Development of a Me₂SO-free cryopreservation medium and its long-term cryoprotection on the CAR-NK cells

Cryopreservation is a crucial step in the supply process of off-the-shelf chimeric antigen receptor engineered natural killer (CAR-NK) cell products. Concerns have been raised over the clinical application of dimethyl sulfoxide (Me₂SO) due to the potential for adverse reactions following infusion and limited cell-specific cytotoxic effects if misapplied. In this study, we developed a Me₂SO-free cryopreservation medium specifically tailored for CAR-NK cells to address this limitation. The cryopreservation medium was formulated using human serum albumin (HSA) and glycerol as the base components. Following initial screening of seven clinically-compatible solutions, four with cryoprotective properties were identified. These were combined and optimized into a single formulation: IF-M. The viability, phenotype, and function of CAR-NK cells were evaluated after short-term and long-term cryopreservation to assess the effectiveness of IF-M, with Me₂SO serving as the control group. The viability and recovery of CAR-NK cells in the IF-M group were significantly higher than those in the Me₂SO group within 90 days of cryopreservation. Moreover, after 1 year of cryopreservation the cytotoxic capacity of CAR-NK cells cryopreserved with IF-M was comparable to that of fresh CAR-NK cells and significantly superior to that of CAR-NK cells cryopreserved in Me₂SO. The CD107a expression intensity of CAR-NK cells in IF-M group was significantly higher than that of Me₂SO group. No statistical differences were observed in other indicators under different cryopreservation times. These results underscore the robustness of IF-M as a suitable replacement for traditional Me₂SO-based cryopreservation medium for the long-term cryopreservation and clinical application of off-the-shelf CAR-NK cells.