If I want to perform an assay with a specific cell line that has been cryopreserved for over 5 years will it affect the results of my study?
The viability might be affected, but it also depends on the typebof cell line, and the viability of the cells while freezing them.
What u can do is before planning the experiment, u can first revive one vial and check its viability and monitor its growth for 2 days, if you feel it us declining, then probably do not use it for your experiment...
Cryopreservation by slow freezing can cause two types of cell damage; physical and molecular. Physical injuries were the first to be identified and include ice nucleation, solution effects, osmotic shock, cold shock as well as cryoprotectant toxicity. Moreover different cell lines show different response under cryopreservation. For example, The viability of the PBMCs was also reduced significantly after cryopreservation (P < 0.0001) but remained stable during the different cryopreservation times, the statistical results showed that 1 m versus 3 m, P = 0.99; 1 m versus 6 m, P = 0.10; 3 m versus 6 m, P = 0.05
Hello Julissa Mejía
It will depend on many factors:
1) The type of storage condition: Cells cryopreserved at -80 degree C will start losing viability after 1 year. Cells cryopreserved in liquid nitrogen may maintain their viability for long term (many years). I have no problem using cells stored in liquid nitrogen for more than 5 years with a viability percent of 90-95%. I have used these cells for bioassays and they have worked very well.
2) Maintenance of storage condition: If the temperature is not maintained up to the mark, then the cells will show reduced viability. For instance, if one fails to maintain the level of liquid nitrogen in the tank, there is a risk of damage to the cells in terms of viability and performance which may be clearly visible when they are thawed on a later date. Similarly, power fluctuations may fail to maintain a constant temperature of -80 degree C for -80 degree C freezer which in turn may have a negative effect on the cells performance in culture.
3) Freezing of mammalian cells: The cells have to be frozen gradually at -1 degree C /minute using cryoprotectant like DMSO in order to protect the cells from damage due to ice crystals. This is best achieved using the cryocooler like Mr. Frosty. The cells have to be frozen when they are in the logarithmic phase or the exponential phase. If the cells have not been frozen as per the standard protocol, viability of cells is likely to reduce and on thawing you may obtain mostly dead cells.
4) Thawing of mammalian cells: Though the thawing process may not be directly related, it is important to perform quick thawing of the cryopreserved mammalian cells in order to avoid recrystallization during rewarming process. Thus thawing of cells also contributes to the viability of cells.
If all the above mentioned factors are taken care of, you should not have any problem in your study thawing the cell line that has been cryopreserved for over 5 years.
Best.
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