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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 Me 2 SO 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., Competing Interests: Declaration of competing interest B.R. has filed a 2017 patent application related to isochoric vitrification, which is under review as of the date of submission of this work. M.J.P.-P. and B.R. have financial stakes in a commercial entity that holds the license to the said patent application. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Isochoric (constant volume) freezing is a novel food preservation technology that has demonstrated the ability to preserve food products at subfreezing temperatures in an unfrozen state, thereby avoiding the detrimental effects of ice formation. It minimizes the quality loss of fresh fruits and juices, increases their nutrient content, and reduces microbial counts. Orange juice (OJ) samples were subjected to conventional freezing (CF) and isochoric freezing (IF) for 7 days and then stored at 4°C for an additional 7 days. We evaluated the microbiological and physicochemical quality of CF and IF OJ before and after storage. The IF was performed at three different conditions: -5°C/73 MPa, -10°C/93 MPa, and -15°C/143 MPa. The results indicate that the total aerobic count of OJ remained below the detection limit after heat treatment, 7 days of CF and 7 days of IF. Yeast and mold counts increased in fresh and CF OJ after 7 days of storage at 4°C, whereas IF OJ remained below the detection limit. Less color difference was observed in IF (-15°C/143 MPa) OJ compared to heat-treated and CF OJ. Heat treatment inactivated 42% of pectin methylesterase (PME), whereas 7-day long IF increased PME activity up to 150%. Additionally, IF (-15°C/143 MPa) OJ showed reduced pulp sedimentation, which can be advantageous, as sedimentation in juices has been a recognized technological issue in the juice industry. Ascorbic acid level was significantly higher in IF (-15°C/143 MPa) OJ compared to fresh and CF OJ after storage., (© 2024 Institute of Food Technologists.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pomegranate juice was treated by isochoric freezing (-15°C/130 MPa) for 24 h and then stored under three different conditions for up to 4 weeks: 4°C/0.1 MPa, 24°C/0.1 MPa, and -10°C/100 MPa. The juice microbiological stability and quality were compared to those using heat treatment at 95°C for 15 s followed by cold storage at 4°C. Heat-treated and isochoric frozen (IF) pomegranate juice stored under isochoric conditions showed no spoilage microorganisms after 4 weeks of storage. Also, IF juice stored at 4 or 24°C for 4 weeks had lower microbial loads than those in fresh pomegranate juice. IF juice stored under isochoric conditions showed greater color stability, antioxidant capacity, and nutrient retention (anthocyanins, ascorbic acid, and total phenolic compounds) than heat-treated juices stored at 4°C. IF juice stored at 4°C also showed greater anthocyanin and ascorbic acid contents compared with heat-treated juice. PRACTICAL APPLICATION: Isochoric freezing storage at -10°C can be used to preserve the quality properties of fresh pomegranate juice. Isochoric freezing at -15°C for 24 h can also be used as a pretreatment to extend the shelf life of refrigerated pomegranate juice since the applied pressures reached total inactivation levels of spoilage microorganisms., (© 2024 Institute of Food Technologists. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Isochoric freezing (IF) at -5°C/77 and -10°C/100 MPa was used to preserve carrot juice for 12 weeks. The juice qualities were compared to those using heat treatment (HT) at 95°C for 15 s followed by cold storage at 4°C. The native population of total aerobic bacteria, yeasts, and molds in isochoric frozen juice remained below the detection limit for 12 weeks. In comparison, microbes started to grow in heat-treated juices after 3 weeks of refrigeration. The color of isochoric frozen juice appeared more deep orange than the fresh juice due to an increase in carotenoid extractability. IF was not effective in reducing the activities of peroxidase, polyphenol oxidase, and pectin methyl esterase compared with HT. However, the isochoric samples showed higher carotenoid content, polyphenol content, and antioxidant capacity compared to the fresh and heat-treated juices. PRACTICAL APPLICATION: Isochoric freezing was used to produce carrot juice with extended shelf life. Isochoric freezing could be a beneficial alternative to conventional heat treatment for carrot juice processing as the applied pressures reached total inactivation levels of spoilage microorganisms. Moreover, the low processing temperatures better retained desirable compounds and quality attributes of fresh juice throughout its shelf life., (© 2024 Institute of Food Technologists. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Introduction: Irreversible electroporation (IRE) is a tissue ablation technology that kills cells with short electrical pulses that do not induce thermal damage, thereby preserving the extracellular matrix. Preclinical research suggests that IRE may be developed as a tool for regenerative surgery by clearing existing host cells within a solid organ and creating a supportive niche for new cell engraftment. We hypothesized that hepatocytes transplanted by injection into the portal circulation would preferentially engraft within liver parenchyma pretreated with IRE., Methods: Transgene-positive β-galactosidase-expressing hepatocytes were isolated from B6.129S7-Gt(ROSA)26Sor/J (ROSA26) mice and transplanted by intrasplenic injection into wild-type littermates that received liver IRE pretreatment or control sham treatment. Engraftment of donor hepatocytes in recipient livers was determined by X-gal staining., Results: Significantly higher numbers of X-gal + donor hepatocytes engrafted in the livers of IRE-treated mice as compared to sham-treated mice. X-gal + hepatocytes persisted in IRE-treated recipients for at least 11 d post-transplant and formed clusters. Immunostaining demonstrated the presence of HNF4A/Ki67/β-galactosidase triple-positive cells within IRE-ablation zones, indicating that transplanted hepatocytes preferentially engrafted in IRE-treated liver parenchyma and proliferated., Conclusions: IRE pretreatment of the liver increased engraftment of transplanted hepatocytes within the IRE-ablation zone. IRE treatment of the host liver may be developed clinically as a strategy to increase engraftment efficiency of primary hepatocytes and/or hepatocytes derived from stem cells in cell transplant therapies., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Directional freezing harnesses crystal growth development to create aligned solid structures or etchable patterns, useful for directed ice growth in cryobiology and cryoprinting for tissue engineering. We have delved into the ice-modulating properties of FucoPol, a fucose-rich, bio-based polysaccharide. Previous research on FucoPol revealed its non-colligative hysteresis in kinetic freezing point, reduced crystal dimensions and cryoprotective effect. Here, FucoPol reshaped developing sharp, anisotropic obloid ice dendrites into linearly-aligned, thin, isotropic spicules or tubules (cooling rate-dependent morphology). The effect was enhanced by increased concentration and decreased cooling rate, but major reshaping was observed with 5 μM and below. These structures boasted remarkable enhancements: uniform alignment (3-fold), tip symmetry (5.9-fold) and reduced thickness (5.3-fold). The ice-modulating capability of FucoPol resembles the Gibbs-Thomson effect of antifreeze proteins, in particular the ice reshaping profiles of type I antifreeze proteins and rattlesnake venom lectins, evidenced by a 52.6 ± 2.2° contact angle ( θ ) and spicular structure generation. The high viscosity of FucoPol solutions, notably higher than that of sucrose, plays a crucial role. This viscosity dynamically intensifies during directional freezing, leading to a diffusion-limited impediment that influences dendritic formation. Essentially, the ice-modulating prowess of FucoPol not only reinforces its established cryoprotective qualities but also hints at its potential utility in applications that harness advantageous ice growth for intentional structuring. For instance, its potential in cryobioprinting is noteworthy, offering an economical, biodegradable resource, of easy removal, sidestepping the need for toxic reagents. Moreover, FucoPol fine-tunes resulting ice structures, enabling the ice-etching of biologically relevant patterns within biocompatible matrices for advanced tissue engineering endeavors.

