Comparison with the theory of the kinetics and extent of ice crystallization and of the glass-forming tendency in aqueous cryoprotective solutions

The glass-forming tendency and stability of the wholly amorphous state of various cryoprotective solutions has been studied in recent years (5-10, 20). A lot of experimental data including heats of ice crystallization at various cooling rates and devitrification temperatures have been given. In this article these data have been compared with analytical expressions using a semiempirical model. The theoretical variation of the total quantity of ice crystallized with the cooling rate fits very well with the experimental data, adjusting only one parameter. Using the same model, theoretical differential scanning calorimeter (DSC) crystallization peaks have been obtained for cooling or rewarming. The general shape, height, and width of the theoretical peaks are very similar to those of the experimental peaks. The differences are comparable to the random variations of the experimental peaks from one experiment to another. The analytical expressions obtained here could be used to study the relationship between the kinetics of ice crystallization and cell damage when ice crystallizes incompletely inside or outside the cells. These expressions have been applied to ice crystallization for applications in cryobiology. But they could also probably be used in other fields of research such as crystallization from silicates or other mineral or organic glasses.     

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 large tissues with dielectric warming: biological problems and some approaches to their solution

If large pieces of tissue and organs are to be successfully stored at low temperatures, some means must be found to minimize the disruption of extracellular structures by the ice that develops during conventional cryopreservation methods. The use of sufficiently high concentrations of cryoprotectant (CPA) to vitrify rather than freeze the tissue is a possible solution to this problem, and the retention of function of embryos and elastic arteries after vitrification suggests that some cells and tissues at least can withstand exposure to the high concentrations of CPA necessary for this process to occur. There are, however, additional problems in applying vitrifying techniques to bulky tissues and organs. These are related to the additional time required for tissue equilibration of CPA to occur and the consequences for toxic injury, the difficulty in achieving sufficiently rapid and uniform cooling rates to produce the required glassy state, and the even more rapid and uniform warming rates that are necessary to avoid devitrification. Non-uniformity of temperature will increase the risk of mechanical stresses and fractures developing in the glass during rapid warming. This paper reviews possible strategies and the progress that has been made in overcoming these problems. This will include the permeation of CPA mixtures into whole tissues and possibilities for reducing their toxicity by the inclusion of adjuncts such as ice inhibitors and sugars. The warming of tissues by dielectric heating is currently the only practical means by which sufficiently rapid rates can be achieved in bulky tissues given that the tolerable limits of CPA concentration will most likely be insufficient to prevent the development of ice nuclei during cooling. The biological effects of microwaves are reviewed and their effectiveness in producing the required uniformity in warming of tissue models of various shapes are discussed.     

Experimental dissection of devitrification in aqueous solutions of 1,3-butanediol

Devitrification is a major problem which must be overcome for successful organ cryopreservation. Devitrification can be initiated on fracture planes and on bubbles, but the focus of attention here is on devitrification by ordinary heterogeneous and homogeneous mechanisms, which are the most relevant for organ preservation by vitrification. The purpose of the present studies was to define the devitrification process: to determine nucleation rates, ice-crystal growth rates, and the distribution of ice-crystal size and to evaluate the applicability of existing quantitative models of these processes which have successfully approximated the behavior of other aqueous systems. The present work was done using differential scanning calorimetry and cryomicroscopy. The amount of ice formed has been estimated for highly concentrated solutions. Kinetic parameters are presented here for isothermal conditions and continuous heating rate experiments. The classical theory based on the Johnson-Avrami equation has been evaluated and the results are compared with the theory of Boutron. The agreement is good for the continuous heating rate conditions, but results differ for the isothermal conditions.     

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.     

Physical aspects of vitrification in aqueous solutions

Vitrification is the process by which a liquid solidifies at temperatures usually far below the normal freezing point, but without the formation of any crystalline phase. The liquid has formed a glass. Occasionally, when the liquid consists of a solution, one of the components freezes to form a crystalline phase during cooling, but the remainder of the solution vitrifies. The product is then a partially crystallized glass. Glass formation, as a feature of aqueous solutions, either of the whole solution or of the remaining fraction after crystallization of ice, is discussed. We focus on the general principles involved in glass formation and also discuss in detail the effect of pressure on the nucleation and vitrification of the solution. In particular we look at the physical processes involved as well as the chemical aspects of the solutes which can be used in vitrifiable aqueous solutions. In any application of vitrification of aqueous solutions the material properties of the resultant glass are also important; these are also briefly considered. Recent work concerning the nature of the devitrification (crystallization during warming) event at high pressures is detailed.     

