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 ∼3.5 M 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.

Limits of pressure-based ice detection during isochoric vitrification.

Vitrification under isochoric (constant-volume or volumetrically confined) conditions has emerged as an intriguing new cryopreservation modality, but the physical complexities of the process confound straight-forward interpretation of experimental results. In particular, the signature pressure-based ice detection used in many isochoric techniques becomes paradoxical during vitrification, wherein the emergence of a sharp increase in pressure reliably indicates the presence of ice, but avoidance of this increase does not necessarily indicate its absence. This phenomenon arises from the rich interplay between thermochemical and thermovolumetric effects in isochoric systems, and muddies efforts to confirm the degree to which a sample has vitrified. In this work, we seek to aid interpretation of isochoric vitrification experiments by calculating thermodynamic limits on the maximum amount of ice that may form without being detected by pressure, and by clarifying the myriad physical processes at play. Neglecting kinetic effects, we develop a simplified thermodynamic model accounting for thermal contraction, cavity formation, ice growth, solute ripening, and glass formation, we evaluate it for a range of chamber materials and solution compositions, and we validate against the acutely limited data available. Our results provide both counter-intuitive insights- lower-concentration solutions may contract less while producing more pressure-undetectable ice growth for example- and a general phenomenological framework by which to evaluate the process of vitrification in isochoric systems. We anticipate that the model herein will enable design of future isochoric protocols with minimized risk of pressure-undetectable ice formation, and provide a thermodynamic foundation from which to build an increasingly rigorous multi-physics understanding of isochoric vitrification.

An entropic theory of homogeneous ice nucleation in non-ionic aqueous solutions.

The nucleation of ice from aqueous solutions is a process essential to myriad environmental and industrial processes, but the physical factors affecting the capacity of different solutes to depress the homogeneous nucleation temperature of ice are yet poorly understood. In this work, we demonstrate that for many binary aqueous solutions of non-ionic solutes, this depression is dominated by the entropy of the liquid phase. Employing the classic Turnbull interpretation of the interfacial free energy γ∼TSliquid-Ssolid and estimating solution entropies with a Flory-style modification of the ideal entropy of mixing that accounts for solute size effects, we demonstrate that mixing entropy alone predicts experimental homogeneous nucleation temperatures across a wide variety of non-ionic solutions. We anticipate that this physical insight will not only enhance a fundamental understanding of homogeneous nucleation processes across fields but also open new avenues to the rational design of aqueous solutions for desired nucleation behaviors.

Effects of Isochoric Freezing on the Quality Characteristics of Raw Bovine Milk.

This study investigated the effects of isochoric freezing (IF) on the shelf-life and quality of raw bovine milk over a 5-week period. The results were compared with conventional refrigeration (RF) and refrigeration after pasteurization (HTST). The IF treatment process entailed storing liquid raw milk in isochoric chambers in thermodynamic equilibrium at -5 °C/77 MPa and -10 °C/96 MPa. Several parameters were analyzed, including microbiology count, physicochemical properties, indigenous enzyme activity, protein content, volatile organic compounds profile, and lipid degradation. Both raw and pasteurized milk experienced increases in the microbial level past the acceptable threshold (≥5.5 log CFU/mL) after 2 weeks and 5 weeks, respectively, leading to the deterioration of other parameters during storage. In comparison, microbiology count decreased significantly during storage for both IF treatment conditions but was more pronounced for the higher pressure (96 MPa) treatment, leading to undetectable levels of microorganism after 5 weeks. IF treatment maintained stable pH, titratable acidity, viscosity, lipid oxidation, volatile profiles, total protein content, and lactoperoxidase activity throughout the storage period. Color was preserved during IF treatment at -5 °C/77 MPa; however, color was impacted during IF treatment at -10 °C/96 MPa. Protein structures were also modified during pressurized storage in both IF treatments. Overall, the study demonstrated that isochoric freezing could significantly increase the shelf-life of milk by reducing microbiology activity, whilst maintaining its nutritional content. These results underscore the potential role of isochoric freezing as a valuable tool in eliminating pathogens while maintaining quality characteristics similar to raw milk over long storage periods.

