Development of a novel GAK solution for efficient hypothermic preservation of NK cell viability and anti-tumor...

Natural killer cells exhibit significant potential within current immunotherapeutic modalities. Hypothermic preservation is a critical step for the clinical transportation and temporary storage of NK cell products. However, during this process, both cell viability and function significantly decrease. The core damage mechanisms remain unclear, and effective, specialized preservation protocols are currently lacking. This study aimed to elucidate the primary modes of cell death and maintain NK cell potency during hypothermic preservation.

This study first evaluated the effects of different intravenous fluids and temperatures on NK cell preservation efficiency. Subsequently, various cell death inhibitors, molecular biomarker assays, and electron microscopy techniques were utilized to systematically elucidate the modes of cell death. Based on these findings, we optimized the best-performing fluids by replacing sodium lactate with glucose to enhance energy supply, adding the antioxidant α-tocopherol to mitigate oxidative stress, and elevating the potassium ion concentration (to 22.5 mM) to suppress excessive nutrient uptake and associated oxidative damage. These optimizations were incorporated into a new formulation designated as the GAK solution. The preservation efficacy was comprehensively assessed through in vitro cell viability and cytotoxicity assays, as well as an in vivo leukemia xenograft model.

Storage at 4 °C was much better than at 25 °C. Among the tested intravenous fluids, lactated Ringer's demonstrated the highest efficacy in preserving NK cell viability and function. NK cells undergo complex death pathways during hypothermic preservation, specifically ferroptosis, pyroptosis, and necroptosis. Our optimized GAK effectively mitigates energy depletion and oxidative stress, significantly maintaining the viability and potent cytotoxic capacity of NK cells against various tumor cells after hypothermic storage. In vivo experiments further demonstrated that NK cells preserved in the GAK solution maintained significant antitumor activity, which contributed to prolonged survival in tumor-bearing mice.

This study revealed multiple death mechanisms of NK cells during hypothermia and successfully developed a well-defined and highly efficient GAK solution. The GAK solution presents a new approach to the hypothermic preservation of NK cells, facilitating the efficient and convenient transport of NK cell products, which has significant practical implications for the accelerated clinical adoption of cellular immunotherapy.