Thermal properties of ethylene glycol aqueous solutions

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

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.     

Modeling of the deformation of flexible tubes using a single law: application to veins of the lower limb in man

The topic of this study mainly concerns a representative model of the behavior of flexible ducts such as elastic tubes or veins. This model is based on a phenomenological approach of the inflation and collapse of the tube. It leads to a single "universal" analytical expression of the tube law, valid fir a wide range of' positive and negative transmural pressures, which presents a significant improvement compared to previous theoretical studies defined with different expressions on restricted ranges of pressure. Moreover, the theoretical approaches most often require simplif'ing hypotheses--no longitudinal tension, no surrounding tissues--which are quite unrealistic both in the physiological case and in the experimental setup. These theoretical models can therefore be expected only roughly to describe the actual behavior of such vessels. The representative model, on the contrary, allows one to account for the deformation--inflating as well as collapse--of elastic tubes or veins with better accuracy. The tube law is a function of six parameters chosen in order to fit the experimental data. A comparison between results obtained in our laboratory using silicone tubes and representative models is presented. The model is then applied to physiological data obtained in vivo on human leg veins.     

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.     

Cryopreservation and biophysical properties of articular cartilage chondrocytes

In order to successfully cryopreserve articular cartilage chondrocytes, it is important to characterize their osmotic response during the cryopreservation process, as the ice forms and the solutes concentrate. In this study, experimental work was undertaken to determine the osmotic parameters of articular cartilage chondrocytes. The osmotically inactive volume of articular cartilage chondrocytes was determined to be 44% of the isotonic volume. The membrane hydraulic conductivity parameters for water were determined by fitting a theoretical water transport model to the experimentally obtained volumetric shrinkage data; the membrane hydraulic conductivity parameter L(Pg) was found to be 0.0633 microm/min/atm, and the activation energy E, 8.23 kcal/mol. The simulated cooling process, using the osmotic parameters obtained in this study, suggests a cooling rate of 80 degrees C/min for the cryopreservation of the articular cartilage chondrocytes of hogs. The data obtained in this study could serve as a starting point for those interested in cryopreservation of chondrocytes from articular cartilage in other species in which there is clinical interest and there are no parameters for prediction of responses.     

The influence of hydroxyethyl starch on ice formation in aqueous solutions

Differential scanning calorimetry, and, in some supplementary experiments, X-ray diffractometry and cryomicroscopy, were applied to study the influence of concentration (< 70 wt%) and cooling/warming rates (< 320 K/min) on ice formation in aqueous solutions of HES. The calorimetric measurements of the quantity of crystallizing water indicated that a mass fraction ? = 0.522 (i.e., grams water per gram HES) remained unfrozen. These results are in good agreement with our earlier extrapolations from ternary phase diagram data and tend to support the proposed cryoprotective mechanism. The value of ? determined during warming was essentially independent of composition up to the corresponding saturation concentration. It was observed that solutions containing 60 wt% HES or more remained wholly amorphous during cooling even at rates as low as 2.5 K/min (down to 120 K). Such glassy solutions are subject to devitrification at temperatures T_d which depend on the warming rate. The concentrations close to 55 wt% HES mark a transitional range exhibiting two crystallization peaks, probably due to different mechanisms of nucleation, the portion of ice formed during cooling being related to the imposed cooling rate. All samples showed a recrystallization transition at 257.5 K which was also observed cryomicroscopically. Glass transitions, however, could not be detected by the methods applied in this study. The X-ray diffraction patterns contained the structure of only one solid phase, namely hexagonal ice. A comparison of various modifications of HES, PEG, and PVP involving bound water and melting temperature did not reveal marked differences. Minimum initial HES concentrations preventing lethal salt enrichment were computed for both binary and ternary mass fractions of NaCl as biologically relevant parameters, yielding 24.1 and 10.8 wt% HES, respectively.     

