(681g) Cell Membrane Permeability Properties for Endothelial Tissue and Computer-Aided Optimization of Cryoprotectant Addition and Dilution Procedures

Cryopreservation is a critical technology for the development of cell- and tissue-based products because it enables long-term preservation for purposes of stockpiling, quality control, and product distribution. The cryopreservation process requires that cells be subjected to extreme conditions, including high solute concentrations, low temperatures, and contact with ice, all of which can cause damage. To mitigate damage, cell membrane permeable chemicals called cryoprotective agents (CPAs) are commonly added to the cryopreservation medium. However, addition of CPA before freezing and removal by dilution after thawing causes water transport across the cell membrane, which may lead to extreme volume changes and concomitant cellular damage. CPA addition and removal protocols have been optimized for several types of isolated cells using cell-membrane mass transfer models. However, the necessary permeability parameters are lacking for tissue. We have adapted a fluorescence quenching technique for in situ measurement of CPA permeability in adherent tissue. Monolayers of bovine pulmonary artery endothelial cells were treated with calcein-AM and exposed to solutions containing CPA within a temperature-controlled flow chamber. The resulting volume-dependent changes in fluorescence intensity were recorded by digital video microscopy and fit with a mathematical model of membrane transport to determine the permeability parameters. Our results indicate that the common cryoprotectant, glycerol, permeates the cell membrane slowly in endothelial tissue, resulting in relatively large cell volume changes during CPA addition and removal. In contrast, DMSO and propylene glycol permeated the endothelial tissue rapidly, yielding minimal volume changes. Therefore, DMSO and propylene glycol are expected to be suitable CPAs for cryopreservation of endothelial tissue, but glycerol is not.