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Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below − 100°C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000°C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard.

Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below -40 degrees C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2007) 158-166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to -25 degrees C, holding them 10 min, rapidly cooling them to -70 degrees C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at -56 degrees C during warming and was complete by -46 degrees C. The present study differs in that the oocytes were warmed rapidly from -70 degrees C to temperatures between -65 and -50 degrees C and held for 3-60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (E(a)) for the blackening process; namely, 27.5 kcal/mol. This translates to about a quadrupling of the blackening rate for every 5 degrees C rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 degrees C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 degrees C. These findings have significant implications both for cryobiology and cryo-electron microscopy.

Mazur and collaborators began examining the validity of initial views regarding mouse oocyte and embryo vitrification and found that most are partially or fully wrong. First, the relative effects of warming and cooling rates on the survival of mouse oocytes subjected to a vitrification procedure were determined. The high sensitivity to warming rate strongly suggests that the lethality of slow warming is a consequence of either the crystallisation of intracellular glassy water during warming or the recrystallisation during slow warming of small intracellular crystals that had formed during cooling. Warming rates of 107°C min–1 were achieved in 0.1-µL drops of ethylene glycol–acetamide–Ficoll–sucrose (EAFS) solution plus a small amount of India ink on Cryotops warmed using an infrared laser pulse. Under these conditions, survival rates of 90% were obtained even when mouse oocytes were suspended in 0.3× EAFS, a concentration that falls in the range that many cells can tolerate. A second important finding was that the survival of oocytes is more dependent on the osmotic withdrawal of much of the intracellular water before vitrification than it is on the penetration of cryoprotective solutes into the cells. Herein we review the roles of internal ice formation, vitrification and recrystallisation. It remains to be seen how widely these findings will be applicable to other types of cells and tissues from other species.

Vitrification is now the main route to the cryopreservation of human and animal oocytes and preimplantation embryos. A central belief is that for success, the cells must be placed in very high concentrations of cryoprotective solutes and must be cooled extremely rapidly. We have shown recently that these beliefs are incorrect. Over 90% of mouse oocytes and embryos survive being cooled relatively slowly even in solutions containing only 1/3rd the normal solute concentrations, provided that they are warmed ultra-rapidly at 107°C/min by a laser pulse. Nearly all vitrification solutions contain both permeating and non-permeating solutes and an important question is whether the former protect because they permeate the cells and promote intracellular vitrification (as is almost universally believed), or because they osmotically withdraw a large fraction of intracellular water prior to cooling. The answer for the mouse system is clearly the latter. When oocytes or embryos are placed in 1 molal concentrations of the impermeable solute sucrose, they osmotically lose ~85% of their cellular water in less than 2 minutes. If the cells are then cooled rapidly to −196°C, nearly 90% remain viable after warming, again provided that the warming is ultra rapid.

We are currently investigating factors that influence intracellular ice formation (IIF) in mouse oocytes and oocytes of the frog Xenopus. A major reason for choosing these two species is that while their eggs normally do not possess aquaporin channels in their plasma membranes, these channels can be made to express. We wish to see whether IIF is affected by the presence of these channels. The present Xenopus study deals with control eggs not expressing aquaporins. The main factor studied has been the effect of a cryoprotective agent [ethylene glycol (EG) or glycerol] and its concentration. The general procedure was to (a) cool the oocytes on a cryostage to slightly below the temperatures at which extracellular ice formation occurs, (b) warm them to just below the melting point, and (c) then re-cool them to -50 degrees C at 10 degrees C/min. In the majority of cases, IIF occurs well into step (c), but a sizeable minority undergo IIF in steps (a) or (b). The former group we refer to as low-temperature flashers; the latter as high-temperature flashers. IIF is manifested as abrupt blackening of the egg, which we refer to as "flashing." Observations on the Linkam cryostage are restricted to Stage I and II oocytes, which have diameters of 200 300 microm. In the absence of a cryoprotective agent, that is in frog Ringers, the mean flash temperature for the low-temperature freezers is -11.4 degrees C, although a sizeable percentage flash at temperatures much closer to that of the EIF (-3.9 degrees C). When EG is present, the flash temperature for the low-temperatures freezers drops significantly to approximately -20 degrees C for EG concentrations ranging from 0.5 to 1.5 M. The presence of 1.5 M glycerol also substantially reduces the IIF temperature of the low-temperature freezers; namely, to -29 degrees C, but 0.5 and 1 M glycerol exert little or no effect. The IIF temperatures observed using the Linkam cryostage agree well with those estimated by calorimetry [F.W. Kleinhans, J.F. Guenther, D.M. Roberts, P. Mazur, Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry, Cryobiology 52 (2006) 128-138]. The IIF temperatures in Xenopus are substantially higher than those observed in mouse oocytes [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53]. Perhaps that is a reflection of their much larger size.

MII mouse oocytes in 1 and 1.5M ethylene glycol(EG)/phosphate buffered saline have been subjected to rapid freezing at 50 degrees C/min to -70 degrees C. When this rapid freezing is preceded by a variable hold time of 0-3 min after the initial extracellular ice formation (EIF), the duration of the hold time has a substantial effect on the temperature at which the oocytes subsequently undergo intracellular ice formation (IIF). For example, in 1M EG, the IIF temperatures are -23.7 and -39.2 degrees C with 0 and 2 min hold times; in 1.5M EG, the corresponding IIF temperatures are -29.1 and -40.8 degrees C.

