Rapid and uniform electromagnetic heating of aqueous cryoprotectant solutions from cryogenic temperatures

Devitrification (ice formation during warming) is one of the primary obstacles to successful organ vitrification (solidification without ice formation). The only feasible approach to overcoming either devitrification or its damaging effects in a large organ appears at present to be the use of some form of electromagnetic heating (EH) to achieve the required high heating rates. One complication of EH in this application is the need for warming within a steel pressure vessel. We have previously reported that resonant radiofrequency (RF) helical coils provide very uniform heating at ambient temperatures and low heating rates and can be modified for coaxial power transmission, which is necessary if only one cable is to penetrate through the wall of the pressure vessel. We now report our initial studies using a modified helical coil, high RF input power, and cryogenic aqueous cryoprotectant solutions [60% (w/v) solution of 4.37 M dimethylsulfoxide and 4.37 M acetamide in water and 50% (w/w) 1,2-propanediol]. We also describe the electronic equipment required for this type of research. Temperatures were monitored during high-power conditions with Luxtron fiberoptic probes. Thermometry was complicated by the use of catheters needed for probe insertion and guidance. The highest heating rates we observed using catheters occurred at temperatures ranging from about -70 to -40 degrees C, the temperature zone where devitrification usually appears in unstable solutions during slow warming. We find that in this range we can achieve measured heating rates of approximately 300 degrees C/min in 30- to 130-ml samples using 200 to 700 W of RF power without overheating the sample at any point. However, energy conservation calculations imply that our measured peak heating rates may be considerably higher than the true heating rates occurring in the bulk of our solutions. We were able to estimate the overall true heating rates, obtaining an average value of about 20 degrees C/min/100 W/100 ml, which implies a heating efficiency close to 100%. It appears that it should be possible to warm vitrified rabbit kidneys rapidly enough under high-pressure conditions to protect them from devitrification.     

A theoretical model of intracellular devitrification

Devitrification of the intracellular solution can cause significant damage during warming of cells cryopreserved by freezing or vitrification. Whereas previous theoretical investigations of devitrification have not considered the effect of cell dehydration on intracellular ice formation, a new model which couples membrane-limited water transport equations, classical nucleation theory, and diffusion-limited crystal growth theory is presented. The model was used to explore the role of cell dehydration in devitrification of human keratinocytes frozen in the presence of glycerol. Numerical simulations demonstrated that water transport during cooling affects subsequent intracellular ice formation during warming, correctly predicting observations that critical warming rate increases with increasing cooling rate. However, for cells with a membrane transport activation energy less than approximately 50 kJ/mol, devitrification was also affected by cell dehydration during warming, leading to a reversal of the relationship between cooling rate and critical warming rate. Thus, for low warming rates (less than 10 degrees C/min for keratinocytes), the size and total volume fraction of intracellular ice crystals forming during warming decreased with decreasing warming rate, and the critical warming rate decreased with increasing cooling rate. The effects of water transport on the kinetics of intracellular nucleation and crystal growth were elucidated by comparison of simulations of cell warming with simulations of devitrification in H(2)O-NaCl-glycerol droplets of constant size and composition. These studies showed that the rate of intracellular nucleation was less sensitive to cell dehydration than was the crystal growth rate. The theoretical methods presented may be of use for the design and optimization of freeze-thaw protocols.     

Implications of storage and handling conditions on glass transition and potential devitrification of oocytes and embryos

Devitrification, the process of crystallization of a formerly crystal-free, amorphous glass state, can lead to damage during the warming of cells. The objective of this study was to determine the glass transition temperature of a cryopreservation solution typically used in the vitrification, storage, and warming of mammalian oocytes and embryos using differential scanning calorimetry. A numerical model of the heat transfer process to analyze warming and devitrification thresholds for a common vitrification carrier (open-pulled straw) was conducted. The implications on specimen handling and storage inside the dewar in contact with nitrogen vapor phase at different temperatures were determined. The time required for initiation of devitrification of a vitrified sample was determined by mathematical modeling and compared with measured temperatures in the vapor phase of liquid nitrogen cryogenic dewars. Results indicated the glass transition ranged from −126 °C to −121 °C, and devitrification was initiated at −109 °C. Interestingly, samples entered rubbery state at −121 °C and therefore could potentially initiate devitrification above this value, with the consequent damaging effects to cell survival. Devitrification times were calculated considering an initial temperature of material immersed in liquid nitrogen (−196 °C), and two temperatures of liquid nitrogen vapors within the dewar (−50 °C and −70 °C) to which the sample could be exposed for a period of time, either during storage or upon its removal. The mathematical model indicated samples could reach glass transition temperatures and undergo devitrification in 30 seconds. Results of the present study indicate storage of vitrified oocytes and embryos in the liquid nitrogen vapor phase (as opposed to completely immersed in liquid nitrogen) poses the potential risk of devitrification. Because of the reduced time-handling period before samples reach critical rubbery and devitrification values, caution should be exercised when handling samples in vapor phase.     

