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.

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.

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.

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.

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.

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.

Covid-19 pandemic impacts on essential transit riders: Findings from a U.S. Survey.

The Covid-19 pandemic has decimated public transit service across the United States and caused significant decreases in ridership. Little is known about the reasons for unevenness in pandemic-era mode shifts and the impacts of pandemic-related transit reductions on riders' day-to-day lives. Using a national survey of U.S. transit riders (n = 500) conducted in fall 2020, this study examines changes in transit use since the pandemic began, the reasons for transit reductions, and the effects of reduced transit use and transit service on transit riders' ability to meet their travel needs. The Covid-19 pandemic has exacerbated existing transportation burdens for those who have limited mobility options, those facing socioeconomic challenges, Hispanic or Latinx riders, and female, non-binary or genderqueer people. We close with recommendations for strengthening transit service for these groups in the long term as we recover from the pandemic.

Electronic Nose With Detection Method for Alcohol, Acetone, and Carbon Monoxide in Coronavirus Disease 2019 Breath Simulation Model.

Electronic nose technology may have the potential to substantially slow the spread of contagious diseases with rapid signal indication. As our understanding of infectious diseases such as Corona Virus Disease 2019 improves, we expect electronic nose technology to detect changes associated with pathogenesis of the disease such as biomarkers of immune response for respiratory symptoms, central nervous system injury, and/or peripheral nervous system injury in the breath and/or odor of an individual. In this paper, a design of an electronic nose was configured to detect the concentration of a COVID-19 breath simulation sample of alcohol, acetone, and carbon monoxide mixture. After preheating for 24 hours, the sample was carried into an internal bladder of the collection vessel for analysis and data was collected from three sensors to determine suitability of these sensors for the application of exhaled breath analysis. Test results show a detection range in parts-per-million within the sensor detection range of at least 10-300 ppm. The output response of an MQ-2 and an MQ-135 sensor to a diverse environment of target gasses show the MQ-2 taking a greater length of time to normalize baseline drift compared to an MQ-135 sensor due to cross interferences with other gasses. The COVID-19 breath simulation sample was established and validated based on preliminary data obtained from parallel COVID-19 breath studies based in Edinburgh and Dortmund. This detection method provides a non-invasive, rapid, and selective detection of gasses in a variety of applications in virus detection as well as agricultural and homeland security.

High thermal conductivity in soft elastomers with elongated liquid metal inclusions.

Soft dielectric materials typically exhibit poor heat transfer properties due to the dynamics of phonon transport, which constrain thermal conductivity (k) to decrease monotonically with decreasing elastic modulus (E). This thermal-mechanical trade-off is limiting for wearable computing, soft robotics, and other emerging applications that require materials with both high thermal conductivity and low mechanical stiffness. Here, we overcome this constraint with an electrically insulating composite that exhibits an unprecedented combination of metal-like thermal conductivity, an elastic compliance similar to soft biological tissue (Young's modulus < 100 kPa), and the capability to undergo extreme deformations (>600% strain). By incorporating liquid metal (LM) microdroplets into a soft elastomer, we achieve a ∼25× increase in thermal conductivity (4.7 ± 0.2 W⋅m-1⋅K-1) over the base polymer (0.20 ± 0.01 W⋅m-1·K-1) under stress-free conditions and a ∼50× increase (9.8 ± 0.8 W⋅m-1·K-1) when strained. This exceptional combination of thermal and mechanical properties is enabled by a unique thermal-mechanical coupling that exploits the deformability of the LM inclusions to create thermally conductive pathways in situ. Moreover, these materials offer possibilities for passive heat exchange in stretchable electronics and bioinspired robotics, which we demonstrate through the rapid heat dissipation of an elastomer-mounted extreme high-power LED lamp and a swimming soft robot.

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.

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.

Preservation of rat hearts in subfreezing temperature isochoric conditions to - 8 °C and 78 MPa.

Isochoric (constant volume) preservation at subfreezing temperatures is being investigated as a novel method for preserving cells and organs. This study is a first initial effort to evaluate the efficacy of this method for heart preservation, and to provide a preliminary outline of appropriate preservation parameters. To establish a baseline for further studies, rat hearts were preserved in a University of Wisconsin (UW) intracellular solution for one hour under isochoric conditions at: 0 °C (atmospheric pressure - 0.1 MPa), - 4 °C (41 MPa), - 6 °C (60 MPa) and - 8 °C (78 MPa). The viability of the heart was evaluated using Langendorff perfusion and histological examination. The physiological performance of hearts preserved at - 4 °C (41 MPa) was comparable to that of a heart preserved on ice at atmospheric pressure, with no statistically significant difference in histological injury score. However, hearts preserved at -4 °C displayed substantially reduced interstitial edema compared to hearts preserved by conventional hypothermic preservation in UW on ice at atmospheric pressure, suggesting significant protection from increased vascular permeability following preservation. Hearts preserved at - 6 °C (60 MPa) suffered injury from cellular swelling and extensive edema, and at - 8 °C (78 MPa) hearts experienced significant morphological disruption. To the best of our knowledge, this is the first publication showing that a mammalian organ can survive low subfreezing temperatures without the use of a cryoprotective additive. Lowering the preservation temperature reduces metabolism and improves preservation quality, and these results suggest that improvements in preservation are possible at subzero temperatures with low to moderate pressures observed at -4 °C. Notably, tissue damage was observed at lower temperatures (-6 °C or below) accompanying further elevation of pressure associated with isochoric preservation that may prove detrimental. Therefore, subfreezing temperature isochoric preservation protocols should optimize, a combination of temperature and pressure that will minimize the negative effects of elevated pressure while retaining the beneficial effect of lower temperatures and reduced metabolism.

