Glass-forming tendency in the system water-dimethyl sulfoxide.

The glass-forming tendency on cooling and the stability of the wholly amorphous state on warming have been previously reported for many cryoprotective solutions. However, unlike the other solutions, those of dimethyl sulfoxide (Me(2)SO) have not been studied on cooling. In this paper, the glass-forming tendency of Me(2)SO aqueous solutions has been measured for solutions containing 40, 43, 45, and 47.5% (w/w) Me(2)SO. At a concentration of 45% (w/w), the glass-forming tendency decreases in the following order: levo-2, 3-butanediol, 1,3-butanediol, 1,2-propanediol, 1,2,3-butanetriol, dimethyl sulfoxide, dimethylformamide, diethylformamide, 1, 4-butanediol, ethylene glycol, glycerol, 1,3-propanediol. New measurements have also been made on warming the Me(2)SO solutions.

Separation of racemic from meso-2,3-butanediol

2,3-Butanediol containing less than 3% of the meso form has been obtained from samples containing up to 50% of the meso form. The diacetate was obtained by esterification with acetic anhydride in the presence of traces of sulfuric acid as a catalyst and was then purified. When the diacetate was held at 4 degrees C, crystals of racemic 2,3-butanediol diacetate formed, and these were separated by filtration. The diacetate was then transformed back to 2, 3-butanediol by transesterification with methanol in the presence of sodium methylate as a catalyst. The resulting 2,3-butanediol contained less than 3% of the meso form. For an original batch of 2, 3-butanediol containing 50% dl and 50% meso, this method can isolate up to 70% of the racemate content. If the original 2,3-butanediol contains too much meso form, racemic 2,3-butanediol diacetate does not crystallize, but 2,3-butanediol containing up to 60% of the meso form can be enriched up to 70% racemate by distillation. Copyright 1999 Academic Press.

Glass-forming tendency and stability of aqueous solutions of diethylformamide and dimethylformamide

The glass-forming tendency on cooling and the stability of the wholly amorphous state on warming of aqueous solutions of diethylformamide and of dimethylformamide have been studied by calorimetry. With diethylformamide, only ice formation is observed except on warming at the lowest rate of 2.5 degreesC/min, where occasionally a hydrate forms also. The hydrate was observed up to 10 degreesC/min with 50% diethylformamide. With dimethylformamide hydrates form even at high warming rates. The last hydrate melts at -47.7 degreesC. The warming thermograms are much more complicated than for diethylformamide. For the glass-forming tendency on cooling, as well as for the stability of the wholly amorphous state on warming, these two compounds, at concentrations of 40, 45, or 50% (w/w) in water, are more efficient than glycerol and ethylene glycol, but less than 1,2-propanediol and levo-2,3-butanediol. On warming, they are comparable to DMSO. Pure diethylformamide could not be crystallized, whereas, conversely, pure dimethylformamide could not be vitrified. Curiously, the glass transition of aqueous solutions of diethylformamide increases and then decreases with the diethylformamide concentration in water, contrary to other cryoprotectants, for which it always increases or decreases. Diethyl- and dimethylformamide could be interesting cryoprotectants if they are not too toxic when added before cryopreservation, and in the case of dimethylformamide, if one can avoid damage due to its hydrates. Copyright 1998 Academic Press.

Critical cooling and warming rates to avoid ice crystallization in small pieces of mammalian organs permeated with cryoprotective agents.

Measurements were made by differential scanning calorimetry on small pieces of rabbit kidney permeated with 2, 3-butanediol containing mainly the levo- and dextro-isomers. The critical cooling rates necessary to vitrify the pieces of organ, and the corresponding critical warming rates which are required to avoid crystallization in the vitrified samples, were determined. The dynamic method used for these determinations is described. The glass-forming tendency and the stability of the amorphous state were both greater in the kidney tissue samples than in the bulk cryoprotective solution. This result is discussed in the context of the lowering of the freezing point of water in emulsions and the promotion of supercooling in hydrogels and porous materials. In corresponding experiments with rat hearts impregnated with 1,2-propanediol, only the critical warming rate was reduced.

