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.     

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% ($\text{w}\text{w}$) 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.     

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.     

Maximum in the stability of the amorphous state in the system water--glycerol--ethanol.

When the proportions of glycerol and ethanol vary for a given water concentration, the stability of the amorphous state of the whole solution in the system water-glycerol-ethanol passes through a maximum. This ternary system may then be much more interesting for cryoprotection than the system water-glycerol-DMSO. The phase transitions on rewarming at several rates after rapid or slow cooling, or after annealing, were observed by calorimetry and the various states between the transitions including the amorphous state were observed by X-ray diffraction. However, this system is much more complicated than the system water-glycerol-DMSO, due to the existence of two ethanol hydrates. Since, beyond a certain ethanol concentration, a first melting occurs before the devitrification, the stability of the amorphous state could no longer be defined by the critical warming rate. It was then defined by the amount of crystals formed on cooling, which was surprisingly reproducible in the present experiments for each cooling rate. The maximum in the stability occurs for rather low ethanol concentrations, which is of interest since ethanol is more toxic that glycerol. The concentration corresponding to the maximum depends on the cooling rate. It occurs at about 20% ($\text{w}\text{w}$) ethanol/(glycerol + ethanol) for the 50 and 55% ($\text{w}\text{w}$) water solutions. It is shifted to 40% ($\text{w}\text{w}$) ethanol/(glycerol + ethanol) for the 60% ($\text{w}\text{w}$) water solutions.     

Ternary systems with 1,2-propanediol—A new gain in the stability of the amorphous state in the system water-1,2-propanediol-1-propanol

Three ternary systems with water and 1,2-propanediol were investigated, where the third component is 1-propanol, ethanol, or glycerol. 1-Propanol and ethanol give hydrates in their aqueous solutions as well as in these ternary systems, while glycerol gives none. No gain in the stability of the amorphous state and glass-formation tendency is obtained, for the same water contents, when 1,2-propanediol is partially replaced by ethanol. The gain is negligible when it is partially replaced by glycerol. On the contrary, a large maximum in the stability of the amorphous state is obtained, with a critical warming rate dropping from 10⁸ to 10⁴ °C/min in the presence of 65% (w/w) water when 15% (w/w) of the 1,2-propanediol is replaced by 1-propanol. The decrease in the glass formation tendency due to this replacement and corresponding to a few hydrate crystallization is small. Not only the higher stability of the amorphous state, but also in some cases the replacement of ice crystallization by clathrate crystallization at lower temperatures could perhaps contribute to a better cryoprotection of cells for some cooling and warming rates. The similarities observed between the ternary systems investigated gives an idea of the general behaviour of these systems     

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

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.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions

Ice formation on warming is of comparable or greater importance to ice formation on cooling in determining survival of cryopreserved samples. Critical warming rates required for ice-free warming of vitrified aqueous solutions of glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol 200 and sucrose have been measured for warming rates of order 10–10⁴ K/s. Critical warming rates are typically one to three orders of magnitude larger than critical cooling rates. Warming rates vary strongly with cooling rates, perhaps due to the presence of small ice fractions in nominally vitrified samples. Critical warming and cooling rate data spanning orders of magnitude in rates provide rigorous tests of ice nucleation and growth models and their assumed input parameters. Current models with current best estimates for input parameters provide a reasonable account of critical warming rates for glycerol solutions at high concentrations/low rates, but overestimate both critical warming and cooling rates by orders of magnitude at lower concentrations and larger rates. In vitrification protocols, minimizing concentrations of potentially damaging cryoprotectants while minimizing ice formation will require ultrafast warming rates, as well as fast cooling rates to minimize the required warming rates.     

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.     

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.     

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.     

Thermal properties of ethylene glycol aqueous solutions

Preventing ice crystallization by transforming liquids into an amorphous state, vitrification can be considered as the most suitable technique allowing complex tissues, and organs cryopreservation. This process requires the use of rapid cooling rates in the presence of cryoprotective solutions highly concentrated in antifreeze compounds, such as polyalcohols. Many of them have already been intensively studied. Their glass forming tendency and the stability of their amorphous state would make vitrification a reality if their biological toxicity did not reduce their usable concentrations often below the concentrations necessary to vitrify organs under achievable thermal conditions. Fortunately, it has been shown that mixtures of cryoprotectants tend to reduce the global toxicity of cryoprotective solutions and various efficient combinations have been proposed containing ethanediol. This work reports on the thermal properties of aqueous solutions with 40, 43, 45, 48, and 50% (w/w) of this compound measured by differential scanning calorimetry. The glass forming tendency and the stability of the amorphous state are evaluated as a function of concentration. They are given by the critical cooling rates v(ccr)above which ice crystallization is avoided, and the critical warming rates v(cwr) necessary to prevent ice crystallization in the supercooled liquid state during rewarming. Those critical rates are calculated using the same semi-empirical model as previously. This work shows a strong decrease of averaged critical cooling and warming rates when ethanediol concentration increases, V(ccr) and V(cwr) = 1.08 x 10 (10) K/min for 40% (w/w) whereas V(ccr) = 11 and V(cwr) = 853 K/min for 50% (w/w). Those results are compared with the corresponding properties of other dialcohols obtained by the same method. Ethylene glycol efficiency is between those of 1,2-propanediol and 1,3-propanediol.     

