Globular Antifreeze\nProtein-Inspired Nanoparticle-Based\nLarge-Scale T‑Cell Cryoprotection System for Lymphoma Immunotherapy

Large-scale biosafe T-cell cryopreservation is required\nto bring\nT-cell therapies to the market, but it remains challenging due to\nthe cytotoxicity of common cryoprotectants [e.g., dimethyl sulfoxide\n(DMSO)] and unavoidable ice injuries to cells. Herein, inspired by\nnatural globular antifreeze proteins, we establish a biocompatible\nzwitterionic magnetic nanoparticle (ZMNP)-based cryoprotection system,\nachieving large-scale cryopreservation of T cells for lymphoma immunotherapy.\nZMNPs could form a globular hydration shell to inhibit water molecule\naggregation as well as ice growth, and the surficial hydration strength–antifreeze\nperformance relationship of ZMNPs was investigated. During the thawing\nprocess, ZMNPs possessed a magnetic field-mediated nanowarming property\nthat enabled rapid heating and also facilitated easy magnetic separation\nfor cell recovery. These combined effects resulted in a high post-thaw\nviability (>80%) of large-scale T-cell cryopreservation (20 mL).\nNotably,\npost-thaw T cells exhibited similar transcript profiles to fresh cells,\nwhile up- or downregulation of 1050 genes was found in the DMSO group.\nIn a mouse E.G7-OVA lymphoma model, ZMNP-system-cryopreserved T cells\nachieved a tumor suppression rate of 77.5%, twice as high as the DMSO\ngroup. This work holds great promise for the application of advanced\ncryopreservation techniques in the development of therapeutic cellular\nproducts.