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.

Background: Tumor Treating Fields (TTFields) is a non-invasive modality for cancer treatment that utilizes a specific sinusoidal electric field ranging from 100 kHz to 300 kHz, with an intensity of 1 V/cm to 3 V/cm. Its purpose is to inhibit cancer cell proliferation and induce cell death. Despite promising outcomes from clinical trials, TTFields have received FDA approval for the treatment of glioblastoma multiforme (GBM) and malignant pleural mesothelioma (MPM). Nevertheless, global acceptance of TTFields remains limited. To enhance its clinical application in other types of cancer and gain a better understanding of its mechanisms of action, this review aims to summarize the current research status by examining existing literature on TTFields' clinical trials and mechanism studies., Conclusions: Through this comprehensive review, we seek to stimulate novel ideas and provide physicians, patients, and researchers with a better comprehension of the development of TTFields and its potential applications in cancer treatment., (© 2023 Xing Li et al., published by Sciendo.)

Temperature-controlled 3D cryoprinting (TCC) is an emerging tissue engineering technology aimed at overcoming limitations of conventional 3D printing for large organs: (a) size constraints due to low print rigidity and (b) the preservation of living cells during printing and subsequent tissue storage. TCC addresses these challenges by freezing each printed voxel with controlled cooling rates during deposition. This generates a rigid structure upon printing and ensures cell cryopreservation as an integral part of the process. Previous studies used alginate-based ink, which has limitations: (a) low diffusivity of the CaCl 2 crosslinker during TCC's crosslinking process and (b) typical loss of print fidelity with alginate ink. This study explores the use of an ink made of agar and alginate to overcome TCC protocol limitations. When an agar/alginate voxel is deposited, agar first gels at above-freezing temperatures, capturing the desired structure without compromising fidelity, while alginate remains uncrosslinked. During subsequent freezing, both frozen agar and alginate maintain the structure. However, agar gel loses its gel form and water-retaining ability. In TCC, alginate crosslinking occurs by immersing the frozen structure in a warm crosslinking bath. This enables CaCl 2 diffusion into the crosslinked alginate congruent with the melting process. Melted agar domains, with reduced water-binding ability, enhance crosslinker diffusivity, reducing TCC procedure duration. Additionally, agar overcomes the typical fidelity loss associated with alginate ink printing.

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., (© 2023. Springer Nature Limited.)

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., (© 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).)