Vitrification of human monocytes

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

Effects of four disaccharides on nucleation and growth of ice crystals in concentrated glycerol aqueous solution

Devitrification has been determined to be one of the major causes of cell death in cryopreservation by vitrification method. Reliable quantification of the nucleation and growth of ice crystals of devitrification is of great importance for the optimization of the vitrification solutions. In the present study, cryomicroscopy was used to investigate the nucleation and growth of ice crystals in concentrated glycerol aqueous solution (60 wt%) in the presence of sucrose, trehalose, maltose and lactose. Results showed that sucrose rather than trehalose seems to be the most effective one to inhibit the nucleation and ice growth, despite the excellent inhibitory ability of trehalose on ice growth that has been confirmed in many researches. Hence, for ice inhibition, sucrose was a more effective disaccharide additive to suppress nucleation and growth of ice crystals that occurred during devitrification in concentrated glycerol solutions.     

Devitrification in glass-forming aqueous solutions

The process by which a metastable glass, or the supercooled liquid obtained by heating the glass above its glass transition temperature, forms the stable crystalline phase or phases is generally termed devitrification. In aqueous-solution glasses the devitrification process has been found to consist mainly of the nucleation and growth of a large number of ice particles and is often most rapid at compositions near the water-rich edge of the glass-forming region of concentrations. This, unfortunately, is also the main regions of interest in the cryobiological application of these glass-forming solutions, and hence a knowledge of devitrification and how best to minimize or avoid it becomes important to this work. In this paper our experimental and theoretical knowledge of the devitrification process in aqueous and other glass-forming systems will be reviewed. Recent experimental and theoretical simulation work will also be discussed. In principle devitrification can be substantially avoided by sufficiently rapid heating; hence the purpose of the simulations is to allow the extrapolation of the experimental data into regions of high heating rates (> 100 °C min⁻¹) which are inaccessible to current experimental observation but may nonetheless be useful in the cryobiological application.     

Measurement of essential physical properties of vitrification solutions

Vitrification is an "ice-free" cryopreservation method that has rapidly developed in recent years and might become the method of choice for oocyte cryopreservation. Five sources of damage should be considered when attempting to achieve successful oocyte cryopreservation by vitrification: (1) Solution effects (2) Crystallization (3) Glass fractures (4) Devitrification and recrystallization (5) Chilling injury. The probability of successful vitrification depends on three major factors: viscosity of the sample; cooling and warming rates; and sample volume. One of the problems associated with the vitrification solution is that it may contain high concentrations of cryoprotectants (CP), which can damage the cells through chemical toxicity and osmotic shock. In the present study, we examined the principal parameters associated with successful vitrification, and attempted to compose guidelines to the most important aspects of the vitrification process. The first step was the selection of a suitable and least toxic vitrification solution. We then evaluated the effects of cooling rate and volume on the probability of vitrification. Reduction of the sample volume, combined with accelerated cooling, enabled reduction of the CP concentration. However, in practice, a delicate balance must be maintained among all the factors that affect the probability of vitrification in order to prevent crystallization, devitrification, recrystallization, glass fractures and chilling injury.     

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage,access and transport

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me₂SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me₂SO and EG, respectively. Concentration changes in EG/Me₂SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.     

Calorimetric determination of inactivation parameters of micro-organisms

AIMS: This study aimed to apply differential scanning calorimetry (DSC) to evaluate the thermal inactivation kinetics of bacteria. METHODS AND RESULTS: The apparent enthalpy (DeltaH) of Escherichia coli cells was evaluated by a temperature scan in a DSC after thermal pretreatment in the calorimeter to various temperatures between 56 and 80 degrees C. Conventional semilogarithmic survival curve analysis was combined with a linearly increasing temperature protocol. Calorimetrically determined D and z values were compared to those obtained from plate count data collected under isothermal conditions to validate the new approach. CONCLUSIONS: The calculated D values using both apparent enthalpy and viability data for cells heat treated in the DSC were similar to the D values obtained from isothermal treatment. Temperatures for 1 through 10-log microbial population reductions, calculated from plate count and enthalpy data, were in agreement within 0.5-2.4 degrees C at a 4 degrees C min-1 heating rate. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel calorimetric method provides an approach to obtain accurate and reproducible kinetic parameters for inactivation. The calorimetric method here described is time efficient and is conducted under conditions similar to food processing conditions.     