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.

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.

Freezing Biological Time: A Modern Perspective on Organ Preservation.

Transporting tissues and organs from the site of donation to the patient in need, while maintaining viability, is a limiting factor in transplantation medicine. One way in which the supply chain of organs for transplantation can be improved is to discover novel approaches and technologies that preserve the health of organs outside of the body. The dominant technologies that are currently in use in the supply chain for biological materials maintain tissue temperatures ranging from a controlled room temperature (+25 °C to +15 °C) to cryogenic (-120 °C to -196 °C) temperatures (reviewed in Criswell et al. Stem Cells Transl Med. 2022). However, there are many cells and tissues, as well as all major organs, that respond less robustly to preservation attempts, particularly when there is a need for transport over long distances that require more time. In this perspective article, we will highlight the current challenges and advances in biopreservation aimed at "freezing biological time," and discuss the future directions and requirements needed in the field.

Novel isochoric impregnation to develop high-quality and nutritionally fortified plant materials (apples and sweet potatoes).

Isochoric impregnation was explored as a novel pressure-assisted infusion technique to fortify plant materials with bioactive compounds. Apple and potato cylinders were impregnated with a sucrose solution containing 4% ascorbic acid (AA) while freezing under isochoric conditions. Isochoric impregnation resulted in greater infusion of AA compared to infusion at atmospheric pressure, which demonstrated the feasibility of this impregnation technology. Processing temperatures (-3°C and -5°C) and processing times (1, 3, and 5 h) significantly affected the AA infusion. The AA content values ranged from 446 to 516 mg/100 g for apples and 322 to 831 mg/100 g for sweet potatoes under isochoric conditions. For both plant materials, isochoric impregnation at -3°C did not cause major changes in texture and microstructure of the biological tissues. These results indicated that isochoric impregnation of solid foods could be a feasible technology for infusion of bioactive compounds without significantly altering their matrix. PRACTICAL APPLICATION: The findings of this study showed that the use of isochoric impregnation as a fortification technique is a promising way to develop fresh-like and value-added products with improved nutrition during preservation at subfreezing temperatures.

Calculation of a temperature-volume phase diagram of water to inform the study of isochoric freezing down to cryogenic temperatures.

Phase diagrams are integral to the application and interpretation of materials thermodynamics, and none is more ubiquitous than the common temperature-pressure diagram of water and its many icy phases. Inspired by recent advances in isochoric thermodynamics, we here employ a simple convex hull approach to efficiently calculate an updated temperature-volume phase diagram for water and five of its solid polymorphs from existing Helmholtz free energy data. We proceed to highlight fundamental similarities between this T-V diagram and conventional binary temperature-concentration (T-x) diagrams, provide the volume coordinates of a variety of three-phase invariant reactions (e.g. "confined" or "volumetric" eutectics, peritectics, etc.) that occur amongst the phases of pure water under isochoric or confined conditions, and calculate the phase fraction evolution of ice Ih with temperature along multiple isochores of interest to experimental isochoric freezing. This work provides a requisite baseline upon which to extend the study of isochoric freezing to cryogenic temperatures, with potential applications in thermodynamic metrology, cryovolcanism, and cryopreservation.

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.

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.

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.

Isochoric supercooling cryomicroscopy.

We introduce an isochoric (constant-volume) supercooling cryomicroscope (ISCM), enabling the ice-free study of biological systems and biochemical reactions at subzero temperatures at atmospheric pressure absent ice. This technology draws from thermodynamic findings on the behavior of water in isochoric systems at subfreezing temperatures. A description of the design of the ISCM and a demonstration of the stability of the supercooled solution in the ISCM is followed by an illustration of the possible use of the ISCM in the preservation of biological matter research. A comparison was made between the survival of HeLa cells in the University of Wisconsin (UW) solution in the ISCM at +4 °C under conventional atmospheric conditions and at -5 °C under isochoric supercooled conditions. Continuous real-time monitoring at cryopreservation temperature via fluorescence microscopy showed that after three days of isochoric supercooling storage, the percentage of compromised cells remained similar to fresh controls, while storage at +4 °C yielded approximately three times the mortality rate of cells preserved at -5 °C.

