Development of Low Immunogenic Antifreeze Peptides for Cryopreservation

The successful application of antifreeze proteins (AFPs) for biospecimen cryopreservation is limited by their immunogenicity. In this work, three low immunogenic antifreeze peptides are developed based on the functional motif of the Tenebrio molitor antifreeze protein (TmAFP). In vivo tests demonstrate their negligible cytotoxicity to hepatic function, myocardial enzymes, and renal function. Meanwhile, molecular dynamics simulations demonstrate that the −OH groups of residues Thr1 and Thr3 perfectly match the lattice of the ice crystal by H-bond interaction, thus resulting in the adsorption of peptides onto the ice-crystal surface. Accordingly, the peptides exhibit activities on ice-recrystallization inhibition (IRI), thermal-hysteresis (TH), and ice-crystal shaping. Furthermore, these peptides can also protect cell membranes. During the cryopreservation of NIH/3T3 cells, the addition of only 0.5% peptides can significantly improve cell survival rates (from 37.86% to 81.32%). This work provides new insights to develop biocompatible agents for cryopreservation.