Ice Nucleating Agent Research Articles

Lipid bilayers as potential ice nucleating agents.

Cellular damage is a key issue in the context of cryopreservation. Much of this damage is believed to be caused by extracellular ice formation at temperatures well above the homogeneous freezing point of pure water. Hence the question: what initiates ice nucleation during cryopreservation? In this paper, we assess whether cellular membranes could be responsible for facilitating the ice nucleation process, and what characteristics would make them good or bad ice nucleating agents. By means of molecular dynamics simulations, we investigate a number of phospholipids and lipopolysaccharide bilayers at the interface with supercooled liquid water. While these systems certainly appear to act as ice nucleating agents, it is likely that other impurities might also play a role in initiating extracellular ice nucleation. Furthermore, we elucidate the factors which affect a bilayer's ability to act as an ice nucleating agent; these are complex, with specific reference to both chemical and structural factors. These findings represent a first attempt to pinpoint the origin of extracellular ice nucleation, with important implications for the cryopreservation process.

Ice induction using Snomax® in the dimethyl-sulfoxide-containing aqueous solution for DSC experiments

Controlling the supercooling state during ice crystallization process is important for differential scanning calorimetry (DSC) analysis. In cryopreservation solutions, the control of the nucleation temperature (Tn) is necessary as it allows the simulation of slow-freezing procedures. Herein, the effect of Snomax® on Tn value in aqueous solutions containing dimethyl-sulfoxide (Me2SO) (H2O + 10 %(v/v)Me2SO) was studied. The effect of Me2SO, a cryoprotectant, on nucleation control was investigated by evaluating λhet, which describes the influence of a molecule on the nucleation behaviour of an ice-nucleating agent (INA). Note that λhet is specific to the molecule added in solution and to the maximum melting temperature of the solution. The knowledge of λhet helps evaluate the ranges of the supercooling magnitude at which the INA in solution can trigger nucleation. This study shows how Snomax can be used in DSC experiments using the H2O + 10 %(v/v)Me2SO solution when ice nucleation must be controlled.

Solute- and concentration-dependent heterogeneous ice nucleation behaviors in AgI composite water droplets

Ice nucleation behaviors were investigated by a differential scanning calorimetry (DSC) of the freezing of emulsified solution droplets with silver iodide (AgI) as an ice-nucleating agent. For various aqueous organic solutions studied, notable concentration-dependent ice nucleation behaviors appeared in the presence of AgI: two heterogeneous nucleation temperature (Tf,het) curves and/or anomalous Tf,het logarithmic increases were confirmed as the function of concentrations. The solute adsorption phenomena were considered to trigger these changes. The increase in supercooling degree with increasing concentration tended to be greater for homogeneous nucleation than for both heterogeneous nucleations. A counterexample was found in the dimethyl sulfoxide system.

Why α-Alumina Is an Effective Ice Nucleus

Experiments have shown α-Al2O3 (corundum) to be a very efficient ice-nucleating agent. Molecular dynamics simulations are used to explore the microscopic origins of its ice-nucleating ability. This...

On certain approaches to the control methods development for the precipitation formation processes in convective clouds

The article aims at searching for the optimal way of emission of ice nucleating agent in convective cloud in order to prevent ‎formation of harmful hail by analyzing simulations of this process within a numerical model ‎of cloud‎. The state of the physics of clouds and active influences on them is discussed. It is noted that at the present time studies of the regularities of the formation and development of clouds as a whole begin taking into account their systemic properties. The main directions of research at the next stage of its development are discussed. The features of the existing methods of active action on convective clouds are noted, the main tasks encountered in the development of methods for controlling sedimentation in convective clouds by introducing reagents are formulated. It is noted that research on the development of methods for active influence on clouds should be conducted on the basis of new and more effective approaches, which should be based on the extensive use of mathematical modeling. Some approaches to solving this problem are discussed. According to the authors, the most promising of them are approaches based on the theory of optimal control and bifurcation theory. Some results of numerical modeling of the active effect on convective clouds are given.

