Development and Storage of Freeze-Dried Red Blood Cells

Blood transfusions are the single most often used lifesaving procedure in hospitals worldwide. Unfortunately, packed red blood cells (RBCs) used for transfusion can only be stored for 42 days at 39 °F (4 °C) before they must be discarded due to irreversible damage that occurs during storage. Any reduction in available RBCs for an extended period, like decreased blood donations during the ongoing COVID-19 crisis, has severe consequences and can lead to shortages of lifesaving transfusion units. To increase the shelf-life of RBCs, we are investigating freeze-drying (lyophilizing) in the presence of the non-toxic sugar trehalose as a method for long-term stabilization. However, freeze-drying of RBCs and water absorbance after processing can compromise the functionality of these cells. Precisely, the oxygen-transport protein hemoglobin can convert via oxidation into the nonfunctional methemoglobin form. We tested different processing solutions and storage conditions to determine the optimal conditions with the lowest formation of methemoglobin. Porcine RBCs were suspended in a solution containing trehalose, snap-frozen at ultra-low temperature using liquid nitrogen at -320 °F, then transferred to a lyophilizer and dried. During storage, the humidity was controlled using air-tight chambers. Spectroscopy was employed to determine hemoglobin\u27s oxidation state after lyophilization and any changes during storage. We found that oxidation of hemoglobin to methemoglobin during freeze-drying is a challenge and methemoglobin concentration increased during storage. This increase occurred during storage at high and low humidity and under vacuum. However, RBCs fortified with vitamin C before lyophilization and stored at 0% relative humidity showed the lowest methemoglobin content. Optimizations to the freeze-drying and storage processes are currently performed to develop this technology further