Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultrastructure of the samples was examined by Cryo Scanning Electron Microscopy (CryoSEM) and freeze substitution, to determine whether intracellular ice formed and to examine alternative mechanisms of cell injury during rapid cooling. No intracellular ice formation was detected at any cooling rate. Differential scanning Calorimetry (DSC) was employed to examine the amount of ice formed at different rate of cooling. It is concluded that cell damage to horse spermatozoa, at cooling rates of up to 3,000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.     

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.     

Effect of warming rate on mouse embryos frozen and thawed in glycerol

Mouse embryos (8-cell) fully equilibrated in 1.5 M-glycerol were cooled slowly (0.5 degrees C/min) to temperatures between - 7.5 and - 80 degrees C before rapid cooling and storage in liquid nitrogen (-196 degrees C). Some embryos survived rapid warming (approximately 500 degrees C/min) irrespective of the temperature at which slow cooling was terminated. However, the highest levels of survival of rapidly warmed embryos were observed when slow cooling was terminated between -25 and -80 degrees C (74-86%). In contrast, high survival (75-86%) was obtained after slow warming (approximately 2 degrees C/min) only when slow cooling was continued to -55 degrees C or below before transfer into liquid N2. Injury to embryos cooled slowly to -30 degrees C and then rapidly to -196 degrees C occurred only when slow warming (approximately 2 degrees C/min) was continued to -60 degrees C or above. Parallel cryomicroscopical observations indicated that embryos became dehydrated during slow cooling to -30 degrees C and did not freeze intracellularly during subsequent rapid cooling (approximately 250 degrees C/min) to -150 degrees C. During slow warming (2 degrees C/min), however, intracellular ice appeared at a temperature between -70 and -65 degrees C and melted when warming was continued to -30 degrees C. Intracellular freezing was not observed during rapid warming (250 degrees C/min) or during slow warming when slow cooling had been continued to -65 degrees C. These results indicate that glycerol provides superior or equal protection when compared to dimethyl sulphoxide against the deleterious effects of freezing and thawing.     

Changes in cell size during the cooling, warming and post-thawing periods of the freeze-thaw cycle.

Chinese Hamster Ovary (CHO) cells were cooled at 1 and 200 °C/min and subsequently thawed, while being studied with a cryomicroscope. Post-thaw size changes were measured with a Quantimet 720 Image Analysing Computer. It was found that the behavior of individuals in a population varied and depended on cooling rate. Cooling at 1 °C/min resulted in cells showing no intracellular ice, whereas cooling at 200 °C/min caused intracellular ice formation in some cells but not in others. In addition, at the slow rate, during cooling, the cells shrank significantly but swelled on thawing to become larger than non-frozen controls. Following swelling, as their temperature rose, the cells shrank to the size of non-frozen controls. At the fast rate, cells showed variation in their amount of intracellular ice and in their degree of shrinkage. Cells containing most ice shrank least. On warming, cells with intracellular ice began to swell at a lower temperature than did those cells without intracellular ice, while after thawing they swelled to a greater extent partly due to widespread blebbing. Corresponding recovery indices were measured, and correlation of these with the above effects suggests that: (i) cells completely filled with intracellular ice are non-viable; (ii) cells partially filled with intracellular ice respond to, or can be rescued by, first warming; (iii) cells without intracellular ice are viable; (iv) viable cells are those which regain their original size following thawing; (v) non-viable cells are those which remain swollen above their original size.     

Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity

The objective of this study was to evaluate the effects of thawing and processing temperatures on post-thaw sperm viability, occurrence of osmotic shock and sperm membrane functional status. The occurrence of osmotic shock, characterized by increased spermatozoa with coiled tails, eventually results in reduced sperm viability and sperm membrane integrity. The effects of different thawing temperatures were assessed by thawing frozen specimens at 37, 21 or 5 degrees C for 1 to 2-min, followed by processing at these temperatures. A subset of frozen specimens were thawed at 37 degrees C for 10 to 15-sec and transferred to a water bath at 21 or 5 degrees C for 1 to 2-min to complete thawing, followed by processing at these temperatures. Sperm processing (washing) consisted of dilution, centrifugation and resuspension to remove glycerol from the medium and to gradually return the spermatozoa to isotonic conditions. Post-thawed specimens (0.5 mL) were slowly diluted 1:1 (v/v) at a rate of 0.1 mL/min, centrifuged, and resuspended to 0.5 mL (37 degrees C). Diluted specimens were equilibrated for 1 to 2-min after dilution and for 5-min after resuspension. The specimens were then incubated for 2-h (37 degrees C) and assessed at 60-min intervals for the percentage of motility, for progressive motility (Grades 0 to 4), for the percentage of spermatozoa with coiled tails, and for the percentage of swollen spermatozoa. The percentage of swollen spermatozoa (measurement of sperm membrane integrity) was assessed by exposing spermatozoa to a modified hypoosmotic swelling (HOS) test. The results obtained seem to indicate that physiological thawing and processing temperatures (37 degrees C) are required to maintain sperm motility. However, thawing and processing at lower temperatures (< 37 degrees C) seems to prevent the occurrence of osmotic shock and to maintain sperm membrane functional integrity. In this study, thawing at 37 degrees C (10 to 15-sec) and transfer to a water bath at 21 degrees C (1-min) to complete thawing, followed by processing at 21 degrees C, yielded better results in terms of increased sperm viability, reduced occurrence of osmotic shock and higher reactivity to the HOS test.     

