Structure-cryoprotective effect relation in non-cell-permeable zwitterions for DMSO-free cell cryopreservation

With the rapid development in cell therapy and biopharmaceutical production in recent years, safe cryopreservation technologies are required to ensure product quality and safety. Dimethyl sulfoxide (DMSO), which is widely used as a conventional cryoprotectant, raises concerns owing to its cell permeability and consequent cytotoxicity. In this study, we focused on non-cell-permeable zwitterions that exhibit low cytotoxicity. We synthesised 21 zwitterions and systematically evaluated the relation between their molecular structures and cryoprotective effects. Elongation of the cationic side chain or spacer decreases the cryoprotective effects, which are associated with cytotoxicity and freezing of the unfrozen fraction. In contrast, ammonium/sulfonate-type zwitterions exhibited strong cryoprotective effects. This suggests their involvement in the modulation of membrane properties and the suppression of ice crystal formation. Notably, one of the synthesized ammonium/sulfonate-type zwitterions achieved cell recovery comparable with that of a commercial DMSO-containing cryoprotectant for human mesenchymal stem cells. This study deepens understanding of cryoprotective mechanisms based on the molecular design of zwitterions and provides guidelines for safe cellular cryoprotectants.