Effect of Saccharides on the Glass-Forming Tendency and Stability of Solutions of 2,3-Butanediol, 1,2-Propanediol, or 1,3-Butanediol in Water, Phosphate-Buffered Saline, Euro-Collins Solution, or Saint Thomas Cardioplegic Solution

The effect of sugars or reduced saccharides trehalose, sucrose, sorbitol, or mannitol on the glass-forming tendency during cooling and the stability of the wholly amorphous state during warming has been studied with 2,3-butanediol, 1,2-propanediol, or 1,3-butanediol in three different carrier solutions. The 2,3-butanediol contained 96.7% (w/w) racemic mixture of the levo and dextro isomers and 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl). The carrier solutions were water, a phosphate-buffered saline, and two organ preservation solutions (Euro-Collins and Saint Thomas). The latter two were chosen because they are often used for kidney and heart preservation, respectively. The concentrations of 2,3-butanediol, 1,2-propanediol, and 1,3-butanediol varied respectively from 25 to 34, 30 to 35, and 30% (w/w). The concentrations of saccharides were 4 or 5% (w/w). In the absence of saccharides, for a given 2,3-butanediol concentration, the glass-forming tendency increased in the following order: water, Saint Thomas, the phosphate buffer, Euro-Collins. Addition of 4 or 5% (w/w) saccharide resulted in a large increase in the glass-forming ability of the solution during cooling and increased the stability of the glass during warming; but replacement of 4 or 5% diol by an equivalent weight (percentage) of a saccharide decreased, though to a lesser extent, these properties.

[Organ cryopreservation: current status of research carried out in Grenoble]. / Cryopréservation d'organes: etat actuel des recherches développées à Grenoble.

After discussing the problem of organ cryopreservation and reviewing the current data available on this subject, the Grenoble project is presented. Physical and biological studies have been combined with experimentation of autologous renal transplantation in rabbits to assess the functional value of the retransplanted organ after treatment and cooling. Renal resistances are measured during perfusion of the kidney with the cryoprotective solution. In order to verify the homogeneity of the cryoprotector concentration in the organ, on NMR spectral imaging test has been developed. A new rapid imaging method now allows real time monitoring of concentration variations during perfusion. In addition to concentration and homogeneity, analysis of local spectra also provides information about the local temperature de the kidney.

Reduction in toxicity for red blood cells in buffered solutions containing high concentrations of 2,3-butanediol by trehalose, sucrose, sorbitol, or mannitol.

Erythrocytes were stored at 4 degrees C in solutions of phosphate-buffered saline containing 2,3-butanediol and 4% (w/w) trehalose, sucrose, sorbitol, or mannitol. The 2,3-butanediol contained 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (2,3-butanediol 97% dl). The concentrations of 2,3-butanediol were 30 and 35% (w/w). A solution of 30% 2,3-butanediol showed relatively low toxicity. Hemolysis was only 2% after 5 h, but increased to 6% after 21 h and reached 60% after 46 h. Adding 4% (w/w) of one of the above compounds drastically decreased the toxicity. The two most efficient were the sugars trehalose and sucrose. With 30% 2,3-butanediol and 4% of any of the four compounds, hemolysis was about 0.6% after 2 days of storage. Furthermore, with trehalose or sucrose, hemolysis remained below 3% for 1 month. With sorbitol or mannitol, hemolysis slowly increased to 2% after 7 days and then increased rapidly. Even with 35% 2,3-butanediol, solutions containing trehalose or sucrose showed low toxicity. Hemolysis was also measured after redilution to buffered solution without 2,3-butanediol and without the additive, to mimic perfusion of organs with cryoprotectants and washing. Minima of hemolysis were observed after a few days of storage. The present solutions also have high glass-forming tendencies. They could be of great interest for organ vitrification.

Cryoprotection of red blood cells by a 2,3-butanediol containing mainly the levo and dextro isomers.

