Measurement of Cryoprotectant Permeability in Adherent Endothelial Cells and Applications to Cryopreservation

Vitrification is a promising approach for cryopreservation of adherent cells because it allows complete avoidance of ice formation. However, high cryoprotectant (CPA) concentrations are required to prevent freezing, and exposure to high CPA concentrations increases the risk of osmotic and toxic damage. Although cell membrane transport modeling can be used for rational design of CPA equilibration procedures, the necessary permeability data is extremely scarce for adherent cells. This study validates a method for in situ measurement of water and CPA permeability in adherent cells based on the fluorescence quenching of intracellular calcein. Permeability parameters for endothelial monolayers were measured during exposure to four common cryoprotectants (dimethyl sulfoxide, ethylene glycol, propylene glycol and glycerol) at temperatures of 4°C, 21°C and 37°C. Propylene glycol exhibited the highest permeability and glycerol the lowest. The data was fit using an Arrhenius model, yielding activation energies ranging from 45 kJ/mol to 61 kJ/mol for water transport and 84 kJ/mol to 99 kJ/mol for CPA transport. These permeability parameters will facilitate the development of mathematically-optimized CPA equilibration procedures for vitrification of adherent endothelial cells. Our results establish calcein fluorescence quenching as an effective method for measurement of CPA permeability in adherent cells.Keywords: adherent cells, vitrification, calcein, fluorescence quenching, membrane permeabilit