Search

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.

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.

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.

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 CaCl2 crosslinker during TCCs 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 CaCl2 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.

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 systems 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.

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.

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.

BackgroundPancreatic 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 methodsPancreatic 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.ResultsPancreatic 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.ConclusionsThis 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.

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.

A three-dimensional (3D) printing technology that facilitates continuous printing along a combination of Cartesian and curvilinear coordinates, designed for in vivo and in situ bioprinting, is introduced. The combined Cartesian/curvilinear printing head motion is accomplished by attaching a biomimetic, flexible, “tendon cable” soft robot arm to a conventional Cartesian three axis 3D printing carousel. This allows printing along a combination of Cartesian and curvilinear coordinates using five independent stepper motors controlled by an Arduino Uno with each motor requiring a microstep driver powered via a 12 V power supply. Three of the independent motors control the printing head motion along conventional Cartesian coordinates while two of the independent motors control the length of each pair of the four “tendon cables” which in turn controls the radius of curvature and the angle displacement of the soft printer head along two orthogonal planes. This combination imparts motion along six independent degrees-of-freedom in Cartesian and curvilinear coordinates. The design of the system is described together with experimental results, which demonstrate that this design can print continuously along curved and inclined surfaces while avoiding the “staircase” effect, which is typical of conventional three axis 3D printing along curvilinear surfaces.

3D Bioprinting is a popular method of fabricating scaffolds for tissue engineering. While soft bioinks such as alginate are useful for reproducing the softest tissues of the human body, it remains difficult to 3D print objects out of bioinks that cannot support their own weight. 3D cryoprinting is a promising method of printing objects out of soft bioinks and creating highly porous scaffolds for tissue engineering. However, crosslinking the frozen objects before they thaw and collapse remains a challenge. Here we investigate a process which we name “freezing-modulated-crosslinking” for crosslinking frozen objects produced by 3D cryoprinting. During freezing-modulated-crosslinking, frozen objects are thawed in a crosslinker bath at a controlled melting rate, so that crosslinking occurs layer by layer and the object can maintain its printed shape. First, we examine the process with a mathematical model to determine the important thermal parameters. Second, we validate our results experimentally by printing a variety of multi-layer alginate objects. By systematically examining this crosslinking approach, we expand the options for 3D cryoprinting. 3D cryoprinted alginate scaffolds can be seeded with cells for use in 3D cell culture or for tissue regeneration.

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.

Three-dimensional (3D) bioprinting is a fabrication method with many biomedical applications, particularly within tissue engineering. The use of freezing during 3D bioprinting, aka "3D cryoprinting," can be utilized to create micopores within tissue-engineered scaffolds to enhance cell proliferation. When used with alginate bio-inks, this type of 3D cryoprinting requires three steps: 3D printing, crosslinking, and freezing. This study investigated the influence of crosslinking order and cooling rate on the microstructure and mechanical properties of sodium alginate scaffolds. We designed and built a novel modular 3D printer in order to study the effects of these steps separately and to address many of the manufacturing issues associated with 3D cryoprinting. With the modular 3D printer, 3D printing, crosslinking, and freezing were conducted on separate modules yet remain part of a continuous manufacturing process. Crosslinking before the freezing step produced highly interconnected and directional pores, which are ideal for promoting cell growth. By controlling the cooling rate, it was possible to produce pores with diameters from a range of 5 μm to 40 μm. Tensile and firmness testing found that the use of freezing does not decrease the tensile strength of the printed objects, though there was a significant loss in firmness for strands with larger pores.

BackgroundTumor 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.MethodsWe 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.ResultsExperiments 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.

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.

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 Ly6CloF4/80hi 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 Ly6Clo monocytes/macrophages at the site of IRE injury, thus establishing conditions of regenerative repair.

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.

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.

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.

ObjectiveAn 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.MethodsChanges in the scattering parameters (S21) 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.ResultsAn 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.SignificanceThis study is an important step towards the goal of building an inexpensive and safe device that can detect an early brain hemorrhagic stroke.

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.

BackgroundThe 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.MethodsElectrical 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.ResultsAt 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.ConclusionNon-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.

BackgroundNonthermal 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.MethodsRats 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.ResultsTreatment 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.ConclusionNonthermal 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.