Tuning, measurement and prediction of the impact of freezing on product morphology: A step toward improved design of freeze-drying cycles

The control of freezing is crucial as it can influence the freeze-drying operation as well as the chemical and physical properties of the lyophilized product such as the specific surface area, dissolution rate, and drug stability. In this review article, we focus on two areas related to lyophilization in the pharmaceutical industry: (1) advances in our understanding of the relationship between the freezing conditions, product morphology and drying efficiency, and (2) the control of nucleation in solutions to be frozen. The final objective is to accelerate the transition toward a more robust design of freeze-drying cycles, based on deep knowledge of the phenomena involved rather than on empirical observations. While these topics are not new, they are areas of significant current interest to the community engaged in drying in the pharmaceutical industry.Abbreviations: FDM: freeze-drying microscope; GMP: good manufacturing practice; INA: ice nucleating agent; QbD: quality by design; PRT: pressure rise test; SSA: specific surface area, m2; UIIN: ultrasound-induced ice nucleation; VIN: vacuum induced nucleation; VISF: vacuum induced surface freezing.

Ice induction in DSC experiments with Snomax®

For differential scanning calorimetry (DSC) analysis, controlling the degree of supercooling at which crystallization begins may be required for several studies, experiments, and applications. In this paper, the use of Snomax, an ice nucleating agent (INA), was evaluated to create ice at a desirable temperature range in a DSC aluminum sample pan. The effect of Snomax on the nucleation temperature (Tn) was studied in pure water. Best practices and methods are described in terms of the Tn dependence on three experimental parameters: (i) the Snomax concentration that controls the Tn value for different groups according to the three classes of the Pseudomonas syringae protein aggregates (from which Snomax originates); (ii) the sample volume that affects the presence probabilities of the different INA subpopulations in the solution and that could also favor their deterioration; and (iii) the cooling rate that does not seem to further affect the Tn value. There is no-evidence of time dependence of the nucleation process promoted by Snomax. The presence of artifacts or disturbances introduced by the addition of Snomax into the solution was evaluated. No major disturbances of the thermodynamic characteristics of these solutions were observed with the addition of Snomax below 103 mg L−1 concentration. This underscores the possible use of Snomax for controlling ice nucleation during DSC experiments.

Unravelling the origins of ice nucleation on organic crystals.

Organic molecules such as steroids or amino acids form crystals that can facilitate the formation of ice - arguably the most important phase transition on earth. However, the origin of the ice nucleating ability of organic crystals is still largely unknown. Here, we combine experiments and simulations to unravel the microscopic details of ice formation on cholesterol, a prototypical organic crystal widely used in cryopreservation. We find that cholesterol - which is also a substantial component of cell membranes - is an ice nucleating agent more potent than many inorganic substrates, including the mineral feldspar (one of the most active ice nucleating materials in the atmosphere). Scanning electron microscopy measurements reveal a variety of morphological features on the surfaces of cholesterol crystals: this suggests that the topography of the surface is key to the broad range of ice nucleating activity observed (from -4 to -20 °C). In addition, we show via molecular simulations that cholesterol crystals aid the formation of ice nuclei in a unconventional fashion. Rather than providing a template for a flat ice-like contact layer (as found in the case of many inorganic substrates), the flexibility of the cholesterol surface and its low density of hydrophilic functional groups leads to the formation of molecular cages involving both water molecules and terminal hydroxyl groups of the cholesterol surface. These cages are made of 6- and, surprisingly, 5-membered hydrogen bonded rings of water and hydroxyl groups that favour the nucleation of hexagonal as well as cubic ice (a rare occurrence). We argue that the phenomenal ice nucleating activity of steroids such as cholesterol (and potentially of many other organic crystals) is due to (i) the ability of flexible hydrophilic surfaces to form unconventional ice-templating structures and (ii) the different nucleation sites offered by the diverse topography of the crystalline surfaces. These findings clarify how exactly organic crystals promote the formation of ice, thus paving the way toward deeper understanding of ice formation in soft and biological matter - with obvious reverberations on atmospheric science and cryobiology.

Ice nucleating agents allow embryo freezing without manual seeding

Embryo slow freezing protocols include a nucleation induction step called manual seeding. This step is time consuming, manipulator dependent and hard to standardize. It requires access to samples, which is not always possible within the configuration of systems, such as differential scanning calorimeters or cryomicroscopes. Ice nucleation can be induced by other methods, e.g., by the use of ice nucleating agents. Snomax is a commercial preparation of inactivated proteins extracted from Pseudomonas syringae. The aim of our study was to investigate if Snomax can be an alternative to manual seeding in the slow freezing of mouse embryos. The influence of Snomax on the pH and osmolality of the freezing medium was evaluated. In vitro development (blastocyst formation and hatching rates) of fresh embryos exposed to Snomax and embryo cryopreserved with and without Snomax was assessed. The mitochondrial activity of frozen-thawed blastocysts was assessed by JC-1 fluorescent staining. Snomax didn't alter the physicochemical properties of the freezing medium, and did not affect embryo development of fresh embryos. After cryopreservation, the substitution of manual seeding by the ice nucleating agent (INA) Snomax did not affect embryo development or embryo mitochondrial activity. In conclusion, Snomax seems to be an effective ice nucleating agent for the slow freezing of mouse embryos. Snomax can also be a valuable alternative to manual seeding in research protocols in which manual seeding cannot be performed (i.e., differential scanning calorimetry and cryomicroscopy).

