded anyway.
If your freezing protocol involves some time in the -20 before transfer to -80, have you ever done a comparison of that method relative to direct placement into -80? I wonder if the difference in recovery is worth the risk of making this mistake again?
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