Evaluating Isochoric Freezing as a Strategy for Storage of Red Blood Cells

Red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation during storage. Preserving RBCs for clinical transfusion remains a challenge due to storage-induced damage and limitations of traditional freezing methods. This study investigates isochoric freezing-a constant-volume, high-pressure cryopreservation technique that suppresses ice formation-as an alternative approach for RBC cryopreservation. RBC samples were preserved under isochoric freezing conditions at -2.5°C to -15°C with corresponding pressures of 31-156 MPa and a comparator supercooled control group. Hemolysis, cell count, morphology, and membrane integrity were assessed using hematological analyses, imaging flow cytometry, and dextran permeability assays. It is reported that hemolysis and morphological deterioration increased with decreasing temperature and rising pressure, with higher membrane damage compared to supercooling. Lower-temperature isochoric freezing resulted in loss of membrane integrity that was irreversible. While isochoric freezing minimized ice formation, elevated pressures adversely affected RBC viability. These findings highlight critical pressure-temperature thresholds necessary for optimizing isochoric freezing protocols for RBC preservation and inform future development of safer, long-term blood storage strategies.