This technical paper introduces a novel organ preservation system based on isochoric (constant volume) supercooling. The system is designed to enhance the stability of the metastable supercooling state, offering potential long-term preservation of large biological organs at subfreezing temperatures without the need for cryoprotectant additives. Detailed technical designs and usage protocols are provided for researchers interested in exploring this field. The paper also presents a control system based on the thermodynamics of isochoric freezing, utilizing pressure monitoring for process control. Sham experiments were performed using whole pig liver sourced from a local food supplier to evaluate the system's ability to sustain supercooling without ice nucleation for extended periods. The results demonstrated sustained supercooling without ice nucleation in pig liver tissue for 24 and 48 h. These findings suggest the potential of this technology for large-volume, cryoprotectant-free organ preservation with real-time control over the preservation process. The simplicity of the isochoric supercooling device and the design details provided in the paper are expected to serve as encouragement for other researchers in the field to pursue further research on isochoric supercooling. However, final evidence that these preserved organs can be successfully transplanted is still lacking.

Temperature-Controlled-Cryoprinting (TCC) is a new 3D bioprinting technology that allows for the fabrication and cryopreservation of complex and large cell-laden scaffolds. During TCC, bioink is deposited on a freezing plate that descends further into a cooling bath, keeping the temperature at the nozzle constant. To demonstrate the effectiveness of TCC, we used it to fabricate and cryopreserve cell-laden 3D alginate-based scaffolds with high cell viability and no size limitations. Our results show that Vero cells in a 3D TCC bioprinted scaffold can survive cryopreservation with a viability of 71%, and cell viability does not decrease as higher layers are printed. In contrast, previous methods had either low cell viability or decreasing efficacy for tall or thick scaffolds. We used an optimal temperature profile for freezing during 3D printing using the two-step interrupted cryopreservation method and evaluated drops in cell viability during the various stages of TCC. Our findings suggest that TCC has significant potential for advancing 3D cell culture and tissue engineering.

This study was motivated by the increasing interest in finding ways to preserve organs in a supercooled state for transplantation. Previous research with small volumes suggests that the isochoric (constant volume) thermodynamic state enhances the stability of supercooled solutions. The primary objective of this study was to investigate the feasibility of storing a large organ, such as the pig liver, in a metastable isochoric supercooled state for clinically relevant durations. To achieve this, we designed a new isochoric technology that employs a system consisting of two domains separated by an interior boundary that can transfer heat and pressure, but not mass. The liver is preserved in one of these domains in a solution with an intracellular composition, which is in osmotic equilibrium with the liver. Pressure is used to monitor the thermodynamic state of the isochoric chamber. In this feasibility study, two pig livers were preserved in the device in an isochoric supercooled state at -2°C. The experiments were terminated voluntarily, one after 24 h and the other after 48 h of supercooling preservation. Pressure measurements indicated that the livers did not freeze during the isochoric supercooling preservation. This is the first proof that organs as large as the pig liver can remain supercooled for extended periods of time in an isotonic solution in an isochoric system, despite an increased probability of ice nucleation with larger volumes. To serve as controls and to test the ability of pressure monitoring to detect freezing in the isochoric chamber, an experiment was designed in which two pig livers were frozen at -2°C for 24 h and the pressure monitored. Histological examination with H&E stains revealed that the supercooled liver maintained a normal appearance, even after 48 h of supercooling, while tissues in livers frozen to -2°C were severely disrupted by freezing after 24 h., Competing Interests: The authors whose names are listed above certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (© 2023 The Authors.)

This is an exploratory study on the effect of electrolysis, delivered during the thawing stage of a cryoablation protocol, on tissue ablation. This treatment protocol, that combines freezing and electrolysis, is named "cryoelectrolysis". In cryoelectrolysis the cryoablation probe is also used as the electrolysis delivering electrode. The study was performed on the liver of Landrace pigs and the tissues were examined 24 hours after treatment (two pigs) and 48 hours after treatment (one pig). The cryoelectrolysis device and different cryoelectrolysis ablation configurations tested are described. This exploratory, non-statistical study shows that the addition of electrolysis expands the ablated area in comparison to cryoablation alone and that there is a substantial difference between the histological appearance of tissue treated by cryoablation alone, tissue treated by cryoablation and electrolysis at the anode and tissue treated by cryoablation and electrolysis at the cathode., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lugnani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Pancreatic islet transplantation via infusion through the portal vein, has become an established clinical treatment for patients with type 1 diabetes. Because the engraftment efficiency is low, new approaches for pancreatic islets implantation are sought. The goal of this study is to explore the possibility that a non-thermal irreversible electroporation (NTIRE) decellularized matrix in the liver could be used as an engraftment site for pancreatic islets., Materials and Methods: Pancreatic islets or saline controls were injected at sites pre-treated with NTIRE in the livers of 7 rats, 16 hours after NTIRE treatment. Seven days after the NTIRE treatment, islet graft function was assessed by detecting insulin and glucagon in the liver with immunohistochemistry., Results: Pancreatic islets implanted into a NTIRE-treated volume of liver became incorporated into the liver parenchyma and produced insulin and glucagon in 2 of the 7 rat livers. Potential reasons for the failure to observe pancreatic islets in the remaining 5/7 rats may include local inflammatory reaction, graft rejection, low numbers of starting islets, timing of implantation., Conclusions: This study shows that pancreatic islets can become incorporated and function in an NTIRE-generated extracellular matrix niche, albeit the success rate is low. Advances in the field could be achieved by developing a better understanding of the mechanisms of failure and ways to combat these mechanisms., (© 2023 Yanfang Zhang, Yanpeng Lv, Yunlong Wang, Tammy T Chang, Boris Rubinsky, published by Sciendo.)

