Cryostasis is the reversible hypothermic preservation of live biological objects.
Living tissues cooled below the freezing point of water are damaged by the dehydration of the cells as ice is formed between the cells.
The mechanism of freezing damage in living tissues has been elucidated by Renfret (1968) (Renfret A.P. Cryobiology: some fundamentals in surgical context. In: Cryosurgery. Rand R.W., Renfret A.P., von Leden H., Eds. Springfield, IL: Charles C. Thomas, 1968) and by Mazur (1984) (Am. J. Physiol., 247:125–147, 1984): ice formation begins in the intercellular spaces. The vapor pressure of the ice is lower than the vapor pressure of the solute water in the surrounding cells and as heat is removed at the freezing point of the solutions, the ice crystals grow between the cells, extracting water from them.
Ice formation in the intercellular space.
As the ice crystals grow, the volume of the cells shrinks, and the cells are crushed between the ice crystals. Additionally, as the cells shrink, the solutes inside the cells are concentrated in the remaining water, increasing the intracellular ionic strength and interfering with the organization of the proteins and other organized intercellular structures. Eventually, the solute concentration inside the cells reaches the eutectic and freezes. The final state of frozen tissues is pure ice in the former extracellular spaces, and inside the cell membranes a mixture of concentrated cellular components in ice and bound water. In general, this process is not reversible.
Clathrate hydrates are a class of solids in which gas molecules occupy "cages" made up of hydrogen-bonded water molecules. These "cages" are unstable when empty, collapsing into conventional ice crystal structure, but they are stabilised by the inclusion of the gas molecule within them. Most low molecular weight gases (including CH4, H2S, Ar, Kr, and Xe) will form a hydrate under some pressure-temperature conditions.
Clathrate formation of the water inside the cells, limiting the formation of ice outside the cells.
Live biological object can be saturated with the clathrate-forming gas(es) by diffusion or perfusion at the appropriate pressure and the clathrate-forming temperature. After saturation, the biological object is cooled, first below the clathrate-forming temperature, but above the freezing point of water, then to a temperature where the clatharate is metastable at ambient pressure, and the pressure allowed to go to ambient. The biological object is then gradually cooled down to some appropriate temperature at normal atmospheric pressure and stored an indefinite time. The method protects biological tissues by retention of water inside the cells by clathrate formation of the water with the introduced gases, limiting the formation of ice to outside the cells.