Glass-forming property of the system diethyl sulphoxide/water and its cryoprotective action on Escherichia coli survival

In this work the thermal properties of diethyl sulphoxide (Et2SO), as well as its cryoprotective ability are studied and related to other well-known cryoprotectant substances, like dimethyl sulphoxide (Me2SO). We have investigated the thermal properties of Et2SO/water systems using Differential Scanning Calorimetry at a very low heating/cooling rate (2 degrees C/min). Liquid/solid or glassy/crystalline transitions have been observed only for the solutions with content of Et2SO ranging from 5 up to 40% w/w and/or greater than 85%. In the 45-75% w/w Et2SO range we have found a noticeable glass-forming tendency and a great stability of the amorphous state to the reheating. In samples with Et2SO content ranging from 80 to 85%, we observed a great stability of the glass forming by cooling, but a lesser stability to the subsequent reheating. The glass-forming tendency of these solutions is discussed in terms of existing competitive interactions between molecules of Et2SO, on the one hand, and Et2SO and water molecules, on the other hand. The results are well explainable on the basis of the model structure of water/Et2SO solutions, deduced by Raman and infrared studies [J. Mol. Struct. 665 (2003) 285-292]. The cryoprotective ability of Et2SO on Escherichia coli survival has been also investigated, and a comparison among Et2SO and other widely used cryoprotectants, like Me2SO and glycerol has been done. Survival of E. coli, determined after freezing-thawing process, was maximal at 45% w/w Et2SO (more than 85% viability). It should be noted that at the same concentration the survival is only about 35% in the presence of Me2SO and not more than 15% in the presence of glycerol. These features are well consisted with the glass-forming properties of Et2SO.     

Characterization of cryobiological responses in TF-1 cells using interrupted freezing procedures

Cryopreservation currently is the only method for long-term preservation of cellular viability and function for uses in cellular therapies. Characterizing the cryobiological response of a cell type is essential in the approach to designing and optimizing cryopreservation protocols. For cells used in therapies, there is significant interest in designing cryopreservation protocols that do not rely on dimethyl sulfoxide (Me₂SO) as a cryoprotectant, since this cryoprotectant has been shown to have adverse effects on hematopoietic stem cell (HSC) transplant patients. This study characterized the cryobiological responses of the human erythroleukemic stem cell line TF-1, as a model for HSC. We measured the osmotic parameters of TF-1 cells, including the osmotically-inactive fraction, temperature-dependent membrane hydraulic conductivity and the membrane permeability to 1 M Me₂SO. A two-step freezing procedure (interrupted rapid cooling with hold time) and a graded freezing procedure (interrupted slow cooling without hold time) were used to characterize TF-1 cell recovery during various phases of the cooling process. One outcome of these experiments was high recovery of TF-1 cells cryopreserved in the absence of traditional cryoprotectants. The results of this study of the cryobiology of TF-1 cells will be critical for future understanding of the cryobiology of HSC, and to the design of cryopreservation protocols with specific design criteria for applications in cellular therapies.     

Devitrification and recrystallization of nanoparticle-containing glycerol and PEG-600 solutions

Nanoparticles in solution offer unique electrical, mechanical and thermal properties due to their physical presence and interaction with the state of dispersion. This work is aimed to study the effects of hydroxyapatite (HA) nanoparticles on the devitrification and recrystallization events of two important cryoprotective solutions used in cell and tissue preservation namely glycerol (60% w/w) and PEG-600 (50% w/w). HA nanoparticles (20, 40 or 60 nm) were incorporated into solutions at the content of 0.1% or 0.5% (w/w), and were studied by differential scanning calorimeter (DSC) and cryomicroscopy. The presence of nanoparticles does not change the glass transition temperatures and melting temperatures of quenched solutions, but significantly affects the behavior of devitrification and recrystallization upon warming. Cryomicroscopic investigation showed the complex interactions among solution type, nanoparticle size and nanoparticle content, which apparently influence ice crystal growth or recrystallization in the quenched dispersions. These findings have significant implications for biomaterial cryopreservation, cryosurgery, and food manufacturing. The complexity of ice crystal growth kinetics in nanoparticle-containing dispersions remains to be poorly understood at the moment.     

Melting point equations for the ternary system water/sodium chloride/ethylene glycol revisited

Partial phase diagrams are of considerable utility in the development of optimized cryobiological procedures. Recent theoretical predictions of the melting points of ternary solutions of interest to cryobiology have caused us to re-examine measurements that our group made for the ethylene-glycol–sodium chloride–water phase diagram. Here we revisit our previous experiments by measuring melting points at five ethylene-glycol to sodium chloride ratios (R values; R = 5, 10, 15, 30, and 45) and five levels of concentration for each ratio. Melting points were averaged from three measurements and plotted as a function of total solute concentration for each R value studied. The new measurements differed from our original experimental values and agreed with predicted values from both theoretical models. Additionally, the data were fit to the polynomial described in our previous report and the resulting equation was obtained:T_m=(38.3-2.145×10^-1R)w+(81.19-2.909×10^-1R)w²,where w is the total solute mass fraction. This new equation provided good fits to the experimental data as well as published values and relates the determined polynomial constants to the R value of the corresponding isopleths of the three dimensional phase diagram, allowing the liquidus curve for any R value to be obtained.     

