Unfrozen Liquid Beds Enable Soft Spheroid Preservation at -25 °C

The clinical adoption of regenerative medicine is limited by the lack of reliable methods for transporting preserved spheroids because their cryopreservation remains challenging due to their large size. Conventional "hard" cryopreservation, slow freezing and vitrification, theoretically causes unavoidable physical damage to "soft" spheroids because of ice formation and cracking. In this study, we proposed "soft" preservation permitting the liquid-state preservation of spheroids at -25 °C, suitable for transportation, using cryoprotective agents. The cryoprotective agents were specifically designed for spheroids in this study, overcoming the limitations of the conventional methods, which have been designed for single cells. We developed nonfreezing, low-cytotoxicity solutions composed of highly entropic and biocompatible zwitterions, with dimethyl sulfoxide and ethylene glycol. The spheroids were preserved with a cell recovery of 96%, whereas it was around 10% for conventional hard cryopreservation. The highly stable liquid state enabled high reproducibility (>85% recovery for all 18 samples), indicating potential clinical adaptability. These results demonstrate the potential of soft preservation as a practical strategy for preserving and transporting three-dimensional cellular systems for regenerative medicine.