Strategies to reduce osmotic stress during cryopreservation of red blood cells when using trehalose

The aim of this study was to determine optimal conditions for cryopreserving red blood cells (RBCs) using trehalose. We assessed the extent of trehalose uptake by RBCs during incubation at 37 °C and following freezing-and-thawing. Additionally, we examined whether betaine could alleviate osmotic stress at various stages of the cryopreservation process, including trehalose loading, freezing, and return to isotonic conditions after thawing. Trehalose uptake during incubation at 37 °C was limited, whereas freezing-and-thawing RBCs in trehalose-containing solutions led to substantially higher intracellular trehalose concentrations. The greatest post-thaw survival was observed with 400 mM trehalose in combination with rapid cooling. When trehalose was combined with betaine, optimal cryosurvival was maintained at a total solute concentration of 400 mM, whereas supplementing 400 mM trehalose with membrane-permeating cryoprotectants such as glycerol or dimethyl sulfoxide (DMSO) further enhanced cell cryosurvival. Membrane permeability studies indicated that betaine acts as a non-permeating solute contradicting literature findings. Direct transfer of trehalose-loaded, cryopreserved RBCs to isotonic conditions after thawing resulted in hemolysis, but this could be reduced by using hypertonic washing solutions. In conclusion, no synergistic protective effect was observed from combining betaine with trehalose for RBC cryopreservation, whereas combinations of trehalose with membrane-permeating agents like glycerol or DMSO appear promising for improving RBC cryopreservation outcomes.