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.     

Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at -196 degrees C and -20 degrees C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (-196 degrees C and -80 degrees C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at -20 degrees C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact

Cutting frozen sections of large (greater than 60 cc) blocks of monkey brain using the conventional procedures of infiltration with 30% sucrose as a cryoprotectant before freezing with pulverized dry ice often produces unacceptable levels of freezing artifact (FA) caused by displacement of tissue by ice crystals. Experiments investigating FA utilized perfusion-fixed brains from 46 monkeys and spanned combinations of cryoprotectants (glycerol, sucrose), freezing methods (dry ice or -75 degrees C isopentane), and fixatives (10% formalin, Karnovsky's or Timm's). The effects were evaluated by rating of FA severity in frozen sections of whole monkey brains. Minor FA appears as enlarged capillaries, more serious FA as large vacuoles, and both first appear midway between the periphery and center of the block. Stronger fixatives increased the severity of freezing artifact. The best method for eliminating FA was graded infiltration with up to 20% glycerol and 2% DMSO (in buffer or fixative), followed by rapid freezing in -75 degrees C isopentane. Although using a glycerol-DMSO infiltration before conventional freezing with pulverized dry ice or using conventional sucrose infiltration before freezing in isopentane gave better results than sucrose infiltration and dry-ice freezing, only the combination of glycerol-DMSO infiltration and freezing in isopentane produced consistently excellent results and virtually eliminated freezing artifact. To determine the effect of freezing with dry ice or isopentane on the rate of cooling in large blocks of CNS tissue, thermocouples were embedded in an 80-cc block of albumin-gelatin and frozen with the two methods. The rate of cooling (-3.5 degrees C/min) was twice as fast using isopentane.     

Review of biomaterial thermal property measurements in the cryogenic regime and their use for prediction of equilibrium and non-equilibrium freezing applications in cryobiology

It is well accepted in cryobiology that the temperature history and cooling rates experienced in biomaterials during freezing procedures correlate strongly with biological outcome. Therefore, heat transfer measurement and prediction in the cryogenic regime is central to the field. Although direct measurement of temperature history (i.e. heat transfer) can be performed, accuracy is usually achieved only for local measurements within a given system and cannot be readily generalized to another system without the aid of predictive models. The accuracy of these models rely upon thermal properties which are known to be highly dependent on temperature, and in the case of significant cryoprotectant loading, also on crystallized fraction. In this work, we review the available thermal properties of biomaterials in the cryogenic regime. The review shows a lack of properties for many biomaterials in the subzero temperature domain, and especially for systems with cryoprotective agents. Unfortunately, use of values from the limited data available (usually only down to −40 °C) lead to an underestimation of thermal property change (i.e. conductivity rise and specific heat drop due to ice crystallization) with lower temperatures. Conversely, use of surrogate values based solely on ice thermal properties lead to an overestimation of thermal property change for most biomaterials. Additionally, recent work extending the range of available thermal properties to −150 °C has shown that the thermal conductivity will drop in both PBS and tissue (liver) due to amorphous/glassy phases (versus crystalline) of biomaterials with the addition of cryoprotective additives such as glycerol. Thus, we investigated the implications of using approximated or constant property values versus measured temperature-dependent values for predicting temperature history during freezing in PBS (phosphate-buffered saline) and porcine liver with and without cryoprotectants (glycerol). Using measured property values (thermal conductivity, specific heat, and latent heat of phase change) of porcine liver, a standard was created which showed that values based on surrogate ice properties under-predicted cooling times, while constant properties (i.e. based on limited data reported near the freezing point) over-predicted cooling times. Additionally, a new iterative numerical method that accommodates non-equilibrium cooling effects as a function of time and position (i.e. crystallization versus amorphous phase) was used to predict temperature history during freezing in glycerol loaded systems. Results indicate that in addition to the increase in cooling times due to the lowering of thermal diffusivity with more glycerol, non-equilibrium effects such as the prevention of maximal crystallization (i.e. amorphous phases) will further increase required cooling times. It was also found that the amplified effect of non-equilibrium cooling and crystallization with system size prevents the thermal history to be described with non-dimensional lengths, such as was possible under equilibrium cooling. These results affirm the need to use accurate thermal properties that incorporate temperature dependence and crystallized fraction. Further studies are needed to extract thermal properties of other important biomaterials in the subzero temperature domain and to develop accurate numerical methods which take into account non-equilibrium cooling events encountered in cryobiology when partial or total vitrification occurs.     

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at −196°C and −20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (−196°C and −80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at −20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

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

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

Relation of ice formation to ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

Ultrastructural observations on the frozen state of pancreatic acinar cells were correlated with results of parallel studies before freezing and after thawing, as to cryoinjury and cryoprotection.Data support an hypothesis of freezing injury based upon intracellular ice and solution effects during rapid and slow freezing, respectively. The basis for superiority of extracellular over intracellular glycerol in cryoprotection was demonstrated in terms of these factors.Evidence is offered to explain the ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum (RER) seen after thawing, relative to the combined effects of freezing rate and glycerol. Slow freezing, in combination with the presence of extracellular glycerol, provided sufficient dehydration to almost completely suppress intracellular ice formation, yielding minimal ultrastructural alteration of RER. Greatest cryoinjury, expressed as extensive conversion of RER into sphere-like vesicles, was induced by the extensive intracellular ice formation which accompanied rapid freezing. A mechanism is suggested to explain physical damage of RER by intracellular ice.     