Vitrification of bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro.

Two experiments were conducted to assess the viability of bovine blastocysts obtained by in vitro fertilization of oocytes matured in vitro (IVM-IVF) and cryopreserved by vitrification. In Expt 1, the optimal concentrations of glycerol and 1,2-propanediol in the basic medium (modified TCM199) for cooling and warming without formation of ice crystals were determined by plunging the solution into liquid nitrogen and then warming it in a water bath at 15 degrees C; when both glycerol and 1,2-propanediol were present in the solution (> 45% v/v), vitrification of the medium was observed. In Expt 2, IVM-IVF blastocysts were equilibrated to the mixture of glycerol and 1,2-propanediol (0% to 45%) at 15 degrees C in a stepwise manner as follows: (i) in one step, for 18 min to the final vitrification solution; (ii) in two steps, for 8 min in the first step and 10 min in the second step; (iii) in four steps, for 4 min in the first three steps and 6 min in the last step; (iv) in eight steps, for 2 min in each step, but 4 min in the last step; and (v) in 16 steps, for 1 min in each step, but 3 min in the last step. After removal of cryoprotectants, the blastocysts were cultured for 24 h in vitro. The survival rates for the embryos equilibrated in 1, 2, 4, 8 and 16 step(s) were 56, 89, 100, 100 and 100%, respectively. The blastocysts equilibrated in 1, 2, 4, 8 and 16 steps were vitrified by plunging the straws containing them into liquid N2, thawed and cultured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Freezing injury to erythrocytes. I. Freezing patterns and post-thaw hemolysis.

The extent of hemolysis of human red blood cells suspended in different concentrations of glycerol and frozen at various cooling rates was investigated on the basis of morphological observation in the frozen state. Hemolysis of the cells in the absence of glycerol showed a V-shaped curve in terms of cooling rates. There was 70% hemolysis at an optimal cooling rate of approximately 10³ °C/min and 100% hemolysis at all other rates tested. Morphologically, a lower than optimal cooling rate resulted in cellular shrinkage, while a higher than optimal rate resulted in the formation of intracellular ice.The cryoprotective effect of glycerol was dependent upon its concentration and on the cooling rate. Samples frozen at 10³ and 10⁴ °C/min showed freezing patterns which differed from cell to cell. The size of intraand extracellular ice particles became smaller, and there was less shrinkage or deformation of cells as the rate of cooling and concentration of glycerol were increased.There was some correlation between the morphology of frozen cells and the extent of post-thaw hemolysis, but the minimum size of intracellular ice crystals which might cause hemolysis could not be estimated. As a cryotechnique for electron microscopy, the addition of 30% glycerol and ultrarapid freezing at 10⁵ °C/min are minimum requirements for the inhibition of ice formation and the prevention of the corresponding artifacts in erythrocytes.     

The effect of cooling and warming rate on cortical cell function of glycerolized rabbit kidneys

Experiments previously reported (I. A. Jacobsen, D. E. Pegg, H. Starklint, J. Chemnitz, C. J. Hunt, P. Barfort, and M. P. Diaper, Cryobiology19, 668, 1982) suggested that rabbit kidneys permeated with 2 M glycerol are least damaged during freezing and thawing if they are cooled very slowly (1 °C/ hr). Using similar techniques of glycerolization, cooling, storage at ?80 °C, rewarming, and deglycerolization, active cell function in cortical tissue slices prepared from such kidneys has now been studied. Oxygen uptake, tissue $\text{K}{}^{\mathrm{+}}\text{Na}{}^{\mathrm{+}}$ ratio after incubation, and slice/medium PAH ratio after incubation were measured. Kidneys cooled at 3.1 °C/min and warmed at 4.2 °C/min gave poor results in the previous studies and the lowest levels of cell function in the present experiments. Kidneys cooled at 1 °C/hr exhibited degrees of slice function that were dependent on warming rate: warming at 1 °C/min was better than warming at either 1 °C/hr or c.20 °C/min. These results refine the previously drawn conclusions, (loc cit) and indicate optimal cooling and warming rates for rabbit kidneys containing 2 M glycerol, in the region of 1 °C/hr cooling and 1 °C/min warming. These rates are much lower than have hitherto been used by others for any system. Some implications of these findings are discussed.     