The dynamical Matryoshka model: 1. Incoherent neutron scattering functions for lipid dynamics in bilayers

Fluid lipid bilayers are the building blocks of biological membranes. Although there is a large amount of experimental data using incoherent quasi-elastic neutron scattering (QENS) techniques to study membranes, very little theoretical works have been developed to study the local dynamics of membranes. The main objective of this work is to build a theoretical framework to study and describe the local dynamics of lipids and derive analytical expressions of intermediate scattering functions (ISF) for QENS. As results, we developed the dynamical Matryoshka model which describes the local dynamics of lipid molecules in membrane layers as a nested hierarchical convolution of three motional processes: (i) individual motions described by the vibrational motions of H-atoms; (ii) internal motions including movements of the lipid backbone, head groups and tails, and (iii) molecule movements of the lipid molecule as a whole. The analytical expressions of the ISF associated with these movements are all derived. For use in analyzing the QENS experimental data, we also derived an analytical expression for the aggregate ISF of the Matryoshka model which involves an elastic term plus three inelastic terms of well-separated time scales and whose amplitudes and rates are functions of the lipid motions. And as an illustrative application, we used the aggregated ISF to analyze the experimental QENS data on a lipid sample of multilamellar bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). It is clear from this analysis that the dynamical Matryoshka model describes very well the experimental data and allow extracting the dynamical parameters of the studied system.     

Stability of the amorphous state in the system water--glycerol--dimethylsulfoxide

In aqueous solutions containing both glycerol and DMSO, the various states during rewarming after quenching have been identified by X-ray diffraction. The amorphous state of the whole solution has been observed at very low temperatures. The eutectic was seen by X rays after rewarming only in the solutions containing mainly DMSO. In the other solutions only pure ice has been seen. It crystallizes directly in the hexagonal system, if enough DMSO is present. Otherwise, a mixture of cubic and hexagonal ice appears first. The temperature of the end of fusion and the devitrification temperature were measured with a scanning differential calorimeter for a wide range of warming rates. From these measurements was deduced the stability of the amorphous state, defined by the critical heating rate above which no crystallization occurs. That stability presents no maximum, but increases from glycerol to DMSO for a given water concentration in agreement with the fact that Ashwood-Smith considers DMSO a better cryoprotector than glycerol. But a small amount of glycerol in a solution of DMSO greatly enhances the difficulty of crystallization of the eutectic, without decreasing the stability of the amorphous state of the whole solution by much. Then those containing about 10% ($\text{w}\text{w}$) glycerol/(glycerol + DMSO) are perhaps better cryoprotectants than those with only DMSO, at least for low cooling or warming rates where the eutectic may have enough time to crystallize, eventually with deleterious effects, outside or inside the cells.     

Cryoprotection of red blood cells by 1,3-butanediol and 2,3-butanediol

1,3-Butanediol and 2,3-butanediol have been used in buffered solutions with 20, 30, or 35% (w/w) alcohol to cool erythrocytes to -196 degrees C at different cooling rates between 1 to 3500 degrees C/min, followed by slow or rapid rewarming. 1,3-butanediol shows the same shapes of red blood cell survival curves as 1,2-propanediol. Having nearly the same physical properties, they have comparable effects on cell survival. The classical maximum of survival for intermediate cooling rates and an increase for the highest cooling rates are observed. This increase seems to be correlated with the glass-forming tendency of the solution. After the fastest cooling rates, a warming rate of 5000 degrees C/min is sufficient to avoid cell damage, but a warming rate of 100-200 degrees C/min is not. Yet both of these rates would be insufficient to avoid the intracellular ice crystallization on warming. The damage on warming after fast cooling seems once again to be correlated with the transition from cubic to hexagonal ice. For all our results, 1,3-butanediol is like a "second" 1,2-propanediol and could be useful as a cryoprotectant for preservation by total vitrification. 2,3-Butanediol always gives extremely low survival rates, though it presents good physical properties. The crystallization of its hydrate seems to be lethal on cooling or on rewarming.     