Controlled ice nucleation using freeze-dried Pseudomonas syringae encapsulated in alginate beads

The control of ice nucleation is of fundamental significance in many process technologies related to food and pharmaceutical science and cryobiology. Mechanical perturbation, electromagnetic fields and ice-nucleating agents (INAs) have been known to induce ice nucleation in a controlled manner. But these ice-nucleating methods may suffer from cumbersome manual operations, safety concerns of external fields, and biocompatibility and recovery issues of INA particles, especially when used in living systems. Given the automatic ice-seeding nature of INAs, a promising solution to overcome some of the above limitations is to engineer a biocomposite that accommodates the INA particles but minimizes their interactions with biologics, as well as enabling the recovery of used particles. In this study, freeze-dried Pseudomonas syringae, a model ice-nucleating agent, was encapsulated into microliter-sized alginate beads. We evaluated the performance of the bacterial hydrogel beads to initiate ice nucleation in water and aqueous glycerol solution by investigating factors including the size and number of the beads and the local concentration of INA particles. In the aqueous sample of a fixed volume, the total mass of the INA particles (m) was found to be the governing parameter that is solely responsible for determining the ice nucleation performance of the bacterial hydrogel beads. The freezing temperature has a strong positive linear correlation with log10m. The findings in this study provide an effective, predictable approach to control ice nucleation, which can improve the outcome and standardization of many ice-assisted process technologies.

Ice nucleation efficiency of AgI: review and new insights

Abstract. AgI is one of the best-investigated ice-nucleating substances. It has relevance for the atmosphere since it is used for glaciogenic cloud seeding. Theoretical and experimental studies over the last 60 years provide a complex picture of silver iodide as an ice-nucleating agent with conflicting and inconsistent results. This review compares experimental ice nucleation studies in order to analyze the factors that influence the ice nucleation ability of AgI. The following picture emerges from this analysis: the ice nucleation ability of AgI seems to be enhanced when the AgI particle is on the surface of a droplet, which is indeed the position that a particle takes when it can freely move in a droplet. The ice nucleation by particles with surfaces exposed to air depends on water adsorption. AgI surfaces seem to be most efficient at nucleating ice when they are exposed to relative humidity at or even above water saturation. For AgI particles that are completely immersed in water, the freezing temperature increases with increasing AgI surface area. Higher threshold freezing temperatures seem to correlate with improved lattice matches as can be seen for AgI–AgCl solid solutions and 3AgI·NH4I·6H2O, which have slightly better lattice matches with ice than AgI and also higher threshold freezing temperatures. However, the effect of a good lattice match is annihilated when the surfaces have charges. Also, the ice nucleation ability seems to decrease during dissolution of AgI particles. This introduces an additional history and time dependence for ice nucleation in cloud chambers with short residence times.

Immersion freezing of supercooled water drops containing glassy volcanic ash particles

The freezing temperatures of hundreds of water drops with radii 20–50μm containing known average concentrations of suspended, mostly micron- to submicron-sized, volcanic ash particles composed of SiO2-rich glass were recorded using optical microscopy. As expected, the ash suppresses supercooling, and in contrast to earlier studies of much larger ash particles, the median freezing temperature clearly scales with the available ash surface area per drop. The heterogeneous nucleation rate coefficient per unit mass of ash (jm) increases exponentially with decreasing temperature (T) (increasing supercooling) with a possible change in the slope of a plot of logjm against T at T=245±1K. Although uncertainties in the ash surface area limit quantitative comparisons, we conclude that volcanic glass is a less effective ice-nucleating agent than feldspar crystals and more similar to other minerals previously studied.

