Membrane permeability of the human granulocyte to water dimethyl sulfoxide glycerol propylene glycol and ethylene glycol

摘要

Granulocytes are currently transfused as soon as possible after collection because they rapidly deteriorate after being removed from the body. This short shelf life complicates the logistics of granulocyte collection, banking and safety testing. Cryopreservation has the potential to significantly increase shelf life; however, cryopreservation of granulocytes has proven to be difficult. In this study, we investigate the membrane permeability properties of human granulocytes, with the ultimate goal of using membrane transport modeling to facilitate development of improved cryopreservation methods. We first measured the equilibrium volume of human granulocytes in a range of hypo- and hypertonic solutions and fit the resulting data using a Boyle-van't Hoff model. This yielded an isotonic cell volume of 378 μm³ and an osmotically inactive volume of 165 μm³. To determine the permeability of the granulocyte membrane to water and cryoprotectant (CPA), cells were injected into well-mixed CPA solution while collecting volume measurements using a Coulter Counter. These experiments were performed at temperatures ranging from 4 to 37 °C for exposure to dimethyl sulfoxide, glycerol, ethylene glycol and propylene glycol. The best-fit water permeability was similar in the presence of all of the CPAs, with an average value at 21 °C of 0.18 μm atm⁻¹ min⁻¹. The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 21 °C was 6.4, 1.0, 8.4 and 4.0 μm/min for dimethyl sulfoxide, glycerol, ethylene glycol and propylene glycol, respectively, and the activation energy for CPA transport ranged between 59 and 68 kJ/mol.