For how long a mammalian cell line containing cryo-vial can be left in -80 degree freezer, provided that cell were frozen following standard protocol but due to unavailability/shortage of liquid N2, vials couldn't be shifted to liquid nitrogen laden canisters?
I have used upto one year at -86. I believe they may survive longer too.
It's a difficult question with many factors. Essentially storing them this way is increasing their susceptibility to cryodamage.. Firstly there is a lot of variability even in cryopreserving in LN2. It will depend partly on how large (H20 content) content your cells are? If they are large then they will be more susceptible to cryodamage. sperm are the smallest mammalian cells and have less water so freeze well. But even in sperm the evaluation of a single sample after freezing, does not guarantee the homogeneity of all the samples (Castilla et al., 2007). Cholesterol content of the cell membrane will be important as the cell membranes of species that exhibit a greater sterol to phospholipid ratio show greater resistance to phase transition during cooling (Drobnis et al., 1993). Comparing -70 and LN2, there is detectable damage at 7 days and more damage at three months (Trummer 1998). Saying that I have heard that cells have been used for ICSI even after extended periods at -20C. So your cells will certainly be damaged, but may still be viable. However, the viability will decrease with time so get them into LN2 if you can.
Castilla, J. A., Sanchez-Lean, M., Garrido, A., Ramirez, J. P., Clavero, A. & Martinez, L. (2007). Procedure control and acceptance sampling plans for donor sperm banks: A theoretical study. Cell and Tissue Banking, 8, 257-265.
Drobnis, E. Z., Crowe, L. M., Berger, T., Anchordoguy, T. J., Overstreet, J. W. & Crowe, J. H. (1993). Cold shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as a model. Journal of Experimental Zoology, 265, 432-437.
If temperature is stable, most cells will keep very high viability, specially for short times. I have used cells with 2 years or more at -80 and never have problems, but as Chey suggested that varies among cell lines. I wouldn’t recommend you to store them for years unless someone from your lab with the same cell line had already tried it, but for a few month it will not be a big issue.
Thank you everyone for your valuable inputs. Chey G Dearing , thank you for the elaborate reply. Actually I have found that HEK293T are loosing viability upon long term storage in -80 deg C freezer, it's not that I'm not getting any cell upon thawing but the number of viable cells upon thawing, are gradually decreasing with increasing duration of keeping them in -80 freezer. I have seen same trend for B16 too. Thank you Diego Raffo for your feedback.
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