Synergistic proline-based deep eutectic solvent enables low-toxicity microdroplet cryopreservation of 3D cell...

Establishing in vitro organoid biobanks-three-dimensional (3D) self-organized microtissues derived from stem cells or patient tissues-holds great value for clinical disease modeling, drug screening, and regenerative medicine applications. However, current cryopreservation approaches are often constrained by the use of chemically harsh cryoprotectants (CPAs) and multi-step operation procedures, which may compromise biological integrity and limit practical applicability. To address these challenges, we report a low-toxicity cryopreservation strategy that integrates an L‑proline‑based deep eutectic solvent (DES) with controlled evaporative dehydration (CED) under negative pressure, thereby reducing reliance on high concentrations of conventional CPAs during the microdroplet cryopreservation of 3D cell spheroids. As a proof-of-concept study, NIH/3T3 spheroids were used as a reproducible 3D model to evaluate the feasibility of this strategy. The amino acid architecture of L-proline DES provides intrinsic biocompatibility, abundant hydrogen-bonding sites, and strong water-structuring capacity, enabling the formation of a protective microenvironment around 3D spheroids while reducing dependence on CPAs. During CED, the L‑proline DES establishes a non‑permeating protective microenvironment around spheroids, significantly alleviating osmotic injury. A low concentration of DMSO was introduced to form a binary synergistic system that optimized cryoprotectant mass transfer and intracellular concentration dynamics, thereby providing coordinated extracellular and intracellular protection during microdroplet quenching. Quenching after 4-6 min of CED was identified as the optimal cryopreservation window. Under these conditions, post-warming viability was approximately 30 percentage points higher than that obtained with conventional slow freezing. Further molecular analyses demonstrated that this L-proline DES system was associated with enhanced antioxidant defense and attenuation of ferroptosis-related oxidative injury. This work establishes an amino acid-derived DES system for CED-based microdroplet cryopreservation and elucidates its cryoprotective mechanisms at macroscopic and microscopic levels. Crucially, it offers an efficient, low-toxicity strategy for preserving complex 3D biological systems. STATEMENT OF SIGNIFICANCE: Cryopreservation of three-dimensional cell spheroids remains challenging because dense multicellular architectures limit cryoprotectant transport and increase sensitivity to osmotic and chemical injury. Here, we develop a biomaterial-enabled cryopreservation strategy combining an L-proline-based deep eutectic solvent with controlled evaporative dehydration (CED). This approach reduces dependence on high DMSO concentrations while enabling ice-free preservation and high post-thaw viability of 3D spheroids. Mechanistically, the DES-rich microenvironment buffers dehydration stress and suppresses ferroptosis-associated lipid peroxidation through regulation of iron homeostasis and GPX4/GSH antioxidant defense. This work provides a low-toxicity materials strategy for preserving complex 3D cellular constructs.