The role of stretching in slow axonal transport

Axonal stretching is linked to rapid rates of axonal elongation. Yet the impact of stretching on elongation and slow axonal transport is unclear. Here, we develop a mathematical model of slow axonal transport that incorporates the rate of axonal elongation, protein half-life, protein density, adhesion strength, and axonal viscosity to quantify the effects of axonal stretching. We find that under conditions where the axon (or nerve) is free of a substrate and lengthens at rapid rates (>4 mm day⁻¹), stretching can account for almost 50% of total anterograde axonal transport. These results suggest that it is possible to accelerate elongation and transport simultaneously by increasing either the axon's susceptibility to stretching or the forces that induce stretching. To our knowledge, this work is the first to incorporate the effects of stretching in a model of slow axonal transport. It has relevance to our understanding of neurite outgrowth during development and peripheral nerve regeneration after trauma, and hence to the development of treatments for spinal cord injury.     

Metallic support films reduce optical heating in cryogenic correlative light and electron tomography

Super-resolved cryogenic correlative light and electron tomography is an emerging method that provides both the single-molecule sensitivity and specificity of fluorescence imaging, and the molecular scale resolution and detailed cellular context of tomography, all in vitrified cells preserved in their native hydrated state. Technical hurdles that limit these correlative experiments need to be overcome for the full potential of this approach to be realized. Chief among these is sample heating due to optical excitation which leads to devitrification, a phase transition from amorphous to crystalline ice. Here we show that much of this heating is due to the material properties of the support film of the electron microscopy grid, specifically the absorptivity and thermal conductivity. We demonstrate through experiment and simulation that the properties of the standard holey carbon electron microscopy grid lead to substantial heating under optical excitation. In order to avoid devitrification, optical excitation intensities must be kept orders of magnitude lower than the intensities commonly employed in room temperature super-resolution experiments. We further show that the use of metallic films, either holey gold grids, or custom made holey silver grids, alleviate much of this heating. For example, the holey silver grids permit 20× the optical intensities used on the standard holey carbon grids. Super-resolution correlative experiments conducted on holey silver grids under these increased optical excitation intensities have a corresponding increase in the rate of single-molecule fluorescence localizations. This results in an increased density of localizations and improved correlative imaging without deleterious effects from sample heating.     

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.     

Melting, glass transition, and apparent heat capacity of α-d-glucose by thermal analysis

The thermal behaviors of α-d-glucose in the melting and glass transition regions were examined utilizing the calorimetric methods of standard differential scanning calorimetry (DSC), standard temperature-modulated differential scanning calorimetry (TMDSC), quasi-isothermal temperature-modulated differential scanning calorimetry (quasi-TMDSC), and thermogravimetric analysis (TGA). The quantitative thermal analyses of experimental data of crystalline and amorphous α-d-glucose were performed based on heat capacities. The total, apparent and reversing heat capacities, and phase transitions were evaluated on heating and cooling. The melting temperature (T_m) of a crystalline carbohydrate such as α-d-glucose, shows a heating rate dependence, with the melting peak shifted to lower temperature for a lower heating rate, and with superheating of around 25 K. The superheating of crystalline α-d-glucose is observed as shifting the melting peak for higher heating rates, above the equilibrium melting temperature due to of the slow melting process. The equilibrium melting temperature and heat of fusion of crystalline α-d-glucose were estimated. Changes of reversing heat capacity evaluated by TMDSC at glass transition (T_g) of amorphous and melting process at T_m of fully crystalline α-d-glucose are similar. In both, the amorphous and crystalline phases, the same origin of heat capacity changes, in the T_g and T_m area, are attributable to molecular rotational motion. Degradation occurs simultaneously with the melting process of the crystalline phase. The stability of crystalline α-d-glucose was examined by TGA and TMDSC in the melting region, with the degradation shown to be resulting from changes of mass with temperature and time. The experimental heat capacities of fully crystalline and amorphous α-d-glucose were analyzed in reference to the solid, vibrational, and liquid heat capacities, which were approximated based on the ATHAS scheme and Data Bank.     

