15. Control of ice formation in biological samples

Abstract

Lowering the temperature of a biological sample reduces the pace of biochemical metabolism and ultimately stops it altogether. This process, in combination with rewarming the sample with minimal tissue damage, is the essence of cryopreservation. A key obstacle to successful cryopreservation is that, as the biological material is cooled, liquid water within the sample may reorganize into a more stable, crystalline form, namely ice. Such crystallization can be devastating for several reasons: it disrupts the structure of membranes, reduces the water content of solutions, and induces mechanical and possibly electrical stresses. Below I list several factors that are relevant to ice formation during cryopreservation, and outline methods and ideas on how to control it. Under the conditions relevant to cryobiology, ice has an open structure with hexagonal symmetry (ice Ih) or, under some conditions, cubic symmetry (ice Ic). As water freezes its volume increases, which can create high pressure in tissues. In addition, gases and solutes are excluded from the ice matrix during freezing, leading to gas bubble formation and to high solute concentrations in the surrounding solution and hence to osmotic stress on cells. The surface energy of an ice crystal plays an important role in its stability. The melting point shifts to lower temperature as the crystal surface curvature increases. This melting point shift drives recrystallization of ice in which small crystals melt while bigger crystals grow at the same temperature, a process that promotes increasingly large ice crystals that are harmful to cells. Because crystals smaller than a certain size cannot grow at a given temperature, water can be supercooled to below its equilibrium melting temperature without nucleation of ice crystals. The equilibrium melting temperature is also influenced by solutes in the water that reduce the water chemical potential and thus lower the melting temperature of the ice. Moreover, because ice crystal growth depends of the diffusion of water molecules to the crystal surface, another important factor is the viscosity of water, which increases with decreasing temperature and increasing solute concentration. Several methods are used, or have been suggested, to control ice growth. First, solutes reduce the melting point and increase the viscosity of a solution, thus reducing the nucleation rate. At high enough concentrations, solutes can lead to vitrification where the solution solidifies into an amorphous solid without crystallization. Alternatively, controlled nucleation—in which nucleation is selectively induced by ice nucleation proteins, electrofreezing, sonication in addition to local cooling—can be implemented to maintain a moderate supercooling while avoiding internal nucleation of ice in cells. In addition, unidirectional solidification can control the build-up of pressure in samples. High pressure methods can lower the melting point, and reduce nucleation rates. Rapid warming is used to avoid ice growth during devitrification and recrystallization. In addition, ice-binding proteins and other ice-active materials can influence ice growth by directly interacting with ice. In this way they inhibit the recrystallization and growth of ice, and influence ice nucleation. A hurdle approach, in which a combination of methods to control ice growth is utilized, will probably prove to be the optimal method for the fine control of ice in cryopreservation.