Evaluation of ice recrystallization inhibitors for preserving the post-thaw function of iPSC-derived natural killer cells

Human induced pluripotent stem cell (iPSC)-derived natural killer cells (iNKs) are promising candidates for scalable, allogeneic ‘off-the-shelf’ cancer immunotherapies. However, effective cryopreservation is required to support their manufacture, storage, distribution, and clinical use, and freeze-thaw processes can adversely affect NK-cell recovery and function. In this study, we evaluated whether supplementation of CryoStor® CS10 with a proprietary small-molecule ice recrystallization inhibitor (IRI) influences the post-thaw properties of iNKs following storage in liquid nitrogen for 1, 3, and 5 months. Immediate post-thaw viability and recovery were evaluated descriptively, while cytotoxic function against MDA-MB-231 breast cancer cells was assessed in three independent experiments. IRI supplementation did not result in an apparent detrimental effect on post-thaw viability or recovery. Although cryopreserved iNKs exhibited reduced cytotoxic activity compared with fresh, non-cryopreserved cells, iNKs cryopreserved with IRI-supplemented CS10 exhibited significantly greater cytotoxic activity than cells cryopreserved in CS10 alone after 3 and 5 months of storage. These preliminary findings suggest that supplementation of conventional cryopreservation media with IRIs may help preserve iNK cytotoxic function following extended cryogenic storage. Additional studies incorporating greater biological replication and broader functional endpoints will be needed to validate these findings.