Mediated trehalose un-loading for reduced erythrocyte osmotic fragility and phosphatidylserine translocation

Recently, high concentrations of intracellular trehalose (>200mM) were employed to enhance the cryoprotection and desiccation protection of human erythrocytes. However, significant challenges must be overcome if this advancement is to be translated into clinical practice. It is here demonstrated that 247 ± 5 mM intracellular trehalose caused the lysis of 60 ± 2 percent of erythrocytes upon resuspension in PBS of physiological osmolality (300 mOsm) and caused surviving cells to swell up to 140 ± 2 percent of isotonic cell volume. Trehalose loaded cells also exhibited 24 ± 1 percent incidence of phosphatidylserine translocation upon resuspension in 300 mOsm PBS, likely due to loading induced cell swelling. Un-loading of trehalose from erythrocytes using the membrane-permeabilizing biopolymer PP-50 was investigated as a technique to mitigate these damaging effects. After erythrocyte un-loading from 247 ± 5 mM to 39 ± 2 mM intracellular trehalose, cell lysis at 300 mOsm PBS was reduced from 60 ± 2 percent to 17 ± 3 percent. Un-loading also reduced cellular incidence of PS translocation in resuspended cells from 24 ± 1 percent to 13 ± 1 percent.