Innocuous intracellular ice formation: Mechanisms and implications

Successful cryopreservation of tissues has been limited by the lack of information on the mechanism by which these systems are damaged as a result of exposure to low temperatures. Intracellular ice formation (IIF), a potentially lethal form of cryoinjury, has been shown to occur more predominantly in biological systems with cell-cell and cell-surface interactions. Innocuous IIF has been proposed when the mechanism of ice formation does not result in permanent damage to the cell.;The work presented in this thesis represents a concerted effort to understand the mechanisms and implications of innocuous intracellular ice formation in tissue model systems. Investigations were conducted to explore the nature of the damage that results when ice forms within cells and the effect this intracellular ice has on the response of cells and tissue models to low temperatures. Fluorescent cryomicroscopy was utilized extensively to document the formation of intracellular ice in tissue models and the role of the plasma membrane in this process. By coupling cryomicroscopic studies with a progressive assessment technique, the effect of intracellular ice on cell viability was determined. The data collected from these experiments was combined into a conceptual model on the formation of intracellular ice in tissue models and the implications tested against a standard cryopreservation protocol.;The cell plasma membrane is a critical site of injury during the formation of intracellular ice. Innocuous IIF occurs when plasma membrane integrity is maintained by the propagation of intracellular ice between adjacent cells. This process of intercellular ice propagation is facilitated by the presence of gap junctions. Innocuous intracellular ice affects the low temperature response of tissue models and protects the constituent cells from slow cooling injury by acting as an intracellular cryoprotectant. In the absence of chemical cryoprotectants, inducing intracellular ice formation is an effective method for the cryopreservation of tissue models.;With a better understanding of the protective mechanism of intracellular ice, development of techniques and procedures to minimize cryoinjury can be explored. These results provide researchers and clinicians new avenues to explore and new techniques to use in the development of the next generation of cryopreservation protocols.