P. Boutron

Stability of the amorphous state in the system water—1,2-Propanediol

Abstract For the same water contents, the stability of the wholly amorphous state of the aqueous solutions of 1,2-propanediol is much greater than that for all the solutions previously studied by us with glycerol, dimethylsulfoxide, ethanol, and ethylene glycol. To the degree that cyroprotection is related to that stability, 1,2-propanediol should be a better cryoprotectant than all these other compounds. The aqueous solutions of 1,2-propanediol have a simple behavior. No hydrate cyrstallizes on cooling, and for intermediate concentrations, on warming, after fast cooling, only ice crystallizes from the wholly amorphous state—first cubic, then hexagonal. The great stability of the amorphous state is shown by the critical warming rates above which no crystallization occurs, as well as by the difficulty in crystallizing on cooling.

Comparison with the theory of the kinetics and extent of ice crystallization and of the glass-forming tendency in aqueous cryoprotective solutions

The glass-forming tendency and stability of the wholly amorphous state of various cryoprotective solutions has been studied in recent years (5-10, 20). A lot of experimental data including heats of ice crystallization at various cooling rates and devitrification temperatures have been given. In this article these data have been compared with analytical expressions using a semiempirical model. The theoretical variation of the total quantity of ice crystallized with the cooling rate fits very well with the experimental data, adjusting only one parameter. Using the same model, theoretical differential scanning calorimeter (DSC) crystallization peaks have been obtained for cooling or rewarming. The general shape, height, and width of the theoretical peaks are very similar to those of the experimental peaks. The differences are comparable to the random variations of the experimental peaks from one experiment to another. The analytical expressions obtained here could be used to study the relationship between the kinetics of ice crystallization and cell damage when ice crystallizes incompletely inside or outside the cells. These expressions have been applied to ice crystallization for applications in cryobiology. But they could also probably be used in other fields of research such as crystallization from silicates or other mineral or organic glasses.

Stability of the amorphous state in the system water-glycerol-dimethylsulfoxide

In aqueous solutions containing both glycerol and DMSO, the various states during rewarming after quenching have been identified by X-ray diffraction. The amorphous state of the whole solution has been observed at very low temperatures. The eutectic was seen by X rays after rewarming only in the solutions containing mainly DMSO. In the other solutions only pure ice has been seen. It crystallizes directly in the hexagonal system, if enough DMSO is present. Otherwise, a mixture of cubic and hexagonal ice appears first. The temperature of the end of fusion and the devitrification temperature were measured with a scanning differential calorimeter for a wide range of warming rates. From these measurements was deduced the stability of the amorphous state, defined by the critical heating rate above which no crystallization occurs. That stability presents no maximum, but increases from glycerol to DMSO for a given water concentration in agreement with the fact that Ashwood-Smith considers DMSO a better cryoprotector than glycerol. But a small amount of glycerol in a solution of DMSO greatly enhances the difficulty of crystallization of the eutectic, without decreasing the stability of the amorphous state of the whole solution by much. Then those containing about 10% (ww) glycerol/(glycerol + DMSO) are perhaps better cryoprotectants than those with only DMSO, at least for low cooling or warming rates where the eutectic may have enough time to crystallize, eventually with deleterious effects, outside or inside the cells.

Stability of the amorphous state in the system water-glycerol-ethylene glycol

Abstract The behavior of the ternary solutions, water-glycerol-ethylene glycol, on warming after quenching is simple. No hydrate crystallizes, contrary to the system water-glycerol-ethanol; on warming after quenching only the glass transition, the devitrification and fusion peaks appear. The stability of the amorphous state was defined by the critical warming rate above which no crystallization occurs. For a given water content, that stability presents no maximum, but increases from glycerol to ethylene glycol.

Glass-forming tendency and stability of the amorphous state in the aqueous solutions of linear polyalcohols with four carbons: I. Binary systems water-polyalcohol

All the aqueous solutions of linear saturated polyalcohols with four carbons have been investigated at low temperature. Only ice has been observed in the solutions of 1,3-butanediol and 1,2,3- and 1,2,4-butanetriol. For same solute concentration, the glass-forming tendency on cooling is highest with 2,3-butanediol, where it is comparable to that with 1,2-propanediol, the best solute reported to date. However, the quantity of ice and hydrate crystallized is particularly high on slow cooling or on subsequent rewarming. The highest stability of the amorphous state is observed on rewarming the 1,2-butanediol and 1,3-butanediol solutions. With respect to this property, these compounds come just after 1,2-propanediol and before all the other compounds studied so far. They are followed by dimethylsulfoxide and 1,2,3-butanetriol. The glass-forming tendency of the 1,3-butanediol solutions is also very high; it is third only to that of 1,2-propanediol and 2,3-butanediol. The glass-forming tendency is a little smaller with 1,2-butanediol, but it is cubic instead of ordinary hexagonal ice which crystallizes on cooling rapidly with 35% 1,2-butanediol. Cubic ice is thought to be innocuous. A gigantic glass transition is observed with 45% of this strange solute. 1,4-Butanediol, 45% also favors cubic ice greatly. Therefore, 1,2- and 1,3-butanediol with comparable physical properties are perhaps as interesting as 1,2-propanediol for cryopreservation of cells or organs by complete vitrification. Together with 1,2-propanediol, 1,2- and 1,3-butanetriol, 1,2,3-butanetriol, and perhaps 2,3-butanediol provide an interesting battery of solutions for cryopreservation by vitrification.

