Abstract
Natural killer (NK) cell-based therapies are under assessment for the treatment of various cancers due to their intrinsic ability to distinguish between malignant and healthy cells in an allogeneic context, enabling off-the-shelf manufacturing possibilities. However, cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. In this study, we evaluated three cryoprotectants (CryoStor 10; ZKCELL FM-01; FBS + DMSO) for the cryopreservation of NK cells. Post-thaw viability, ATP levels, and cytotoxicity were assessed and found to have persistent differences between cryopreserved and fresh cells. Transmission electron microscopy, flow cytometry, and Western blot analysis revealed a complex mode of cell death in cryopreserved cells, which could be partially mitigated by adding some death inhibitors. We further investigated the effects of centrifugation on thawed cells, identifying lysosomal stability as a key determinant of cell death. Pretreatment with low-dose LLOMe prior to cryopreservation induced stress granule formation, stabilizing lysosomes and improving cell recovery rates without compromising effector functional capacity. These findings offer new insights for optimizing NK cell cryopreservation and facilitating their clinical application.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 82101929), Collaborative Innovation Projects of Heilongjiang Province, China (Grant No. LJGXCG2023-003), and the Outstanding Youth Funding of the First Affiliated Hospital of Harbin Medical University (Grant No. 2021J02).
Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 82101929), Collaborative Innovation Projects of Heilongjiang Province, China (Grant No. LJGXCG2023-003), and the Outstanding Youth Funding of the First Affiliated Hospital of Harbin Medical University (Grant No. 2021J02).
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Human blood related work has been approved by the Ethics Committee of First Affiliated Hospital of Harbin Medical University. All human subjects provided written informed consent prior to enrollment, and peripheral blood samples were collected in accordance with approved institutional ethical protocols.
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Liu, Y., Liu, X., Wu, G. et al. Induction of stress granules alleviates programmed cell death induced by lysosomal damage during NK cell cryopreservation. Cell Death Discov. (2026). https://doi.org/10.1038/s41420-026-03149-0
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DOI: https://doi.org/10.1038/s41420-026-03149-0