Poly(vinyl alcohol) Cryogel Formation Using Biocompatible Ice Nucleating Agents

AbstractTo control the influence of supercooling on the preparation of poly(vinyl alcohol) (PVA) cryogel, various biocompatible ice nucleating agents, including, two fatty alcohols, and eight different amino acids are investigated for their ice nucleating efficiency in PVA aqueous solutions. The L‐aspartic acid is found to be the most efficient ice nucleating agent with the ability to induce freezing at ∼−5.5 °C in 8.3 wt% aqueous PVA solution as compared to ∼−20 °C without L‐aspartic acid. The PVA cryogels are prepared with L‐aspartic acid as the heterogeneous ice nucleating agent and investigated for their rheological properties. Finally, it is found that freezing temperature close to the sol–gel transition temperature produces cryogel with the high tack important for various biomedical applications.

The microscopic features of heterogeneous ice nucleation may affect the macroscopic morphology of atmospheric ice crystals

It is surprisingly difficult to freeze water. Almost all ice that forms under "mild" conditions (temperatures > -40 degrees C) requires the presence of a nucleating agent--a solid particle that facilitates the freezing process--such as clay mineral dust, soot or bacteria. In a computer simulation, the presence of such ice nucleating agents does not necessarily alleviate the difficulties associated with forming ice on accessible timescales. Nevertheless, in this work we present results from molecular dynamics simulations in which we systematically compare homogeneous and heterogeneous ice nucleation, using the atmospherically important clay mineral kaolinite as our model ice nucleating agent. From our simulations, we do indeed find that kaolinite is an excellent ice nucleating agent but that contrary to conventional thought, non-basal faces of ice can nucleate at the basal face of kaolinite. We see that in the liquid phase, the kaolinite surface has a drastic effect on the density profile of water, with water forming a dense, tightly bound first contact layer. Monitoring the time evolution of the water density reveals that changes away from the interface may play an important role in the nucleation mechanism. The findings from this work suggest that heterogeneous ice nucleating agents may not only enhance the ice nucleation rate, but also alter the macroscopic structure of the ice crystals that form.

Cryopreservation of Encapsulated Liver Spheroids for a Bioartificial Liver: Reducing Latent Cryoinjury Using an Ice Nucleating Agent

Acute liver failure has high mortality due to donor organ shortages. A bioartificial liver could "bridge the gap" to transplant or spontaneous recovery. Alginate encapsulation of HepG2 cells enables cell spheroid formation, thus providing sufficient functional biomass. Cryopreservation (CryoP) of these spheroids would allow an off-the-shelf capability for unpredictable emergency use. Cell death during CryoP often results from intracellular ice formation, after supercooling. An ice nucleating agent (INA), crystalline cholesterol, was trialled to reduce supercooling and subsequent cryoinjury. Spheroids were cooled in a controlled rate freezer in 12% dimethylsulfoxide/Celsior +/- INA, and sample temperatures were recorded throughout. Viability was assessed using fluorescent staining with image analysis, cell number by nuclei count, function using assays to detect liver-specific protein synthesis and secretion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, and broad-spectrum cytochrome P450 activity. Spheroids cryopreserved without INA displayed latent cryoinjury in the first 6 h after thawing. INA reduced supercooling during CryoP and also latent cryoinjury. Cell numbers, viability, and function as measured over 72 h post-thaw were all improved when INA was present during CryoP.

Preparation of nano-silver iodide powders and their efficiency as ice-nucleating agent in weather modification

Nano-meter-sized silver iodide (n-AgI) powders were prepared by precipitation method at room temperature. The size, structure and specific surface area of the n-AgI powders were characterized by transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscope and Brunner–Emmett–Teller, respectively. The n-AgI powders obtained have an average particle size of ∼90 nm, similar surface lattice structure with ice crystal (H 2O) and larger specific surface area than conventional micro-meter-sized silver iodide (m-AgI) powders. The cloud chamber experiment revealed that the n-AgI powders exhibit higher ice nucleation efficiency and higher threshold temperature value than conventional m-AgI powders.

A Study of the Impact of Freezing on the Lyophilization of a Concentrated Formulation with a High Fill Depth

