Dual-Bioinspired Micro Eutecto-Vesicles (MEVs) for Glycerol-Free Green Cryopreservation

Preserving biological functionality under cryogenic conditions remains essential across biotechnology, food, and healthcare sectors, yet conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol pose significant cytotoxic and environmental concerns. Herein, a sustainable cryopreservation strategy based on hydrocolloid-like micro eutecto-vesicles (MEVs), fabricated via a facile centrifugal process using glucose/fructose (GF), proline/glucose (PG), and proline/sorbitol (PS) based natural deep eutectic solvents (NADESs), and sodium alginate (SA), was developed, which was dual bioinspired by cold-tolerant organisms and frog eggs. The results showed that the introduction of NADESs can improve the colloidal stability and uniformity of the MEVs preparation process, and the diameter of the MEVs obtained at 1000 rpm centrifugal speed was about 300 μm. The toxicity test for yeast showed that the components used were less toxic than glycerol and DMSO in terms of proliferation activity and ROS accumulation. For the evaluation of post-thaw cell viability, the cryoprotection of SA/PG based MEVs can achieve a survival rate of yeast cells higher than 92%, while ensuring the integrity of cell structure and excellent proliferation activity. The subsequent mechanism exploration showed that the colloidal vesicle structure of MEVs can reduce the formation of intercellular ice crystals during freezing and inhibit the recrystallization of ice crystals. This study presents a biocompatible, scalable, environmentally responsible, and food grade hydrocolloid-based platform for the long-term stabilization of biological systems, with direct implications for sustainable probiotic production, cold-chain logistics, and bioresource preservation.