Levo- and dextro-2,3-butanediol and their racemic mixture : very efficient solutes for vitrification

Binary systems with water and levo- or dextro-2,3-butanediol have a much larger glass-forming tendency on cooling and stability of the wholly amorphous state on warming, for same solute contents, than any previously studied binary and ternary systems, including all those containing 1,2-propanediol. For the first time, complete vitrification could be obtained with only 30% (w/w) solute. With 35% (w/w) of these isomers, a cooling rate of 20 °C/min is sufficient to obtain complete vitrification, compared with 300 °C/min with 35% 1,2-propanediol in water. The warming rate necessary to avoid ice crystallization from the wholly amorphous solution is about 104 °C/min, compared with about 108 °C/min with 35% 1,2-propanediol. The aqueous solutions of the racemic mixtures of these two isomers have almost the same properties as those of separate isomers and are as interesting. These solutes could perhaps allow the solution of the problem of organ cryopreservation by vitrification.

More accurate determination of the quantity of ice crystallized at low cooling rates in the glycerol and 1,2-propanediol aqueous solutions: comparison with equilibrium

It is generally assumed that when cells are cooled at rates close to those corresponding to the maximum of survival, once supercooling has ceased, above the eutectic melting temperature the extracellular ice is in equilibrium with the residual solution. This did not seem evident to us due to the difficulty of ice crystallization in cryoprotective solutions. The maximum quantities of ice crystallized in glycerol and 1,2-propanediol solutions have been calculated from the area of the solidification and fusion peaks obtained with a Perkin-Elmer DSC-2 differential scanning calorimeter. The accuracy has been improved by several corrections: better defined baseline, thermal variation of the heat of fusion of the ice, heat of solution of the water from its melting with the residual solution. More ice crystallizes in the glycerol than in the 1,2-propanediol solutions, of which the amorphous residue contains about 40 to 55% 1,2-propanediol. The equilibrium values are unknown in the presence of 1,2-propanediol. With glycerol, in our experiments, the maximum is first lower than the equilibrium but approaches it as the concentration increases. It is not completely determined by the colligative properties of the solutes.

Structural model for amorphous solid water

We construct two continuous random network models for amorphous solid water and compute their x‐ray and neutron diffraction properties. The results for one type of network accord well with experiment. This implies that there are important relatively long‐range, as well as short‐range, positional correlations in the solid amorphous phase of water.

Comparison of the cryoprotection of red blood cells by 1,2-propanediol and glycerol

Red blood cells are cooled in buffered solutions containing 10, 15, 20, 30, or 35% (w/w) 1,2-propanediol or glycerol. Cell survival is measured after cooling to -196 degrees C at rates between 1 and 3500 degrees C/min, followed by rewarming rapidly, except in a few cases. At low cooling rates, where the injuries are due to solution effects, for the same (w/w) concentrations of 15 or 20% (w/w), 1,2-propanediol protects erythrocytes better than glycerol. Differences are still observed when the two cryoprotectants are compared on a mole-fraction basis. At high cooling rates the survival passes through a minimum and then increases again. For the same concentrations, the minimum occurs at much lower cooling rates with 1,2-propanediol than with glycerol, in agreement with the better glass-forming tendency of 1,2-propanediol solutions. These cooling rates almost coincide with those at which the quantity of ice crystallized begins to decrease in the corresponding solutions. Thus, survival seems to be closely related to the glass-forming tendency at the survival minimum, and at higher cooling rates. After the fastest cooling rates, the warming rates necessary to avoid damage on warming are much smaller than those necessary to avoid devitrification. Therefore, in the present experiments the survivals are not related to the stability of the wholly amorphous state. However, injury follows the presumed transition from cubic to hexagonal ice, in erythrocytes as well as in other kinds of cells.

The influence of hydroxyethyl starch on ice formation in aqueous solutions

Differential scanning calorimetry, and, in some supplementary experiments, X-ray diffractometry and cryomicroscopy, were applied to study the influence of concentration (< 70 wt%) and cooling/warming rates (< 320 K/min) on ice formation in aqueous solutions of HES. The calorimetric measurements of the quantity of crystallizing water indicated that a mass fraction ϑ = 0.522 (i.e., grams water per gram HES) remained unfrozen. These results are in good agreement with our earlier extrapolations from ternary phase diagram data and tend to support the proposed cryoprotective mechanism. The value of ϑ determined during warming was essentially independent of composition up to the corresponding saturation concentration. It was observed that solutions containing 60 wt% HES or more remained wholly amorphous during cooling even at rates as low as 2.5 K/min (down to 120 K). Such glassy solutions are subject to devitrification at temperatures Td which depend on the warming rate. The concentrations close to 55 wt% HES mark a transitional range exhibiting two crystallization peaks, probably due to different mechanisms of nucleation, the portion of ice formed during cooling being related to the imposed cooling rate. All samples showed a recrystallization transition at 257.5 K which was also observed cryomicroscopically. Glass transitions, however, could not be detected by the methods applied in this study. The X-ray diffraction patterns contained the structure of only one solid phase, namely hexagonal ice. A comparison of various modifications of HES, PEG, and PVP involving bound water and melting temperature did not reveal marked differences. Minimum initial HES concentrations preventing lethal salt enrichment were computed for both binary and ternary mass fractions of NaCl as biologically relevant parameters, yielding 24.1 and 10.8 wt% HES, respectively.