The history and principles of cryopreservation.

The ability of glycerol to protect cells from freezing injury was discovered accidentally. The subsequent development of cryopreservation techniques has had a huge impact in many fields, most notably in reproductive medicine. Freezing injury has been shown to have two components, direct damage from the ice crystals and secondary damage caused by the increase in concentration of solutes as progressively more ice is formed. Intracellular freezing is generally lethal but can be avoided by sufficiently slow cooling, and under usual conditions solute damage dominates. However, extracellular ice plays a major role in tissues. Cryoprotectants act primarily by reducing the amount of ice that is formed at any given subzero temperature. If sufficient cryoprotectant could be introduced, freezing would be avoided altogether and a glassy or vitreous state could be produced, but osmotic and toxic damage caused by the high concentrations of cryoprotectant that are required then become critical problems. The transport of cryoprotectants into and out of cells and tissues is sufficiently well understood to make optimization by calculation a practical possibility but direct experiment remains crucial to the development of other aspects of the cryopreservation process.