Individual Microparticle Manipulation Using Combined Electroosmosis and Dielectrophoresis through a Si3N4 Film with a Single Micropore.

Porous dielectric membranes that perform insulator-based dielectrophoresis or electroosmotic pumping are commonly used in microchip technologies. However, there are few fundamental studies on the electrokinetic flow patterns of single microparticles around a single micropore in a thin dielectric film. Such a study would provide fundamental insights into the electrokinetic phenomena around a micropore, with practical applications regarding the manipulation of single cells and microparticles by focused electric fields. We have fabricated a device around a silicon nitride film with a single micropore (2-4 µm in diameter) which has the ability to locally focus electric fields on the micropore. Single microscale polystyrene beads were used to study the electrokinetic flow patterns. A mathematical model was developed to support the experimental study and evaluate the electric field distribution, fluid motion, and bead trajectories. Good agreement was found between the mathematic model and the experimental data. We show that the combination of electroosmotic flow and dielectrophoretic force induced by direct current through a single micropore can be used to trap, agglomerate, and repel microparticles around a single micropore without an external pump. The scale of our system is practically relevant for the manipulation of single mammalian cells, and we anticipate that our single-micropore approach will be directly employable in applications ranging from fundamental single cell analyses to high-precision single cell electroporation or cell fusion.

Isochoric supercooled preservation and revival of human cardiac microtissues.

Low-temperature biopreservation and 3D tissue engineering present two differing routes towards eventual on-demand access to transplantable biologics, but recent advances in both fields present critical new opportunities for crossover between them. In this work, we demonstrate sub-zero centigrade preservation and revival of autonomously beating three-dimensional human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues via isochoric supercooling, without the use of chemical cryoprotectants. We show that these tissues can cease autonomous beating during preservation and resume it after warming, that the supercooling process does not affect sarcomere structural integrity, and that the tissues maintain responsiveness to drug exposure following revival. Our work suggests both that functional three dimensional (3D) engineered tissues may provide an excellent high-content, low-risk testbed to study complex tissue biopreservation in a genetically human context, and that isochoric supercooling may provide a robust method for preserving and reviving engineered tissues themselves.

Effects of Isochoric Freezing Conditions on Cut Potato Quality.

Isochoric freezing is a pressure freezing technique that could be used to retain the beneficial effects of food storage at temperatures below their freezing point without ice damage. In this study, potato cylinders were frozen in an isochoric system and examined using full factorial combinations of three processing procedures (immersed in water, vacuum-packed and immersed in ascorbic acid solution), four freezing temperatures/pressures (-3 °C/37 MPa, -6 °C/71 MPa, -9 °C/101 MPa and -15 °C/156 MPa) and two average compression rates (less than 0.02 and more than 0.16 MPa/s). The effects of process variables on critical quality attributes of frozen potatoes after thawing were investigated, including mass change, volume change, water holding capacity, color and texture. Processing procedure and freezing temperature/pressure were found to be highly significant factors, whereas the significance of the compression rate was lower. For the processing procedures, immersion in an isotonic solution of 5% ascorbic acid best preserved quality attributes. At the highest pressure level of 156 MPa and low compression rate of 0.02 MPa/s, potato samples immersed in ascorbic acid retained their color, 98.5% mass and 84% elasticity modulus value. These samples also showed a 1% increase in volume and 13% increase in maximum stress due to pressure-induced hardening.

Preservation of grape tomato by isochoric freezing.

