Expression of antifreeze proteins in transgenic plants

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. Theafa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.     

Viability of deformed cells

Most of the researchers in the field of cryobiology believe that the mechanism of damage during freezing with low cooling rates is chemical and related to the hypertonicity of the extracellular solution. However, there is some evidence to indicate that cells may be destroyed during freezing also by compression between ice crystals. We have developed an experimental procedure to study the effect of cell compression on viability. Using human prostate primary adenoma cancer cells we show that cell viability decreases steeply when cells are compressed to 30% of their original diameter. If uniform expansion of cell membrane is assumed, this corresponds to a 50% increase in the cell membrane surface area. A simple mathematical model shows that the temperature at which the compression effect may cause cell damage is related to the spacing between ice crystals. When the ice crystals are spaced at distances comparable to the cell diameter the model combined with our experimental data predicts compression damage at about -1.8 degrees C. This is consistent with experimental observation on frozen cell destruction in the presence of antifreeze proteins.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

The stability during low-temperature storage of an antifreeze protein isolated from the roots of cold-acclimated carrots

Natural antifreeze proteins (AFPs) not only inhibit freezing at high subzero temperatures; they have the additional properties of inhibiting the recrystallization of ice during warming and of preventing devitrification. The natural AFP that occurs in the roots of cold-acclimated carrots can be extracted reasonably simply and is non-toxic: it was selected for study as a possible ingredient of the vitrification mixtures that are being developed for use in tissue cryopreservation. For this application, it would be essential for the AFP to remain active during prolonged storage at very low temperatures. For logistic reasons, it would also be essential to have an effective method of storage of the purified AFP itself. In this study, carrot AFP was isolated and purified, and its ability to inhibit recrystallization was monitored over 40 weeks of storage at -80 or -196 degrees C. The data revealed a progressive decrease in activity during storage, reaching half the original activity in 10-20 weeks and only 2-3% of the original activity at 40 week. These data suggest that carrot AFP will not be effective in tissue cryopreservation.     

Formation of Ice Microparticles in the Ovarian Fluid and Homogenates of Unfertilized Russian Sturgeon Eggs during Cooling to –196°C

Cryopreservation of fish and amphibian eggs is still an unsolved problem. The formation of ice crystals inside and outside cells acts as a main detrimental factor during a deep freezing of fish eggs, as well as crystal growth (recrystallization and repeated crystallization). Designing efficient cryoprotective media is necessary in order to avoid egg injury from freezing. Additional components that are present in a cryoprotective medium and reduce the thermomechanical stress and cracks of frozen tissues might increase oocyte survival after freezing–thawing. Natural components of eggs and the ovarian fluid are promising as such additives. The formation of ice microparticles was studied in thin layers (0.2 mm) of the ovarian fluid and components of Russian sturgeon egg homogenates upon their cooling to a liquid nitrogen temperature (–196°C). The processes of freezing, ice cracking, and microparticle formation were observed as the temperature was decreased gradually. The shape and size of ice microparticles were found to depend on the composition of the freezing solution. Certain fractions of egg homogenate were assumed to be suitable as components of a cryoprotective medium.

     

Ice Morphology: Fundamentals and Technological Applications in Foods

Freezing is the process of ice crystallization from supercooled water. Ice crystal morphology plays an important role in the textural and physical properties of frozen and frozen-thawed foods and in processes such as freeze drying, freeze concentration, and freeze texturization. Size and location of ice crystals are key in the quality of thawed tissue products. In ice cream, smaller ice crystals are preferred because large crystals results in an icy texture. In freeze drying, ice morphology influences the rate of sublimation and several morphological characteristics of the freeze-dried matrix as well as the biological activity of components (e.g., in pharmaceuticals). In freeze concentration, ice morphology influences the efficiency of separation of ice crystals from the concentrated solution. The cooling rate has been the most common variable controlling ice morphology in frozen and partly frozen systems. However, several new approaches show promise in controlling nucleation (consequently, ice morphology), among them are the use of ice nucleation agents, antifreeze proteins, ultrasound, and high pressure. This paper summarizes the fundamentals of freezing, methods of observation and measurement of ice morphology, and the role of ice morphology in technological applications.     

