Cryoprotective Compounds

Cryoprotection

Cryoprotective additives, chemicals that reduce the injury of cells during freezing and thawing, are usually separated into two broad classes based on their ability to diffuse across cell membranes. Penetrating cryoprotectants are able to move across cell membranes whereas non-penetrating agents cannot.

Mazur's two-factor hypothesis of cryoinjury postulates two distinct mechanisms of cell injury during freezing and thawing; one occuring at cooling rates where the cell remains close to osmotic equilibrium and the other at rates in which there is supercooled water within the cell. In light of this dichotomy in the injury, it seems unlikely that cryoprotectants would provide equal protection from both forms of damage. The two forms of injury can be conveniently separated using McGann's graded freezing technique, thereby providing a convenient method for investigating the relative protection afforded by penetrating and non-penetrating compounds to cryoinjury caused by both slow cooling and rapid cooling.

Graded Freezing

The graded freezing technique cools samples slowly and then at several predetermined temperatures, two samples are removed from slow cooling and one is thawed in a 37C water bath and the other is plunged into liquid nitrogen (and then later thawed in a 37C water bath). The following diagram shows this schematically:

Fig. 8.1.1
At each of several temperatures, samples are removed from the slow cooling protocol. One sample is thawed directly in a 37C water bath and the other is plunged in liquid nitrogen.

As the cells are slow cooled to lower temperatures, they become more and more dehydrated, following the equilibrium water loss curve. This loss of cell water makes intracellular freezing less likely. Therefore, at the start of the protocol, the effects of intracellular freezing are demonstrated with very little injury from solution effects (slow cooling injury). As slow cooling progresses, the injury shifts toward being solely due to solution effects. The two types of injury can be clearly seen in the following survival curves for hamster fibroblasts in the absence of a cryoprotectant.

Fig. 8.1.2
Survival of hamster fibroblasts as a function of the temperature of termination of slow cooling. (McGann 79)

The upper curve shows the recovery of cells that were warmed in the 37C water bath after being removed from the slow cooling apparatus. The recovery starts high since the cells have only been exposed to the freeze-concentrated solution for a short period of time (the first point is held for 5 min at -5C after ice is seeded). As the temperature drops, the cells are exposed to ever more concentrated solutions and their total time of exposure to the freeze-concentrated solution also increases. Concomitant with that is the drop in recovery that falls to near zero by -50C. The lower curve shows the recovery for the cells that were plunged in liquid nitrogen once they had been slow-cooled to the prescribed temperature. This group starts off with near zero recovery because the cells have lost very little water and are susceptible to intracellular freezing when they are plunged in liquid nitrogen. As the temperature is lowered, the cells become progressively dehydrated and therefore more able to be rapidly frozen without the lethal injury that accompanies intracellular freezing. This effect is countered by the accumulated slow-cooling injury, however, by the time the temperature at which the cells are removed gets to -20C. Since the plunge group must necessarily be exposed to the identical slow- cooling injury as the warmed group, the lower curve cannot cross the upper curve. Below -20C, the damage in both groups is due to the solution effects injury that accumulated during slow cooling.

Penetrating Cryoprotectants

By adding a penetrating cryoprotectant such as dimethyl sulfoxide (DMSO), we can observe the change in the two curves to see the relative differences between injury due to slow cooling and rapid cooling in the presence of the cryoprotectant.

Fig. 8.1.3
Effect of DMSO concentration on the survival of cells cooled at 1C/min to various temperatures before rapid warming or rapid cooling to -196C followed by rapid warming. (McGann 79)

Figure 8.1.3(a) shows the effect of adding 1% DMSO to the cell suspension. The upper curve now starts at 100% recovery and drops monotonically to about 50% recovery at -60C. The penetrating cryoprotectant has clearly mitigated the slow cooling injury although there is still a significant loss of cells when warming from low temperatures. The lower curve starts off at zero and climbs as in 8.1.2 but the recovery is higher.

Figure 8.1.3(b) shows the effect of adding 5% DMSO. The recovery in the upper curve remains above 85% throughout the slow cooling protocol, indicating that 5% DMSO has almost completely eliminated this type of injury. The lower curve shows that cell water loss is essentially complete by -30C and so no more intracellular ice forms below that temperature.

Non-Penetrating Cryoprotectants

Fig. 8.1.4
Survival of cells in hydroxyethyl starch cooled at 1C/min to various temperatures before rapid warming or rapid cooling to -196C followed by rapid warming. (McGann 79)

Hydroxyethyl starch is a polysaccharide that is commonly used as a non- penetrating cryoprotectant. It is a large molecule that can only be taken up by cells through endocytosis. The upper curve shows that it has no effect on slow cooling injury; this curve being essentially identical to the upper curve in 8.1.2. Comparing the lower curve with 8.1.2, however, shows a significant effect on rapid cooling injury. The recovery at -20C is almost completely due to slow cooling injury even though the sample was plunged into liquid nitrogen.

