Red Blood Cell Cryopreservation with Minimal Post-Thaw Lysis Enabled by a Synergistic Combination of a Cryoprotecting Polyampholyte with DMSO/Trehalose

[Image: see text] From trauma wards to chemotherapy, red blood cells are essential in modern medicine. Current methods to bank red blood cells typically use glycerol (40 wt %) as a cryoprotective agent. Although highly effective, the deglycerolization process, post-thaw, is time-consuming and results in some loss of red blood cells during the washing procedures. Here, we demonstrate that a polyampholyte, a macromolecular cryoprotectant, synergistically enhances ovine red blood cell cryopreservation in a mixed cryoprotectant system. Screening of DMSO and trehalose mixtures identified optimized conditions, where cytotoxicity was minimized but cryoprotective benefit maximized. Supplementation with polyampholyte allowed 97% post-thaw recovery (3% hemolysis), even under extremely challenging slow-freezing and -thawing conditions. Post-thaw washing of the cryoprotectants was tolerated by the cells, which is crucial for any application, and the optimized mixture could be applied directly to cells, causing no hemolysis after 1 h of exposure. The procedure was also scaled to use blood bags, showing utility on a scale relevant for application. Flow cytometry and adenosine triphosphate assays confirmed the integrity of the blood cells post-thaw. Microscopy confirmed intact red blood cells were recovered but with some shrinkage, suggesting that optimization of post-thaw washing could further improve this method. These results show that macromolecular cryoprotectants can provide synergistic benefit, alongside small molecule cryoprotectants, for the storage of essential cell types, as well as potential practical benefits in terms of processing/handling.