Mechanism Unraveling of Scalable Antifreeze Oligopeptides for Enhanced Cryopreservation

Cryopreservation is fundamental to cell-based therapeutics but is severely limited by ice formation and growth, which causes irreversible membrane rupture, osmotic imbalance, and chilling injury. Antifreeze proteins and conventional cryoprotectants like gold standard dimethyl sulfoxide (DMSO) struggle to offer a cost-effective and biocompatible strategy fitting various cellular storage. Herein, we design and fabricate a class of oligopeptides exhibiting potent ice recrystallization inhibition (IRI) activity, achieving a 55.5% reduction in the mean largest grain size at a concentration of less than 0.1 wt %. The side-chain functional groups (e.g., hydroxyl and amine groups) and length (<10 amino acids) are meticulously optimized to avoid the thermal hysteresis (TH) activity that causes sudden burst of fatal needle-like ice crystals. Simulation and experimental results illustrate that ice inhibition mechanism of oligopeptides involves binding to the ice crystal surface and disrupting the ordering of water molecules, thereby preventing the formation of well-structured ice crystals. Notably, the employment of oligopeptides as cryoprotectants maintains cell proliferation and differentiation capabilities while having a high cell viability of 90-95%, comparable to 10% DMSO.