Infrared spectroscopic analysis of hydrogen-bonding interactions in cryopreservation solutions

BackgroundIn this study we investigated hydrogen bonding interactions in hydrated and frozen solutions of different cryoprotective agents (CPAs) including dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, and trehalose. We also investigated the effect of CPAs on ice crystal growth during storage and correlated this with storage stability of liposomes.MethodsFTIR spectroscopy was used to study hydrogen bonding interactions in CPA solutions in H₂O and D₂O, and their thermal response was analyzed using van ’t Hoff analysis. The effect of CPAs on ice crystal growth during storage was investigated by microscopy and correlated with storage stability of liposomes encapsulated with a fluorescent dye.ResultsPrincipal component analyses demonstrated that different CPAs can be recognized based on the shape of the OD band region only. Chemically similar molecules such as glycerol and ethylene glycol closely group together in a principal component score plot, whereas trehalose and DMSO appear as condensed separated clusters. The OH/OD band of CPA solutions exhibits an overall shift to higher wavenumbers with increasing temperature and changed fractions of weak and strong hydrogen interactions. CPAs diminish ice crystal formation in frozen samples during storage and minimize liposome leakage during freezing but cannot prevent leakage during frozen storage.ConclusionsCPAs can be distinguished from one another based on the hydrogen bonding network that is formed in solution. DMSO-water mixtures behave anomalous compared to other CPAs that have OH groups. CPAs modulate ice crystal formation during frozen storage but cannot prevent liposome leakage during frozen storage.     

Ice Recrystallization Behavior of Corn Starch/Sucrose Solutions: Effects of Addition of Corn Starch and Antifreeze Protein III

Knowledge of the behavior of corn starch during frozen storage is necessary to understand more complex systems. In the present study, ice recrystallization in corn starch (0.3% and 3%, w/w)/sucrose (40%, w/w) solution was investigated at −10 °C based on the theory of Ostwald ripening. The addition of corn starch to the sucrose solution increased the ice recrystallization (IR) rate constant. To explore the mechanism causing higher IR rate constant, fluorescence microscopy was used to analyze the distribution of corn starch molecules. Fluorescence micrograph showed corn starch distributed homogenously in the freeze-concentrated phase. Ice crystal size distribution assessment showed that at the same average radius, the addition of corn starch increased the standard deviation of ice crystal size distribution. The findings revealed that the addition of corn starch widened the distribution of ice crystal size, which may be the mechanism causing higher IR rate constant. To inhibit the ice recrystallization process, antifreeze protein type III (AFP III) was added to sucrose solutions with and without corn starch. In the presence of corn starch, 0.01-mg/mL AFP III was enough to significantly reduce the IR rate. Conversely, the samples without corn starch did not show a significant reduction in IR rate constant at the same AFP III concentration. The outcomes revealed that corn starch enhanced the activity of AFP III. The results of this study showed that corn starch increased the IR rate constant, and AFP III supplemented with corn starch was synergistically more efficient in retarding IR rate constant.

     

Ice crystals formed in tissue during cryosurgery. II. Electron microscopy

Tissues frozen by means of a cryosurgical probe have been examined by electron microscopy following techniques designed to preserve the ice crystal spaces.Ice crystals appeared similar whether tissues were quenched or not following cryosurgery and the various techniques of dehydration resulted in similar ice crystal architecture.Ice crystal spaces in the area deep to the freezing probe were intracellular both in epithelium and muscle although in the muscle zone some fibers contained large and others small crystal spaces. It is suggested that this might be due to variations in the local blood supply.At the periphery of the frozen area ice crystals were usually extracellular producing gross distortion of the cells which, however, retained intracellular structural integrity. These results are consistent with the belief of many workers that intracellular ice is lethal while extracellular ice is not, but no evidence of penetration of cell membrane by ice crystals was seen.     

Theoretical analysis of the ice crystal size distribution in frozen aqueous specimens.

To estimate theoretically how suited different freezing techniques are for freezing of freeze-etch specimens, it is necessary to know the relationship between specimen cooling rate and the resulting average ice crystal size. Using a somewhat simplified theoretical analysis, we have derived the approximate ice crystal size distribution of nonvitrified frozen aqueous specimens frozen at different cooling rates. The derived size distribution was used to calculate the relationship between relative change in average ice crystal size, (delta l/l), and relative change in specimen cooling rate delta (dT/dt)/(dT/dt). We found this relationship to be (delta l/l) = -k X delta (dT/dt)/(dT/dt) where k = 1.0 when specimen solidification takes place at about -6 degrees C, and k congruent to 1.3 when it takes place at about -40 degrees C.     

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

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

The effect of ice formation on the function of smooth muscle tissue stored at −21 or −60 °C

The mechanisms by which single cells are injured during freezing are relatively well understood, but it is likely that additional factors apply to tissues and organs, factors that may be responsible for the poor suecess of attempts to cryopreserve complex multicellular systems. One such factor may be the formation of extracellular ice.