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. [ABSTRACT FROM AUTHOR]

Non-thermal irreversible electroporation (NTIRE) is a biophysical phenomenon in which certain electric fields delivered across the cell membrane in tissue, cause cell death, without affecting the extracellular matrix. "Minimally invasive regenerative surgery" is a new medical modality for treatment of end-stage organ or tissue failure in which exogenous cells are implanted in a decellularized niche in tissue, formed by the delivery of NTIRE electric fields across a targeted volume of tissue. We anticipate that the success of the procedure will depend on the time of implantation relative to the application of NTIRE. This study was performed to elucidate the histological and molecular events that occur within 24 h after NTIRE, in the context of optimal criteria for the time of implantation. To this end, we examined the histology of NTIRE treated rat liver with H&E, Masson trichrome and TUNEL staining. Western blot was used to examine pro and cleaved caspase-3 (marker for apoptosis), pro and cleaved caspase-1 and gasdermin D (markers for pyroptosis), and RIP3 and MLKL (markers for necroptosis). The key findings are that, complete hepatocytes disintegration within an intact extracellular matrix is seen at 6 h and, new hepatocytes are seen in the treated region at 24 h, after NTIRE. There is no evidence of apoptotic cell death from NTIRE, contrary to commonly made claims in the NTIRE literature. However, molecular pathways of pyroptosis and necroptosis, programed necrosis associated with inflammation, are activated at 6 h after NTIRE and are not evident at 24 h after NTIRE. These are fundamental new findings of basic value to the field of NTIRE in all its applications. Taken together the results suggest the hypothesis that an optimal time for implantation is about 24 h after NTIRE. Future studies in which exogenous cells are implanted at different times after NTIRE are required to examine this hypothesis.

It was recently shown that electrolysis may play a substantial detrimental role in microfluidic electroporation. To overcome this problem, we have developed a non-electrolytic micro/nano electroporation (NEME) electrode surface, in which the metal electrodes are coated with a dielectric. A COMSOL based numerical scheme was used to simultaneously calculate the excitation frequency and dielectric material properties dependent electric field delivered across the dielectric, fluid flow, electroporation field and Clausius-Mossotti factor for yeast and E. coli cells flowing in a channel flow across a NEME surface. A two-layer model for yeast and a three-layer model for E. coli was used. The numerical analysis shows that in NEME electroporation, the electric fields could induce electroporation and dielectrophoresis simultaneously. The simultaneous occurrence of electroporation and dielectrophoresis gives rise to several interesting phenomena. For example, we found that a certain frequency exists for which an intact yeast cell is drawn to the NEME electrode, and once electroporated, the yeast cell is pushed back in the bulk fluid. The results suggest that developing electroporation technologies that combine, simultaneously, electroporation and dielectrophoresis could lead to new applications. Obviously, this is an early stage numerical study and much more theoretical and experimental research is needed.

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo. We treated the livers of mice with NTIRE and subsequently implanted exogenous congenic hepatocytes within the zone of cell ablation. Donor hepatocytes engrafted and integrated with host liver parenchyma pre-treated with NTIRE. This new approach should have value for studying the effects of a native matrix scaffold on in vivo cell growth and may pioneer a new type of minimally-invasive regenerative surgery.

BackgroundCryoelectrolysis is a new minimally invasive tissue ablation surgical technique that combines the ablation techniques of electrolytic ablation with cryosurgery. The goal of this study is to examine the hypothesis that electrolysis can take place in a frozen aqueous saline solution.MethodTo examine the hypothesis we performed a cryoelectrolytic ablation protocol in which electrolysis and cryosurgery are delivered simultaneously in a tissue simulant made of physiological saline gel with a pH dye. We measured current flow, voltage and extents of freezing and pH dye staining.ResultsUsing optical measurements and measurements of currents, we have shown that electrolysis can occur in frozen physiological saline, at high subzero freezing temperatures, above the eutectic temperature of the frozen salt solution. It was observed that electrolysis occurs when the tissue resides at high subzero temperatures during the freezing stage and essentially throughout the entire thawing stage. We also found that during thawing, the frozen lesion temperature raises rapidly to high subfreezing values and remains at those values throughout the thawing stage. Substantial electrolysis occurs during the thawing stage. Another interesting finding is that electro-osmotic flows affect the process of cryoelectrolysis at the anode and cathode, in different ways.DiscussionThe results showing that electrical current flow and electrolysis occur in frozen saline solutions imply a mechanism involving ionic movement in the fluid concentrated saline solution channels between ice crystals, at high subfreezing temperatures. Temperatures higher than the eutectic are required for the brine to be fluid. The particular pattern of temperature and electrical currents during the thawing stage of frozen tissue, can be explained by the large amounts of energy that must be removed at the outer edge of the frozen lesion because of the solid/liquid phase transformation on that interface.ConclusionElectrolysis can occur in a frozen domain at high subfreezing temperature, probably above the eutectic. It appears that the most effective period for delivering electrolytic currents in cryoelectrolysis is during the high subzero temperatures stage while freezing and immediately after cooling has stopped, throughout the thawing stage.

