Insights into Cryoprotective Mechanisms of Food-Derived Antifreeze Peptides: Coordinated Regulation of Intra- and Extra-Cellular Protection

Cell cryopreservation is essential for food biotechnology and cell-based bioprocessing. However, the widely used cryoprotectant dimethyl sulfoxide (DMSO) shows dose-dependent cytotoxicity, motivating safer alternatives. Here, food-derived antifreeze peptides isolated from Takifugu obscurus skin (TsAFP) were evaluated as primary cryoprotectants using stem cells as a representative cellular model. At 15 mg/mL, TsAFP achieved a 70% replacement of DMSO while maintaining post-thaw cell growth, metabolic activity, adhesion, and multilineage differentiation. TsAFP increased solution viscosity and reduced water activity, effectively inhibiting ice growth and recrystallization. It also adsorbed onto cell membranes to form a protective interfacial layer. Molecular dynamics simulations revealed that TsAFP disrupted water ordering through stable hydrogen-bond interactions. Together, TsAFP provides extracellular and interfacial protection, while low-dose DMSO contributes intracellular protection, resulting in coordinated cryoprotection. TsAFP offers a safer, food-derived strategy to reduce DMSO reliance.