The major limiting obstacle to reversible cryopreservation of complex organs is cryoprotectant toxicity. Elimination of ice formation through vitrification requires high concentrations of cryoprotective agents. These high concentrations of cryoprotectants can be toxic to tissues. Over the years, major advances by the cryobiology research company 21st Centrury Medicine have been made to reduce the toxicity of vitrification agents, culminating in the least toxic vitrification agent to date, M22.
In 2004, Fahy et al. published a landmark paper that proposed a model to predict general cryoprotectant toxicity. Although the authors speculate about the mechanisms of cryoprotectant toxicity in the discussion section of the paper, the emphasis of their investigations is to formulate less toxic vitrification solutions. Whereas general cryoprotectant toxicity is proposed to reflect cryoprotectant-induced perturbation of intracellular water, the mechanisms underlying specific cryoprotectant toxicity involve the effects of individual cryoprotective agents on macromolecules (for example, metabolic conversion of glycerol to a toxic compound).
A number of viability measures are available to investigate the toxicity of cryoprotective agents. One such measure is the potassium/sodium ratio. In complex organs such as the brain, other viability measures are possible such as measuring electrical activity after vitrification and rewarming. These viability measures can be used to improve vitrification agents but they do not throw much light on the actual mechanisms of cryoprotectant toxicity. More “sophisticated” viability assays such as measurements of post-vitrification gene expression are available to help elucidating those mechanisms. Another technique that may hold promise for investigating cryoprotectant toxicity is cryoenzymology.
Cryoenzymology is the study of of enzymes at subzero temperatures in fluid solvents. The study of enzymes at low subzero temperatures overcomes two problems in studying enzyme reactions in steady state conditions: 1) the rapidity of the reactions and 2) the low concentrations of intermediates present. By starting enzyme-catalyzed transactions at low subzero temperatures the progressive transformation of intermediates into a subsequent one can be studied as the temperature is gradually increased. This method can produce detailed structural and kinetic information of substrate-enzyme reactions which are not available at room temperature.
Because cryoenzymology requires a fluid aqueous environment at low subzero temperatures, organic cosolvents are used to prevent ice formation. Because the organic solvents used in cryoenzymology serve a similar function as cryoprotectants in vitrification, it is not surprising that we often find the use of the same solvents such as DMSO and ethylene glycol. An ideal solvent for cryoenzymology should inhibit ice formation without adverse effects on the structure or kinetics of the molecules that need to be studied. Researchers in cryoenzymology have also found that the presence of high concentration of organic solvents decreases the temperature at which proteins denaturate. Similarly, in cryobiology, there is a need to expose biological tissues to low subzero temperatures without causing cryoprotectant-induced protein denaturation.
Although an ideal organic solvent for cryoenzymology is not necessarily an ideal cryoprotectant, observations of the interaction of organic solvents and proteins at subzero temperatures can throw light on phenomena such as solvent-induced versus temperature-induced protein denaturation, chilling injury, cold shock, and solvent-water-protein interactions. The field of cryoenzymology also had to address a lot of challenges encountered in cryobiological research such as selection of proper buffers for use with organic solvents at cryogenic temperatures and the effect of solvent on solution viscosity.
Cryoenzymology is also of interest to other areas in biology such as the study of life under extreme conditions. The study of extremophiles is flourishing because of its relevance to astrobiology, the study of life (or the potential for it) in the universe.
Review papers on cryoenzymology:
Fink AL: Cryoenzymology: the use of sub-zero temperatures and fluid solutions in the study of enzyme mechanisms (1976)
Fink AL, Geeves MA: Cryoenzymology: the study of enzyme catalysis at subzero temperatures (1979)
Douzou P: Cryoenzymology (1983)
Travers F, Barman T: Cryoenzymology: how to practice kinetic and structural studies (1995)