Cryoprotectant permeability parameters for cells used in a bioengineered human corneal equivalent and applications for cryopreservation

Abstract

A human corneal equivalent is being developed with applications in pharmaceutical testing and biomedical research, but the distribution of this engineered tissue, depends on successful cryopreservation. Cryopreservation of tissues depends on the presence of cryoprotectants, their addition and removal, and exposure to conditions during freezing and thawing, all of which depend on cellular membrane permeabilities to water and cryoprotectant. This study defines the permeability properties that define the rate of water and cryoprotectant movement across the plasma membrane of isolated human corneal endothelial, keratocyte, and epithelial cells. Cells were transferred from isotonic conditions (300 mosm/kg) to 0.5, 1, or 2 M dimethyl sulfoxide and propylene glycol solutions at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after cryoprotectant exposure allowed calculation of hydraulic conductivity ( L p), cryoprotectant permeability ( P s), and the reflection coefficient ( σ). Experimental values for L p and P s at 4, 13, 22, and 37 °C were used to determine the Arrhenius activation energy ( E a). Defining the permeability parameters and temperature dependencies allows simulation of responses of human corneal cells to addition and removal of cryoprotectants and to freezing conditions, allowing amount of supercooling, intracellular electrolyte concentration, and intracellular cryoprotectant concentration to be calculated. Simulations also show that the constituent cells in the bioengineered cornea respond differently to addition and removal of cryoprotectants and to freezing. This study has defined the requirements during cryopreservation for the corneal cells; future work will define the matrix requirements which will allow the development of a cryopreservation protocol.