Freeze-drying of red blood cells at ultra-Low temperatures

The hemolysis of human red blood cells (RBCs) after freeze-drying and resuspension depends on the vacuum-drying temperature. In an experimental study, RBCs were first solidified based on a modified high-yield cryopreservation protocol in the presence of hydroxyethyl starch and maltose. Afterward, they were vacuum-dried in a special low-temperature freeze-drying device at selected shelf temperatures between -5 and -65 degrees C. Subsequently, the dried samples were resuspended in an isotonic, phosphate-buffered saline solution. The hemolysis was determined according to a modified saline stability test. It decreases with a decreasing shelf temperature until a minimum is reached at -35 degrees C. A further decrease of the shelf temperature has no beneficial effect; the hemolysis even increases. To interpret these results, we assume that the hemolysis depends on two contrary damaging effects: (1) the higher the shelf temperature, the higher the probability of structural damages occurring during drying; (2) the lower the shelf temperature, the lower the driving force for water transport; this may lead to an incomplete intracellular dehydration which means that the cells are not in a glassy state at ambient temperature.