Hello,
I need your help. I am working on a SARS-Cov2 seroprevalence study. I will isolate the plasmas using a ficoll gradient; I would also like to keep the PBMCs for future studies. I want to store them at -80°C as I do not have a liquid nitrogen tank. According to your experiments, how long could I keep the cells at -80°C and especially what medium should I use for freezing.
Thank you.
Hello Thiam Fatou
You can use the Freezing medium (90% FBS + 10% DMSO) for freezing PBMCs. PBMCs can be stored at -80 degree C for 1-3 weeks. Long term storage of PBMCs in liquid nitrogen would be preferable.
You can also refer to the protocol for the isolation and cryopreservation of PBMCs given below.
https://www.immunetolerance.org/sites/default/files/Separation%20of%20mononuclear%20cells%20from%20whole%20blood%20using%20Ficoll%20gradients.pdf
Best.
Hello Malcolm Nobre
Thank you for your answer. It's really nice.
Best regards
Look up pentastarch freezing medium - Pat Stiff was the first author and it was published in Experimental Hematology back in the late 80s/early 90s. I am no longer in the lab, but my memory is that it is 5% pentastarch, 5% HSA and 10% DMSO, add equal volume to your PBMC suspension; do not exceed 10^8 PB-MNC per ml final concentration. Freeze at -80oC. We did this with PB-MNC collected from cancer patients mobilized with G-CSF. I was able to recover 92% of CFU-GM frozen 14-25 years post-freeze. Viability of non-HSC [by 7-AAD exclusion] was lower and more variable, but ran 40-63%.
I've routinely used the FBS/DMSO medium described by Malcolm Nobre for freezing cell lines. Typically, they are good for 3 years at -80oC, then the viability drops.
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