Formation of ice microparticles in cryoprotective solutions

The formation of ice microparticles in 0.2-mm thin layers of aqueous solutions of cryoprotective agents cooled to temperatures reaching–196°C was studied. The shape and size of ice microparticles were determined by cryomicroscopy. At temperatures below 0°C, the formation of solid ice masses was observed; further cooling caused ice fracturing induced by emerging thermomechanical stresses and the formation of microparticles. The shape and size of the particles depended on the composition of the frozen solution and on the rate of cooling. The components of cryoprotective solutions (cryoprotectants, egg yolk, sugars, and lipids) significantly changed the shape and size of the ice microparticles.     

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.     

Peculiarities of state diagrams of aqueous solutions of cryoprotective agents

The phase transitions in aqueous solutions of glycerol and PEO-1500 within the temperature range of +30 to −150 °C have been studied using the methods of thermoplastic analysis and volumetric scanning tensodilatometry. We present the revealed phenomenon of cluster cyrystallization of these solutions as well as principles of describing this phenomenon using state diagrams, containing the intervals of concentration corresponding to the existence of amorphous and cryocolloid fractions. We note that for the cryocolloid fraction, a low temperature association of molecules of cryoprotective agents leads the formation of ice nanocrystals either close to or directly inside the aggregations. These fractions exist in cooled cryoprotective solutions near the vitrification temperatures of the liquid phase and may contribute to the initiation of damaging events in cryopreserved biological systems. Our data may be helpful in explaining the peculiarities observed during crystallization of cryoprotective solutions and may further contribute to a broader understanding of the principles of protection and protocol optimization of biological materials at temperatures approaching vitrification.     

Comparative crystallization behavior of polymers and aqueous solutions.

This paper gives a short introduction to the modes of crystallization of polymers and shows, by a series of micrographs, that the resulting morphology is very similar to that of ice crystals growing from aqueous solutions. The similarity is explained by similar conditions of nucleation and growth, leading in both cases to a hindered and partial crystallization. It is shown that the resulting crystal patterns can be qualitatively explained by estimating the relative growth rates in different directions on a developing crystal face as a function of supercooling and concentration.     

Influence of lipids on ice formation in cryoprotective media

Cryomicroscopic analysis demonstrated that two lipid preparations from marine vertebrates (<0.1%) and liposomes prepared from rainbow trout sperm lipids (<0.5%) efficiently hindered the growth of ice crystals during freezing of multicomponent cryoprotective media used for trout sperm cryopreservation. At higher lipid concentrations, crystals either did not form at all or had altered shape and blurred boundaries. Addition of egg yolk (10%) together with these lipids increased the size of crystal structures and markedly changed their shape.     

Hot spot kinetics of the sonolysis of aqueous acetate solutions

Water and acetate solutions were irradiated under argon by 300 kHz ultrasonic waves. Oxygen was found to be generated besides the products H2 and H2O2, already known. In the presence of acetate the O2 yield decreased rapidly while that of H2O2 decreased more slowly. Succinic acid was found as a product of the attack of OH radicals on acetate. Appreciable amounts of glyoxylic and glycolic acid and smaller amounts of formaldehyde and carbon dioxide were also detected. They resulted from the reaction of sonolytically generated oxygen with CH2CO2- radicals, produced upon attack of OH on acetate. Methane was a minor product of sonolysis. At acetate concentrations above 0.4 mol dm-3 CO2 and CO became the predominant products of sonolysis. This is explained by a second kind of action of ultrasound on dissolved acetate, i.e. by a thermal decomposition. This decomposition is possibly facilitated by radical attack on acetate. The results are discussed in terms of a 'structured hot spot' model, in which three regions for the occurrence of chemical reactions are postulated: a hot gaseous nucleus, an interfacial region with radial gradient in temperature and local radical density; and the bulk solution at ambient temperature.     

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.     

Nucleation rates of ice in undercooled water and aqueous solutions of polyethylene glycol

Differential calorimetry has been employed in two different instrumental modes to measure ice nucleation rates in undercooled water and aqueous solutions of polyethylene glycol (PEG) as a function of temperature. The results are consistent with the classical theory of homogeneous nucleation kinetics. It appears that the inhibition of ice nucleation by PEG results mainly from a marked perturbation of the diffusional freedom of water molecules by the polymer.     

