Loading Trehalose into Red Blood Cells by Improved Hypotonic Method

As a first step toward long-term preservation of red blood cells (RBCs) by using freeze-drying, we present an improved hypotonic method for loading RBCs with trehalose. The method is based on the osmotic pressure difference and the concentration difference between the inside and the outside of the RBCs. RBCs were dehydrated in hypertonic glucose solutions to increase the osmotic pressure inside the cells, and then incubated in hypotonic trehalose solutions to allow trehalose to enter into RBCs. The data showed that trehalose uptake by RBCs increased with osmotic pressure difference across the cell membrane, 43.2 mM intracellular trehalose was observed when osmotic pressure difference reached 1369.8 mOsm/L. Hematology analysis indicated that RBCs shrank in hypertonic glucose solution and swelled in hypotonic trehalose solution, resulting in the release of a few cytoplasmic hemoglobins (Hbs). The level of ATP in trehalose-loaded RBCs recovered to normal value after 4-h incubation. Osmotic fragility data showed that trehalose-loaded RBCs are osmotically stable after dehydration and loading processes.