Natural Deep Eutectic Solvents Enable Programmable Cooling-Free Cryopreservation

This study systematically evaluates two natural deep eutectic solvents (NADES)─PS21 (l-proline/sucrose, 2:1 molar ratio) and PS31 (l-proline/sucrose, 3:1 molar ratio)─as low-toxicity, programmable cooling-free cryoprotectants. Through comprehensive physicochemical characterization (FTIR, DSC, cryomicroscopy) and biological assays, we demonstrate that both PS21/PS31 variants significantly inhibit ice crystallization, modify ice morphology, and reduce recrystallization damage, and their unfrozen water content at 5 wt % (0.23 for PS21, 0.18 for PS31) was significantly higher than that of 10% dimethyl sulfoxide (DMSO, 0.10); cryomicroscopy revealed that NADES restrict ice crystal growth to 400-1000 μm² (far smaller than the 4000-6000 μm² in PBS) and modify ice morphology to reduce recrystallization damage. In biological assays using A549 cells: after 24 h of coculture, cell viability with 5 wt % PS31 (57.7%) was significantly higher than that with 10% DMSO(20.3%), and even at 20 wt %, PS21(18.6%) maintained viability comparable to DMSO. By optimizing cooling protocols (e.g., Method 2 with a cooling rate of -6.2 °C/min), high post-thaw performance was achieved: the highest survival rate (88.2%) was observed in the 15% PS31 group, and the 24 h proliferation rate of the 10% PS31 group reached 74.25%, significantly exceeding that of the DMSO group.These findings establish NADES as scalable, green alternatives for biobanking, offering streamlined workflows and enhanced sample quality while aligning with sustainable cryopreservation practices.