Although fish embryos have been used in a number of slow-freezing cryopreservation experiments, they have never been successfully cryopreserved. In part this is because little is known about whether ice forms within the embryo during the slow-freezing dehydration process. Therefore, we examined the temperature of intraembryonic ice formation ( T IIF ) and the temperature of extraembryonic ice formation ( T EIF ), using a cryomicroscope. We used both unmodified zebrafish embryos and those with water channels (aquaporin-3 or AQP3) inserted into their membranes to increase permeability to water and cryoprotectants, examined at 100% epiboly to the 6-somite stage. In these experiments we examined: (1) the spontaneous freezing of (external) solutions; (2) the spontaneous freezing of solutions containing embryos; (3) the effect of preloading the embryos with cryoprotectants on T IIF ; (4) whether preloading the embryos with cryoprotectant helps in survival after nucleating events in the solution; and (5) the damaging effects of extracellular nucleation events versus solution toxicity on the embryos. The solutes alone (embryo medium—EM, sucrose culture medium, 1 M propylene glycol in EM, and 1 M propylene glycol in a sucrose culture medium) froze at −14.9 ± 1.1, −17.0 ± 0.3, −17.8 ± 1.0, and −17.7 ± 1.4, respectively. There was no difference amongst these means ( P > 0.05), thus adding cryoprotectant did not significantly lower the nucleation point. Adding embryos (preloaded with cryoprotectant or not) did not change the basic freezing characteristics of these solutes. In all these experiments, ( T EIF ) equaled ( T IIF ), and there was no difference in the freezing point of the solutions with or without the embryos ( P > 0.05). Additionally, there was no difference in the freezing characteristics of embryos with and without aquaporins ( P > 0.05). The formation of intraembryonic ice was lethal to the zebrafish embryos in all cases. But this lethal outcome was not related to solution injury effects, because 88–98% of embryos survived when exposed to a higher solute concentration with no ice present. Taken together, these data suggest that slow-freezing is not a suitable option for zebrafish embryos. The mechanism of this high temperature nucleation event in zebrafish embryos is still unknown.

Mouse spermatozoa in 18% raffinose and 3.8% Oxyrase in 0.25× PBS exhibit high motilities when frozen to −70°C at 20–130°C/min and then rapidly warmed. However, survival is

Osmotic tolerance of spermatozoa is a critical determinant of functional survival after cryopreservation. This study first tested the hypothesis that mouse spermatozoa behave as linear osmometers, using an electronic particle counter to measure the change in sperm volume in response to anisosmotic solutions. The resulting Boyle-van't Hoff plot was linear (r 2 = 0.99) from 75 to 1200 mOsmolal and indicates that 60.7% of the total cell volume is osmotically inactive. Next, mouse sperm tolerance to osmotic stress was determined by assessment of plasma membrane integrity, mitochondrial viability, and motility. Each functional endpoint was measured after exposure to anisosmotic solutions and again after return to isosmolality. The dual fluorescent stains-carboxyfluorescein diacetate with propidium iodide and Rhodamine 123 with propidium iodide-were used to determine membrane integrity and functional mitochondria, respectively. Motility was measured by video microscopy in the range of 1-2400 mOsmolal and was further analyzed from 140 to 600 mOsmolal using computer-assisted semen analysis. The data indicate that motility is substantially more sensitive to osmotic stress than either mitochondrial viability or membrane integrity and that mouse spermatozoa should be maintained within 76-124% of their isosmotic volume during cryopreservation in order to maintain > 80% of pretreatment motility.

Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to −7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to −70 °C. IIF that occurred during the 20 °C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were −34, −40, −35, and −25 °C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10–15 °C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-strandedAqp3RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.

The osmotic and permeability parameters of a cell membrane are essential physico-chemical properties of a cell and particularly important with respect to cell volume changes and the regulation thereof. Here, we report the hydraulic conductivity, L(p), the non-osmotic volume, V(b), and the Arrhenius activation energy, E(a), of mammalian COS-7 cells. The ratio of V(b) to the isotonic cell volume, V(c iso), was 0.29. E(a), the activation energy required for the permeation of water through the cell membrane, was 10,700, and 12,000 cal/mol under hyper- and hypotonic conditions, respectively. Average values for L(p) were calculated from swell/shrink curves by using an integrated equation for L(p). The curves represented the volume changes of 358 individually measured cells, placed into solutions of nonpermeating solutes of 157 or 602 mOsm/kg (at 0, 10 or 22°C) and imaged over time. L(p) estimates for all six combinations of osmolality and temperature were calculated, resulting in values of 0.11, 0.21, and 0.10 µm/min/atm for exosmotic flow and 0.79, 1.73 and 1.87 µm/min/atm for endosmotic flow (at 0, 10 and 22°C, respectively). The unexpected finding of several fold higher L(p) values for endosmotic flow indicates highly asymmetric membrane permeability for water in COS-7. This phenomenon is known as rectification and has mainly been reported for plant cell, but only rarely for animal cells. Although the mechanism underlying the strong rectification found in COS-7 cells is yet unknown, it is a phenomenon of biological interest and has important practical consequences, for instance, in the development of optimal cryopreservation.