Cryopreservation of Plasmodium chabaudi. II. Cooling and warming rates

Various cooling and warming rates were investigated to determine the optimum conditions for cryopreserving the intraerythrocytic stages of Plasmodium chabaudi. Infected blood, equilibrated in 10% v/v glycerol at 37 degrees C or in 15% v/v Me2SO at 0 degree C for 10 min, was cryopreserved using cooling rates between 1 and 5100 degrees C min-1. After overnight storage in liquid nitrogen the samples were warmed at 12,000 degrees C min-1. Warming rates between 1 and 12,000 degrees C min-1 were investigated using samples previously cooled at 3600 degrees C min-1. After thawing, the glycerol and Me2SO were removed by dilution in 15% v/v glucose-supplemented phosphate-buffered saline. Survival was assayed by inoculation of groups of five mice each with 10(6) infected cells and the time taken to reach a level of 2% parasitemia estimated. The optimum cooling rate was 3600 degrees C min-1 for parasites frozen using either 10% glycerol or 15% Me2SO; the pre-2% patent periods were 0.90 and 1.01 days above control values (representing survival levels of 21 and 17.5%, respectively). The optimum warming rate was 12,000 degrees C min-1; the pre-2% patent periods were 1.01 and 1.32 days above control values, respectively (18 and 10% survival), for glycerol and Me2SO. With ethanediol (5% v/v) and sucrose (15% w/v) as cryoprotectants the optimum warming rates were also 12,000 degrees C min-1 while the optimum cooling rates were 330 and 3600 degrees C min-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting the cryosurvival of mouse two-cell embryos

A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.     

Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice

Successful cryopreservation demands there be little or no intracellular ice. One procedure is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One took place in oocytes that were partly dehydrated by an initial hold for 12 min at -25 degrees C. They were then cooled rapidly to -70 degrees C and warmed slowly, or they were warmed rapidly to intermediate temperatures and held. These oocytes underwent no IIF during cooling but blackened from IIF during warming. The blackening rate increased about 5-fold for each five-degree rise in temperature. Upon thawing, they were dead. The second aspect involved oocytes that had been vitrified by cooling to -196 degrees C while suspended in a concentrated solution of cryoprotectants and warmed at rates ranging from 140 degrees C/min to 3300 degrees C/min. Survivals after warming at 140 degrees C/min and 250 degrees C/min were low (<30%). Survivals after warming at > or =2200 degrees C/min were high (80%). When warmed slowly, they were killed, apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing are discussed.     

Interactions of cooling rate, warming rate, glycerol concentration, and dilution procedure on the viability of frozen-thawed human granulocytes

