Direct comparison of isobaric and isochoric vitrification of two aqueous solutions with photon counting X-ray computed tomography.

Vitrification is a promising approach for ice-free cryopreservation of biological material, but progress is hindered by the limited set of experimental tools for studying processes in the interior of the vitrified matter. Isochoric cryopreservation chambers are often metallic, and their opacity prevents direct visual observation. In this study, we introduce photon counting X-ray computed tomography (CT) to compare the effects of rigid isochoric and unconfined isobaric conditions on vitrification and ice formation during cooling of two aqueous solutions: 50 wt% DMSO and a coral vitrification solution, CVS1. Previous studies have only compared vitrification in isochoric systems with isobaric systems that have an exposed air-liquid interface. We use a movable piston to replicate the surface and thermal boundary conditions of the isochoric system yet maintain isobaric conditions. When controlling for the boundary conditions we find that similar ice and vapor volume fractions form during cooling in isochoric and isobaric conditions. Interestingly, we observe distinct ice and vapor cavity morphology in the isochoric systems, possibly due to vapor outgassing or cavitation as rapid cooling causes the pressure to drop in the confined systems. These observations highlight the array of thermal-fluid processes that occur during vitrification in confined aqueous systems and motivate the further application of imaging techniques such as photon counting X-ray CT in fundamental studies of vitrification.

Experimental observation of cavity-free ice-free isochoric vitrification via combined pressure measurements and photon counting x-ray computed tomography.

Isochoric (constant-volume or volumetrically confined) vitrification has shown potential as an alternative cryopreservation-by-vitrification technique, but the complex processes at play within the chamber are yet poorly characterized, and recent investigations have prompted significant debate around whether a truly isochoric vitrification process (in which the liquid remains completely confined by solid boundaries) is indeed feasible. Based on a recent thermomechanical simulation of a high-concentration Me2SO solution, Solanki and Rabin (Cryobiology, 2023, 111, 9-15.) argue that isochoric vitrification is not feasible, because differential thermal contraction of the solution and container will necessarily drive generation of a cavity, corrupting the rigid confinement of the liquid. Here, we provide direct experimental evidence to the contrary, demonstrating cavity-free isochoric vitrification of a ∼3.5M vitrification solution by combined isochoric pressure measurement (IPM) and photon-counting x-ray computed tomography (PC-CT). We hypothesize that the absence of a cavity is due to the minimal thermal contraction of the solution, which we support with additional volumetric analysis of the PC-CT reconstructions. In total, this study provides experimental evidence both demonstrating the feasibility of isochoric vitrification and highlighting the potential of designing vitrification solutions that exhibit minimal thermal contraction.

An extreme value statistics model of heterogeneous ice nucleation for quantifying the stability of supercooled aqueous systems.

The propensity of water to remain in a metastable liquid state at temperatures below its equilibrium melting point holds significant potential for cryopreserving biological material such as tissues and organs. The benefits conferred are a direct result of progressively reducing metabolic expenditure due to colder temperatures while simultaneously avoiding the irreversible damage caused by the crystallization of ice. Unfortunately, the freezing of water in bulk systems of clinical relevance is dominated by random heterogeneous nucleation initiated by uncharacterized trace impurities, and the marked unpredictability of this behavior has prevented the implementation of supercooling outside of controlled laboratory settings and in volumes larger than a few milliliters. Here, we develop a statistical model that jointly captures both the inherent stochastic nature of nucleation using conventional Poisson statistics as well as the random variability of heterogeneous nucleation catalysis through bivariate extreme value statistics. Individually, these two classes of models cannot account for both the time-dependent nature of nucleation and the sample-to-sample variability associated with heterogeneous catalysis, and traditional extreme value models have only considered variations of the characteristic nucleation temperature. We conduct a series of constant cooling rate and isothermal nucleation experiments with physiological saline solutions and leverage the statistical model to evaluate the natural variability of kinetic and thermodynamic nucleation parameters. By quantifying freezing probability as a function of temperature, supercooled duration, and system volume while accounting for nucleation site variability, this study also provides a basis for the rational design of stable supercooled biopreservation protocols.

Methods to stabilize aqueous supercooling identified by use of an isochoric nucleation detection (INDe) device.

Stable aqueous supercooling has shown significant potential as a technique for human tissue preservation, food cold storage, conservation biology, and beyond, but its stochastic nature has made its translation outside the laboratory difficult. In this work, we present an isochoric nucleation detection (INDe) platform for automated, high-throughput characterization of aqueous supercooling at >1 mL volumes, which enables statistically-powerful determination of the temperatures and time periods for which supercooling in a given aqueous system will remain stable. We employ the INDe to investigate the effects of thermodynamic, surface, and chemical parameters on aqueous supercooling, and demonstrate that various simple system modifications can significantly enhance supercooling stability, including isochoric (constant-volume) confinement, hydrophobic container walls, and the addition of even mild concentrations of solute. Finally, in order to enable informed design of stable supercooled biopreservation protocols, we apply a statistical model to estimate stable supercooling durations as a function of temperature and solution chemistry, producing proof-of-concept supercooling stability maps for four common cryoprotective solutes.

