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  • 1
    Publication Date: 2004-11-16
    Description: A method for freeze-drying red blood cells (RBCs) while maintaining a high degree of viability, has important implications in blood transfusion and clinical medicine. The disaccharide trehalose, found in animals capable of surviving dehydration can aid in this process. We are reporting a method for loading RBCs with trehalose followed by subsequent freeze-drying and rehydration. The loading of erythrocytes is based on the thermal properties of the RBC plasma membranes and provides efficient uptake of the sugar at 37°C in a time span of 7 hours. The data show that RBCs can be loaded with trehalose from the extracellular medium through a combination of osmotic imbalance and the phospholipid phase transition, producing an intracellular trehalose concentration of about 40 mM. Freeze-drying of trehalose loaded RBCs results in water contents in the range between 2 and 4 % and a level of survival of around 37 %, as measured by the extent of hemolysis. Surprisingly, freeze-dried and rehydrated RBCs showed high levels of ATP and 2,3-DPG and low methemoglobin. Biochemical analysis demonstrated that the activities of superoxide dismutase, catalase and acetylcholine esterase in freeze-dried RBCs are similar to those in fresh RBCs. These data provide an important step toward a stable erythrocyte product, which will be invaluable for transfusion and clinical applications. Supported by DARPA grant N66001-03-1-9827
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
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  • 2
    Publication Date: 2004-11-16
    Description: A wide variety of medical procedures require transfusion of red blood cells (RBCs). RBCs are currently preserved either at 4°C, at a higher hematocrit (70%) for up to 7–12 weeks or in a frozen state in the presence of glycerol at −80°C for several years. However, each procedure has its demerits. Storage in the dry state offers a possibility for storing the cells for long periods of time under conditions that are far easier to maintain (i.e. room temperature), making the transport to sites of immediate need feasible. We developed a method for freeze-drying RBCs using 15% hematocrit, resulting in a survival of 40% after rehydration, as assessed by the percent hemolysis. In this work, we report the effect of cell hematocrit, concentration of trehalose, salts and overall osmolality of the freeze-drying medium on the survival after freeze-drying and rehydration. Decreasing the percent hematocrit and trehalose in the freeze-drying buffer resulted in about 20% improvement in the post-rehydration survival. Freeze-dried and rehydrated RBCs showed high levels of ATP, 2,3-DPG and low percent methemoglobin. These data are discussed in terms of the glass transition properties of the freeze-drying buffer. This work provides an important step in formulating a freeze drying medium that will provide optimum RBC survival after freeze drying and rehydration.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
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  • 3
    Publication Date: 2004-11-16
    Description: 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
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
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