Difficulties in the successful freezing of human granulocytes could lie at two levels. One is that critical cryobiological variables have not yet been identified, the other is that the inconsistent results may be due to unusual biological aspects of the cell. This paper is concerned with the former. A prerequisite for the successful freezing of mammalian cells is the ability of the cell to tolerate cryoprotective levels of additive. The additive studied here was glycerol. Based on fluorescent staining with fluorescein diacetate, we found that 1 and 2 M concentrations are in fact chemically toxic at 22 degrees C. Superimposed on this toxicity is some osmotic sensitivity to the removal of the additive by other than slow dilution. The dilution procedure was selected on the basis of computer modeling of the osmotic response of the cells. The model requires a value for the permeability coefficient for glycerol. The value (4 X 10(-5) cm/min) was obtained by measuring the rate of increase of the volume of cells in hyperosmotic glycerol. The response of human granulocytes to freezing to -196 degrees C and thawing in 1 or 2 M glycerol was not unusual. The optimum cooling rate was 1-3 degrees C/min, and cooling at 10 degrees C/min or faster was especially deleterious if warming was slow (1 degree C/min) rather than rapid (188 degrees C/min). The FDA assay showed that some 75% of the cells survived freezing and thawing at optimum rates in 1 or 2 M glycerol; and some 50-60% remained viable after the glycerol had been removed, provided that the cells remained at 0 degrees C. However, granulocytes normally function at 37 degrees C. Because chemotaxis is considered a good assay of normal function, we developed a modified procedure capable of discriminating among random migration, enhanced random migration (chemokinesis), and directed cell migration (true chemotaxis). When frozen-thawed-diluted cells were incubated for 60 min at 37 degrees C, their survival, based both on the FDA assay and on the chemotaxis assay, was zero. In fact, a prior exposure of the cells to 2 M glycerol at 0 degrees C, even in the absence of freezing, resulted in a rapid loss in FDA viability when the cells were subsequently held at 37 degrees C for up to 60 min. Survivals based on FDA are usually reported to be considerably higher than survivals based on functional assays such as chemotaxis or phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cryomicroscopic observations of cattle embryos during freezing and thawing

A cryomicroscope was used to observe changes in the appearance of day 6 1 2 to 7 1 2 cattle embryos during cooling and warming in 1.4M glycerol/PBS. Embryos were cooled at various rates between 0.2 and 25 degrees C/min to temperatures between -25 and -60 degrees C and then cooled rapidly ( approximately 250 degrees C/min) to temperatures below -140 degrees C. The volume of the embryos calculated from the cross-sectional area during slow cooling decreased at -25 degrees C to about 50% of the isotonic volume. Fracture planes could be observed in the extracellular ice matrix surrounding the embryos after rapid cooling to approximately -140 degrees C. The fracture planes often touched the zona pellucida and sometimes caused cracks in the zona. Cracks in the zona pellucida were observed more often after rapid cooling from temperatures between -20 to -35 degrees C (9 13 ) than from temperatures between -36 to -60 degrees C (2 7 ). When embryos were warmed rapidly ( approximately 250 degrees C/min) from temperatures below -140 degrees C, no change was observed in the appearance of either the embryo or its surroundings except the melting of the extracellular ice. However, when embryos were warmed slowly (2 or 5 degrees C/min), a series of events was observed; first, at approximately -70 degrees C the cytoplasm and the extracellular space gradually darkened and reached maximum darkness at approximately -55 degrees C. Then, on continued slow warming, the dark material gradually disappeared and finally the large extracellular ice crystals melted.     

Effect of cooling and warming rate on glycerolized rabbit kidneys

Cooling and warming rates are known to be important determinants of viability for cryopreserved cells, but optimal rates have not previously been determined for any whole organ. In this study, rabbit kidneys, permeated with 2 M glycerol were cooled to -80 degrees C at four rates varying from 1 degrees C/hr to 3.1 degrees C/min and then rewarmed at four rates from 1 degrees C/hr to 4.2 degrees C/min, giving 16 experimental treatments. After gradual deglycerolization at 10 degrees C, each kidney was autografted and observed for 30 min. Assessment was by measurement of vascular resistance, immediate post-thaw lactate dehydrogenase (LDH) release, gross appearance, light- and electron microscopy, and tissue K+/Na+ ratio 30 min after transplantation. The best results were obtained after cooling at 1 degrees C/hr; warming rate had little apparent influence on the criteria used to assess function with the exception of LDH release, which indicated a preferred warming rate around 1 degrees C/min. Histological studies revealed extensive vascular damage, notably to the glomerular capillaries, that was minimized by very slow cooling. Freeze substitution, carried out on samples removed at -80 degrees C, demonstrated extensive ice formation in the interstitial space and, at the faster cooling rates, in the glomerular capillaries. Intracapillary ice formation was reduced in the kidneys cooled at 1 degrees C/hr.     

Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling

The formation of ice crystals within cells (IIF) is lethal. The classical approach to avoiding it is to cool cells slowly enough so that nearly all their supercooled freezable water leaves the cell osmotically before they have cooled to a temperature that permits IIF. An alternative approach is to cool the cell rapidly to just above its ice nucleation temperature, and hold it there long enough to permit dehydration. Then, the cell is cooled rapidly to -70 degrees C or below. This approach, often called interrupted rapid cooling, is the subject of this paper. Mouse oocytes were suspended in 1.5M ethylene glycol (EG)/PBS, rapidly cooled (50 degrees C/min) to -25 degrees C and held for 5, 10, 20, 30, or 40 min before being rapidly cooled (50 degrees C/min) to -70 degrees C. In cells held for 5 min, IIF (flashing) occurred abruptly during the second rapid cool. As the holding period was increased to 10 and 20 min, fewer cells flashed during the cooling and more turned black during warming. Finally, when the oocytes were held 30 or 40 min, relatively few flashed during either cooling or warming. Immediately upon thawing, these oocytes were highly shrunken and crenated. However, upon warming to 20 degrees C, they regained most of their normal volume, shape, and appearance. These oocytes have intact cell membranes, and we refer to them as survivors. We conclude that 30 min at -25 degrees C removes nearly all intracellular freezable water, the consequence of which is that IIF occurs neither during the subsequent rapid cooling to -70 degrees C nor during warming.     

Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing

Cryopreservation protocols for umbilical cord blood have been based on methods established for bone marrow (BM) and peripheral blood stem cells (PBSC). The a priori assumption that these methods are optimal for progenitor cells from UCB has not been investigated systematically. Optimal cryopreservation protocols utilising penetrating cryoprotectants require that a number of major factors are controlled: osmotic damage during the addition and removal of the cryoprotectant; chemical toxicity of the cryoprotectant to the target cell and the interrelationship between cryoprotectant concentration and cooling rate. We have established addition and elution protocols that prevent osmotic damage and have used these to investigate the effect of multimolar concentrations of Me(2)SO on membrane integrity and functional recovery. We have investigated the effect of freezing and thawing over a range of cooling rates and cryoprotectant concentrations. CD34(+) cells tolerate up to 60 min exposure to 25% w/w (3.2M) Me(2)SO at +2 degrees C with no significant loss in clonogenic capacity. Exposure at +20 degrees C for a similar period of time induced significant damage. CD34(+) cells showed an optimal cooling range between 1 degrees C and 2.5 degrees C/min. At or above 1 degrees C/min, increasing the Me(2)SO concentration above 10% w/w provided little extra protection. At the lowest cooling rate tested (0.1 degrees C/min), increasing the Me(2)SO concentration had a statistically significant beneficial effect on functional recovery of progenitor cells. Our findings support the conclusion that optimal recovery of CD34(+) cells requires serial addition of Me(2)SO, slow cooling at rates between 1 degrees C and 2.5 degrees C/min and serial elution of the cryoprotectant after thawing. A concentration of 10% w/w Me(2)SO is optimal. At this concentration, equilibration temperature is unlikely to be of practical importance with regard to chemical toxicity.     

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.     

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.     

Protection of cryopreserved Onchocerca microfilariae (nematoda) from dilution shock by the use of serum

This paper describes the use of newborn calf serum during the cooling and warming/dilution phases of the cryopreservation of Onchocerca gutturosa microfilariae using an interrupted slow cool to ?196 °C in the presence of 5% (v/v) methanol. Serum proved detrimental at concentrations above 20% (v/v) in the cooling medium unless it was also present in high concentrations, 60% (v/v) in the warming/ dilution medium.Damage to the organisms occurred predominantly during the thawing/dilution phase of cryopreservation and not the cooling phase and could be reduced greatly by the presence of high serum concentrations when thawing. This indicates that the major protective action of serum is that of reducing dilution shock—shock produced by a rapid influx of water and/or the effects of high solute concentrations established during cooling.     