Effect of the rate of temperature increase of the dynamic method on the heat tolerance of fishes

Heat tolerance is commonly determined by exposing organisms to increasing temperatures until they show symptoms of thermal stress or death. Here we carried out an experiment on a blenny species (Acantemblemaria hancocki; Pisces: Chaenopsidae) and reviewed the literature to evaluate the extent to which variations in the rate at which temperature is increased in experimental trials affects thermal tolerance of fishes. For the blenny species, we found that thermal tolerance decreases significantly from an intermediate heating rate of ∼1 °C/h towards quicker and slower heating rates. In the literature we found very few comparisons of thermal tolerance among heating rates (i.e. eight fish species) and although such comparisons were done over narrow ranges of heating rates, overall they appear to follow the pattern described for the blenny species. We discuss a variety of factors including variations in the levels of acclimation, energy use and body quality among heating rates as the causes for this pattern. However, available data are still limited and further research will be necessary to determine the generality and causes of the pattern we found here. Nevertheless, our results indicate the need for caution in the extrapolation of thermal tolerance data when assessing the tolerance of organisms to environmental phenomena that vary in their rates of warming.     

Vitrification of rabbit tissues with propylene glycol and trehalose

A previous study had suggested the use of a mixture of propanediol and trehalose for the preservation of tissues by vitrification. In this paper, we describe experiments in which stepwise procedures were developed for adding these cryoprotectants to high final concentrations in two rabbit tissues—carotid artery and cornea. The tissue concentration of the additives was measured at the end of each step so that the temperature of the next step could be chosen to reduce toxicity but avoid freezing. This process was arrested when a concentration had been reached that should permit vitrification if the tissues were cooled rapidly to −175 °C. They were stored at that temperature; warmed rapidly by conduction; the cryoprotectants removed by stepwise dilution; and appropriate active functions measured. These were contraction and relaxation for arteries and endothelial integrity and ability to control stromal swelling for the corneas. In control experiments the exposure and functional assays were carried out without vitrification. It was shown that the tissue concentration of propanediol was 33%w/w in artery and 30% in cornea. These permitted cooling to −175 °C without freezing but devitrification occurred during the warming of the arteries, though not of the corneas, despite the lower tissue concentration reached in the cornea. The function of the vitrified arteries was severely reduced but the endothelium of the corneas was substantially intact although we were unable to demonstrate any ability to control stromal swelling during normothermic perfusion. It appears that concentrations of cryoprotectants sufficient to prevent freezing in these tissues during cooling were well tolerated so long as appropriate stepwise means of addition and removal were used. Devitrification during warming remained a major problem with arteries, but not with corneas. We suggest that the composition of the aqueous phase in the tissue with respect to components other than the vitrifying agents may be crucial here and that the search for agents that will suppress devitrification is an important avenue for further study.     

Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization

Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth.     

Thermal analysis of marginal conditions to facilitate cryopreservation by vitrification using a semi-empirical approach

This study aims at the thermal analysis of marginal conditions leading to cryopreservation by vitrification, which appears to be the only alternative for indefinite preservation of large-size tissues and organs. The term “marginal conditions” here refers to cooling rates in close range with the so-called critical cooling rate, above which crystallization is avoided. The analysis of thermal effects associated with partial crystallization during vitrification is associated with the coupled phenomena of heat transfer and kinetics of crystallization. This study takes a practical, semi-empirical approach, where heat transfer is analyzed based on its underlying theoretical principles, while the thermal effects associated with partial crystallization are taken into account by means of empirical correlations. This study presents a computation framework to solve the coupled problem, while presenting a proof-of-concept for DP6 as a representative cryoprotective agent. The thermal effects associated with crystallization at various relevant cooling rates are measured in this study by means of differential scanning calorimetry. Results of this study demonstrate that, due to the thermal effects associated with partial crystallization, the cooling rate at the center of a large organ may lag behind the cooling rate in its surroundings under some scenarios, but may also exceed the surroundings cooling rate in other scenarios, leading to counter-intuitive effects associated with partial crystallization.     

Calorimetric study of crystal growth of ice in hydrated methemoglobin and of redistribution of the water clusters formed on melting the ice.