A 2,3-butanediol containing 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl) has been used for the cryoprotection of red blood cells. The erythrocytes were cooled to -196 degrees C at rates between 2 and 3500 degrees C/min, followed by slow or rapid warming. Up to 20% (w/w) of this polyalcohol, only the classical peak of survival is observed, as with up to 20% (w/w) 1,2-propanediol or 1,3-butanediol. Twenty percent 2,3-butanediol 97% dl can protect red blood cells very efficiently. The maximum survival, of 90%, as with 20% glycerol, is a little lower than with 20% 1,2-propanediol and higher than with 20% 1,3-butanediol. Fifteen percent 2,3-butanediol protects fewer red blood cells than 15% glycerol or 1,2-propanediol, with a maximum survival of about 80%. The best cryoprotection by 30% 2,3-butanediol 97% dl is obtained at the slowest cooling and warming rates, where survival approaches 90%. After a minimum, an increase of survival is observed at the fastest cooling rates, which would correspond to complete vitrification. These rates are lower than with 30%, 1,2-propanediol or 1,3-butanediol, in agreement with the higher glass-forming tendency of 2,3-butanediol 97% dl solutions. In agreement with the remarkable physical properties of its aqueous solutions, the present experiments also suggest that 2,3-butanediol containing mainly the levo and dextro isomers could be a very useful cryoprotectant for organ cryopreservation. However, it would perhaps be better to use it in combination with other cryoprotectants, since it is a little more toxic than glycerol or 1,2-propanediol at high concentrations.

Theoretical prediction of devitrification tendency: determination of critical warming rates without using finite expansions.

Previously, critical warming rates vcr above which ice did not have enough time to crystallize had been roughly evaluated for many wholly amorphous aqueous solutions. These evaluations were obtained by extrapolation of the linear variation of the devitrification temperature Td with log v, where v is the warming rate, observed experimentally between 2.5 and 80 degrees C/min. Theory also gives such a linear variation, but only using the first term of a finite expansion. The other terms can be neglected only for small variations of Td. These evaluations were sufficient for classification of the solutions, but large errors were made in vcr. A new and more accurate method of determination of the variation of Td with v is presented here. The general equation giving in our models the derivative of the quantity of ice formed versus temperature T is differentiated, instead of integrated using a finite expansion. This gives an explicit expression of v versus Td assuming that the ratio xd of the quantity of ice formed at Td to the total quantity of ice formed on warming is constant. Experimentally, xd is constant within a good approximation. Theoretical curves representing the variation of Td with v have been drawn for solutions of 35 or 45% (w/w) 1,2-propanediol in water. Td never reaches the temperature of the end of melting Tm, but as v tends toward infinity, Td tends toward an asymptotic value of 0.96Tm for 35% solute. For that solution, above about 10(3) degrees C/min, Td deviates appreciably from linearity with log v, but 1/Td remains almost linear with log v up to Td congruent to 0.95Tm. Therefore, systematic comparison of the theoretical variation of Td with v with a linear variation of 1/Td with log v has been done, varying the parameters of the equations within the entire experimental range. Similar conclusions can be given for all the solutions. Experimentally for Td = 0.95Tm, the quantity of ice crystallized is generally less than 0.1% of the solution, reaching 1% only once. Therefore, a new definition of the critical warming rate vcr has been used, corresponding to extrapolation of the linear variation of 1/T with log v up to Td = 0.95Tm. New values of vcr have been calculated for all the binary systems previously studied. The order of the solutions is almost the same, but the new values of vcr are significantly smaller than the former.

Cryoprotection of red blood cells by 1,3-butanediol and 2,3-butanediol.

1,3-Butanediol and 2,3-butanediol have been used in buffered solutions with 20, 30, or 35% (w/w) alcohol to cool erythrocytes to -196 degrees C at different cooling rates between 1 to 3500 degrees C/min, followed by slow or rapid rewarming. 1,3-butanediol shows the same shapes of red blood cell survival curves as 1,2-propanediol. Having nearly the same physical properties, they have comparable effects on cell survival. The classical maximum of survival for intermediate cooling rates and an increase for the highest cooling rates are observed. This increase seems to be correlated with the glass-forming tendency of the solution. After the fastest cooling rates, a warming rate of 5000 degrees C/min is sufficient to avoid cell damage, but a warming rate of 100-200 degrees C/min is not. Yet both of these rates would be insufficient to avoid the intracellular ice crystallization on warming. The damage on warming after fast cooling seems once again to be correlated with the transition from cubic to hexagonal ice. For all our results, 1,3-butanediol is like a "second" 1,2-propanediol and could be useful as a cryoprotectant for preservation by total vitrification. 2,3-Butanediol always gives extremely low survival rates, though it presents good physical properties. The crystallization of its hydrate seems to be lethal on cooling or on rewarming.

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.

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.

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.

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.