Enhanced Cryopreservation of Genetically Engineered Porcine Red Blood Cells Using a Novel Chitosan-Based Cryoprotectant Combination

초록

Sustaining a stable blood supply is increasingly challenged due to declining donations and rising demand. Cryopreservation of genetically engineered porcine red blood cells (pRBCs) is one of the promising strategies to support transfusion shortages. However, the state-of-art for RBC cryopreservation is high glycerol method (HGM), which has limitations including cytotoxicity and a time-consuming post-thaw process. Here, we developed a novel cryoprotectant (CPA) formulation, DPC, composed of dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), and chitosan oligosaccharides (COS) and compared it with DMSO–PEG–Trehalose (DPT) and DMSO–PEG (DP).Triple-knockout (TKO) pRBCs were cryopreserved by a slowfreezing method and evaluated for post-thaw recovery, hemoglobin (Hb) retention, oxidative stress markers, intracellular ATP levels, phosphatidylserine (PS) exposure, macrophage-mediated phagocytosis, and thermal properties via differential scanning calorimetry (DSC). During cryopreservation, thawing and washing, DPC-treated pRBCs yielded significantly higher post-thaw recovery (84.8%) than DP or DPT. In addition, DPC showed reduced lipid peroxidation and reactive oxygen species (ROS), increased ATP levels, and decreased PS exposure, resulting in macrophage uptake rates comparable to fresh RBCs. Indeed, DSC analysis revealed a lower melting point (–5.28 °C) and reduced enthalpy change in DPC, indicating enhanced ice recrystallization inhibition. Despite the stringent washing process, pRBC morphology was preserved with ~15% cell loss, outperforming DP and DPT. The DPC formulation provides superior cryoprotection for TKO pRBCs through membrane stabilization, oxidative stress mitigation, and thermal protection. These findings support DPC as a promising alternative CPA formulation for RBC cryopreservation and further in vivo validation for xenotransfusion applications.

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