How should we refreeze cells? We thawed lymphocytes after receiving them from another lab, then tried to freeze some of the sample again to use at a later time. When we thawed the cells that we froze again, we saw very low viability compared to the cells that were only thawed once. Is this to be expected?
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The Dos and Don’ts of Cryopreservation
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Despite our best efforts to optimize CPA and the other numerous variables within a freezing/thawing protocol, cryopreservation is a traumatic process for cells. Therefore, observing some loss of viability after the freezing/thawing process is to be expected, and repeated cycles may result in progressive cell loss. Generally speaking, there are several factors that you need to consider when observing low cell viability: cell health and density when freezing, avoiding prolonged exposure to CPA or room temperature during harvesting, controlled and appropriate cooling rate, avoiding exposure to warm temperatures during transfer to cryogenic storage, and quick thawing and proper removal of CPAs. We have outlined this in more detail in a previous question. I would also suggest that you also check cell viability under microscopy before freezing in order to check dead cell populations in your vessels. To remove the dead cells, you can spin the PBMC with low speed centrifugation during the washing step.
Specifically for PBMCs, progressive loss of cell viability can occur as storage time increases(1). In addition, the disease of the individual from whom the PBMCs come also affects cryopreservation. According to literature(2), PBMCs from pediatric patients who are naturally infected with Dengue virus showed reduction of cell viability after cryopreservation, compared to fresh PBMCs from healthy children using the TB exclusion method.
I have included some PBMC protocols for your review. I would pay particular attention to PBMC centrifugation, DMSO and serum concentration, and freezing medium temperature(3,4).
(1) https://www.ncbi.nlm.nih.gov/p
(2) https://www.ncbi.nlm.nih.gov/p
(3) https://www.ncbi.nlm.nih.gov/p
(4) https://journals.plos.org/plos
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