A Novel Supercooling-Based Nonfreezing Preservation Method for Early Embryos Using Plant-Derived Substances to Maintain Cellular Integrity

Maintaining cellular integrity during storage is essential for the stable handling and quality control of living cells in future sensor-integrated biological systems. In this study, we investigated whether plant-derived substances improve the effectiveness of a supercooling-based nonfreezing preservation method for early embryos while maintaining cellular integrity. Mouse 2-cell embryos were used as a model system, and the University of Wisconsin (UW) solution was used as the basal preservation medium. Coffee lees extract was examined as a plant-derived preservation agent, and its representative components, caffeine and chlorogenic acid, were evaluated. Embryos preserved in UW solution alone at -6°C for 72 h showed residual membrane deformation, whereas supplementation with coffee lees extract, particularly at 10%, improved post-preservation morphology. Caffeine showed concentration-dependent protective and adverse effects, with the intermediate concentration providing the most favorable preservation outcome. Additional supporting experiments demonstrated that coffee lees extract and caffeine maintained stable supercooling without freezing under prolonged storage conditions and improved cytoskeletal integrity and post-preservation developmental competence, as assessed by blastocyst development after culture. These findings demonstrate that natural plant-derived substances enhance the nonfreezing preservation of early embryos under supercooling conditions while maintaining cellular integrity. The proposed method may provide a practical basis for the storage, transport, and quality control of living cells and support future applications involving sensor-integrated biological systems.