Intracellular freezing of glycerolized red cells.

The response of glycerolized human red blood cells to freezing has been evaluated in terms of the thermodynamic state of the frozen intracellular medium. The physiochemical conditions requisite for intracellular freezing, characterized by the cooling rate and the degree of extracellular supercooling, are altered appreciably by the prefreezing addition of glycerol to the cells.Fresh human erythrocytes were suspended in an isotonic glycerol solution yielding a final cryophylactic concentration of either 1.5 or 3.0 m. Subsequently the cell suspension was frozen on a special low temperature stage, mounted on a light microscope, at controlled constant cooling rates with varying degrees of extracellular supercooling (ΔT_sc). The formation of a pure intracellular ice phase was detected by direct observation of the cells.The addition of glycerol produced several significant variations in the freezing characteristics of the blood. As in unmodified cells, the incidence of intracellular freezing increased with the magnitudes of both the cooling rate and the extracellular supercooling. However, the glycerolized cells exhibited a much greater tendency to supercool prior to the initial nucleation of ice. Values of ΔT_sc > ?20 °C were readily obtained. Also, the transition from 0 to 100% occurrence of intracellular ice covered a cooling rate spectrum in excess of 300 to 600 °K/min, as compared with 10 °C/min for unmodified cells. Thus, the incidence of intracellular ice formation was significantly increased in glycerolized cells.     

Cooling of rabbit kidneys permeated with glycerol to sub-zero temperatures.

The aims of this study were to investigate if kidney preservation could be enhanced by cooling of the organs to high sub-zero temperatures after depression of their freezing points by addition of glycerol, and to study whether the added amounts of this compound would confer protection to the organs during freezing and thawing at slow rates.Glycerol was added and removed gradually by continuous, hypothermic perfusion, and the post-preservation viability was assessed by autotransplantation.Brief cooling to ?5 °C of kidneys perfused with 3 m glycerol was found to be compatible with life-sustaining posttransplant function, whereas no kidneys stored at that temperature for 5 days survived.Slow cooling af kidneys glycerolized to 3 m to ?80 °C was associated with a marked increase in vascular resistance after thawing, and none of such frozen kidneys functioned after transplantation. They showed immediately after revascularization severe impairment of the circulation, and vascular damage was observed by light microscopy. The use of 5 m glycerol for cryoprotection attenuated this rise in vascular resistance and reduced the release of the endocellular enzyme, lactate dehydrogenase after thawing, indicating less cellular damage although no kidneys functioned after grafting.It is suggested that the mechanical effect of interstitial and intravascular ice formation is a major factor in damage to intact organs during freezing, and that further injury is produced by incomplete removal of the cryoprotectant before transplantation.     

The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.     

Vitreous cryopreservation maintains the function of vascular grafts

Avoidance of ice formation during cooling can be achieved by vitrification, which is defined as solidification in an amorphous glassy state that obviates ice nucleation and growth. We show that a vitrification approach to storing vascular tissue results in markedly improved tissue function compared with a standard method involving freezing. The maximum contractions achieved in vitrified vessels were >80% of fresh matched controls with similar drug sensitivities, whereas frozen vessels exhibited maximal contractions below 30% of controls and concomitant decreases in drug sensitivity. In vivo studies of vitrified vessel segments in an autologous transplant model showed no adverse effects of vitreous cryopreservation compared with fresh tissue grafts.     

Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi

One hundred and ninety five strains of fungi were observed during freezing and thawing using a cryogenic light microscope. There was no obvious link between taxonomic position and their morphological response to freezing and thawing. The viability of seven of these strains was examined following freezing and thawing in the presence or absence of the cryoprotectants glycerol and dimethyl sulphoxide. Intracellular ice and hyphal shrinkage were not necessarily lethal events, but in many cases they affected the rate and quality of growth. Both cryoprotectants reduced shrinkage, shifted the cooling rate where intracellular ice formed in many cases, and improved the recovery of strains. The results presented aid the development of successful cryopreservation protocols.     

Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi

One hundred and ninety five strains of fungi were observed during freezing and thawing using a cryogenic light microscope. There was no obvious link between taxonomic position and their morphological response to freezing and thawing. The viability of seven of these strains was examined following freezing and thawing in the presence or absence of the cryoprotectants glycerol and dimethyl sulphoxide. Intracellular ice and hyphal shrinkage were not necessarily lethal events, but in many cases they affected the rate and quality of growth. Both cryoprotectants reduced shrinkage, shifted the cooling rate where intracellular ice formed in many cases, and improved the recovery of strains. The results presented aid the development of successful cryopreservation protocols.     

