Cryopreservation of Avian Erythrocytes Using Macromolecular Cryoprotectants

Erythrocyte (red blood cell) cryopreservation is an essential biotechnology for healthcare (transfusions), diagnostics, conservation, and fundamental research. Mature mammalian erythrocytes are anucleate, do not divide, and cannot undertake protein biosynthesis. In contrast, avian erythrocytes have a distinct evolutionary pathway, retaining their nucleus, and are capable of other limited functions, such as protein biosynthesis, but have a shorter shelf life than mammalian erythrocytes. Here, we report the successful cryopreservation of avian red blood cells using a polyampholyte (macromolecular cryoprotectant), which can reduce intracellular ice formation. Under optimized conditions with 10% DMSO and 60 mg·mL^-1 of polyampholyte, ∼90% recovery could be achieved, outperforming a conventional glycerol-based formulation, especially when longer-term storage (up to 6 months) was tested. Scanning electron microscopy confirmed that the cells' morphology was retained. Confocal fluorescence microscopy showed reduced apoptosis compared to glycerol cryopreservation and confirmed increased recovery of actin (cytoskeleton) polymerization post-thaw. Overall, these results show the potential of macromolecular cryoprotectants to be deployed in nonhuman cryobiology for animal health and potentially species conservation.