BACKGROUND:Freezing is commonly used for food preservation. It is usually done under constant atmospheric pressure (isobaric). While extending the life of the produce, isobaric freezing has detrimental effects. It causes loss of food weight and changes in food quality. Using thermodynamic analysis, we have developed a theoretical model of the process of freezing in a constant volume system (isochoric). The mathematical model suggests that the detrimental effects associated with isobaric freezing may be reduced in an isochoric freezing system. To explore this hypothesis, we performed a preliminary study on the isochoric freezing of a produce with which our group has experience, the potato. METHOD:Experiments were performed in an isochoric freezing device we designed. The device is robust and has no moving parts. For comparison, we used a geometrically identical isobaric freezing device. Following freezing and thawing, the samples were weighed, examined with colorimetry, and examined with microscopy. RESULTS:It was found that potatoes frozen to -5 °C in an isochoric system experienced no weight loss and limited enzymatic browning. In contrast the -5 °C isobaric frozen potato experienced substantial weight loss and substantial enzymatic browning. Microscopic analysis shows that the structural integrity of the potato is maintained after freezing in the isochoric system and impaired after freezing in the isobaric system. DISCUSSION:Tissue damage during isobaric freezing is caused by the increase in extracellular osmolality and the mechanical damage by ice crystals. Our thermodynamic analysis predicts that during isochoric freezing the intracellular osmolality remains comparable to the extracellular osmolality and that isochoric systems can be designed to eliminate the mechanical damage by ice. The results of this preliminary study seem to confirm the theoretical predictions. CONCLUSION:This is a preliminary exploratory study on isochoric freezing of food. We have shown that the quality of a food product preserved by isochoric freezing is better than the quality of food preserved to the same temperature in isobaric conditions. Obviously, more extensive research remains to be done to extend this study to lower freezing temperatures and other food items.

BackgroundElectrolytic ablation and electroporation based ablation are minimally invasive, non-thermal surgical technologies that employ electrical currents and electric fields to ablate undesirable cells in a volume of tissue. In this study, we explore the attributes of a new tissue ablation technology that simultaneously delivers a synergistic combination of electroporation and electrolysis (E2).MethodA new device that delivers a controlled dose of electroporation field and electrolysis currents in the form of a single exponential decay waveform (EDW) was applied to the pig liver, and the effect of various parameters on the extent of tissue ablation was examined with histology.ResultsHistological analysis shows that E2 delivered as EDW can produce tissue ablation in volumes of clinical significance, using electrical and temporal parameters which, if used in electroporation or electrolysis separately, cannot ablate the tissue.DiscussionThe E2 combination has advantages over the three basic technologies of non-thermal ablation: electrolytic ablation, electrochemical ablation (reversible electroporation with injection of drugs) and irreversible electroporation. E2 ablates clinically relevant volumes of tissue in a shorter period of time than electrolysis and electroporation, without the need to inject drugs as in reversible electroporation or use paralyzing anesthesia as in irreversible electroporation.

