Relevance of controlled cooling and freezing phases in T-cell cryopreservation

When cells are cryopreserved, they go through a freezing process with several distinct phases (i.e., cooling until nucleation, ice nucleation, ice crystal growth and cooling to a final temperature). Conventional cell freezing approaches often employ a single cooling rate to describe and optimize the entire freezing process, which neglects its complexity and does not provide insight into the effects of the different freezing phases. The aim of this work was to elucidate the impact of each freezing phase by varying different process parameters per phase. Hereto, spin freezing was used to freeze Jurkat T cells in either a Me₂SO-based or Me₂SO-free formulation. The cooling rates before ice nucleation and after total ice crystallization impacted cell viability, resulting in viability ranging from 26.7% to 52.8% for the Me₂SO-free formulation, and 22.5%–42.6% for the Me₂SO-based formulation. Interestingly, the degree of supercooling upon nucleation did not exhibit a significant effect on cell viability in this work. However, the rate of ice crystal formation emerged as a crucial factor, with viability ranging from 2.4% to 53.2% for the Me₂SO-free formulation, and 0.3%–53.2% for the Me₂SO-based formulation, depending on the freezing rate. A morphological study of the cells post-cryopreservation was performed using confocal microscopy, and it was found that cytoskeleton integrity and cell volume were impacted, depending on the formulation-process parameter combination. These findings underscore the importance of scrutinizing all cooling and freezing phases, as each phase impacted post-thaw viability in a distinct way, depending of the specific formulation used.