Demography,recombination hotspot intensity,and the block structure of linkage disequilibrium

BACKGROUND: Effective gene mapping based on genetic association data will require detailed knowledge of patterns of linkage disequilibrium (LD) in human populations. It has been recently suggested that linkage disequilibrium in humans may be organized in a block-like structure, with islands of high LD separated by regions of rapid breakdown of LD due to recombination hotspots. The experimental data to date, however, are limited, and fundamental questions remain about the implications of recombination rate heterogeneity. Here, we use computer simulations to evaluate how such heterogeneity influences patterns of LD, and we develop formal criteria to assess whether the patterns are functionally block like in the context of association mapping.RESULTS: Our analyses suggest that, even in models of extreme recombination rate heterogeneity, some human populations will have a functionally block-like structure to the pattern of LD, but others will not, depending on their precise demographic histories. In fact, for many models, we find that, following an LD-generating event, populations may move through discrete phases that can be functionally described as pre-block, block, and post-block. An analysis of observed and expected patterns of LD surrounding hotspots within the MHC Class II region confirms these theoretical expectations.CONCLUSIONS: Even if highly punctuated patterns of recombination are the rule, patterns of LD are still likely to show differences among populations and among genomic regions that are of practical importance in the design of genetic association studies. The notion that the average extent of LD is a useful concept for the design of association studies must be abandoned in light of the experimental and theoretical evidence.     

Effects of tissue trauma on the characteristics of microdialysis zero-net-flux method sampling neurotransmitters

Microdialysis has been used for studying neurochemistry in brain regions that respond to afferent inputs or administered drugs. As the knowledge derived from and concerning microdialysis grows, so do the concerns over its invasiveness and, hence, the credibility of resulting data. Recent experimental and theoretical studies impugned the validity of the microdialysis zero-net-flux (ZNF) method in measuring brain extracellular neurotransmitters, suggesting that the tissue trauma resulting from probe implantation seriously compromises its worth. This paper developed a theoretical model to study the influences of two categories of tissue trauma on microdialysis ZNF operation: (1) morphological alterations in tissue extracellular structure and (2) physiological impairment of neurotransmitter release and uptake processes. Model results show that alterations of tissue extracellular structure negligibly affect the accuracy of the ZNF method in determining the basal level of extracellular neurotransmitter but do affect the fundamental characteristics of microdialysis: the extraction efficiency and relative recovery. An inhibited or damaged neurotransmitter uptake process always decreases the efficiency of microdialysis extraction, but rise of the relative recovery of neurotransmitters with the same uptake inhibition/damage occurs only when there is far more damage to the neurotransmitter release than to the uptake process in the tissue. A criterion for this rising trend of microdialysis relative recovery is discussed in terms of trauma parameters and neurotransmitter uptake inhibition.     

Effect of incubation temperature after devitrification on quality parameters in human sperm cells

Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 °C) and (ii) incubated at 37 °C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (ΔΨM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and ΔΨM compared to the control, while the incubation at 37 °C promoted a decrease in these parameters. The ROS levels were increased at both incubation conditions. The progressive motility was decreased in all experimental groups and the decrease was more pronounced under incubation at RT. No increase in phosphatidylserine externalization was observed. In conclusion, prior to use in assisted reproduction procedures, devitrified spermatozoa at RT conserve a better viability and ΔΨM than at 37 °C.     

Principes de préparation et de congélation des spermatozoïdes testiculaires humains

The advantages and feasibility of human testicular spermatozoa cryoconservation for intracytoplasmic sperm injection (ICSI) have now been clearly demonstrated. However, the freezing protocol is based on empirical knowledge obtained from freezing of ejaculated spermatozoa. Testicular spermatozoa may not be fully mature gametes and may also be retrieved in only limited quantities. Little research has been conducted to determine whether they have the same cryobiological requirements as ejaculated spermatozoa. A better understanding of their cryobiological features and assessment of possible subcellular changes after thawing would help to optimize testicular preparations for cryopreservation (whole biopsies, seminiferous tubules, shredded suspension, single spermatozoa, etc.), freezing-thawing procedure, freezing media, and storage. Finally, there is a growing need for welldefined criteria (nuclear quality, etc.) to evaluate the tolerance of testicular spermatozoa to freezing-thawing procedure for ICSI     

生物催化剂发现与改造的研究进展

生物催化是指将酶或生物有机体用于有用的化学转化的过程,在人们对传统化学催化的环境影响抱有忧虑的情况下,生物催化提供了一种有吸引力的选择。在过去的几十年里,对生物催化剂的研究每出现一次大的进步,生物催化的发展就会出现一次高潮。因此,生物催化剂的发现与改造已成为当今研究的热点。宏基因组文库技术的出现克服了许多微生物不可培养的障碍,人们能够从自然资源中获得丰富的潜在的生物催化剂。而基于理性设计的分子改造技术的发展,可以使得人们对潜在的生物催化剂进行快速而有效的改造以满足工业化生产的需求。随着生物催化剂发现与改造的手段不断进步,更多的优良生物催化剂得到了广泛的应用,生物催化在工业生产中也得到了更深入的应用。结合作者的研究工作,总结了生物催化剂发现与改良的一些研究进展,以为获得更多优良的、能够实现工业应用的生物催化剂奠定理论基础。