Intracellular ice formation

Figure 3. Intracellular freezing of 8-cell mouse embryos cooled at 20 °C/min in 2 M DMSO. The black "flashing" occurring in cells at -31 °C to -46 °C is characteristic of intracellular ice formation, and is caused by the scattering of light by many small highly branched ice crystals. (Modified from Rail et al., 1983.)...

Toner M, CravaUio EG, Karel M (1990) Thermodynamics and kinetics of intracellular ice formation during freezing of biological ceUs. J Appl Phys 67 1582-1593... 

The controlled-rate freezer is preprogrammed to cool the embryos from -7°C to -35°C. At the start temperature of -7°C the embryos must be seeded to induce ice nucleation, which helps prevent lethal intracellular ice formation. This is simply done by using forceps precooled in liquid nitrogen (contained in the small Dewar flask) to touch each straw briefly (1-2 sec) until a small ice nucleus can... 

CHANDRASEKARAN M and PITT RE (1992) On the use of nucleation theory to model intracellular ice formation. CryoLett, 13,261-272. 

The intracellular ice formation (IIF) has been thought to be the main source of viability loss for preserved biological systems. It is postulated that the ice crystals in the cytosol mechanically disrupt the... 

Frost-resistant (hardy) plants are less sensitive than others to damage by low temperatures that is caused by water loss and intracellular, ice-crystal formation. Production of such highly hydrophilic proteins as glycoproteins would constitute a potential mechanism, through the formation of hydrogen... 

Cryoinjury to the specimen is caused directly by extra- or intracellular ice crystal formation as well as by ice-induced solution effects during cryopreservation. Ice crystals seriously deform cell components. Another disadvantage of the formation of ice crystals near the specimen surface is slowing the cooling rate in areas below the surface because their thermal conductivity is about half that of solid water in a noncrystalline state. Furthermore, ice crystal formation is accompanied by the generation of latent heat, which also slows down the freezing rate. 

Much as liquid water is essential for life, frozen water, ice, is frequently lethal, especially if ice formation occurs within the cell. Upon formation of ice, loss of liquid water may impair or preclude the four basic water-related functions listed above. In particular, the structures and the activities of macromolecules and membranes may be severely damaged. In fact, the harmful effects of ice formation are due to a suite of physical and chemical effects. Physical damage from ice crystals that form within a cell can lead to rupture of membranes and the consequent dissipation of concentration gradients between the cell and external fluids or between membrane-bounded compartments within the cell. Ice formation in the extracellular fluids also can lead to damage to membranes as well as to lethal dehydration of the cell, as water moves down its concentration gradient from the intracellular space to the now depleted pool of liquid water in the extracellular space. Dehydration of the cell not only deprives it of water, but also leads to harmful and perhaps lethal increases in the concentrations of inorganic ions, which remain behind in the cell. Because the activities and structures of nucleic acids and proteins are affected by the concentrations of ions in their milieu, dehydration is expected to lead to perturbation of macromolecular structure and metabolic activity. It should come as no surprise, therefore, that with rare exceptions such as the fat body cells of certain cold-tolerant insects (Lee et al., 1993b Salt, 1962), ice formation within cells is lethal. 

The threats posed by ice formation in the intracellular and extracellular fluids have favored selection in diverse types of organisms for mechanisms that either prevent the formation of ice or minimize the potential for enlargement of any small ice crystals that do form in the internal fluids. These mechanisms are under intense study, both because of their inherent interest to evolutionary and ecological physiol-... 

A different picture emerges when formation of solid-state water in extracellular spaces is examined. Here, ice formation commonly occurs. Because extracellular fluids lack the complex membrane systems found within the cell, the potential for physical disruption of structures is much less than in cells. The tolerance of extracellular protein systems to increased solute concentration may also be greater than those of typical intracellular proteins, whose coordinated enzymatic functions generally are very sensitive to solute composition and concentration (chapter 6). 

Destruction of Cell Structure by Intracellular Ice Crystal Formation... 

Two possibly related phenomena have been found to be dependent on the flux of monovalent ions. The hypothetical common link is represented by a newly discovered family of intracellular proteases whose activity may be influenced by concentrations. Interleukin converting enzyme (ICE) is the best studied member of this family. Efflux of from monocytes leads to activation of ICE, so that the cells rapidly process and export IL-16 (Walev etal., 1995). An ICE-related protease is involved in regulating programmed cell death, which may be the reason why formation of K -permissive pores by alpha-toxin in human T-lymphoctes causes apoptosis (Jonas et a/., 1994). Both apoptosis and ICE-activation are inhibited when alpha-toxin treated cells are suspended in K" -rich medium. It is of interest that simultaneous flooding of cells with Ca , such as occurs when larger pores are formed in lymphocytes (e.g. at high alpha-toxin concentrations or with . coli hemolysin) counteracts the apoptosis-promoting effect of K -efflux (Jonas et a/., 1994). 

Thus, strong dehydration of bovine gametes (0.1 g/g) and subsequent partial hydration (0.3 g/g) result in an increase in the content of SBW unfrozen at low temperature (i.e., less dangerous for cells due to the formation of ice crystallites). Addition of weakly polar CDCI3 or strongly polar DMSO to partially hydrated cells allows controlling the structure of intracellular water, i.e., amounts of WBW and SBW and WAW and SAW. 

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