Cryopreservation of the promastigote form of Leishmania tropica var major at different cooling rates

Callow L. L. and Farrant J. 1973. Cryopreservation of the promastigote form of Leishmania tropica var major at different cooling rates. International Journal for Parasitology, 3: 77–88. An investigation of the optimal conditions for the preservation of Leishmania tropica var major by freezing was undertaken because it was important to obtain a high yield when the thawed organisms were cultured. As a prerequisite for comparing different conditions, assay methods were devised. One method was based on the reduced growth of promastigotes in diphasic medium that was found to follow inoculation of relatively few organisms. The other employed serial ten-fold dilutions of suspensions of organisms, and the inoculation of medium at each dilution stage. Viability was related to the time taken for flagellates to be found in the medium. A 1·0 m solution of glycerol in the flagellate suspension inhibited growth when diphasic medium was inoculated. This effect was removed by separating the organisms from the glycerol before inoculation, or by diluting the suspension approximately one hundred-fold. A similar inhibition was not observed for dimethylsulphoxide (DMSO). Glycerol (1·0 m), DMSO (1·5 m), polyvinylpyrrolidone (10% w/v) and sucrose (0·3 m) were not obviously detrimental to the organisms. Normal growth in diphasic medium resulted when these additives were removed after being in contact with organisms for 5 h in an ice bath. In freezing experiments, flagellates survived freezing and thawing while they were still in contact with their nutrient medium, and also after they had been separated, washed and resuspended in isotonic saline with 10 mm of glucose. The survival rate was much greater when either 1·5 m DMSO or 1·0 m glycerol was added. These additives were compared at one rate only, $\text{0·3°C}\text{min}$, and DMSO gave better protection. With 1·5 m DMSO, maximal survival was obtained at a cooling rate of $\text{1·9°C}\text{min}$. Relatively high rates of cooling, that is, over $\text{400°C}\text{min}$ were detrimental to the organisms.     

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

Sensitivity of human embryonic stem cells to different conditions during cryopreservation

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me₂SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding.     

Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

Influence of various cooling and warming temperatures on survival after thawing of cryopreserved Plasmodium berghei

Heavy concentrations of viable P. berghei in the natural milieu [20% ($\text{v}\text{v}$) parasitized red blood cells, or 20% ($\text{w}\text{v}$) homogenate of splenic tissue in which malarial cells sequestered wer suspended in a serum-free, protective medium. Various rates of cooling are designated as low (1.3 °C/min) and intermediate (4 °C/ min) on exposure in cold gas evolving from liquid nitrogen refrigerant to ?70 °C, and this followed by direct immersion in the low temperature refrigerant (?196 °C). Cooling designated high was accomplished by abrupt immersion of the sealed vials with the live malaria-bearing tissue in the liquid nitrogen refrigerant. Rates of warming and thawing were designated low (after slow rewarming of frozen tissue in air at 25.5 °C) and high (after rapid rewarming and thawing in a water bath at 40 °C). Strip chart recordings were made of the complete cooling and freezing wave patterns of the suspending medium to ?70 ° C. The functional survivals of the freeze-thaw P. berghei malaria were measured by a special infectivity titration method.None of the cooling and freezing treatments adversely influenced the parasite survivals. Our data showed the optimum cooling velocity that maximally protected this highly lethal P. berghei strain within the host erythrocyte matrix was 1.3 ° C/min to ?70 to ?196 ° C. The functional survivals of two RBC stabilates with P. berghei, after retrieval from 25 days storage in the liquid nitrogen refrigerant, excelled by more than 100-fold the infectivity titer found by viability assay in the pool of the 0-days nonfrozen infected RBC.The precise factors favoring the maximal survivals of the freeze-thaw P. berghei are unclear. Several factors, singly or in combination, may have played key roles in protecting the living P. berghei from the freeze-thaw damage. These factors are: The composition of the suspending medium fortified by additions of bicarbonate, glucose, lactalbumin hydrolysate and yeastolate; the presence of naturally occurring peptide-containing materials surrounding the parasites in the host red cell milieu; and the protective glycerol agent. Any of these constituents singly or combined possess potential for reducing freeze-thaw injury to the parasites to produce maximal survivals.