Hyperosmotic stress, which cells experience during the freezing process, and its release during the warming process are both related to cryoinjury. To define optimal cooling or warming rates and prevent osmotic injury to human sperm, information is required regarding the osmotic tolerance of the cells as a function of 1) time, 2) temperature, 3) type of solute, and 4) solute concentration. Human sperm samples were divided into three aliquots. The aliquots were equilibrated at 0, 8, and 22 degrees C, respectively. Different hyperosmotic solutions were prepared by addition of either a permeating cryoprotective agent (glycerol) or nonpermeating solutes (sucrose, non-ionic; or NaCl, ionic) to isotonic Mann's Ringer solution. Aliquots of the prepared solutions were equilibrated at 0, 8, and 22 degrees C, respectively. A small volume (2.5 microliters) of each sperm aliquot was quickly mixed with 50 microliters of each hyperosmotic solution at the corresponding temperature. After times ranging from 5 s to 5 min, 10 microliters of each hyperosmotic cell suspension was abruptly returned to an isosmotic environment by mixing with 500 microliters of Mann's Ringer solution at the corresponding temperature. The plasma membrane integrity of cells after exposure to hyperosmotic stress and after return to isosmotic conditions was measured by a dual staining (carboxyfluoroscein diacetate and propidium iodide) technique and flow cytometry. The morphology of the treated cells was observed by scanning electron microscopy of freeze-substituted sperm. The results indicate that human spermatozoa exhibited a significant posthypertonic lysis/injury, i.e., loss of membrane integrity, when returned to isosmotic conditions after exposure to hyperosmotic solutions of NaCl or sucrose. The higher the hyperosmolality, the more serious the cell injury. The majority of the cells (> 50%) lost membrane integrity when the osmolality was > or = 2000 mOsm. In contrast, if the sperm were not returned to isosmotic conditions, the majority of the sperm in the hyperosmotic solutions appeared to maintain membrane integrity. For a given higher hyperosmolality (> 1000 mOsm), posthypertonic spermolysis was reduced with a decrease of temperature. Cell survival was also affected by time of cell exposure to hyperosmotic environments before cells were returned to the isotonic condition. The shorter the time, the higher the cell survival. When exposed to hyperosmotic glycerol solutions that were isotonic with respect to electrolytes, few cells lost their membrane integrity if the osmolality of glycerol was 3000 mOsm), the lower the temperature, the higher the percentage spermolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to -50 degrees C or below occurs at temperatures where a critical fraction of the external water remains unfrozen [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53; P. Mazur, I.L. Pinn, F.W. Kleinhans, The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature, Cryobiology 54 (2007) 223-233]. For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. [E.J. Woods, M.A.J. Zieger, D.Y. Gao, J.K. Critser, Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/Water and their application to cryopreservation., Cryobiology 38 (1999) 403-407]. Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic [F.W. Kleinhans, P. Mazur, Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest, Cryobiology 54 (2007) 212-222]. Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. [J.F. Guenther, S. Seki, F.W. Kleinhans, K. Edashige, D.M. Roberts, P. Mazur, Extra-and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401-416] on IIF in oocytes of the frog Xenopus. It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol.

The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from -14 degrees C in buffered isotonic saline (PBS) to -41 degrees C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at -3.9 to -7.8 degrees C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above -30 degrees C, flashing occurs in a fraction of a second. Below -30 degrees C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best correlates with IIF.

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C. The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.

The goals of the centrifugation of cell suspensions are to obtain the maximum yield of cells with minimum adverse effects of centrifugation. In the case of mechanically sensitive cells such as mouse sperm, the two goals are somewhat contradictory in that g-forces sufficient to achieve high yields are damaging, and g-forces that yield high viability produce low yields. This paper mathematically analyzes the factors contributing to each goal. The total yield of pelleted cells is determined by the sedimentation rate governed by Stokes' Law, and depends on the relative centrifugal force, centrifugation time, size and shape of the cells, density of the cells and medium, viscosity of the medium, and the length of the column of suspension. Because in the situation analyzed the column is short relative to the rotor radius, the analysis considers the centrifugal field to be quasi-homogeneous. The assumption is that cells are not damaged during sedimentation, but that they become injured at an exponential rate once they are pelleted, a rate that will depend on the specific cell type. The behavior is modeled by the solution of coupled differential equations. The predictions of the analysis are in good agreement with experimental data on the centrifugation of mouse sperm.

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have identified several likely underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (0.3 M) than the approximately 1 M concentrations that are optimal for most cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have identified two contributors to damage from higher concentrations of glycerol, namely, chemical toxicity proportional to concentration and exposure time and osmotic damage arising from too rapid an addition and removal of the glycerol. When toxicity is minimized by restricting the exposure time to 1 or 5 min and osmotic shock is minimized by adding and removing the glycerol stepwise, relatively high percentages of the sperm survive contact with 0.8 M glycerol. Free-radical damage in mouse sperm is known to be proportional to the oxygen concentration. We have determined the consequences of reducing the oxygen to

Summary Biological time ceases at the temperature of liquid nitrogen (−196°C), a fact that permits the long-term preservation of cells, provided that they can withstand the dangerous roundtrip voyage to such low temperatures. The cell is poised between Scylla and Charybdis. If it is cooled too rapidly, its water freezes internally with lethal consequences. If it is cooled too slowly, intracellular freezing is avoided, but the cell becomes exposed to enormous concentrations of solutes and narrowing channels of liquid as the water in the external medium is converted to ice. Certain cryoprotective solutes such as glycerol and dimethyl sulfoxide have been found to ameliorate this latter damage either by reducing the concentration of electrolytes or by inhibiting cell-ice and cell-cell contacts. In essence, freezing subjects cells to extremes of osmotic pressure. Consequently, their response to freezing can often be satisfactorily described by the physical-chemical relations that govern the normal osmotic movement of water and solutes through plasma membranes. When one knows the responses, one can take measures to avoid or minimize damaging consequences and thereby achieve the stoppage of biological time. This fundamental approach has led to the successful cryobiological preservation of a variety of single cells and simpler multicellular systems. It will be an important step in developing methods for the successful preservation of more complex tissues and organs.