Survival of corneal endothelium following exposure to a vitrification solution

Corneal endothelium, a monolayer of cells lining the inner surface of the cornea, is particularly susceptible to freezing injury. Ice formation damages the structural and functional integrity of the endothelium, and this results in a loss of corneal transparency. Instead of freezing, an alternative method of cryopreservation is vitrification, which avoids damage associated with ice formation. Vitrification at practicable cooling rates, however, requires exposure of tissues to very high concentrations of cryoprotectants, and this can cause damage through chemical toxicity and osmotic stress. The effects of a vitrification solution (VS1) containing 2.62 mol/liter (20.5%, w/v) dimethyl sulfoxide, 2.62 mol/liter (15.5%, w/v) acetamide, 1.32 mol/liter (10%, w/v) propane-1,2-diol, and 6% (w/v) polyethylene glycol were studied on corneal endothelium. Endothelial function was assessed by monitoring corneal thickness during 6 hr of perfusion at 35 degrees C with a Ringer solution supplemented with glutathione and adenosine. Various dilutions of the vitrification solution were introduced and removed in a stepwise manner to mitigate osmotic stress. Survival of endothelium after exposure to VS1 or a solution containing 90% of the cryoprotectant concentrations in VS1 (90% VS1) was dependent on the duration of exposure, the temperature of exposure, and the dilution protocol. The basic dilution protocol was performed at 25 degrees C: corneas were transferred from 90% VS1 or VS1 into 50% VS1 for 15 min, followed by 25% VS1 for 15 min and finally into isosmotic Ringer solution. Using this protocol, corneal endothelium survived exposure to 90% VS1 for 15 min at -5 degrees C, but 5 min in VS1 at -5 degrees C was harmful and resulted in some very large and misshapen endothelial cells. This damage was not ameliorated by using a sucrose dilution technique; but endothelial function was improved when the temperature of exposure to VS1 was reduced from -5 to -10 degrees C. Exposure to VS1 for 5 min at -5 degrees C was well tolerated, however, when the temperature of the first dilution step into 50% VS1 was reduced from 25 to 0 degree C. The large, misshapen cells were not observed under these conditions nor after exposure to VS1 at -10 degrees C. These results suggested that damage was the result of cryoprotectant toxicity rather than osmotic stress. Thus, corneal endothelium survived exposure to two solutions of cryoprotectants, namely, 90% VS1 and VS1, that were sufficiently concentrated to vitrify. Whether corneas can be cooled fast enough in these solutions to achieve vitrification and warmed fast enough to avoid devitrification remains to be determined.     

Cryopreservation of isolated hepatocytes: intracellular ice formation under various chemical and physical conditions.

Kinetics of intracellular ice formation (IIF) for isolated rat hepatocytes was studied using a cryomicroscopy system. The effect of the cooling rate on IIF was investigated between 20 and 400 degrees C/min in isotonic solution. At 50 degrees C/min and below, none of the hepatocytes underwent IIF; whereas at 150 degrees C/min and above, IIF was observed throughout the entire hepatocyte population. The temperature at which 50% of hepatocytes showed IIF (50TIIF) was almost constant with an average value of -7.7 degrees C. Different behavior was seen in isothermal subzero holding temperatures in the presence of extracellular ice. 50TIIF from isothermal temperature experiments was approximately -5 degrees C as opposed to -7.7 degrees C for constant cooling rate experiments. These experiments clearly demonstrated both the time and temperature dependence of IIF. On the other hand, in cooling experiments in the absence of extracellular ice, IIF was not observed until approximately -20 degrees C (at which temperature the whole suspension was frozen spontaneously) suggesting the involvement of the external ice in the initiation of IIF. The effect of dimethyl sulfoxide (Me2SO) on IIF was also quantified. 50TIIF decreased from -7.7 degrees C in the absence of Me2SO to -16.8 degrees C in 2.0 M Me2SO for a cooling rate of 400 degrees C/min. However, the cooling rate (between 75 and 400 degrees C/min) did not significantly affect 50TIIF (-8.7 degrees C) in 0.5 M Me2SO. These results suggest that multistep protocols will be required for the cryopreservation of hepatocytes.     

Vitrification of human monocytes

Human monocytes purified from peripheral blood by counterflow centrifugal elutriation were cryopreserved in a vitreous state at 1 atm pressure. The vitrification solution was Hanks' balanced salt solution (HBSS) containing (w/v) 20.5% Me2SO, 15.5% acetamide, 10% propylene glycol, and 6% polyethylene glycol. Fifteen milliliters of this solution was added dropwise to 1 ml of a concentrated monocyte suspension at 0 degrees C. Of this, 0.8 ml was drawn into silicone tubing and rapidly cooled to liquid nitrogen temperature, stored for various periods, and rapidly warmed in an ice bath. The vitrification solution was removed by slow addition of HBSS containing 20% fetal calf serum. The numerical cell recovery was about 92% and most of these retained normal phagocytic and chemotactic ability. Differential scanning calorimeter records of the solution show a glass transition at -115 degrees C during cooling and warming, but no evidence of ice formation during cooling. Devitrification occurs at about -70 degrees C during warming at rates as rapid as 80 degrees C/min. The amount of devitrification is dependent upon the warming rate. Freeze-fracture freeze-etch electron microscope observations revealed no ice either intra- or extracellularly in samples rapidly cooled to liquid nitrogen temperatures except for small amounts in some cellular organelles. However, if these cell suspensions were warmed rapidly to -70 degrees C and then held for 5 min, allowing devitrification to occur, the preparation contained significant amounts of both intra- and extracellular ice. Biological data showed that this devitrification was associated with severe loss of cell function.