This study investigated the potential of isochoric freezing to preserve tomatoes. Isochoric freezing is an emerging technology that preserves biological matter at subfreezing temperatures without any ice damage. Isochoric freezing was compared with freezing under isobaric conditions and with preservation techniques used in the food industry: cold storage at 10 °C and individual quick freezing (IQF). Physicochemical and nutritional properties were evaluated weekly for four weeks. Preservation under isochoric conditions maintained the mass, color, nutrient content (ascorbic acid, lycopene and phenolics) and antioxidant activity of the fresh tomatoes. Also, isochoric preservation led to minimal texture damage. In comparison, mass loss of tomatoes stored at 10 °C for 3 weeks contributed to changes in overall visual quality and firmness as well as significant losses in nutrient content. The greatest mass, texture, and nutrients losses were obtained for tomatoes subjected to IQF and isobaric freezing. The results show that isochoric freezing has the potential to preserve tomatoes while maintaining physicochemical and nutritional properties similar to those of fresh tomatoes which might find application in the commercial preservation of tomatoes.

Effect of isochoric freezing on quality aspects of minimally processed potatoes.

The enhanced interest in greater convenience foods has recently led to the expansion of minimally processed potato products. This study investigated the effects of isochoric freezing on pre-peeled potato cubes, including quality attributes (microstructure, texture, and color), nutritional value (ascorbic acid (AA) content, total phenolic content, and antioxidant capacity), and polyphenol oxidase activity. Isochoric freezing (-3 °C/30 MPa) was compared with isobaric freezing (-3 °C/0.1 MPa) and individual quick freezing followed by frozen storage at -20 °C for 4 weeks. The isochoric sample had lower drip loss and volume shrinkage as well as better preserved texture and microstructure than the other samples. All freezing methods caused an increase in total phenolic content and antioxidant capacity, but a decrease in AA content. Also, all freezing methods caused browning of the thawed potatoes, but isochoric freezing delayed its onset for more than 1 week. PRACTICAL APPLICATION: Results showed that isochoric freezing of pre-peeled and cut potatoes caused less freeze damage than isobaric and individual quick freezing, which might find application in the commercial preservation of minimally processed food products.

Thermodynamic Theory and Experimental Validation of a Multiphase Isochoric Freezing Process.

Freezing of the aqueous solutions that comprise biological materials, such as isotonic physiological saline, results in the formation of ice crystals and the generation of a hypertonic solution, both of which prove deleterious to biological matter. The field of modern cryopreservation, or preservation of biological matter at subfreezing temperatures, emerged from the 1948 discovery that certain chemical additives such as glycerol, known as cryoprotectants, can protect cells from freeze-related damage by depressing the freezing point of water in solution. This gave rise to a slew of important medical applications, from the preservation of sperm and blood cells to the recent preservation of an entire liver, and current cryopreservation protocols thus rely heavily on the use of additive cryoprotectants. However, high concentrations of cryoprotectants themselves prove toxic to cells, and thus there is an ongoing effort to minimize cryoprotectant usage while maintaining protection from ice-related damage. Herein, we conceive from first principles a new, purely thermodynamic method to eliminate ice formation and hypertonicity during the freezing of a physiological solution: multiphase isochoric freezing. We develop a comprehensive thermodynamic model to predict the equilibrium behaviors of multiphase isochoric systems of arbitrary composition and validate these concepts experimentally in a simple device with no moving parts, providing a baseline from which to design tailored cryopreservation protocols using the multiphase isochoric technique.

Isochoric conditions enable high subfreezing temperature pancreatic islet preservation without osmotic cryoprotective agents.

While biological systems are typically studied under isobaric (constant pressure) conditions, recent reports on the bio-thermodynamics of isochoric (constant volume) systems point to their potential for subfreezing-temperature preservation of biological matter. This preliminary study, in which we report that pancreatic islets can survive multi-day preservation at high subfreezing temperatures in an isochoric chamber without osmotic cryoprotective agents (CPA), highlights the potential of isochoric cryopreservation in an application of clinical value.