Bioinspired Cryoprotectants Enabled by Binary Natural Deep Eutectic Solvents for Sustainable and Green Cryopreservation

Abstract

Cryopreservation plays an important role in prolonging the cell viability, but non-negligible cytotoxicity of cryoprotectants restrains its further development in many areas with safety concerns such as the food industry. Inspired by cold-tolerant animals, three binary natural deep eutectic solvents (NADESs) were prepared and used as cryoprotectants for achieving cytotoxicity-free and green cryopreservation, whose cryoprotective efficiency was compared with those of glycerol and dimethyl sulfoxide (DMSO), using yeast Saccharomyces cerevisiae, a common commercial microorganism in the food industry, as the cryopreservation object. Cytotoxicity evaluation involving reactive oxygen species accumulation and cell viability showed that the NADESs could be safe and biocompatible. In addition, cytomorphology indicated that the cells cryoprotected by NADESs show plump profiles with smooth cytoderms, while rough cytoderms or even severe cell distortion was partially observed for the ones cryopreserved by glycerol and DMSO. The live/apoptosis/death test of resuscitated cells based on flow cytometry illustrated that the NADES-cryopreserved cells could show higher cell viability (75–80%) with almost no apoptosis (<1.0%). Mechanisms of NADES-supported cryopreservation were further investigated concerning antifreezing capacities and ice recrystallization inhibition activities of NADESs, which suggested that NADESs could exhibit reinforced hydrogen-bonding strengths when decreasing temperature and correspondingly restrain the water molecular motion, making NADESs effectively inhibit ice recrystallization and prevent cells from ice-induced mechanical damage. The excellent cryoprotective efficiency of NADESs may help to open up new avenues for green cryopreservation and attract more concerns for NADESs as emerging cryoprotectants.