This study was designed to discover whether ice formation as such is detrimental to the contractile recovery of pieces of mammalian smooth muscle after storage at subzero temperatures. Strips of taenia coli muscle were equilibrated with 2.56 M Me₂SO in a buffered solution, cooled at either 0.3 or 2 °C/min to ?21 °C and then held at this temperature in the frozen state. Other muscle strips were bathed in a solution the composition of which mimicked that of the unfrozen phase of the previous solution at ?21 °C; it contained 4.49 M Me₂SO and 1.75 times the normal concentration of salts, and muscles equilibrated with this solution were also cooled at either 0.3 or 2 °C/min to ?21 °C, and then held unfrozen for the same length of time.It was shown that exposure to ?21 °C and the increased concentration of solutes had little effect on the contractile recovery of the muscles, whereas ice formation was damaging. Furthermore, the rate of cooling had a marked effect upon functional recovery in the frozen muscles, and this could be correlated with the known effect of these cooling rates on the pattern of ice formation in the tissue. The effect was also seen in muscles frozen at ?60 °C. Improved buffering increased the functional recovery of all groups, but the effect of ice, and of cooling rate in the presence of ice, was confirmed. These findings may have significant implications for attempts to cryopreserve complex tissues and organs.     

Freezing preservation of adult mammalian heart at high subzero temperatures

The present study adapted the overwintering strategy employed by freeze-tolerant amphibians and reptiles to freeze-preserve the isolated rat heart. The heart was flushed with a cardioplegic solution and supercooled to -1.2 and -3 degrees C. Then freezing was induced by inoculation of ice crystal. The viability of the heart explant was assessed after reanimation by the isolated working heart perfusion. There was no recovery of function in hearts flushed with solution containing 0.28 mM CaCl2. Lowering the concentration of CaCl2 to 0.15 mM, however, rendered good functional return. Furthermore, inclusion of 50 mM glycerol in the flush solution dramatically improved functional preservation. Under the best conditions defined here, the recoveries of aortic flow, coronary flow, cardiac output, systolic pressure, and work in hearts stored at -1.2 degrees C for 3 h were 72.8 +/- 6.8, 87.2 +/- 4.2, 77.6 +/- 5.4, 83.4 +/- 2.8, and 66.6 +/- 5.9% (mean +/- SEM, n = 8) of the unstored control levels, respectively. The myocardial ice content was 18.6 +/- 5.4% (n = 5) of tissue water. Prolonging the storage time to 5 h increased the ice content to 45.3 +/- 8.1% and reduced the recovery of cardiac output to 23 +/- 11% of the control value (mean +/- SEM, n = 5). Hearts frozen at -3 degrees C for 1.5 h showed 29.4 +/- 8.7% (n = 3) of control cardiac output during reperfusion. This novel approach may provide an opportunity to advance our knowledge about freezing preservation of not only the heart but other solid organs as well.     

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5–6% dimethyl sulfoxide (Me₂SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at −80 °C. Alternatively, platelets were frozen with 5–6% Me₂SO at −30 °C or with 3% Me₂SO at −80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me₂SO (p = 0.0461) but not at −30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me₂SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at −30 °C or with 3% Me₂SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at −30 °C or with 3% Me₂SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.     

Antifreeze proteins and their potential use in frozen foods

Antifreeze proteins (AFPs) are proteins that have the ability to modify the growth of ice, resulting in the stabilization of ice crystals over a defined temperature range and in the inhibition of the recrystallization of ice. AFPs are found in a wide range of organisms, including bacteria, fungi, plants, invertebrates and fish. Moreover, multiple forms of AFPs are synthesized within each organism. As a result, it should be possible to select an AFP with appropriate characteristics and a suitable level of activity for a particular food product. Antifreeze proteins may improve the quality of foods that are eaten while frozen by inhibiting recrystallization and maintaining a smooth texture. In foods that are frozen only for preservation, AFPs may inhibit recrystallization during freezing, storage, transport and thawing, thus preserving food texture by reducing cellular damage and also minimizing the loss of nutrients by reducing drip. Antifreeze proteins are naturally present in many foods consumed as part of the human diet. However, AFPs may be introduced into other food products either by physical processes, such as mixing and soaking, or by gene transfer.     

