The red blood cell as a model for cryoprotectant toxicity

Various approaches are available to investigate cryoprotectant toxicity, ranging from theoretical work in organic chemistry to  cryopreservation of complete animals. Because resuscitation of complex organisms after cryopreservation is not feasible at the moment, such investigations need to be confined to viability assays of individual cells and tissues or ultrastructural  studies.

One simple model that allows for “high throughput” investigations of cryoprotectant toxicity are red blood cells (erythrocytes). Although the toxic effects of various cryoprotective agents may differ between red blood cells, other cells, and organized tissues, positive results in a red blood cell model can be considered the first experimental hurdle that needs to be cleared before the agent is considered for testing in other models.  Because red blood cells are widely available for research, this model eliminates the need for animal experiments for initial studies. It also allows researchers to investigate human cells. Other advantages include the reduced complexity of the model  (packed red blood cells can be obtained as an off-the-shelf product) and lower costs.

Red bloods cells can be subjected to a number of different tests after exposing them to  a cryoprotective agent. The most basic test is gross observation of the red blood cells in a cryoprotectant solution. When high concentrations of a cryoprotectant are used (such as in vitrification), a stepwise approach is necessary to avoid  osmotic  injury. If a cryoprotectant solution is extremely toxic rapid hemolysis will follow, which often can be observed by a noticeable change of the color of the solution,  red cell debris sinking to the bottom of the test tube, or negligible difference between the pellet (if there is one at all) and the supernatant after centrifugation. But if the researcher is interested in agents that are not extremely toxic, or wants to compare variants of  similar agents with each other, quantitative methods and detailed observations are required using respectively spectrophotometry and light microscopy.

In 1996, Bakaltcheva et al. used the red blood cell model for an elegant and thoughtful study  of cryoprotective toxicity. The authors did not only use spectrophotometry to measure hemolysis  but also used microscopy to study the morphology of the red blood cell after exposure to various agents at different temperatures. The results of these different measurements were in turn correlated with each other in order to determine if there are general properties  affecting cryoprotectant toxicity. The authors propose that reduced toxicity can be achieved by keeping the dialectric constant of the medium and membrane close to that of an aqueous solution without solutes.  These findings can also explain why cryoprotective mixtures  of various agents (such as DMSO and formamide) can reduce toxicity.  A general rule of thumb for formulating vitrification agents with reduced toxicity seems to be to maintain most properties of water but eliminating the posibility of ice formation. It should not be a surprise that such an approach has guided the choice of solvents in areas such as cryoenzymology.

Cryoenzymology and cryoprotectant toxicity

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)