Natural cryoprotectants combinations of l-proline and trehalose for red blood cells cryopreservation

Cryopreservation of red blood cells (RBCs) holds great potential benefits for supplying transfusion timely in emergencies. Currently, glycerol is the main cryoprotectant permitted in clinical therapy for RBCs cryopreservation, but its broad application is limited by the toxicity and complex deglycerolization process. Successful cryopreservation of RBCs using more effective materials should be studied to reduce freezing damage, increase biocompatibility, and save processing time. Herein, a simple protocol using natural cryoprotectants combinations of l-proline and trehalose attains a low degree of hemolysis (11.2 ± 2.73%) after thawing compared to glycerol. Furthermore, the morphology of RBCs and the activities of Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase maintain well. Further mechanism study shows that l-proline plays an important role in decreasing the freezing points and inhibiting the growth of ice crystal by permeating into cells during the freezing process. While trehalose works as an inhibitor of ice growth in the freezing process and ice recrystallization in the thawing process. This simple l-proline & trehalose combinations protocol is a promising method to replace current time-consuming and labor-intensive cryopreservation methods of RBCs.