Although sperm were successfully cryopreserved in 1950, another 22 years elapsed before the successful preservation of a mammalian embryo. In 1963, I had carried out physical–chemical analyses indicating that if cells were cooled too rapidly, they would undergo lethal intracellular ice formation (IIF), and defined what is meant by “too rapidly” for a particular cell. To avoid IIF, the cell had to dehydrate during cooling, and the required rate of cooling depended chiefly on the permeability of a particular cell to water and on the temperature coefficient of that permeability. Between 1968 and 1972, Stanley Leibo and I turned our attention to the survival of yeast, mouse marrow stem cells, and Chinese Hamster tissue culture cells as functions of cooling rate and temperature. We found that plots of survival vs. cooling rate formed an inverted “U”, with high survivals at a medium rate, and lower survivals after very slow and very rapid cooling. We proposed a “Two-Factor” hypothesis which stated that the right hand arm of the inverted U was a result of IIF at high cooling rates. Interestingly, the optimum cooling rate for maximum survival varied over a wide range for the different cells. This was chiefly because the cells differed widely in water permeability and the temperature coefficient of that permeability. In fact if the known values of these parameters were used to generate the theoretical curves, the predicted relation between IIF and cooling rate agreed quite closely with the observed drop in survival vs. cooling rate. In 1971 David Whittingham reported the successful cryopreservation of mouse 8-cell embryos cooled at 50 °C/min in PVP. Leibo and I were excited but perplexed about these results – perplexed because our computations indicated that mouse embryos would undergo lethal IIF when cooled at that rate. We calculated that the cooling rate would have to be about 1 °C/min.. That turned out to be the case. With that change and the substitution of Me 2 SO for PVP, David, Stanley and I obtained very good in vitro and in vivo survival of 8-cell embryos cooled to −196 °C (or −269 °C). The ability to cryopreserve mouse embryos quickly had important applied consequences and important fundamental consequences. Perhaps, the most important direct application was that it permitted the long-term maintenance of thousands of mutant lines of mice in the form of preimplantation embryos in a few liquid nitrogen tanks. The indirect applications stemmed from the fact that essentially the same procedures worked for early embryos of over 20 other mammals, including humans. In the last decade, clinical interest has been emphasizing the cryopreservation of human oocytes. They have proved more difficult to freeze and so interest has shifted somewhat to cryopreserving them by vitrification. As discussed, one way to avoid lethal IIF is to cool cells slowly enough so that the freezable water in the cell flows out of the cell and freezes externally. An alternative approach is to avoid intracellular ice by vitrifying cellular water. Vitrification involves converting water into a non-crystalline or amorphous glass. To achieve this conversion, it has been the strong belief that cells have to be in solutions containing a very high concentration of glass-inducing solutes and have to be cooled very rapidly. In recent work, however, we have found that neither of these is true for mouse oocytes. The important factor is not the cooling rate – it is the warming rate, and the warming rate has to be very high. Furthermore, if the warming rate is very high, one can cut in half the concentration of solutes in the vitrification solution and still obtain high survivals.

The permeability of human spermatozoa to glycerol and its activation energy were determined using electron paramagnetic resonance (EPR) techniques. EPR was used to monitor the aqueous cell volume change vs. time during the glycerol permeation process using the aqueous spin label 15N-tempone and the membrane impermeable broadening agent potassium trioxalatochromiate (chromium oxalate). The permeation process was completed in tens of seconds, requiring the use of a stopped-flow methodology. The glycerol permeability coefficient (Pg) was determined by fitting a simple theoretical model to the experimental data. The permeabilities of human spermatozoa in 1 molar and 2 molar glycerol at 20 degrees C are (10.3 +/- 0.3).10(-4) cm/min (mean +/- S.D.) and (6.0 +/- 1.4).10(-4) cm/min, respectively. The permeabilities of human spermatozoa in 2 molar glycerol at 30, 20, 10, and 0 degrees C are (8.3 +/- 1.3).10(-4) cm/min, (6.0 +/- 1.4).10(-4) cm/min, (2.1 +/- 0.4).10(-4) cm/min, and (1.1 +/- 0.3).10(-4) cm/min, respectively. The activation energy (Ea) for glycerol permeation between 30 degrees C and 0 degrees C was found to be 11.6 kcal/mol.

Drosophila embryos manifest unusually high sensitivity to chilling in that they are killed with increased rapidity by exposure to temperatures between 0 and −25 °C in the absence of ice formation. Thus, 50% of 15-h eggs succumb in 35, 4, and 1 h at 0, −9, and −15 °C, respectively. The sensitivity becomes substantially greater in embryos at stages of development earlier than 12 h, especially at 3 and 6 h. The killing kinetics at given subzero temperatures between 0 and −25 °C are characterized by a shoulder followed by a more-or-less linear decrease in survival with time. The lower the temperature, the shorter the shoulder and the faster the postshoulder decline. The rate of both components follows Arrhenius kinetics, i.e., plots of log rate vs 1/absolute temperature are linear, the slopes being proportional to the activation energy. In both cases the activation energy is high and negative; namely, −46.5 kcal/mol for the shoulder length and −24.7 kcal/mol for the postshoulder inactivation. Negative activation energies are unusual, and according to absolute reaction rate theory, they exist only when the entropy of activation is negative, which suggests that the activated state is more ordered. By combining the duration of the shoulder as a function of time and temperature with the rate of postshoulder inactivation, one can compute survival as a function of temperature for embryos cooled at various rates. For those cooled at ⩽ 1 °C/min, the computed curve of survival vs temperature agrees closely with observed survivals. But for embryos cooled at ~10 °C/min, the drop in survival occurs some 7 to 10 ° above that computed. Embryos exposed to 0 °C for >5 min undergo conditioning that renders them more resistant to subsequent exposure to lower temperatures, and those cooled at 10 °C/min presumably lack sufficient time at 0 °C to undergo such conditioning; hence the discrepancy between observed and computed survivals. As a test of the possibility that chilling injury is a consequence of the loss of synchrony of coupled reactions involved in embryological development, embryos were rendered anoxic prior to chilling, a treatment that has been shown by Foe and Alberts to reversibly halt development of early stages. Although anoxia somewhat reduced chilling injury in 6-h eggs, it had no effect on 15-h eggs. The sensitivity of embryos to chilling injury below −25 °C cannot be experimentally assessed because differential thermal analysis shows that intact eggs freeze between −27 and −32 °C with lethal consequences. But death from chilling occurs so rapidly between −15 and −25 °C that the use of orthodox slow freezing procedures to remove intracellular water prior to entering the intraembryonic nucleation zone will not succeed; the embryos will succumb to chilling even before they have reached that zone. Unless ways can be found to reduce the accelerating rate of chilling injury as cooling progresses, the only solution is to “outrace” it by cooling at high rates and hope that intraembryonic ice formation can be avoided by the introduction of solutes that enhance vitrification.