Anomalous exchange kinetics of peptide amide protons in aqueous solutions

Reports concerning anomalous rates of exchange of some amides in oxytocin, alumichrome, and gramicidin S are reexamined through systematic analysis of the exchange data as a function of pH and primary structure. It is shown that such an analysis can provide useful information on secondary structure when the degree of hydrogen bonding to both the NH undergoing exchange and the neighboring carbonyl group are taken into consideration.     

Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions

Ice formation on warming is of comparable or greater importance to ice formation on cooling in determining survival of cryopreserved samples. Critical warming rates required for ice-free warming of vitrified aqueous solutions of glycerol, dimethyl sulfoxide, ethylene glycol, polyethylene glycol 200 and sucrose have been measured for warming rates of order 10–10⁴ K/s. Critical warming rates are typically one to three orders of magnitude larger than critical cooling rates. Warming rates vary strongly with cooling rates, perhaps due to the presence of small ice fractions in nominally vitrified samples. Critical warming and cooling rate data spanning orders of magnitude in rates provide rigorous tests of ice nucleation and growth models and their assumed input parameters. Current models with current best estimates for input parameters provide a reasonable account of critical warming rates for glycerol solutions at high concentrations/low rates, but overestimate both critical warming and cooling rates by orders of magnitude at lower concentrations and larger rates. In vitrification protocols, minimizing concentrations of potentially damaging cryoprotectants while minimizing ice formation will require ultrafast warming rates, as well as fast cooling rates to minimize the required warming rates.     

Influence of lipids on ice formation during the freezing of cryoprotective medium

The influence of lipids on ice formation during the freezing of cryoprotective medium for the semen of rainbow trout has been studied by the cryomicroscopy technique. It was shown that the lipids extracted from marine vertebrates and liposomes from the lipids of trout sperm effectively inhibit the ice formation in cryoprotective solutions during freezing, fundamentally changing the form and size of ice crystals. At high concentrations of lipids, either the crystallization does not occur in the cryoprotective medium or, even if ice crystals are formed, they have a broken shape and blurred borders. The addition of egg yolk sligthly increases the size and essentially changes the shape of ice crystals during the freezing of solution.     

Congelation of cryoprotective solutions and cryopreservation of fish sperm

A study was made of the formation of ice microparticles (IMP) during freezing of cryoprotective media and the survival of fish sperm in cryopreservation. The IMP shape and size were determined by cryomicroscopy. It is shown that with an increase of the cooling rate the IMP area and perimeter decrease. Under ultrafast cooling (3000–4000 deg/min) in a thin layer (0.1 mm) the medium is vitrified. Additional components (egg yolk, sugars and lipids) substantially alter the shape and size of IMP. The intactness of sperm after cryopreservation does not always correlate with the IMP size and shape in the frozen cryoprotective solution. Formation of rounded particles with blurred boundaries correlates with sperm survival in cryopreservation.     

Ultrasonic study of the kinetics of counterion site binding in aqueous solutions of polyelectrolytes

The excess ultrasonic absorption due to counterion binding has been studied as a function of frequency for a series of polysalts in the range 1–150 MHz. All the relaxation spectra can be represented by a relaxation equation with two relaxation terms. The relaxation frequencies appear concentration independent and the relaxation amplitudes seem proportional to concentration. The low frequency relaxation process appears to depend mainly on the nature of the counterion while the high frequency relaxation process seems to be mostly dependent on the nature of the polyion. These results are quite similar to those obtained in ultrasonic studies of ion-pairing in solutions of divalent sulfates. The kinetic model used for the quantitative analysis of these results has been modified for polysalts through introducing the concept of“counterion condensation”. In this modified model the excess absorption is assigned to the perturbation by the ultrasonic waves of the equilibria between the three states of hydration of ths complex formed by a counterion and that part of the polyion where it is bound. Analytical expressions of the relaxation amplitudes have been derived using classical procedures for this modified kinetic model. In the case of cobalt-polyphosphate (Co-PP), the ultrasonic data together with the results of NMR measurements on either Co²⁺ or Co-PP have been used for the evaluation of the volume changes, the rate constants and the fractions of counterions in the three states of hydration involved in the binding equilibria. The volume changes obtained in this manner depend only slightly on the method of calculation and appear to be consistent with volume changes for outer-sphere and inner-sphere complex formation. These results are discussed.