Abstract
Natural killer (NK) cells are being developed as therapeutic agents targeting hematological malignancies and solid tumors. However, the lack of an optimal cryopreservation medium poses a significant challenge, as cryopreservation often reduces NK cell recovery, viability, and function, hindering their use in cellular therapies. This study investigated the effects of varying concentrations of Dimethyl sulfoxide (Me(2)SO), Proline, Trehalose, and Dextran 40 commonly used in cell cryopreservation on NK cell recovery, viability, and cytotoxic activity. Additionally, we conducted a screening of 19 cryoprotective agents (CPAs) to enhance NK cell cytotoxic activity after freeze-thawing. We found that reducing Me(2)SO concentration significantly decreased NK cell cytotoxic activity, and the combination of Proline, Trehalose, and Dextran 40 was insufficient to prevent this decline. A negative interaction effect between Trehalose and Dextran 40 on NK cell cytotoxic activity was also observed. Screening results identified Betaine, Glycine, Polyvinylpyrrolidone (PVP), α-tocopherol, Poloxamer 188, and Creatine as effective in enhancing NK cell cytotoxic activity after freeze-thawing. These findings provide new insights into the interaction effects of CPAs on NK cell cytotoxic activity and contribute to improving NK cell quality in pharmaceutical manufacturing. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-025-00865-w.