The cryoprotectant glycerol provides a unique window on the mechanism of action of cryoprotectants since it is a penetrating agent if added at physiological temperatures but is essentially impermeant if added at 0C (the permeability is so low at that temperature that almost no glycerol will cross the membrane over the time course of these experiments).

Fig. 8.1.5
Influence of the permeation of glycerol into cells on survival following cooling at 1C/min to various temperatures before rapid warming or rapid cooling to -196C followed by rapid warming. (McGann 79)

Figure 8.1.5(a), in which glycerol was allowed to penetrate is similar to 8.1.3(b) where 5% DMSO was used. Glycerol on both sides of the membrane dramatically reduces slow cooling injury. Figure 8.1.5(b), though, shows that glycerol on only the outside of the membrane provides almost no protection against slow cooling injury or rapid cooling injury. The lower curve shows slightly more recovery at -20C than in the case with no cryoprotectant although it is not nearly as protective as HES.

Mechanisms of Cryoprotection - Penetrating Agents

Lovelock proposed that the mechanism of action of cryoprotectants was due to their colligative properties (that is to say the collective properties that a solution has when these compounds are present--as opposed to chemical properties). In particular, it was the reduction in salt concentration at a given temperature that allowed cells to suffer less injury at that temperature. In the case that Lovelock was investigating, there was a critical salt concentration at which lethal injury began to accumulate. By shifting the temperature at which cells saw this critical salt concentration lower, the rate of any biochemical reactions caused by the high salt concentration was reduced. This in turn led to a reduction in the extent of irreversible injury.

In this view, an effective penetrating cryoprotectant should provide colligative properties in which the salt is buffered down to low temperatures. It should also be freely permeable across the cell membrane so that it can buffer the intracellular salt as well. Such a prediction can be readily tested by comparing similar molecules that have very different phase diagrams. If we look at the two molecules dimethyl sulfoxide and dimethyl sulfone, they have a similar structure and both are able to penetrate cell membranes.


Dimethyl Sulfoxide (DMSO) - Dimethyl Sulfone (DMSO2)

The phase diagrams for these two molecules are very different, however.

Fig. 8.1.6
Phase diagram for DMSO and DMSO2 in 150 mM saline solution.

DMSO2 has a eutectic just below -2C and therefore precipitates out of solution below that point rather than depressing the freezing point colligatively. This should make it a poor choice for a cryoprotectant.

The cryoprotective action of these two compounds has been compared using a graded freezing protocol on a mouse fibroblast cell line.

Fig. 8.1.7
Recovery of Emt6 mouse fibroblasts from graded freezing to -40C.

Fig. 8.1.8
Recovery of mouse fibroblasts from graded freezing in 5% dimethyl sulfoxide.

Fig. 8.1.9
Recovery of mouse fibroblasts from graded freezing in 5% dimethyl sulfone.

The results using dimethyl sulfone are even worse than no cryoprotectant whatever, whereas the results of using DMSO show the characteristic cryoprotective action of a penetrating cryoprotectant. Thus the mechanism of cryoprotective action of penetrating cryoprotectants can be fully explained by their colligative properties.

Some other common penetrating cryoprotectants:


. . . . . .Propylene Glycol . . . . . . . . . . . . .Ethylene Glycol . . . . . . . . . . . . . Glycerol

Mechanisms of Cryoprotection - Non-Penetrating Agents

Non-penetration cryoprotectants are thought to act by dehydrating the cell at high sub-freezing temperatures, thereby allowing them to be rapidly cooled before the solution effects injury of slow cooling can lead to extensive damage. These compounds are generally polymers that form extensive hydrogen bonds with water, reducing the water activity to a much greater extent than would be predicted by their molar concentration (they do not obey Raoult's law).

Some common non-penetrating cryoprotectants:


Hydroxy-ethyl-starch (HES)


Polyvinyl Pyrrolidone (PVP)


Polyethylene Oxide (PEO)


References

McGann LE. Differing Actions of Penetrating and Nonpenetrating Cryoprotective Agents. Cryobiology 15: 382-390. 1978.

McGann LE. Optimal Temperature Ranges for Control of Cooling Rate. Cryobiology 16: 211-216. 1979.

McGann LE and Walterson M. 1984.


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Document last updated Nov. 24, 1998.
Copyright © 1998, Ken Muldrew.