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., (© 2022 Institute of Food Technologists. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

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., (Copyright © 2022 by ASME.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

Background: Tumor Treating Fields (TTFields), are a novel, non-invasive tissue ablation technology for treatment of cancer. Tissue ablation is achieved through the continuous delivery of a narrow range of electromagnetic fields across a tumor, for a period of months. TTFields are designed to affect only cells that divide and to interfere with the cell division process. The therapy is monitored with MRI imaging, performed every couple of months. Current technology is unable to assess the treatment effectiveness in real time., Methods: We propose that the effect of the treatment can be assessed, in real time, by continuously measuring the change in electrical impedance across the TTFields delivery electrode arrays. An in vitro anatomic skull experimental study, with brain and tumor mimics phantom tissues was conducted to confirm the potential value of the proposed monitoring system., Results: Experiments show that measuring the change in the impedance amplitude between opposite TTFields electrode arrays, at a typical TTFields treatment frequency of (200 kHz), can detect changes in the tumor radius with a sensitivity that increases with the radius of the tumor. The study shows that TTFields electrode arrays can be used to assess the effectiveness of TTFields treatment on changes in the tumor dimensions in real time, throughout the treatement. This monitoring system may become a valuable addition to the TTFields cancer treatment technology. It could provide the means to continuously assess the effectiveness of the treatment, and thereby optimize the design of the treatment protocol., Competing Interests: Boris Rubinsky is an Academic Editor for PeerJ. The other co-authors declare that they have no competing interests., (©2022 Li et al.)

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.

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., (© 2021. The Author(s).)

Irreversible electroporation (IRE) is a non-thermal tissue ablative technology that has emerging applications in surgical oncology and regenerative surgery. To advance its therapeutic usefulness, it is important to understand the mechanisms through which IRE induces cell death and the role of the innate immune system in mediating subsequent regenerative repair. Through intravital imaging of the liver in mice, we show that IRE produces distinctive tissue injury features, including delayed yet robust recruitment of neutrophils, consistent with programmed necrosis. IRE treatment converts the monocyte/macrophage balance from pro-inflammatory to pro-reparative populations, and depletion of neutrophils inhibits this conversion. Reduced generation of pro-reparative Ly6C lo F4/80 hi macrophages correlates with lower numbers of SOX9 + hepatic progenitor cells in areas of macrophage clusters within the IRE injury zone. Our findings suggest that neutrophils play an important role in promoting the development of pro-reparative Ly6C lo monocytes/macrophages at the site of IRE injury, thus establishing conditions of regenerative repair., (© 2021. The Author(s).)

Multi-scale macroalgae growth models are required for the efficient design of sustainable, economically viable, and environmentally safe farms. Here, we develop a multi-scale model for Ulva sp. macroalgae growth and nitrogen sequestration in an intensive cultivation farm, regulated by temperature, light, and nutrients. The model incorporates a range of scales by incorporating spatial effects in two steps: light extinction at the reactor scale (1 m) and nutrient absorption at the farm scale (1 km). The model was validated on real data from an experimental reactor installed in the sea. Biomass production rates, chemical compositions, and nitrogen removal were simulated under different seasons, levels of dilution in the environment and water-exchange rate in the reactor. This multi-scale model provides an important tool for environmental authorities and seaweed farmers who desire to upscale to large bioremediation and/or macroalgae biomass production farms, thus promoting the marine sustainable development and the macroalgae-based bioeconomy.

