Hello, anyone.
I have a technical questions related with cell lines recovery after it was found in a complete thawing condition, so, long story short, I have a technical accident where the liq N2 tank in my laboratory was found empty of the liq N2 due to the leakage of the tank. Then, I found most of all the cell lines are already in a thawing condition inside the cryovial and in its cryopreservation medium as well, in which as we know, containing the cryoprotectant agent i.e. DMSO.
I've tried to rescue the cells by seeding it into Complete DMEM with 20% FBS but, until today, the cells were still not giving a sign of recovered. Attached below is one example of the cells where I think it is already giving a sign of recovery in the form of morphological structure, yet since it is a type of an adherent cells, unfortunately, it still not attach to the surface of the culture dish.
Is there any idea of how we can boost cells viability for the recovery from the cryogenic phase? and help enhancing the cells attachment post recovery?
Thank you.
Leakage of N2 is a disaster in cell culture that you do not want to encounter. In my previous lab, we therefore installed an automatic filling system. The problem is that this also failed once. So you have to check at regular intervals to prevent defrosting and optimaly spread several vails over several nitrogen storage tanks.We stored samples in three separate storage vessels. That is a pretty intensive work but prevents potential loss of samples.
Unfortunately, once defrosted, you must immediately proceed to the process of eliminating the cryoprotectants in order to grow your cells. Thawing probably occured hours before you discovered your samples.
The only thing you still can do is looking into the nuclei or gDNA although that might also be uestionable. In another lab I obtained prenatal samples which were overheated (50-60°C). There we still could do FISH for trisomies but we could not revive them anymore.
I hope you can rebuild your collection pretty fast and that you had a fall back option.
thank you for your sharing Stefan Joris Vermeulen so, do you think that the chance of the cells can survive from its critical phase once it has been defrosted is really low? there are not even an additional nutritional support (medium supplementation) could help it from recover? especially in the process to help it recover into the cells adhesion?
If there are I am not aware of them. Yes I indeed think there exists no real wonderdrug to resurect dead cells. DMSO is used as a cryoprotectant but in itself it is toxic. So if cells remain for to long in that environment, i;e. for a couple of hours, and I guess you do not really now when the cryovessel thawed than cells will have died. You could opt for 20% FCS in growth medium. The HT29 is a robust cell line. There are definitely a number of research papers available on the cell induced death by DMSO.
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