Cryopreservation of articular cartilage. Part 3: The liquidus-tracking method

Although it is relatively straightforward to cryopreserve living isolated chondrocytes, at the present time there is no satisfactory method to preserve surgical grafts between the time of procurement or manufacture and actual use. In earlier papers we have established that the cryoprotectants dimethyl sulphoxide or propylene glycol do penetrate into this tissue very rapidly. Chondrocytes are not unusually susceptible to osmotic stress; in fact they appear to be particularly resistant. It appears that damage is associated with the formation of ice per se, even at cooling rates that are optimal for the cryopreservation of isolated chondrocytes. We then showed that current methods of cartilage cryopreservation involve the nucleation and growth of ice crystals within the chondrons rather than ice being restricted to the surrounding acellular matrix. This finding established the need to avoid the crystallization of ice—in other words, vitrification. Song and his colleagues have published a vitrification method that is based on the use of one of Fahy’s vitrification formulations. We confirmed the effectiveness of this method but found it to be very dependent on ultra rapid warming. However, we were able to develop a ‘liquidus-tracking’ method that completely avoids the crystallization of ice and does not require rapid warming. The ability of cartilage preserved in this way to incorporate sulphate into newly synthesized glycosaminoglycans (GAGs) approached 70% of that of fresh control cartilage. In this method the rates of cooling and warming can be very low, which is essential for any method that is to be used in Tissue Banks to process the bulky grafts that are required by orthopaedic surgeons. Work is continuing to refine this method for Tissue Bank use.     

Cryopreservation of articular cartilage. Part 3: the liquidus-tracking method

Although it is relatively straightforward to cryopreserve living isolated chondrocytes, at the present time there is no satisfactory method to preserve surgical grafts between the time of procurement or manufacture and actual use. In earlier papers we have established that the cryoprotectants dimethyl sulphoxide or propylene glycol do penetrate into this tissue very rapidly. Chondrocytes are not unusually susceptible to osmotic stress; in fact they appear to be particularly resistant. It appears that damage is associated with the formation of ice per se, even at cooling rates that are optimal for the cryopreservation of isolated chondrocytes. We then showed that current methods of cartilage cryopreservation involve the nucleation and growth of ice crystals within the chondrons rather than ice being restricted to the surrounding acellular matrix. This finding established the need to avoid the crystallization of ice—in other words, vitrification. Song and his colleagues have published a vitrification method that is based on the use of one of Fahy’s vitrification formulations. We confirmed the effectiveness of this method but found it to be very dependent on ultra rapid warming. However, we were able to develop a ‘liquidus-tracking’ method that completely avoids the crystallization of ice and does not require rapid warming. The ability of cartilage preserved in this way to incorporate sulphate into newly synthesized glycosaminoglycans (GAGs) approached 70% of that of fresh control cartilage. In this method the rates of cooling and warming can be very low, which is essential for any method that is to be used in Tissue Banks to process the bulky grafts that are required by orthopaedic surgeons. Work is continuing to refine this method for Tissue Bank use.     

Current-voltage relations and steady-state characteristics of Na+-Ca2+ exchange: characterization of the eight-state consecutive transport model.

An analytical expression for Na+-Ca2+ exchange currents in cardiac cells has been obtained for an eight-state model. The equation obtained has been used to derive theoretical expressions for current-voltage relationships, maximum Na+-Ca2+ exchange currents, and half-saturating concentrations for Na+ and Ca2+. These equations were analyzed over a wide range of cytoplasmic and extracellular Na+ and Ca2+ concentrations, under forward and reverse "zero-trans" conditions. Correspondence of theoretical results with those obtained from giant excised patch experiments are presented. Rate constants from published reports were used to evaluate turnover rates for Na+-Ca2+ exchange in the forward and reverse directions. A factor, epsilon, is introduced that permits prediction of the extent to which the Na+-Ca2+ exchange cycle is under voltage or diffusion control. This factor can be conveniently used for data interpretation and comparison. The derived equations also provide a foundation for continuing experimental evaluation of the fidelity of this model.     

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.     

Competitive and uncompetitive inhibitors simultaneously acting on an autocatalytic zymogen activation reaction

The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.     

Competitive and uncompetitive inhibitors simultaneously acting on an autocatalytic zymogen activation reaction

The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.     

