Probing mechanisms of cryopreservation damage to natural killer cells

Background aims: Natural killer (NK) cells show significant potential in targeting hard to treat cancers, but these cells need effective preservation methods to maintain viability and efficacy after cryopreservation. Traditional methods of preserving NK cells result in low post-thaw recovery and function. Dimethyl sulfoxide (DMSO) is a very common cryoprotectant for preserving NK cells, but its infusion into patients post-thaw can cause dose-dependent adverse effects, including nausea, discomfort and cardiac arrest. The aim of this work was to evaluate low DMSO- and osmolyte-based cryopreservation solutions across multiple steps in the cryopreservation process for NK cells. Methods: This study investigated NK cell membrane responses to cryoprotectants, dependence of cell survival on cooling rate and nucleation temperature and influence of osmotic shock and pH changes with regard to freezing NK cells using the NK cell line NK-92 as a model system. Results: Exposure to cryoprotectants reduced the membrane fluidity and NK cell-induced cytotoxicity of the cells before freezing, but combinations of osmolytes mitigated this loss. The introduction of cryoprotectants did not reduce perforin or granzyme content, and slow or rapid dilution after thawing did not reduce viability, recovery or proliferation. Controlled rate freezing and Raman cryomicroscopy studies revealed that NK cells tolerated fast cooling rates, and the optimal cooling rate for NK cells was 4–5°C/min. Raman cryomicroscopy mapped the distribution of cryoprotectants and ice of frozen NK cells at –50°C, showing a reduction in cytotoxic granule signal. Conclusions: The large, osmotically inactive volume of NK cells demonstrates cell sensitivity to cryoprotectants and freezing. Exposure to cryoprotectants can reduce NK cell-induced cytotoxicity and membrane fluidity. We hypothesize that cell dehydration and freezing disrupt cytolytic granules, causing NK intracellular damage. These data emphasize the importance of developing robust techniques to enhance the cryopreservation of NK cells and indicate the points where cell damage occurs.