As suspensions of cells freeze, the electrolytes and other solutes in the external solution concentrate progressively, and the cells undergo osmotic dehydration if cooling is slow. The progressive concentration of solute comes about as increasing amounts of pure ice precipitate out of solution and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. The consensus has been that slow freezing injury is related to the composition of the solution in these channels and not to the amount of residual liquid. The purpose of the research reported here was to test this assumption on human erythrocytes. Ordinarily, solute concentration and the amount of liquid in the unfrozen channels are inversely coupled. To vary them independently, one must vary the initial solute concentration. Two solutes were used here: NaCl and the permeating protective additive glycerol. To vary the total initial solute concentration while holding the mass ratio of glycerol to NaCl constant, we had to allow the NaCl tonicity to depart from isotonic. Specifically, human red cells were suspended in solutions with weight ratios of glycerol to NaCl of either 5.42 or 11.26, where the concentrations of NaCl were 0.6, 0.75, 1.0, 2.0, 3.0, or 4.0 times isotonic. Samples were then frozen to various subzero temperatures, which were chosen to produce various molalities of NaCl (0.24-3.30) while holding the fraction of unfrozen water constant, or conversely to produce various unfrozen fractions (0.03-0.5) while holding the molality of salt constant. (Not all combinations of these values were possible). The following general findings emerged: (a) few cells survived the freezing of greater than 90% of the extracellular water regardless of the salt concentration in the residual unfrozen portion. (b) When the fraction of frozen water was less than 75% the majority of the cells survived even when the salt concentration in the unfrozen portion exceeded 2 molal. (c) Salt concentration affected survival significantly only when the frozen fraction lay between 75 and 90%. To find a major effect on survival of the fraction of water that remains unfrozen was unexpected. It may require major modifications in how cryobiologists view solution-effect injury and its prevention.

A central tenet in cryobiology is that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to estimate the amount of solute that has permeated a cell prior to freezing. The amount in bovine red cells was estimated from the flux equation $${{ds} \mathord{\left/ {\vphantom {{ds} {dt}}} \right. \kern-\nulldelimiterspace} {dt}} = P_\gamma A[(activity external solute) - (activity internal solute)].$$ Solving the equation required estimates ofPγ, the permeability constant for the solute. Estimates for glycerol in bovine red cells were made in two ways: (1) by measuring the time to 50% hemolysis of red cells suspended in isosmotic or hyperosmotic (1 to 3m) solutions of glycerol that were hypotonic with respect to NaCl, and (2) by measuring the time required for red cells in hyperosmotic solutions of glycerol in isotonic salinebuffer to become susceptible to osmotic shock upon 10-fold dilution with isotonic saline-buffer. The measurements were made at 0, 10, 15 and 20°C. The values by the second technique ranged from 2.3×10−6 cm/min to 2.7×10−6 cm/min at 20°C, depending on the concentration of glycerol. The values by the first technique were 0 to 30% lower. Both techniques yielded about the same activation energy for permeation between 0 and 20°C, 21 kcal/mole. This is equivalent to a halving of the permeation rate for every 5° drop in temperature.

The cause of slow freezing injury and the basis of the protection by solutes like glycerol are subjects of debate. During slow freezing, cells are sequestered in unfrozen channels between ice crystals that grow by removing pure water from the channels. As a consequence, the solute concentration in the channels rises and the volume of liquid in the channels progressively decreases. The rise in solute concentration, in turn, causes the cells to progressively shrink osmotically. Until recently cryobiologists have ascribed slow freezing injury to either the rise in solute (electrolyte) concentrations in the channels or to the consequent cell shrinkage, rather than to the decrease in the of the channels. Although ordinarily reciprocally coupled, it is possible to separate the composition of the channels from their size, or more precisely from the magnitude of the unfrozen fraction, by suspending cells in NaCl/cryoprotectant solutions in which the mole ratio of the two is held constant, but the molality of the NaCl is allowed to vary below and above isotonic. When human red cells are frozen in such solutions to temperatures that produce given NaCl concentrations (ms), but varying unfrozen fractions (U), survival at low U is found to be strongly dependent on U but independent of ms. At higher values of U, survival becomes inversely dependent on both ms and U. Although cell volume during freezing is independent of the NaCl tonicity in the solution, the cells in the several solutions differ in volume both prior to the onset of freezing and after the completion of thawing. We have now examined and compared the effect of returning the thawed cells to isotonic solutions and isotonic volume or nearly so, and find that there is little change in survival after exposure to low U, but that survival after exposure to high U values exhibits substantially increased sensitivity to ms, a sensitivity that is probably a manifestation of posthypertonic hemolysis. Low values of U were in general attained by the use of solutions with low tonicities of NaCl, and as a consequence cells frozen to low U values had larger volumes prior to freezing than cells frozen to higher U values. The significance of this confounding is discussed.

One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that protective additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M. L. Shepard et al. ( Cryobiology, 13:9–23, 1976), in combination with the data on human red cell survival vs. subzero temperature presented here and in the companion study of Souzu and Mazur ( Biophys. J., 23:89–100), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the red cell freezing experiments was obtained by interpolation of the liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of red cell survival indicate that the correlation between the temperature that yields 50% hemolysis (LT 50 ) and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. For example, the LT 50 for cells frozen in the absence of glycerol corresponds to a NaCl concentration of 12 weight percent (2.4 molal), while for cells frozen in 1.75 M glycerol in buffered saline the LT 50 corresponds to 3.0 weight percent NaCl (1.3 molal). The data, in combination with other findings, lead to two conclusions: ( a ) The protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but ( b ) the protection is less than that expected from colligative effects; apparently glycerol itself can also be a source of damage, probably because it renders the red cells susceptible to osmotic shock during thawing.