A mathematical model for the freezing process in biological tissue

A mathematical model has been developed to study the process of freezing in biological organs. The model consists of a repetitive unit structure comprising a cylinder of tissue with an axial blood vessel (Krogh cylinder) and it is analysed by the methods of irreversible thermodynamics. The mathematical simulation of the freezing process in liver tissue compares remarkably well with experimental data on the structure of tissue frozen under controlled thermal conditions and the response of liver cells to changes in cooling rate. The study also supports the proposal that the damage mechanism responsible for the lack of success in attempts to preserve tissue in a frozen state, under conditions in which cells in suspension survive freezing, is direct mechanical damage caused by the formation of ice in the vascular system.     

Extra- and intracellular ice formation in mouse oocytes

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.     

Freezing preservation of the mammalian cardiac explant. II. Comparing the protective effect of glycerol and polyethylene glycol.

We compared the cryoprotective ability of glycerol and polyethylene glycol (PEG) during freezing. Isolated rat hearts were flushed with one of three cardioplegic solutions (CP-14, CP-15, and CP-16), frozen at -1.4 degrees C, and reperfused after thawing to assess function. After 3 h freezing, cardiac output (CO) in CP-14-flushed hearts recovered to 58.1% of control. CP-16 (CP-14 with 5% PEG) improved CO to 77.5%. Five hours of freezing abolished recovery in CP-14 hearts, but CP-15 (CP-14 with 50 mM glycerol) and CP-16 hearts produced 40.0 and 49.0% CO, respectively. With 6 h freezing, CP-15 hearts did not recover, whereas CP-16 hearts recovered 37.5% CO. In CP-14 hearts frozen for 3 h, 37.4% of the tissue water was ice that increased to 44.7% with 5 h freezing. CP-15 and CP-16 hearts had 34.4 and 30.9% tissue ice, respectively, after 5 h freezing. Tissue water contents in CP-14 and CP-15 hearts (3.83 to 3.96 g H2O/g dry) were 14 to 24% higher than that in CP-16 hearts. Six hours of freezing elevated AMP and ADP contents and reduced ATP levels in CP-15 and CP-16 hearts. Total adenine nucleotide (TAN) content of CP-15 hearts was 72% of control, while that of CP-16 hearts was normal. In conclusion, both glycerol and PEG offered cryoprotection by reducing tissue ice formation. PEG was superior by reducing tissue ice content further via dehydration and by better preserving TAN content.     

Correlated x-ray diffraction and freeze-fracture studies on membrane model systems. Perturbations induced by freeze-fracture preparative procedures.

Lipid-water and protein-lipid-water phases have been examined by X-ray methods before and after freezing. Frozen samples have been subsequently fractured and replicated, thus permitting an evaluation of the nature of structural perturbations in samples examined by freeze-fracture electron microscopy. Important results are summarized: (1) Freezing low water content (approx. less than 25%) phases causes perturbations in the packing of hydrocarbon chains. The results suggest that freezing liquied paraffin chains produces a condensed "glass-like" packing. (2) Additional perturbations occur in high water content samples. After freezing, much smaller lamellar repeat distances, intense ice reflections, and extensive perturbation of fracture faces are consistant with the expulsion of water from between lamellae. Presence of glycerol generally relieves these perturbations but in some cases introduces additional lattice disorder. (3) Surprisingly, cooling by a stream of cold N2 gas (-140 degrees C) produces qualitatively the same results as rapid cooling in liquid Freon-22 (-160 degrees C). (4) Complex perturbations occur in phases containing integral membrane proteins. Interesting results have been obtained with cytochrome b5-lecithin lamellar associations which display both smooth and rough fracture faces without clearly defined particles.     

The process of freezing and the mechanism of damage during hepatic cryosurgery

Experiments were performed to correlate the structures of liver tissue frozen during cryosurgery, liver frozen at various constant cooling rates, and unfrozen, dried normal liver. The results show that during freezing of tissue ice forms and propagates along the vascular system, expanding during freezing at low cooling rates. This expansion occurs over most of the region frozen during cryosurgery and may be one of the mechanisms of damage to tissue during cryosurgery.     

Basic investigations on the freezing of human lymphocytes

Human lymphocytes were frozen at constant cooling rates in the range 2.4 to 1000 degrees K/min without cryoadditive on the cold stage of a thermally defined cryomicroscope. The volume loss due to water efflux was quantified optically for the cooling rates 2.4, 12, 48, and 120 degrees K/min. The likelihood of the formation of intracellular ice was determined as function of the cooling rate. Intracellular crystallization temperatures were obtained for ice formation during both cooling and rewarming. A theoretical analysis of the cell volume loss during freezing was compared to the experimental data and used for an indirect determination of the water permeability of the cells. A relative optimum of the cooling rate is predicted theoretically under the assumption of a critical level of intracellular salt concentration near the eutectic temperature. The dependence of survival and cooling rate was determined cryomicroscopically by simultaneously applying the FDA/EB fluorescence viability test. The optimal cooling rate of about 35 degrees K/min was also found for 2-ml samples frozen within the range of cooling rates of interest. The results show that for freezing in physiological saline solution (1) the optimum of the cooling rate is theoretically predictable, (2) cryomicroscopical data are significant for freezing of samples of larger volume, and (3) the lethal type of intracellular crystallization is cooling rate dependent and distinguishable from innocuous types.     

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.