Intracellular ice formation in tissue constructs and the effects of mass transport across the cell membrane
Higgins, Adam Zachary
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Long-term storage of tissue by cryopreservation is necessary for the efficient mass production of tissue engineered products, and for reducing the urgency and cost of organ transplantation procedures. The goal of this work was to investigate the physical processes thought result in damage during tissue cryopreservation towards development of tissue cryopreservation strategies. Although mathematical models of cell dehydration and intracellular ice formation (IIF) have been successfully used to optimize cryopreservation procedures for cell suspensions, it is not currently possible to use this approach with tissue because of the lack of tissue-specific permeability parameters for predicting cell dehydration during tissue freezing, and because of the increased complexity of the IIF process in tissue. We have measured the membrane permeability properties of tissue comprising a cell monolayer using a fluorescence quenching technique, and compared the results to the corresponding cell suspensions, revealing significant differences in the membrane transport kinetics between monolayers and suspensions. These data enabled the prediction cell dehydration during freezing of cell monolayers. Whereas the mechanisms of IIF are relatively well understood in cell suspensions, tissue is susceptible to new IIF mechanisms. In particular, cell-cell interactions have been shown to increase the IIF probability by enabling the propagation of ice between neighboring cells. We investigated the effect of cell-cell interactions on IIF using genetically modified cells expressing different levels of intercellular junction proteins. A new IIF mechanism was observed in these cells associated with penetration of extracellular ice into the cell-cell interface, and the incidence of this IIF mechanism was reduced in cells expressing the tight junction protein occludin. In addition, we investigated the effect of the cytoplasm supercooling and viscosity on the kinetics of IIF in tissue. We found that increasing the viscosity or decreasing the supercooling significantly decreased the kinetics of IIF, suggesting that IIF protocols for tissue can be optimized by modulating the cytoplasm supercooling and viscosity. Together, these data represent an important step towards developing cryopreservation strategies for tissue.