Post-rehydration membrane properties are an important indicator of effective freeze-drying of mammalian blood cells. In general, cellular membranes are extremely susceptible to damage during freezing and drying. The disaccharide trehalose, naturally occurring in anhydrobiotic organisms, has been shown to prevent such damage through direct interactions with the phospholipids and proteins, and by forming an amorphous glassy matrix around the membranes. Thermal properties and lipid composition of plasma membranes from freeze-dried trehalose loaded RBCs were studied using Fourier Transform Infrared Spectroscopy (FTIR) and analytical thin layer chromatography (TLC). FTIR results showed that plasma membranes from freeze-dried and rehydrated RBCs have major phase transitions between 10 and 20°C, and between 30 and 40°C, similarly to membranes isolated from fresh RBCs. Analysis of the lipid composition of freeze-dried RBCs showed profiles of the freeze-dried RBCs that are very similar to the ones of membranes from freshly isolated RBCs, suggesting that the freeze-drying procedure did not affect the lipid composition. SDS-PAGE and immunoblotting analysis showed that the cytoskeletal components of the membrane such as spectrin, ankyrin, band 3 and glycophorin A which form a flexible meshwork of proteins underlying the lipid bilayer are also well preserved in the freeze-dried RBCs. The study demonstrates that freeze-drying of trehalose loaded human erythrocytes does not result in significant changes in the lipid and protein structure. These findings have immediate applications in clinical medicine for long-term storage of RBCs. Supported by DARPA grant N66001-03-1-9827

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