The freezing of a living cell involves a complex physicochemical process of heat and water transport between the cell and its surrounding medium. Embryos survive cryopreservation only in the presence of a cryoprotectant in concentrations between 1 and 2M. During the addition and dilution of a permeating cryoprotectant, the cell undergoes osmotic changes in cell size. As a consequence, if the addition or particularly the dilution are carried out inappropriately, the viability of cells can be affected. Equations which model the influx and efflux of cryoprotectants in cells can be used to calculate the optimum and most practical addition and removal method. However, the equations require the permeability coefficient of the cryoprotectant, a quantity that has only experimentally determined for a few of the developmental stages of two species.

Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.

The survival of yeast cells after exposure to subzero temperatures was affected by the cooling and warming velocity, temperature itself, and the physical state of the water surrounding the cells. The cells were injured only when the external medium was frozen and then only when the temperature was -10 degrees or below. Survival dropped abruptly between -10 degrees and -30 degrees regardless of whether the cells were suspended in water or 0.1 M solutions of KH(2)PO(4), NaC1, or CaC1(2). The critical temperature range of -10 degrees to -30 degrees was unrelated to the temperatures at which the suspending fluids completely solidified, these temperatures being -0.3 degrees , -11 degrees , -30 degrees , and -71 degrees for the four liquids, respectively. Survival at -30 degrees or below was greatly affected by the rate at which the cells were cooled or warmed, with higher survivals being obtained with slow cooling and with rapid warming. Length of exposure at -30 degrees was not a factor; injury was inflicted within 1 minute. The results are interpreted as indicating that death is a result of intracellular ice formation. Internal freezing is believed to occur when external ice crystals grow through aqueous channels in the cell wall or membrane and seed supercooled water in the cell interior.

Measurements of survival and buoyant densities of bacteriophages T4B, T4Bo 1 , and T4D have demonstrated the following: ( a ) After suspension in a concentrated salt solution, T4B and T4D are sensitive both to osmotic shock and to subsequent exposure to low monovalent salt concentrations. ( b ) Sensitivity of T4B to dilution from a concentrated salt solution is dependent on dilution rate, that of T4D is less dependent, and that of T4Bo 1 is independent. ( c ) Sensitivity of all three phages to low salt concentrations depends on initial salt concentrations to a variable extent. ( d ) Density gradient profiles indicate that nearly half of osmotically shocked T4B retain their DNA. Similar analysis demonstrates that few, if any, T4Bo 1 lose DNA when subjected to a treatment causing 90% loss of infectivity. ( e ) The effective buoyant densities of T4B and T4Bo 1 depend significantly on the dilution treatments to which the phages are subjected prior to centrifugation in CsCl gradients. These data are explicable in terms of the different relative permeabilities of the phages to water and solutes, and of alterations in the counterion distribution surrounding the DNA within the phage heads.

1. 1. The recovery of Pasteurella tularensis cells suspended in a solution of gelatin-saline depended on ( a ) the rate of cooling, ( b ) the minimum temperature attained, ( c ) the time at a given temperature, and ( d ) the rate of subsequent warming. 2. 2. When cells were cooled to no lower than −30 °C., survival was comparatively high regardless of rate of cooling, exposure time, or rate of warming. 3. 3. When cells were cooled to −45 °C. or below, the slower the cooling and the more rapid the warming, the higher the recovery. 4. 4. The results are consistent with the view that death was associated primarily with slow warming or melting of intracellular ice.

When Chinese hamster tissue-culture cells are frozen in a variety of suspending media, the percentage of cells surviving is maximal at optimum cooling rates, rates that are 2–4 orders of magnitude lower than those used to freeze cells for subsequent processing by the electron microscopy techniques of freeze-cleaving and freeze-substitution. The existence of such optima suggests that at least two factors dependent on cooling rate interact to determine the ultimate survival of a frozen-thawed cell. Other data are consistent with the view that the causes of injury in rapidly and slowly frozen cells are different. First, cells frozen rapidly in 0.4 M solutions of sucrose, glycerol, and dimethyl sulfoxide, or in 0.004 M polyvinylpyrrolidone, are inactivated to a much greater extent by slow warming than are cells frozen slowly in those solutions; that is, cells frozen at rates greater than the optimum are considerably more sensitive to slow warming. Second, the inactivation rate of cells frozen rapidly in glycerol is greater at −40 °C than that of cells frozen slowly. Third, the temperatures at which cells are killed as they are slowly frozen are very different from those observed during the slow warming of rapidly frozen cells. The precise nature of the two factors remains uncertain, but indirect evidence suggests that cells cooled slower than optimum are killed by alterations in the properties of the extracellular and intracellular solution induced by ice formation (e.g., high solute concentrations), and that cells cooled faster than optimum are killed by the formation of intracellular ice and its subsequent recrystallization during warming. Such intracellular recrystallization may be a potentially serious source of artifacts in frozen material processed for electron microscopy at temperatures above −60 °C, and perhaps even above −100 °C.

