Programmable Cooling-Free Cryopreservation via Multi-Parameter Optimization of Natural Deep Eutectic Solvents

Natural deep eutectic solvents (NADES) represent a class of low-toxicity cryoprotectants with promising application prospects, serving as sustainable alternatives to conventional cryoprotective agents. This study systematically evaluated the cryoprotective performance of two NADES formulations composed of l-proline and glucose at molar ratios of 1:1 (PG11) and 5:3 (PG53). Through systematic investigation of their cytotoxicity, ice inhibition capacity, viscosity, and cellular permeability─key physicochemical and biological properties─combined with coordinated optimization of the cooling rate and loading time, we successfully developed an efficient cryopreservation strategy that eliminates the need for programmable cooling equipment. FTIR-ATR spectroscopy confirmed the presence of extensive hydrogen-bonding networks within the NADES systems, accounting for their exceptional ice recrystallization inhibition capacity and high unfrozen water content. By designing customized freezing protocols based on their permeation characteristics, optimal cryoprotection was achieved using 5% NADES cooled at 5.28 °C/min in a precooled -80 °C environment. This integrated optimization approach yielded post-thaw cell viability comparable to that of conventional dimethyl sulfoxide (DMSO) while significantly reducing cytotoxicity. Our research not only validates the potential of NADES as highly efficient and biocompatible cryoprotectants, but it also establishes a straightforward and effective cryopreservation protocol system based on their fundamental biophysical characteristics.