Natural killer cell cryopreservation: Influence of composition, temperature, and granules on post-thaw survival and function.

Natural killer (NK) cells are promising off-the-shelf cancer immunotherapies, but their scalable deployment requires effective cryopreservation. Conventional dimethyl sulfoxide (DMSO)-based methods are associated with toxicity and persistent post-thaw functional impairment, and the mechanisms underlying NK cell cryoinjury remain incompletely understood. We investigated NK cell dysfunction following cryopreservation and evaluated DMSO-free cryoprotectant formulations as alternative strategies. DMSO-free formulations were optimized using a differential evolution algorithm. Cryopreserved NK-92 cells were assessed post-thaw for viability, recovery, proliferation, and cytotoxicity over 5 days. Raman cryomicroscopy and flow cytometry were used to characterize cytolytic granule localization and integrity during cooling and freezing. Pre-freezing chemical degranulation was performed to probe granule-mediated injury. Both DMSO-based and DMSO-free formulations had immediate post-thaw recovery and viability over 80% but had notable cell loss over 24 h. Although proliferation resumed by 48 h, expansion remained attenuated, and cytotoxic function was reduced by ≥ 20% and did not recover after 3-5 days. Imaging revealed temperature- and cryoprotective agent (CPA)-induced granule redistribution and destabilization. Pre-freeze degranulation improved short-term recovery and proliferation. These findings support cytolytic granule redistribution and destabilization as contributors to persistent NK cell dysfunction and may inform future cryopreservation strategies for NK cell therapies.