Freeze Drying of Red Blood Cells: The Use of Directional Freezing and a New Radio Frequency Lyophilization Device

Abstract

Red blood cell (RBC) units are administered routinely into patients expressing a wide range of acute and chronic conditions (e.g., anemia, traumatic bleeding, chronic diseases, and surgery). The modern blood banking system has been designed to answer this need and assure a continuous, high quality blood supply to patients. However, RBCs units can be stored under hypothermic conditions for only up to 42 days, which leads to periodic shortages. Cryopreservation can solve these shortages, but current freezing methods employ high glycerol concentrations, which need to be removed and the cells washed prior to transfusion, resulting in a long (more than 1 hour) and cumbersome washing step. Thus, frozen RBCs have limited use in acute and trauma situations. In addition, transportation of frozen samples is complicated and costly. Freeze drying (lyophilization) of RBCs has been suggested as a solution for these problems, since it will allow for a low weight sample to be stored at room temperature, but reaching this goal is not a simple task. We studied the effect of different solutions (IMT2 and IMT3) containing trehalose and antioxidants or trehalose and human serum albumin, respectively, on freezing/thawing and freeze drying of RBCs. In addition, we evaluated the effect of cells concentrations and cooling rates on the post thaw and post rehydration recoveries of the RBCs. Finally, we developed a new radio frequency (RF) lyophilization device for a more rapid and homogeneous sublimation process of the frozen RBCs samples. Recovery and free Hb were measured as well as oxygen association/dissociation and cell's deformability. We found that IMT3 (0.3 M trehalose and 10% HSA) solution that was directionally frozen at a rapid interface velocity of 1 mm/sec (resulting in a cooling rate of 150°C/min) yielded the best results (better than IMT2 solution and slow interface velocity). Freeze thawing gave 100% survival, while freeze drying followed by rehydration with 20% dextran-40kDa solution resulted in 75% survival. However, recovery following freeze drying was possible only when 20% Dextran-40 solution was used as the rehydration medium. The rehydrated cells were not stable upon an eight-fold dilution. The RF lyophilization system increased the sublimation rate more than twice compared to conventional drying and maintained a high survival rate of the RBCs after partial drying.