Intracellular ice formation in tissue constructs and the effects of mass transport across the cell membrane.

摘要

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 prediction 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, representing a significant step toward developing cryopreservation strategies for tissue. 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, revealing that tight junctions may have a protective effect during freezing. A new IIF mechanism was observed associated with penetration of extracellular ice into the paracellular space at the cell-cell interface, suggesting that IIF is caused by mechanical interaction with the extracellular ice. In addition, we investigated the effect of cytoplasm composition, which changes during freezing because of cell dehydration, on the kinetics of IIF in tissue. We found that increasing the viscosity or decreasing the supercooling significantly decreased the rates of independent IIF and intercellular ice propagation, 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.