Relating Metabolism Suppression and Nucleation Probability During Supercooled Biopreservation.

Aqueous supercooling provides a method by which to preserve biological matter at subfreezing temperatures without the deleterious effects of ice formation. The extended longevity of the preserved biologic is a direct result of a reduction in the rate of metabolism with decreasing temperature. However, because the nucleation of ice from a supercooled solution is a stochastic process, supercooled preservation carries the risk of random ice nucleation. Theoretical supercooled biopreservation research to date has largely treated these biological and thermophysical phenomena separately. Here, we apply a statistical model of stochastic ice nucleation to demonstrate how the possible reduction in metabolic rate is inherently related to supercooling stability (i.e., the likelihood of ice nucleation). We develop a quantitative approach by which to weigh supercooling stability versus potential metabolic reduction, and further show how the stability-metabolism relationship varies with system size for two assumed modes of nucleation. Ultimately, this study presents a generalizable framework for the informed design of supercooled biopreservation protocols that considers both phase transformation kinetics and biochemical or biophysical kinetics.

Cryopreservation and revival of Hawaiian stony corals using isochoric vitrification.

Corals are under siege by both local and global threats, creating a worldwide reef crisis. Cryopreservation is an important intervention measure and a vital component of the modern coral conservation toolkit, but preservation techniques are currently limited to sensitive reproductive materials that can only be obtained a few nights per year during spawning. Here, we report the successful cryopreservation and revival of cm-scale coral fragments via mL-scale isochoric vitrification. We demonstrate coral viability at 24 h post-thaw using a calibrated oxygen-uptake respirometry technique, and further show that the method can be applied in a passive, electronics-free configuration. Finally, we detail a complete prototype coral cryopreservation pipeline, which provides a platform for essential next steps in modulating post-thaw stress and initiating long-term growth. These findings pave the way towards an approach that can be rapidly deployed around the world to secure the biological genetic diversity of our vanishing coral reefs.

Design and operation of a molten salt electrochemical cell.

Molten salts such as 2LiF-BeF2 (FLiBe) have been proposed as coolants for advanced nuclear fission and fusion reactors. Critical to the design, licensing and operation of these reactors is characterization and understanding of the chemical behavior and mass transport of activation and fission products, corrosion products, and other solutes in the coolant. Electrochemical techniques are a powerful suite of tools for probing these phenomena. The design of an experimental cell for molten salt electrochemistry is described herein. As a demonstration of this design, details of the experimental methods used to conduct electrochemical experiments with molten FLiBe with addition of LiH are provided. Decommissioning of the cell is considered from the point of view of decontamination and waste generated. Main features of the cell include:•Suitable for operation up to 800 °C; suitable for operation inside and outside of a glovebox.•Enables sweep gas, gas sampling and analysis; enables addition of solid and liquid materials during operation.•Supports a variety of electrode materials and arrangements.

Enhanced Control over Ice Nucleation Stochasticity Using a Carbohydrate Polymer Cryoprotectant.

Metastable supercooling has emerged as a transformative technique for ice-free biopreservation, but issues of stability inherent to the stochastic nature of ice formation have thus far limited its translation out of the laboratory. In this work, we explore the influence of the bio-based carbohydrate polymer FucoPol on aqueous supercooling using an isochoric nucleation detection technique. We show that FucoPol, a high-molecular-weight, fucose-rich polysaccharide, which has previously been shown to reduce average ice crystal sizes after nucleation, also induces a concentration-dependent stabilization of metastable supercooled water, as evidenced by both a significant reduction in nucleation stochasticity (i.e., the spread in temperatures over which the system will nucleate upon cooling) and a corresponding increase in the predicted induction time of nucleation. FucoPol is found to confine the stochasticity of ice nucleation to a narrow, well-defined band of temperatures roughly one-third as wide as that of pure water under identical conditions. Importantly, this substantial reduction in stochasticity is accompanied by only a minimal (<1 °C) change in the average nucleation temperature, suggesting that this effect is distinct from colligative freezing point depression. Reducing and characterizing the stochasticity of aqueous supercooling is essential to the engineering design of practical biopreservation protocols, and the results reported herein suggest that high-viscosity polymer systems may provide a powerful and largely unexplored lever by which to manipulate metastable-equilibrium phase change kinetics at subzero temperatures.