Mazur, Peter (Oak Ridge National Laboratory, Oak Ridge, Tenn.). Manifestations of injury in yeast cells exposed to subzero temperatures. I. Morphological changes in freeze-substituted and in “frozen-thawed” cells. J. Bacteriol. 82: 662–672. 1961.—When cells of the yeast Saccharomyces cerevisiae are cooled rapidly to −30 C or below, fewer than 0.01% survive. In contrast, when they are cooled slowly, up to 50% survive. The effect of cooling rate on survival was reflected in the morphological appearance of cells both before and after thawing. Appearance before thawing was observed by fixing the cells at subzero temperatures by freeze-substitution with cold ethanol. Slowly cooled freeze-substituted cells were considerably smaller and more flattened than those cooled rapidly. The differences in appearance and the differences in survival are both consistent with the view that intracellular ice formation occurs more extensively in rapidly cooled cells and is responsible for their higher mortality. In spite of the high mortality (more than 99.99% killed), rapidly cooled cells remained as intact morphological entities when they were allowed to warm and thaw instead of undergoing freeze-substitution. However, they did differ from normal living yeast in two major respects: Their volume was halved and they lacked the large vacuole found in almost all the untreated living cells. The possession of a vacuole was closely correlated with survival. Suspensions warmed after slow cooling to −30 C contained cells of normal appearance and also nonvacuolate smaller ones. The admixture of morphological types was consistent with the fact that slow cooling yielded a higher percentage of viable cells than did rapid cooling.

Summary The usual procedure for preserving the viability of nucleated mammalian cells by freezing involves suspending them in a presumably permeating additive such as glycerol and cooling them at about 1°C per min. This procedure was found to be highly deleterious to Chinese hamster cells. The optimal cooling velocity when 0.1-ml quantities of cell suspension were rapidly thawed was closer to 100°C per min than to 1°C per min. Furthermore, about 25% of rapidly cooled cells survived freezing to −196°C without any additive. However, even in the presence of an additive, the 0.1-ml samples of rapidly cooled cells survived poorly when warming was slower than 1100°C per min. “Rapid” warming with quantities larger than 0.1 ml is often considerably slower than 1100°C per min, a fact that might make the sequence of rapid cooling and “rapid” warming risky to use as a routine procedure for cell preservation. The highest survivals of slowly cooled cells were obtained not by using glycerol, but by using two additives (PVP and sucrose) that are apparently unable to penetrate the cells. Furthermore, one of these (PVP) has too high a molecular weight for one to be able to ascribe its ability to protect to its molar concentration.

Haplopappus gracilis and Acer saccharum tissue culture cells are extremely sensitive to freezing injury, and exhibit a decrease in survival from 98% at -1 C to 4% at -3 C (Haplopappus) and 92% at -3 C to 13% at -5 C (Acer) when suspended in distilled H(2)O, seeded at -1 C, and then cooled by 0.1 C/minute. Similar results are obtained when cells are suspended in growth medium. The extent of shrinkage of cells during freezing can be duplicated by exposure of the cells to plasmolyzing solutions of nonpenetrating substances (Delta T(f) = 1.86 phivm). Solutions of sucrose and glycerol that produce extensive plasmolysis cause a decrease in survival within 3 to 5 minutes at room temperature, and the higher the molality to which the cell is exposed the greater the injury. Also, the rate of rehydration of the plasmolyzed cell and of the frozen cell affects its survival, with the slower rate being more beneficial. The close correlation between the decrease in survival at subzero temperatures and the decrease in survival when cells are placed in solutions having osmolalities, which could produce the same extent of shrinkage as these killing temperatures, suggests that this shrinkage is related to freezing injury in tissue culture cells.

To avoid intracellular freezing and its usually lethal consequences, cells must lose their freezable water before reaching their ice-nucleation temperature. One major factor determining the rate of water loss in the temperature dependence of the water permeability,L p (hydraulic conductivity). Because of the paucity of water permeability measurements at subzero temperatures, that temperature dependence has usually been extrapolated from above-zero measurements. The extrapolation has often been based on an exponential dependence ofL p on temperature. This paper compares the kinetics of water loss based on that extrapolation with that based on an Arrhenius relation betweenL p and temperature, and finds substantial differences below −20 to −25°C. Since the ice-nucleation temperature of mouse ova in the cryoprotectants DMSO and glycerol is usually below −30°C, the Arrhenius form of the water-loss equation was used to compute the extent of supercooling in ova cooled at rates between 1 and 8°C/min and the consequent likelihood of intracellular freezing. The predicted likelihood agrees well with that previously observed. The water-loss equation was also used to compute the volumes of ova as a function of cooling rate and temperature. The computed cell volumes agree qualitatively with previously observed volumes, but differ quantitatively.

This laboratory has previously reported that the survival of frozen-thawed human erythrocytes is determined more by the fraction of the extracellular solution that remains unfrozen than by the salt concentration in that fraction, especially when the cells are frozen at low hematocrit. To determine the extent to which these findings are applicable to nucleated mammalian cells, we have studied the survival of some 3300 mouse embryos as a function of the unfrozen fraction and the concentration of salt in that unfrozen fraction. Also varied in the study was the weight percentage ratio of glycerol to salt. The concentration of embryos in these experiments (i.e., the cytocrit) was so low that embryo-embryo contacts should have been rare during the freezing. As in the case of the red cells at low hematocrit, we find that the survival of slowly frozen eight-cell embryos is not affected by the high concentrations of salt produced by freezing, at least up to 3.3 molal NaCl, and therefore is not affected by the extent to which the cells shrink below their isotonic volume, nor in general is survival influenced by the temperature at which given salt concentrations and unfrozen fractions are attained or by the glycerol concentration at those temperatures. On the other hand, the attainment of low values of the unfrozen fraction (U) is damaging, but the damage appears in part to be due to the fact that low values of U had to be achieved by placing embryos in solutions hypotonic with respect to NaCl, which caused their volume to be greater than isotonic prior to freezing.