There is growing interest in the use of isochoric (constant volume) freezing for cryopreservation of biological matter. The goal of this study is to generate fundamental experimental data on the pressure temperature relation during the freezing of an isochoric system of aqueous solutions of two compounds, glucose and glycerol. Glucose and glycerol are commonly used as cryoprotectants in conventional isobaric (constant pressure) cryopreservation protocols. Earlier studies have shown that the increase in pressure during isochoric freezing is detrimental to biological matter and limits the range of temperatures in which isochoric freezing can be used for preservation to temperatures corresponding to pressures below 40 MPa. In physiological saline solution this pressure corresponds to a temperature of - 4 °C. Our new experimental data shows that the addition of 2 M glycerol to the saline solution lowers the temperature at which the isochoric freezing pressure is 40 MPa to -11 °C, 3 M glycerol to - 16.5 °C, and 4 M glycerol to - 24.5 °C, thereby substantially expending the range of temperatures in which cryopreservation by isochoric freezing can be practiced., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This paper is a theoretical study of a protocol for transport of high concentrations of cryoprotectants into biological matter, using isochoric freezing. Unlike isobaric freezing, where the entire system freezes at temperatures lower than the freezing temperature, in isochoric freezing a substantial portion of the system remains unfrozen at temperatures below freezing. In isochoric freezing cryopreservation, the system is designed in such a way that the biological matter remains unfrozen and surrounded by an unfrozen solution. The protocol in this study involves the freezing of an isochoric systems along the "liquidus line" at which water and ice are in thermodynamic equilibrium. Rejection of solutes by ice increases the concentration of the solutes in the unfrozen solution surrounding the unfrozen biological matter, leading, thereby, to transport of increasingly higher concentrations of cryoprotectants into the biological matter, as the temperature of the system is lowered and the toxicity of the cryoprotectants is reduced., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Clinical studies show that low intensity (single V/cm), intermediate-frequency (100 kHz-300 kHz) electric fields inhibit the growth of cancer cells, while the mechanism is not yet understood. We examine the hypothesis that electric fields modify the cell membrane potential of dividing cancer cells in a way that correlates with cells growth inhibition., Methods: A Schwan based mathematical model calculates the changes in HeLa cells membrane potential due to single V/cm electric fields and frequencies from 0.1 to 1 MHz. An experimental study examines the effect of these electric fields on the inhibition of HeLa cells growth in an incubator., Results: The theoretical calculation shows that the effects of these electric fields on cell membrane potential decrease with an increase in frequency. The HeLa cells experiments verified the inhibitory effect of these fields on cell growth. The inhibitory effect is decreasing with an increase in frequency, in a way that is similar to the frequency dependent effect of these fields on the cell membrane potential., Conclusions: The superposition of the theoretical results and the experimental results suggest a correlation between the effect of these fields on the cell membrane potential and inhibition of cancer cell growth. It should be emphasized that correlations do not prove causality, however, they suggest an area for future research., Significance: These findings have value for the understanding of the mechanisms of cancer cells growth inhibition with electric fields and suggest an interesting area of research on the interaction between electromagnetic fields and cancer cells.

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., (Published by Elsevier Ltd.)

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.

Objective: An experimental study for testing a simple robust algorithm on data derived from an electromagnetic radiation device that can detect small changes in the tissue/fluid ratio in a realistic head configuration., Methods: Changes in the scattering parameters (S 21 ) of an inductive coil resulting from injections of chicken blood in the 0-18 ml range into calf brain tissue in a human anatomical skull were measured over a 100-1,000 MHz frequency range., Results: An algorithm that combines amplitude and phase results was found to detect changes in the tissue/fluid ratio with 90% accuracy. An algorithm that estimated the injected blood volume was found to have a 1-4 ml average error. This demonstrates the possibility of the inductive coil-based device to possess a practical ability to detect a change in the tissue/fluid ratio in the head., Significance: This study is an important step towards the goal of building an inexpensive and safe device that can detect an early brain hemorrhagic stroke., Competing Interests: Boris Rubinsky is an Academic Editor for PeerJ., (© 2020 Oziel et al.)

Tumor-treating fields (TTFields) are low-intensity and intermediate-frequency alternating electric fields that have been found to inhibit tumor cell growth. While effective, the mechanism by which TTFields affect cell growth is not yet clearly understood. Although numerous mathematical studies on the effects of electromagnetic fields on single cells exist, the effect of TTFields on single cells have been analyzed less frequently. The goal of this study is to explore through a mathematical analysis the effects of TTFields on single cells, with particular emphasis on the thermal effect. We examine herein two single-cell models, a simplified spheroidal model and a simulation of a U-87 MG glioblastoma cell model obtained from microscopic images. A finite element method is used to analyze the electric field distribution, electromagnetic loss, and thermal field distribution. The results further prove that the electric field in the cytoplasm is too weak and its thermal damage can be excluded as a mechanism for cell death in TTFields. Bioelectromagnetics. 2020;41:438-446. © 2020 Bioelectromagnetics Society., (© 2020 Bioelectromagnetics Society.)

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., (© 2020 Institute of Food Technologists®.)

Objective: A theoretical study on the mechanisms through which Tumor Treating Fields (TTFields) affect dividing tumor cells., Methods: Numerical analysis was used to revisit two previously proposed mechanisms and introduce a third. We examine the previous hypotheses that: a) TTFields generate a moment that affects microtubule assembly during early mitosis, and b) dielectrophoretic (DEP) forces cause neutral particles to move toward the cleavage furrow during the telophase stage. We further introduce a new hypothesis that TTFields modify cell membrane potential in dividing tumor cells., Results: a) The Brownian energy is several orders of magnitude larger than the moment induced by TTFields on tubulin dimers. b) Adding Stokes drag forces to DEP forces shows that the motion of the particles in the cytoplasm is very slow, approximately 0.003 µm/s, and therefore, unless the duration of the telophase is long enough there will be no substantial effect from the DEP forces. c) The Schwan equation shows that electric fields at the frequencies of clinical TTFields can cause a 10%-17% change in tumor cell membrane potential., Conclusion: Our studies find limited support for the previously suggested hypotheses and suggest that the TTFields affect ion channels by inducing cell membrane potential change could be a mechanism of tumor cell death., Significance: Previously suggested mechanisms of tumor cell death from TTFields are found lacking. The effect of TTFields on the tumor cell membrane potential warrants further research.

