Ice formation and recrystallization exert severe impairments to cellular cryopreservation. In light of cell-damaging washing procedures in the current glycerol approach, many researches have been devoted to the development of biocompatible cryoprotectants for optimal bioprotection of human erythrocytes. Herein, we develop a novel ACTIVE glycopeptide of saccharide-grafted ε-poly­(L-lysine), that can be credited with adsorption on membrane surfaces, cryopreservation with trehalose, and icephilicity for validity of human erythrocytes. Then, by Borch reductive amination or amidation, glucose, lactose, maltose, maltotriose, or trehalose was tethered to ε-polylysine. The synthesized ACTIVE glycopeptides with intrinsic icephilicity could localize on the membrane surface of human erythrocytes and improve cryopreservation with trehalose, so that remarkable post-thaw cryosurvival of human erythrocytes was achieved with a slight variation in cell morphology and functions. Human erythrocytes (∼50% hematocrit) in cryostores could maintain high cryosurvival above 74%, even after plunged in liquid nitrogen for 6 months. Analyses of differential scanning calorimetry, Raman spectroscopy, and dynamic ice shaping suggested that this cryopreservation protocol combined with the ACTIVE glycopeptide and trehalose could enhance the hydrogen bond network in nonfrozen solutions, resulting in inhibition of recrystallization and growth of ice. Therefore, the ACTIVE glycopeptide can be applied as a trehalose-associated “chaperone”, providing a new way to serve as a candidate in glycerol-free human erythrocyte cryopreservation.

Currently, glycerol is a conventional cryoprotectant of human red blood cells (hRBCs), but the time-consuming thawing and deglycerolization processes are essential before transfusion. Much of the research up to now...

Cryopreservation of human erythrocytes via suitable cryoprotectants is essential for transfusion at emergency, but the conventional glycerolization method requires a tedious thawing-deglycerolization process. Alternatively, trehalose, a nonreducing disaccharide, has gained...

As a nonreducing disaccharide, trehalose can be used as a biocompatible cryoprotectant for solvent-free cell cryopreservation, but the membrane-impermeability limits its cryoprotective efficiency. Herein, a series of aromatic monoamines with...

Intracellular delivery of freezing-tolerant trehalose is crucial for cryopreservation of red blood cells (RBCs) and previous strategies based on membrane-disruptive activity usually generate severe hemolysis. Herein, a dynamic membrane-active glycopeptide is developed by grafting with 25% maltotriose and 50% p-benzyl alcohol for the first time to effectively facilitate entry of membrane-impermeable trehalose in human RBCs with low hemolysis. Results of the mechanism acting on cell membranes suggest that reversible adsorption of such benzyl alcohol-grafted glycopeptide on cell surfaces upon weak perturbation with phospholipids and dynamic transition toward membrane stabilization are essential for keeping cellular biofunctions. Furthermore, the functionalized glycopeptide is indicative of typical 𝜶-helical/𝜷-sheet structure-driven regulations of ice crystals during freeze−thaw, thereby strongly promoting efficient cryopreservation. Such all-in-one glycopeptide enables achieving both high cell recovery post-thaw >85% and exceptional cryosurvival >95% in direct freezing protocols. The rationally designed benzyl alcohol-modified glycopeptide permits the development of a competent platform with high generality for protection of blood cells against freeze-stress.

Red blood cell (RBC) preservation is very important in human health. The RBCs are usually preserved at 4 ± 2 °C without freezing or at a very low temperature (−80 °C or liquid nitrogen) with deep freezing. Herein, non freezable preservation of RBCs at a subzero temperature is reported to prolong the preservation time compared with that at 4 ± 2 °C. By adding glycerol and poly­(ethylene glycol) (PEG) (average number molecular weight 400, PEG-400) into the preservation solution, the freezing point is decreased and the hemolysis is kept low. The cell metabolism of stored RBCs at −8 °C is reduced, and the shelf life of RBCs extends up to at least 70 days. At the end of preservation, the pH decreases a little bit to demonstrate the low metabolic rate of RBCs stored at subzero temperatures. After quick washing, the RBC survival rate is ca. 95%. The adenosine triphosphate, 2,3-diphosphoglycerate, and cell deformation ability of the washed RBCs are maintained at a high level, while the malondialdehyde is relatively low, which verifies the high quality of RBCs stored at this condition.

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