Escherichia coli viability in an isochoric system at subfreezing temperatures.

In comparison with isobaric (constant pressure) freezing, isochoric (constant volume) freezing reduces potential mechanical damage from ice crystals and exposes stored biological matter to a lower extracellular concentration, at the price of increased hydrostatic pressure. This study evaluates the effects of isochoric freezing to low temperatures and high pressures on Escherichia coli (E. coli) survival. The viability of E. coli was examined after freezing to final temperatures between -5 °C and -20 °C for periods from 0.5 h to 12 h, with recovery periods from 0 h to 24 h. Freezing for up to two hours to -10 °C and -15 °C had little effect on the percentage of viable E. coli, relative to the controls. However, after two hours of exposure at -20 °C, when left to recover for 24 h, a 75% reduction in survival is observed. Furthermore, after 12 h of isochoric freezing at -15 °C and -20 °C, E. coli population is reduced by 2.5 logs while freezing to these temperatures in conventional isobaric atmospheric conditions reduces population by only one log. This suggests that the combination of low temperature and high pressure experienced during isochoric freezing close to the triple point may be more detrimental to biological matter survival than the combination of elevated concentration, low temperature, and ice crystallization experienced during conventional freezing, and that this effect may be related to the time of exposure to these conditions.

Isochoric vitrification: An experimental study to establish proof of concept.

Ice-free vitreous cryopreservation (vitrification) is regarded as the principal method for avoiding ice crystallization damage in cryopreserved tissues and organs. We previously established the fundamental thermodynamics of isochoric (constant volume) systems for cryopreservation, and now extend this novel approach to vitrification in an isochoric system. This was achieved by measuring pressure changes in a 2 ml isochoric chamber containing a variety of aqueous solutions of the ubiquitous cryoprotective additives (CPA), dimethyl sulfoxide (Me2SO) and Propane-diol. The CPAs, ranging in concentrations from 0 to 49%(w/v), were prepared in a proprietary preservation solution (Unisol®) in anticipation of future applications to tissue and organ banking. Pressures developed in the system were monitored as a function of CPA concentration and cooling rate when the isochoric chamber was cooled to cryogenic temperature (-160 °C). This study corroborated our previous findings that pressure increases in accordance with the thermodynamics of partially frozen systems of low concentrations of CPAs. A key finding of this study was that in an isochoric system of higher concentrations of CPA, which vitrifies, there is no increase in pressure. In fact, an increase in pressure is a measure of failure to vitrify and a measure of devitrification. Comparison with results from the literature show that the concentration of CPAs needed for vitrification in an isochoric chamber is substantially lower than that needed for vitrification in isobaric systems at 1 atm and hyperbaric systems at 1000 atm. In addition, isochoric chambers are much more effective in promoting vitrification than hyperbaric pressure chambers, and are less expensive, easier to design, and implement.

Getting groceries during the pandemic: How transit remained important despite the rise of e-delivery.

Background: We model the use of public transit to reach grocery stores and the use of online delivery services to get groceries, before and during the COVID-19 pandemic among people who used transit regularly prior to the crisis. Methods: We draw upon a panel survey of pre-pandemic transit riders in Vancouver and Toronto. We conduct multivariable two-step tobit regression models that predict the likelihood of a respondent using transit as their primary mode for getting groceries before the pandemic (step 1) and then during the pandemic (step 2). Models are conducted for two survey waves, May 2020 and March 2021. We also conduct zero-inflated negative binomial regression models predicting the frequency respondents ordered groceries online. Results: Transit riders over the age of 64 were more likely to use transit to reach groceries before the pandemic and more likely to continue to do so during the pandemic (wave 1, OR, 1.63; CI, 1.24-2.14; wave 2, OR, 1.35; CI, 1.03-1.76). Essential workers were more likely to continue using transit to reach groceries during the pandemic (wave 1, OR, 1.33; CI, 1.24-1.43; wave 2, OR, 1.18; CI, 1.06-1.32). Walking distance to the nearest grocery store was positively associated with using transit to get groceries pre-pandemic (wave 1, OR, 1.02; CI, 1.01-1.03; wave 2, OR, 1.02; CI, 1.01-1.03), and in May 2020 (wave 1, OR 1.01; (1.00-1.02). During the pandemic, people who stopped using transit to get groceries were less likely to have made zero online grocery purchases (wave 1, OR, 0.56; CI, 0.41-0.75; wave 2, OR, 0.62; CI, 0.41-0.94). Discussion: People still physically commuting to work were more likely to still use transit to get groceries. Among transit riders, older adults and those living far walking distances from grocery stores are more likely to use transit to get groceries. Older transit riders and those with higher incomes were also more likely to use grocery delivery services, while female, Black, and immigrant riders were less likely to do so.

Public transport access to drug treatment before and during COVID-19: Implications for the opioid epidemic.

Public transport disruptions caused by the COVID-19 pandemic had wide-ranging impacts on the ability of individuals to access health care. Individuals with opioid use disorder represent an especially vulnerable population due to the necessity of frequent, supervised doses of opioid agonists. Focused on Toronto, a major Canadian city suffering from the opioid epidemic, this analysis uses novel realistic routing methodologies to quantify how travel times to individuals\220 nearest clinics changed due to public transport disruptions from 2019 to 2020. Individuals seeking opioid agonist treatment face very constrained windows of access due to the need to manage work and other essential activities. We find that thousands of households in the most materially and socially deprived neighbourhoods crossed 30 and 20-minute travel time thresholds to their nearest clinic. As even small changes to travel times can lead to missed appointments and heighten the chances of overdose and death, understanding the distribution of those most impacted can help inform future policy measures to ensure adequate access to care.

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.

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.