Detecting bacteria in samples and differentiating between Gram-negative and Gram-positive species is an important challenge, and the most common method, Gram staining, is very time consuming. The aim of this study was to evaluate the electrical bioimpedance spectroscopy (EBIS) technique as an inexpensive and practical tool for real-time detection of bacteria and differentiation between Gram-positive and Gram-negative species. The relevant sensitivity for differentiating between species was found in the magnitude and phase at frequencies of 158,489 and 5248 Hz, respectively, at a bacterial concentration of 1 μg/μl. Subsequently, the sensitivity was estimated as a function of bacterial concentration. Our results demonstrated that EBIS can potentially distinguish between presence and absence of bacteria as well as between different types of bacteria.

This review summarizes the use of high-voltage electrical pulses (HVEPs) in clinical oncology to treat solid tumors with irreversible electroporation (IRE) and electrochemotherapy (ECT). HVEPs increase the membrane permeability of cells, a phenomenon known as electroporation. Unlike alternative ablative therapies, electroporation does not affect the structural integrity of surrounding tissue, thereby enabling tumors in the vicinity of vital structures to be treated. IRE uses HVEPs to cause cell death by inducing membrane disruption, and it is primarily used as a radical ablative therapy in the treatment of soft-tissue tumors in the liver, kidney, prostate, and pancreas. ECT uses HVEPs to transiently increase membrane permeability, enhancing cellular cytotoxic drug uptake in tumors. IRE and ECT show immunogenic effects that could be augmented when combined with immunomodulatory drugs, a combination therapy the authors term electroimmunotherapy . Additional electroporation-based technologies that may reach clinical importance, such as gene electrotransfer, electrofusion, and electroimmunotherapy, are concisely reviewed. HVEPs represent a substantial advancement in cancer research, and continued improvement and implementation of these presented technologies will require close collaboration between engineers, interventional radiologists, medical oncologists, and immuno-oncologists., (© RSNA, 2020.)

Irreversible electroporation (IRE) employs brief, high-electric field pulses to ablate tumors while preserving the extracellular matrix. Recently, we showed that combining short high-voltage (SHV) IRE pulses and long low-voltage (LLV) IRE pulses can enlarge the tissue ablation region, presumably through a synergistic effect., Objective: The goal of this study is to further investigate the effect of this combination on a 2-D cell layer tumor model., Methods: 2-D layers of tumor cells are exposed to various SHV and LLV combinations, and the results of propidium iodide (PI) and fluorescein diacetate staining are examined to correlate treatment protocols with the ensuing irreversible and reversible electroporation areas., Results: The combination of SHV+LLV pulses produces a larger area of electroporation and ablation than LLV+SHV pulses, LLV pulses alone, and SHV pulses alone., Conclusion: Judiciously combining SHV and LLV pulses can produce a synergistic effect that enlarges the electroporation-induced ablation area. A hypothetical explanation for this effect is that it involves a combination of pore expansion and electrolysis induced by LLV pulses in the area that had been reversibly permeabilized by the SHV pulses., Significance: This paper is valuable for the design of improved IRE protocols and provides a hypothesis for the mechanisms involved.

Background: Percutaneous image-guided tissue ablation (IGA) plays a growing role in the clinical management of solid malignancies. Electroporation is used for IGA in several modalities: irreversible electroporation (IRE), and reversible electroporation with chemotoxic drugs, called electrochemotherapy (ECT). It was shown that the combination of electrolysis and electroporation-E2-affords tissue ablation with greater efficiency, that is, lower voltages, lower energy and shorter procedure times than IRE and without the need for chemotoxic additives as in ECT., Methods: A new E2 waveform was designed that delivers optimal doses of electroporation and electrolysis in a single waveform. A series of experiments were performed in the liver of pigs to evaluate E2 in the context of clinical applications. The goal was to find initial parameter boundaries in terms of electrical field, pulse duration and charge as well as tissue behavior to enable real time tissue ablation of clinically relevant volumes., Results: Histological results show that a single several hundred millisecond long E2 waveform can ablate large volume of tissue at relatively low voltages while preserving the integrity of large blood vessels and lumen structures in the ablation zone without the use of chemotoxic drugs or paralyzing drugs during anesthesia. This could translate clinically into much shorter treatment times and ease of use compared to other techniques that are currently applied., Competing Interests: Boris Rubinsky is an Academic Editor for PeerJ. Enric Guenther, Nina Klein, Paul Mikus, Michael Stehling, Boris Rubinsky are employees of InterScience GmbH, Lucerne, Switzerland (a company in the field of tissue ablation by electroporation). Franco Lugnani and Matteo Macchioro are employed by Hippocrates D.O.O. (a medical office/company). All other authors have no competing interests., (© 2020 Gunther et al.)

