130 Membrane permeability properties of human granulocytes

Abstract

Granulocytes are a type of circulating white blood cell with high medical relevance because of their potential use in the treatment of infections and possibly even for treatment of cancer. However, granulocytes rapidly deteriorate after being removed from the body. As a result, granulocytes are typically collected from the donor on site and used in a transfusion shortly afterwards. Cryopreservation has the potential to significantly increase shelf life, which would facilitate granulocyte collection, banking and safety testing. 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 μ m 3 and an osmotically inactive volume of 165 μ m 3 . 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 20 °C of 0.17 μ m atm −1 min −1 . The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 20 °C was 5.9, 0.95, 7.7 and 3.6 μ m/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. Source of funding: National Science Foundation Grant #1150861. Conflict of interest: None declared. adam.higgins@oregonstate.edu