Large Ice Crystals in the Nucleus of Rapidly Frozen Liver Cells

Evidence in the literature shows that ice crystals that form in the nucleus of many rapidly cooled cells appear much larger than the ice crystals that form in the surrounding cytoplasm. We investigated the phenomenon in our laboratory using the techniques of freeze substitution and low temperature scanning electron microscopy on liver tissue frozen by liquid nitrogen plunge freezing. This method is estimated to cool the tissue at 1000°C/min. The results from these techniques show that the ice crystal sizes were statistically significantly larger in the nucleus than in the cytoplasm. It is our belief that this finding is important to cryobiology considering its potential role in the process of freezing and the mechanisms of damage during freezing of cells and tissues.     

Induced Synthesis of Mitochondrial RNA in Sweet Potato Root after Wounding

Ice structure was photographically analyzed for frozen soy protein curd and egg albumin gel frozen under various conditions. Dendritic ice structure was observed growing from the cooling plate parallel to the direction of the heat flux. The change in the ice structure size was analyzed at different locations from the cooling plate in the plane perpendicular to the direction of heat flux. In accordance with the theoretical relationship proposed by us before, the mean ice structure size was inversely proportional to the moving speed of the freezing front and the proportionality constant was not very much different from the diffusion coefficient of water, showing the important role of the molcular diffusion mechanism in the process of ice crystal growth. For the freezing accompanied with supercooling, the ice structure became very small, reflecting the very rapid moving speed of the freezing front when supercooling ceased. The theoretical model by us had advantages over the models proposed in the literature for its simple theoretical basis and wider applicability.     

Lipid peroxidation in fish muscle microsomes in the frozen state

The microsomal fraction from fish muscle has previously been shown to catalyze the oxidation of its lipid. In this study we have studied the rate of the reaction in the frozen state. The rate was dependent on temperature, decreasing with decreasing temperature. When the microsomes were frozen in the presence of NaCl there was greater activity than when they were frozen in the presence of KCl. The specific activity of the oxidation decreased with increasing protein concentration. This is possibly due to the limitation of oxygen in the frozen system. Lipid oxidation is a complex reaction and both initial products (lipid hydroperoxides) and breakdown products (those reacting with malondialdehyde) were measured. This ratio was relatively constant over a variety of conditions indicating that the rate-limiting step of the reaction occurred prior to the formation of lipid hydroperoxide. A study of the reaction at above-freezing temperatures and below-freezing temperatures in the presence of miscible solvents to prevent freezing at temperatures below 0 °C gave results which were consistent with the hypothesis that ice crystal formation had an accelerating effect on the reaction. Presumably this is due to concentration of reactants since freezing and thawing of the microsomes did not affect their rates of lipid oxidation. Potent inhibitors of the lipid oxidation reaction were found in the soluble fraction of the muscle tissue. These were both high-molecular and low-molecular-weight compounds. The low-molecular-weight inhibitors were more effective in the frozen state while the high-molecular-weight compounds were relatively more effective in the reaction catalyzed at temperatures above freezing.     

Supercooling and freeze-tolerance in the European wall lizard,Podarcis muralis,with a revisional history of the discovery of freeze-tolerance in vertebrates

Summary Wall lizards were collected in the fall of 1988 from a population introduced in 1951 into Cincinnati, OH. They were acclimated to 5 °C for several weeks prior to testing at sub-zero temperatures. Eleven super-cooled lizards were removed from the cooling chamber prior to crystallization after between 15 min and 26 h at body temperatures ranging from -2.2 to -5.9 °C. With the exception of one individual supercooled to-5.0 °C, all lizards recovered fully. The crystallization temperatures of 15 lizards which froze ranged from -0.6 to -6.4 °C. Frozen lizards were stiff with a distinct blue color, which faded upon thawing at 3 °C. The ice contents of frozen lizards were determined calorimetrically and/or estimated from a theoretical model, the two methods being generally in close agreement. Remarkably, five individuals recovered fully from exposures as long as 2 h and with as much as 28% of their body water frozen. Although these animals are not as tolerant as certain other vertebrates they are clearly able to withstand freezing under some circumstances. Failure to survive freezing was attributed either to excessive ice accumulation during a prolonged freeze or to excessive supercooling prior to freezing, which induced a large initial surge of ice formation upon crystallization. Our results accord with those of Weigmann (1929). We accordingly recognize him as the first to demonstrate freeze-tolerance in vertebrates, and we further recognize P. muralis as the first vertebrate known to survive freezing.     

Freezing of tissue-limits for the autoradiographic localization of diffusible substances

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.