The present study is part of an ongoing effort to develop a simple diagnostic technology for detecting internal bleeding in the brain, which can be used in lieu or in support of medical imaging and thereby reduce the cost of diagnostics in general, and in particular, would make diagnostics accessible to economically disadvantaged populations. The study deals with a single coil inductive device to be used for detecting cerebral hemorrhage. It presents a first-order experimental study that examines the predictions of our recently published theoretical study. The experimental model employs a homogeneous cylindrical phantom in which internal head bleeding was simulated by way of a fluid inclusion. We measured the changes in amplitude and phase across the coil with a network vector analyzer as a function of frequency (100-1,000 MHz), volume of blood simulating fluid, and the site of the fluid injection. We have developed a new mathematical model to statistically analyze the complex data produced in this experiment. We determined that the resolution for the fluid volume increase following fluid injection is strongly dependent on frequency as well as the location of liquid accumulation. The experimental data obtained in this study supports the predictions of our previous theoretical study, and the statistical analysis shows that the simple single coil device is sensitive enough to detect changes due to fluid volume alteration of two milliliters. Bioelectromagnetics. 2020;41:21-33 © 2019 Bioelectromagnetics Society., (© 2019 Bioelectromagnetics Society.)

Real time monitoring of cell division, mitosis, at the single cell level, has value for many biomedical applications; such as developing optimal cancer treatments that target the cell division process. The goal of this theoretical study is to explore the feasibility of using Micro Electrical Impedance Tomography (MEIT) for real time monitoring of mitosis in a single cell, through imaging. MEIT employs a micro (single cell) scale electrode cage with electrodes placed around the cell. The electrodes deliver subsensory current and the consequential voltages on the electrodes are measured. An inverse image reconstruction algorithm uses the electric data from the electrodes to generate a map of electrical conductivity distribution in the chamber, which is the image. EIT is a well-known medical imaging technology that is simple to use but lacks good resolution. Therefore, it is not a-priori obvious that EIT has sufficient resolution to monitor single cell mitosis. To accomplish the goal of this study we have developed a mathematical model of MEIT of single cell mitosis, in which an in silico experiment provided the data for the MEIT image reconstruction. This theoretical study shows that MEIT can detect the outlines of the dividing cell during the various stages of mitosis (metaphase, anaphase and telophase) and, therefore, has potential as a technology for real time monitoring of single cell mitosis.

Background: The safety and efficacy of irreversible electroporation (IRE) in treating hepatic, biliary, and pancreatic malignancies are active areas of clinical investigation. In addition, recent studies have shown that IRE may enable regenerative surgery and in vivo tissue engineering. To use IRE effectively in these clinical applications, it is important to understand how different tissue microenvironments impact the response to IRE. In this study, we characterize the electrical and histological properties of non-fibrotic and fibrotic liver parenchyma before and after IRE treatment., Methods: Electrical resistivity and histology of fibrotic liver from C57BL/6 mice fed a 0.1% 3,5-diethylcarbonyl-1,4-dihydrocollidine (DDC) diet were compared to those of non-fibrotic liver from matched control mice before and after IRE treatment., Results: At baseline, the electrical resistivity of fibrotic liver was lower than that of non-fibrotic liver. Post-IRE, resistivity of non-fibrotic liver declined and then recovered back to baseline with time, correlating with hepatocyte repopulation of the ablated parenchyma without deposition of fibrotic scar. In contrast, resistivity of fibrotic liver remained depressed after IRE treatment, correlating with persistent inflammation., Conclusion: Non-fibrotic and fibrotic liver respond to IRE differently. The underlying tissue microenvironment is an important modifying factor to consider when designing IRE protocols for tissue ablation., (Copyright © 2019 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 by ASME.)

Irreversible electroporation (IRE), is a new non-thermal tissue ablation technology in which brief high electric field pulses are delivered across the target tissue to induce cell death by irreversible permeabilization of the cell membrane. A deficiency of conventional IRE is that the ablation zone is relatively small, bounded by the irreversible electroporation isoelectric field margin. In the previous studies we have introduced a new treatment protocol that combines few short high voltage (SHV) pulses with long low-voltage (LLV) pulses. In the previous studies, we also have shown that the addition of few SHV pulses increases by almost a factor of two the area ablated by a protocol that employs only the LLV pulses. This study employs potato and gel phantom to generate a plausible explanation for the mechanism. The study provides circumstantial evidence that the mechanism involved is the production of electrolytic compounds by the LLV pulse sequence, which causes tissue ablation beyond the margin of the irreversible electroporation isoelectric field generated by the SHV pulses, presumable to the reversible electroporation isoelectric field margin generated by the SHV pulses.