Calorimetric studies of the melting patterns of ice in hydrated methemoglobin powders containing between 0.43 and 0.58 (g water)/(g protein), and of their dependence on annealing at subzero temperatures and on isothermal treatment at ambient temperature are reported. Cooling rates were varied between approximately 1500 and 5 K min-1 and heating rate was 30 K min-1. Recrystallization of ice during annealing is observed at T > 228 K. The melting patterns of annealed samples are characteristically different from those of unannealed samples by the shifting of the melting temperature of the recrystallized ice fraction to higher temperatures toward the value of "bulk" ice. The "large" ice crystals formed during recrystallization melt on heating into "large" clusters of water whose redistribution and apparent equilibration is followed as a function of time and/or temperature by comparison with melting endotherms. We have also studied the effect of cooling rate on the melting pattern of ice with a methemoglobin sample containing 0.50 (g water)/(g protein), and we surmise that for this hydration cooling at rates of > or = approximately 150 K min-1 preserves on the whole the distribution of water molecules present at ambient temperature.     

Simulation of Steady State and Transient Polymer Growth Kinetics. Application to PEEK

Abstract

A simple two-dimensional rate-theory model demonstrates the principal effects of the Sadler/Gilmer theory of isothermal polymer crystallization. In addition it can be used to describe specific polymers by setting its parameters accordingly. We report on recent results of modelling the aromatic thermoplastic poly(aryl-ether-ether-ketone) (PEEK), in particular its isothermal growth rates, lamellar thicknesses and melting point. An extension of this model has made possible for the first time the study of transient processes such as heating scans and annealing. We report on first results.     

Une évaluation des besoins en transplantation d'organes au Québec.

OBJECTIVES: To evaluate the demand for organs for transplantation and to recommend a reorganization of transplantation services in Quebec. DESIGN: Retrospective study. SETTING: Province of Quebec, 1988 to 1992. PATIENTS: All patients on waiting lists for organ transplantation and patients who received transplants registered in national data banks. MAIN OUTCOME MEASURES: The actual annual demand for organ transplantation and the rate of transplantations performed. RESULTS: The rates of heart transplantation were lower than the actual annual demand, which resulted in many patients dying while awaiting transplantation. The actual annual demand for heart transplantation decreased during the last 5 years from 10.9 per million people in 1987 to 6.7 in 1992. The rates of heart transplantation in Quebec were higher than the Canadian average. The actual demand for lung transplantation was only 2.9 per million people on average in 1992. Demand for liver transplantation increased annually, reaching 8.6 per million in 1992. The rate of transplantation increased likewise but remained insufficient. The demand for kidney transplantation reached 27.2 per million people in 1992, and the transplantation rate was 17.8. CONCLUSIONS: Taking into account the actual demand for and supply of organ transplantation, to insure high-quality service and to control costs associated with organ transplantation, we recommend that the present system in Quebec be reorganized so that transplantations are performed in 12 centres: 7 for kidney transplantation, 2 for hearts, 2 for livers and 1 for lungs.     

Multiple steady-state phenomena within enzyme reactors: The enzyme reaction with two substrates

The theoretical dynamic characteristics of an isothermal continuous flow stirred tank enzyme reactor (CFSTER) operating on two substrates are investigated. Under certain conditions multiple steady states are possible; namely, with an enzyme which binds with the two substrates sequentially. The occurrence of multiple steady states is found to be primarily dictated by three dimensionless parameters which incorporate rate law constants. The global stability of certain steady states is examined by numerically solving the transient material balance on the CFSTER. The effect of recycle on the dynamics of an isothermal plug flow enzyme reactor (PFER) is also studied. A general conclusion indicated by this work is that any open isothermal reaction system wherein the reaction rate law passes through a maximum with increasing substrate concentration and where back mixing occurs with exhibit multiple steady-state behavior in some operating range.     

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.     

Nonisothermal heat resistance determinations with the thermoresistometer Mastia

Aims:  To design and build a thermoresistometer, named Mastia, which could perform isothermal and nonisothermal experiments.
Methods and Results:  In order to evaluate the thermoresistometer, the heat resistance of Escherichia coli vegetative cells and Alicyclobacillus acidoterrestris spores was explored. Isothermal heat resistance of E. coli was characterized by D _60°C = 0·38 min and z =  4·7°C in pH 7 buffer. When the vegetative cells were exposed to nonisothermal conditions, their heat resistance was largely increased at slow heating and fast cooling rates. Isothermal heat resistance of A. acidoterrestris was characterized by D _95°C = 7·4 min and z =  9·5°C in orange juice. Under nonisothermal conditions, inactivation was reasonably well predicted from isothermal data.
Conclusions:  The thermoresistometer Mastia is a very suitable instrument to get heat resistance data of micro-organisms under isothermal and nonisothermal treatments.
Significance and Impact of the Study:  The thermoresistometer Mastia can be a helpful tool for food processors in order to estimate the level of safety of the treatments they apply.