23. Innocuous intracellular ice formation: Ice growth in complex systems

Abstract

The governing principle in the cryopreservation of tissues and organs is that intracellular ice formation (IIF) is lethal. During freezing, efforts are taken to either cool very rapidly and avoid ice formation (vitrification), or to cool slowly and minimize the probability of intracellular ice forming. Unfortunately, as IIF has been shown to occur more predominantly in biological systems with cell–cell and cell–surface interactions, ice avoidance can be problematic. The literature shows that conditions exist where intracellular ice is not lethal. It is of interest to note that most of the documented cases involving the innocuous formation of intracellular ice have been in natural systems composed of cell aggregates or intact tissues. Advocates of innocuous IIF suggest that the amount of ice or size of the ice crystals and the location of the ice formed are more critical then the actual presence of intracellular ice. The cell plasma membrane is a critical site of injury during the formation of intracellular ice. It has been proposed that innocuous IIF occurs when plasma membrane integrity and cell viability are maintained by the propagation of intracellular ice across or between adjacent cells. Concerted efforts to understand the mechanisms and implications of innocuous IIF in tissue model systems have been undertaken. High-speed video cryomicroscopy has been a valuable tool to examine para-, intra- and intercellular ice formation. We have proposed that 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 has been shown to be an effective method for the cryopreservation of tissue models. These results provide researchers new avenues to explore and new techniques for the preservation of complex biological systems. The discordance of opinions on lethal and innocuous IIF is influenced by a lack of understanding of the differences between the low temperature response of cells and tissues. By understanding the mechanism by which intracellular ice forms, and the means by which it can remain innocuous, the potential exists for the development of novel approaches for the cryopreservation of tissues and organs. However, mainstream acceptance of the concept of innocuous intracellular ice formation needs to occur first. Detailed examination of intracellular ice formation in tissues and organs is therefore warranted to determine the nature of damage caused by the presence of ice, to examine limits to the amounts of tolerable ice, and to develop strategies for cryopreservation.