Globular Antifreeze Protein-Inspired Nanoparticle-Based Large-Scale T-Cell Cryoprotection System for Lymphoma Immunotherapy

Large-scale biosafe T-cell cryopreservation is required to bring T-cell therapies to the market, but it remains challenging due to the cytotoxicity of common cryoprotectants [e.g., dimethyl sulfoxide (DMSO)] and unavoidable ice injuries to cells. Herein, inspired by natural globular antifreeze proteins, we establish a biocompatible zwitterionic magnetic nanoparticle (ZMNP)-based cryoprotection system, achieving large-scale cryopreservation of T cells for lymphoma immunotherapy. ZMNPs could form a globular hydration shell to inhibit water molecule aggregation as well as ice growth, and the surficial hydration strength-antifreeze performance relationship of ZMNPs was investigated. During the thawing process, ZMNPs possessed a magnetic field-mediated nanowarming property that enabled rapid heating and also facilitated easy magnetic separation for cell recovery. These combined effects resulted in a high post-thaw viability (>80%) of large-scale T-cell cryopreservation (20 mL). Notably, post-thaw T cells exhibited similar transcript profiles to fresh cells, while up- or downregulation of 1050 genes was found in the DMSO group. In a mouse E.G7-OVA lymphoma model, ZMNP-system-cryopreserved T cells achieved a tumor suppression rate of 77.5%, twice as high as the DMSO group. This work holds great promise for the application of advanced cryopreservation techniques in the development of therapeutic cellular products.