Objective: This numerical study was designed to evaluate the feasibility of using an inductive coil for monitoring the changes in the volume of a hematoma in the head in situ and to compare the inductive coil performance to that of a spiral antenna based on the radar principle., Methods: Numerical analysis was used to solve the complete set of Maxwell's equations in full three-dimensional anatomical model of a head and brain with data on clinical occurrence of hematomas from the clinical literature, for frequencies of 100 MHz, 500 MHz, and 1 GHz., Results: 1) The analysis shows that the spiral radar antenna provides a better resolution when the antenna can be placed exactly facing the center of the volume of blood. Under any other circumstance, the inductive coil has a better resolution at both 500 MHz and 1 GHz. 2) The induction coil is more sensitive to rotation artifacts than the spiral antenna. 3) Single frequency measurements do not provide conclusive results., Conclusion: The inductive coil has the ability to monitor small changes in the volume of a hematoma in the head. However, multifrequency measurements are required for correct diagnostic., Significance: This study provides a new, low-cost alternative to the conventional medical imaging for monitoring the hematoma increase.

In engineering and medicine, there is a growing interest in using textiles made of composites with enhanced thermal properties. One such type of textile is fabric impregnated with ceramics and mineral particles. This material has high emissivity in the infrared range and may have therapeutic benefits for treatments of diseases, like Raynaud's syndrome. While there is significant clinical and commercial interest, there is an evident lack of fundamental studies on the heat transfer aspects of these fabrics. The goal of this technical brief is to present results from a fundamental study examining the thermal effects of fabric with ceramics and minerals (produced by Nanobionic, Inc., Athens, Greece) on the temperatures of the hands. With a confidence level of 90%, the results show that the textile with ceramics and minerals has an enhanced thermal effect on warming a cold hand in comparison to a placebo fabric without ceramics or minerals. Much more research is needed to increase the level of confidence and develop a fundamental understanding of the mechanism., (Copyright © 2019 by ASME.)

This study examined the temporal physiological and molecular events following the treatment of the liver with a tissue ablation modality that combined electroporation with electrolysis (E2). Rat liver was treated with an E2 waveform and the tissue examined, 1 h, 3 h, 6 h and 24 h with: H&E, Masson Trichrome, TUNEL stains and Western blot. H&E and TUNEL stains have shown that cell death began to be evident 3 h and hepatocyte regeneration was seen 24 h after treatment. H&E and Masson trichrome have shown that the extracellular matrix and the large lumens, appeared intact after E2. Western blot has shown the following molecular events after E2: cleaved caspase 3-downgraded at 1 h, upgraded at 24 h (apoptosis); cleaved Caspase 1 and cleaved GSDMD-upgraded at 6 h (pyroptosis), RIP3-upgraded at 1 h, MLKL-upgraded at 3 h (necroptosis). The mechanism of cell death was possible initiated by necroptosis pathway. Pyroptosis pathway was also activated. The observation that cell death from E2 was by programed necrosis and the details on the temporal molecular pathways, may have value for the recent attempt to combine electroporation mediated ablation with immunological treatment, by demonstrating that the cell death from E2 involves an inflammatory response and by providing data that could be used to design the optimal timing for the injection of immunological adjuvants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Nonthermal irreversible electroporation is a minimally invasive surgery technology that employs high and brief electric fields to ablate undesirable tissues. Nonthermal irreversible electroporation can ablate only cells while preserving intact functional properties of the extracellular structures. Therefore, nonthermal irreversible electroporation can be used to ablate tissues safely near large blood vessels, the esophagus, or nerves. This suggests that it could be used for thyroid ablation abutting the esophagus. This study examines the feasibility of using nonthermal irreversible electroporation for thyroid ablation., Methods: Rats were used to evaluate the effects of nonthermal irreversible electroporation on the thyroid. The procedure entails the delivery of high electric field pulses (1-3 kV/cm, 100 microseconds) between 2 surface electrodes bracing the thyroid. The right lobe was treated with various nonthermal irreversible electroporation pulse sequences, and the left was the control. After 24 hours of the nonthermal irreversible electroporation treatment, the thyroid was examined with hemotoxylin and eosin histological analysis. Mathematical models of electric fields and the Joule heating-induced temperature raise in the thyroid were developed to examine the experimental results., Results: Treatment with nonthermal irreversible electroporation leads to follicular cells damage, associated with cell swelling, inflammatory cell infiltration, and cell ablation. Nonthermal irreversible electroporation spares the trachea structure. Unusually high electric fields, for these types of tissue, 3000 V/cm, are needed for thyroid ablation. The mathematical model suggests that this may be related to the heterogeneous structure of the thyroid-induced distortion of local electric fields. Moreover, most of the tissue does not experience thermal damage inducing temperature elevation. However, the heterogeneous structure of the thyroid may cause local hot spots with the potential for local thermal damage., Conclusion: Nonthermal irreversible electroporation with 3000 V/cm can be used for thyroid ablation. Possible applications are treatment of hyperthyroidism and thyroid cancer. The highly heterogeneous structure of the thyroid distorts the electric fields and temperature distribution in the thyroid must be considered when designing treatment protocols for this tissue type.