Phospholipid vesicles increase the survival of freeze-dried human red blood cells

In a previous report [Z. Török, G. Satpathy, M. Banerjee, R. Bali, E. Little, R. Novaes, H. Van Ly, D. Dwyre, A. Kheirolomoom, F. Tablin, J.H. Crowe, N.M. Tsvetkova, Preservation of trehalose loaded red blood cells by lyophilization, Cell Preservation Technol. 3 (2005) 96-111.], we presented a method for preserving human red blood cells (RBCs) by loading them with trehalose and then freeze-drying. We have now improved that method, based on the discovery that addition of phospholipid vesicles to the lyophilization buffer substantially reduces hemolysis of freeze-dried RBCs after rehydration. The surviving cells synthesize 2,3-DPG, have low levels of methemoglobin, and have preserved morphology. Among the lipid species we studied, unsaturated PCs were found to be most effective in suppressing hemoglobin leakage. RBC-vesicle interactions depend on vesicle size and structure; unilamellar liposomes with average diameter of less than 300 nm were more effective in reducing the hemolysis than multilamellar vesicles. Trehalose loaded RBCs demonstrated high survival and low levels of methemoglobin during 10 weeks of storage at 4 degrees C in the dry state when lyophilized in the presence of liposomes.