A Tardigrade-Derived CAHS-Motif Enables Glycerol-Free Red Blood Cell Cryopreservation with Enhanced Low-Temperature Trehalose Loading and Synergistic Suppression of Ice Damage.

High-concentration glycerol, the clinical standard for red blood cell (RBC) cryopreservation, requires laborious deglycerolization and causes significant hemolysis, whereas trehalose is biocompatible but poorly permeable to the RBC membrane. Herein, we report a tardigrade-inspired, glycerol-free strategy for RBC cryopreservation based on a conserved CAHS-derived peptide motif. Systematic sequence analysis identified a minimal CAHS-motif that possesses an intrinsic helical propensity and adopts a stabilized amphipathic α-helical conformation under dehydration-mimicking conditions. Under the 4 °C loading condition, CAHS-motif and trehalose co-incubation was associated with increased membrane fluidity and increased intracellular trehalose accumulation while maintaining low pre-freeze hemolysis. In parallel, the CAHS-motif acts synergistically with trehalose to suppress ice-associated damage during freezing and thawing. Under the combined effect, RBCs cryopreserved with the CAHS-motif and trehalose formulation achieved a post-thaw recovery of 89.0 ± 0.6% and excellent blood compatibility (99.0 ± 0.7%), outperforming the conventional glycerol-based method. Post-thaw RBCs retained normal morphology, volume, and key functional activities in vitro and effectively corrected anemia in an APH-induced hemolytic anemia mouse model without detectable pro-inflammatory responses. These results establish a clinically relevant, bioinspired platform for high-recovery RBC cryopreservation and highlight the translational potential of extremophile-derived motifs in transfusion medicine.