Sea ice phenology and timing of primary production pulses in the Arctic Ocean

Arctic organisms are adapted to the strong seasonality of environmental forcing. A small timing mismatch between biological processes and the environment could potentially have significant consequences for the entire food web. Climate warming causes shrinking ice coverage and earlier ice retreat in the Arctic, which is likely to change the timing of primary production. In this study, we test predictions on the interactions among sea ice phenology and production timing of ice algae and pelagic phytoplankton. We do so using the following (1) a synthesis of available satellite observation data; and (2) the application of a coupled ice‐ocean ecosystem model. The data and model results suggest that, over a large portion of the Arctic marginal seas, the timing variability in ice retreat at a specific location has a strong impact on the timing variability in pelagic phytoplankton peaks, but weak or no impact on the timing of ice‐algae peaks in those regions. The model predicts latitudinal and regional differences in the timing of ice algae biomass peak (varying from April to May) and the time lags between ice algae and pelagic phytoplankton peaks (varying from 45 to 90 days). The correlation between the time lag and ice retreat is significant in areas where ice retreat has no significant impact on ice‐algae peak timing, suggesting that changes in pelagic phytoplankton peak timing control the variability in time lags. Phenological variability in primary production is likely to have consequences for higher trophic levels, particularly for the zooplankton grazers, whose main food source is composed of the dually pulsed algae production of the Arctic.     

Analysis of isochoric subcooling

Because ice-I is less dense than water, the formation of an ice nucleus in an isochoric (constant volume) chamber will cause an increase in pressure. This analysis shows that the energy required to overcome such a pressure increase makes homogeneous ice nucleation thermodynamically improbable in an isochoric system at temperatures above -109 degrees C. By suppressing ice nucleation, isochoric cooling is expected to significantly promote vitrification. Because water has a higher freezing temperature and a lower glass-transition temperature than physiological solutions, this analysis represents a scenario for avoiding ice crystallization during the preservation of biological substances. While isochoric cryopreservation has not yet been put into practice, this theoretical, first-order analysis suggests that if attainable it could make organ preservation significantly more effective and practical.     

Critical cooling rates for aqueous cryoprotectants in the presence of sugars and polysaccharides.

The technique of isothermal emulsion differential scanning calorimetry was used to determine time-temperature-transformation (TTT) curves for aqueous glycerol and butane-2,3-diol in the presence of various polysaccharides and sugars. The critical cooling rate required to avoid the crystallization of ice in these solutions was then calculated from the experimental TTT curves. The polysaccharides used in this study included starch hydrolysis products and dextrans of various molecular weights. The sugars used here were sucrose, glucose, trehalose, and raffinose. The results show that the critical cooling rates of butane-2,3-diol and glycerol are reduced by varying amounts by the addition of such materials but that the reduction is not as great as is achieved by the addition of polyethylene glycol with a molecular weight of 400.     

Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts

A method to obtain a high metaphase index and thereafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine-treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast-derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by "monochromatid chromosome." This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.     

Spectrum of the nonstationary electromyographic signal modelled with integral pulse frequency modulation and its application to estimating neural drive information

The spectrum of nonstationary electromyographic signal (EMG) is investigated, from which the error for neural drive information estimation from nonstationary EMG is studied in terms of signal-to-noise ratio (SNR), in analytical, numerical simulation, and experimental work. The signal refers to the neural drive information embedded within the nonstationary EMG, and noise refers to other portions of EMG that induce error in the estimation. The analytical expressions for the SNRs of force-modulated EMG with both single and multiple motor units (MU) are derived based on a sinusoidal integral pulse frequency modulation (IPFM) model. It is shown that the previously developed SNR expressions for stationary (unmodulated) EMG are special cases of the formulas presented here. The SNR results obtained from numerical simulated EMG agree very well with the analytical result. Results from nonstationary (modulated) surface EMG obtained from seven subjects also match the analytical and simulation results reasonably well. The results obtained from this work establish an analytical framework in studying and estimating the neural drive information contained in the EMG in the context of anisotonic and isometric contractions. Through the analytical study, the effects of different physiological parameters are identified, thus providing theoretical guidelines for developing advanced signal processing methods for nonstationary EMG in applications such as prosthesis control.