The objective of this study was to evaluate the impact of freezing on the lyophilization of a concentrated formulation with a high fill depth. A model system consisting of a 15-mL fill of 15% (w/w) sulfobutylether 7-beta-cyclodextrin (SBECD) solution in a 30-mL vial was selected for this study. Various freezing methods including single-step freezing, two-step freezing with a super-cooling holding, annealing, vacuum-induced freezing, changing ice habit using tert-butyl-alcohol (TBA), ice nucleation with silver iodide (AgI), as well as combinations of some of the methods, were used in the lyophilization of this model system. This work demonstrated that the freezing process had a significant impact on primary drying rate and product quality of a concentrated formulation with a high fill depth. Annealing, vacuum-induced freezing, and addition of either TBA or an ice nucleating agent (AgI) to the formulation accelerated the subsequent ice sublimation process. Two-step freezing or addition of TBA improved the product quality by eliminating vertical heterogeneity within the cake. The combination of two-step freezing in conjunction with an annealing step was shown to be a method of choice for freezing in the lyophilization of a product with a high fill depth. In addition to being an effective method of freezing, it is most applicable for scaling up. An alternative approach is to add a certain amount of TBA to the formulation, if the TBA-formulation interaction or regulatory concerns can be demonstrated as not being an issue. An evaluation of vial size performed in this study showed that although utilizing large-diameter vials to reduce the fill depth can greatly shorten the cycle time of a single batch, it will substantially decrease the product throughput in a large-scale freeze-dryer.

Influence of water activity on the ice-nucleating activity of Pseudomonas syringae

Pseudomonas syringae is known as a biological ice-nucleating agent. The bacterium has the unusual property of increasing the temperature at which water freezes by a few degrees. However, the ice-nucleating activity (INA) always remains lower for in vitro cultivated cells, than for cells grown in planta. We examined the effects of the hydrophobic environment and of water availability, on the in vitro growth and INA of P. syringae. The hydrophobic environment was modified by addition of fatty acids, vegetable oils or silicone oil to the culture medium. Addition of olive oil (1%), or traces of silicone oil in the culture medium had a positive effect upon the expression of INA. Variations in water activity from 0.990 to 0.988 by addition of sugar beet fibres or sodium chloride in the culture medium were followed by an increase in INA. This study suggested that control of the medium's water activity must be considered as an important parameter for optimization of INA in P. syringae.

Anatomic Site of Application of Ice-Nucleating Active Bacteria Affects Supercooling in the Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Most overwintering insects do not survive internal freezing and must avoid low temperatures or enhance the capacity of their body fluids to supercool to survive low temperature exposure. Recent reports have demonstrated that the application of ice-nucleating active microorganisms markedly diminishes supercooling. Topical application of as little as 20 ppm of a suspension of a freeze-dried preparation of the ice-nucleating active bacterium Pseudomonas syringae van Hall was sufficient to elevate the mean supercooling point of the overwintering adults of the Colorado potato beetle, Leptinotarsa decemlineata (Say), from −8.7 to −4.7°C. Previous reports have demonstrated that topical application of these biological ice nucleators to insects whose mouths have been sealed still reduces supercooling capacity; however, the anatomical route by which these agents make contact with body water is unclear. Application of the P. syringae suspension to the ventral abdomen did not significantly increase the supercooling point (−5.5°C) compared with beetles treated with the non-ice-nucleating active (control) bacterium Escherichia coli (Migula). However, application of the ice-nucleating agent to the thoracic spiracle, ventral cervix, or abdominal spiracle elevated supercooling point values above those of beetles treated on the ventral abdomen. These data are instructive in the development of methods for the use of ice-nucleating active microorganisms for the biological control of overwintering pests.

Differential scanning calorimetry studies on the cysts of the potato-cyst nematode Globodera rostochiensis during freezing and melting

Differential scanning calorimetry (DSC) was used to determine the thermal events associated with freezing and melting of the cysts of the potato-cyst nematode Globodera rostochiensis. There were no thermal events during the cooling of dry cysts from 5 to -60 °C and warming back to 5 °C, indicating the absence of water freezing in the dry cysts. During heating of dry cysts from 5 to 80 °C, two overlapping endothermic events were observed at 55 °C, indicating the irreversible destruction of the permeability barrier of the eggshell by the melting of the lipids which constitute the lipid layer. The first exothermic event (T=-9 °C) during the cooling of hydrated cysts indicates the presence of an ice-nucleating agent. A broad exotherm at -38 °C is due to the freezing of eggs. DSC thus confirms that the eggshell prevents exogenous ice nucleation and allows the eggs to supercool in the presence of external ice. The enthalpy of the egg exotherm increased during hydration of the cysts. The temperature of the egg exotherm was elevated after heating of the sample to 70 °C. This is thought to be due to the loss of trehalose from the eggs following the destruction of the permeability barrier of the eggshell. During melting, three endothermic events were observed. These became merged after the destruction of the permeability barrier of the eggshell by heating, and only two peaks were observed in isolated eggs. The sample is thus considered to consist of three freezable compartments: (1) the water surrounding the cyst, (2) the solution between the cyst wall and the eggs and (3) the egg contents.