Human granulocytes are injured when returned to isotonic conditions after exposure at 0 degree C to hyperosmotic solutions of NaCl or sucrose with osmolalities above 0.6 osmolal. The damage was expressed as a loss of membrane integrity [fluorescein diacetate (FDA) assay] only after 60-90 min incubation at 37 degrees C. Survival after exposure to a 1.4-osmolal solution at 0 degree C was dependent on the extent of subsequent dilution. Dilution to below 0.6 osmolal was damaging, but cells could be returned to near-osmotic conditions provided that the solute concentration was increased again to 0.64 osmolal before the cells were incubated at 37 degrees C. Granulocyte cell volumes were measured under various osmotic conditions by computer-assisted micrometry. The cells did not display a minimum volume but behaved as osmometers over the observed range of 0.2-1.4 osmolal. Granulocyte volume at a given osmolality was independent of whether the cells had first been exposed to a strongly hyperosmotic medium, indicating that no solute loading occurred in hyperosmotic sucrose solutions. Even though the cells did not survive sequential exposure to greater than 0.6 osmolal solutions, subsequent return to isotonicity, and incubation at 37 degrees C, neither cell lysis nor loss in FDA-positive cells occurred after the first two steps. This finding is not consistent with the critical-surface area-increment theory of freezing injury. The mechanism of cell injury in hyperosmotic solutions is thus not known. However, the results show that osmotic stress is potentially a major damaging factor both in the equilibration of cells with protective additives and during freezing and thawing.

This study concerned the response to freezing of Neurospora crassa conidia in four different states: air-dry, hydrated in water, hydrated in Vogel medium lacking only sucrose, or hydrated in complete Vogel medium. All hydrated conidia were incubated in one of the above media for various times before freezing and were then washed and frozen in distilled water. Viability was estimated by three techniques, and the agreement among them was good. Hydration of air-dry conidia was found to be very rapid and, once hydrated, the conidia were much more sensitive to rapid freezing than they were before hydration. Rapidly cooled conidia survived freezing to a much higher extent when the warming rate was rapid than when it was slow; slowly cooled conidia showed little or no dependence on the warming rate. This sensitivity to rapid cooling and slow warming was attributed to the effects of intracellular ice. The sensitivity to freezing could be reversed by dehydrating the conidia in vacuo before freezing; thus, it was concluded that the presence or absence of water is the determining factor in the initial sensitivity due to freezing. In water, the sensitivity remained constant from 2 min to 15 days after hydration. Although conidia hydrated in growth medium lacking sucrose remained metabolically inactive, their sensitivity to rapid freezing decreased as a function of time in the medium before freezing. The reason for this decreased sensitivity is not understood. Conidia hydrated in complete growth medium (i.e., containing sucrose) became metabolically active and, after the initial sensitivity associated with hydration, became increasingly more sensitive to freezing as a function of their time in the medium. Drying itself was deleterious to metabolically active conidia, and those that survived dehydration did not exhibit a large absolute increase in resistance to subsequent freezing. The increase in sensitivity to freezing and to drying seems associated with the presence of metabolic activity; however, the precise cause of the sensitization remains obscure.

Bovine red cells, like other cells, exhibit maximum survival when frozen at certain optimum rates. Cells cooled more slowly are apparently injured by alterations in the cytoplasm or surrounding medium such as the increased concentration of solutes induced by extracellular ice formation. Additives like glycerol protect against this “slow” freezing injury. It has been generally believed that such protection requires permeation by the additive, but we have found that this supposition is not valid for the bovine red cell. Cells were suspended in 1, 2 or 3m glycerol at 20, 15 or 0°C for 0.7 to 30 min or more and then frozen to −196°C at 43 or 1.7°C/min. In nearly all cases, the percentage survival after thawing was as high for cells held in glycerol for 1 min or less prior to freezing as for cells held in glycerol for 30 min, and it was as high for cells held at 0°C as for cells held at 20°C. Survivals were the same for these times and temperatures of exposure in spite of the fact that the osmolal ratio of glycerol to salts in the cell after 30 min at 20°C, for example, was as much as 800 times greater than that in cells held at 0°C for 0.7 min. In addition, the survival after a contact of 1 or 30 min with 2.3 osmolal sucrose was the same as that after exposure to 2.3 osmolal glycerol even though the bovine red cell is impermeable to sucrose. Although exposures of 1 and 30 min to glycerol yielded similar survivals, exposures for intermediate times produced a transitory but dramatic decrease in survival. The dip occurred after longer periods of incubation when the concentration of glycerol was increased and when the incubation temperature was decreased. No dip was evident in cells chilled to 0°C or in cells frozen in sucrose. Thus, the dip seems to be associated in some way with partial permeation of glycerol prior to freezing.

The survival of various cells subjected to low temperature exposure is higher when they are cooled slowly. This increase is consistent with the view that slow cooling decreases the probability of intracellular freezing by permitting water to leave the cell rapidly enough to keep the protoplasm at its freezing point. The present study derives a quantitative relation between the amount of water in a cell and temperature. The relation is a differential equation involving cooling rate, surface-volume ratio, membrane permeability to water, and the temperature coefficient of the permeability constant. Numerical solutions to this equation give calculated water contents which permit predictions as to the likelihood of intracellular ice formation. Both the calculated water contents and the predictions on internal freezing are consistent with the experimental observations of several investigators.

Freeze-cleaving can be used as a direct probe to examine the ultrastructural alterations of biological material due to freezing. We examined the thesis that at least two factors, which are oppositely dependent upon cooling velocity, determine the survival of cells subjected to freezing. According to this thesis, when cells are cooled at rates exceeding a critical velocity, a decrease in viability is caused by the presence of intracellular ice; but cells cooled at rates less than this critical velocity do not contain appreciable amounts of intracellular ice and are killed by prolonged exposure to a solution that is altered by the presence of ice. As a test of this hypothesis, we examined freeze-fractured replicas of the yeast Saccharomyces cerevisiae after suspensions had been cooled at rates ranging from 1.8 to 75,000 degrees C/min. Some of the frozen samples were cleaved and replicated immediately in order to minimize artifacts due to sample handling. Other samples were deeply etched or were rewarmed to -20 degrees C and recooled before replication. Yeast cells cooled at or above the rate necessary to preserve maximal viability ( approximately 7 degrees C/min) contained intracellular ice, whereas cells cooled below this rate showed no evidence of intracellular ice.