ObjectivePreservation of biological materials at subzero Centigrade temperatures, cryopreservation, is important for the field of tissue engineering and organ transplantation. Our group is studying the use of isochoric (constant volume) systems of aqueous solution for cryopreservation. Previous studies measured the pressure-temperature relations in aqueous isochoric systems in the temperature range from 0°C to - 20°C. The goal of this study is to expand the pressure-temperature measurement beyond the range reported in previous publications.Materials and methodsTo expand the pressure-temperature measurements beyond the previous range, we have developed a new isochoric device capable of withstanding liquid nitrogen temperatures and pressures of up to 413 MPa. The device is instrumented with a pressure transducer than can monitor and record the pressures in the isochoric chamber in real time. Measurements were made in a temperature range from - 5°C to liquid nitrogen temperatures for various solutions of pure water and Me2SO (a chemical additive used for protection of biological materials in a frozen state and for vitrification (glass formation) of biological matter). Undissolved gaseous are is carefully removed from the system.ResultsTemperature-pressure data from - 5°C to liquid nitrogen temperature for pure water and other solutions are presented in this study. Following are examples of some, temperature-pressure values, that were measured in an isochoric system containing pure water: (- 20°C, 187 MPa); (-25°C, 216 MPa); (- 30°C, 242.3 MPa); (-180°C, 124 MPa). The data is consistent with the literature, which reports that the pressure and temperature at the triple point, between ice I, ice III and water is, - 21.993°C and 209.9 MPa, respectively. It was surprising to find that the pressure in the isochoric system increases at temperatures below the triple point and remains high to liquid nitrogen temperatures. Measurements of pressure-temperature relations in solutions of pure water and Me2SO in different concentrations show that, for concentrations in which vitrification is predicted, no increase in pressure was measured during rapid cooling to liquid nitrogen temperatures. However, ice formation either during cooling or warming to and from liquid nitrogen temperatures produced an increase in pressure.ConclusionsThe data obtained in this study can be used to aid in the design of isochoric cryopreservation protocols. The results suggest that the pressure measurement is important in the design of "constant volume" systems and can provide a simple means to gain information on the occurrence of vitrification and devitrification during cryopreservation processes of aqueous solutions in an isochoric system.

BackgroundThis theoretical study examines the use of radar to continuously monitor "accumulation of blood in the head" (ACBH) non-invasively and from a distance, after the location of a hematoma or hemorrhage in the brain was initially identified with conventional medical imaging. Current clinical practice is to monitor ABCH with multiple, subsequent, conventional medical imaging. The radar technology introduced in this study could provide a lower cost and safe alternative to multiple conventional medical imaging monitoring for ACBH.Materials and methodsThe goal of this study is to evaluate the feasibility of using radar to monitor changes in blood volume in the brain through a numerical simulation of ACBH monitoring from remote, with a directional spiral slot antennae, in 3-D models of the brain. The focus of this study is on evaluating the effect of frequencies on the antennae reading. To that end we performed the calculations for frequencies of 100 MHz, 500 MHz and 1 GHz.Results and discussionThe analysis shows that the ACBH can be monitored with radar and the monitoring resolution improves with an increase in frequency, in the range studied. However, it also appears that when typical clinical dimensions of hematoma and hemorrhage are used, the variable ratio of blood volume radius and radar wavelength can bring the measurements into the Mie and Rayleigh regions of the radar cross section. In these regions there is an oscillatory change in signal with blood volume size. For some frequencies there is an increase in signal with an increase in volume while in others there is a decrease.ConclusionsWhile radar can be used to monitor ACBH non-invasively and from a distance, the observed Mie region dependent oscillatory relation between blood volume size and wavelength requires further investigation. Classifiers, multifrequency algorithms or ultra-wide band radar are possible solutions that should be explored in the future.

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. [ABSTRACT FROM AUTHOR]

Nonthermal irreversible electroporation is a new tissue ablation technique that consists of applying pulsed electric fields across cells to induce cell death by creating permanent defects in the cell membrane. Nonthermal irreversible electroporation is of interest because it allows treatment near sensitive tissue structures such as blood vessels and nerves. Two recent articles report that electrolytic reaction products at electrodes can be combined with electroporation pulses to augment and optimize tissue ablation. Those articles triggered a concern that the results of earlier studies on nonthermal irreversible electroporation may have been tainted by unaccounted for electrolytic effects. The goal of this study was to reexamine previous studies on nonthermal irreversible electroporation in the context of these articles. The study shows that the results from some of the earlier studies on nonthermal irreversible electroporation were affected by unaccounted for electrolysis, in particular the research with cells in cuvettes. It also shows that tissue ablation ascribed in the past to irreversible electroporation is actually caused by at least 3 different cytotoxic effects: irreversible electroporation without electrolysis, irreversible electroporation combined with electrolysis, and reversible electroporation combined with electrolysis. These different mechanisms may affect cell and tissue ablation in different ways, and the effects may depend on various clinical parameters such as the polarity of the electrodes, the charge delivered (voltage, number, and length of pulses), and the distance of the target tissue from the electrodes. Current clinical protocols employ ever-increasing numbers of electroporation pulses to values that are now an order of magnitude larger than those used in our first fundamental nonthermal irreversible electroporation studies in tissues. The different mechanisms of cell death, and the effect of the clinical parameters on the mechanisms may explain discrepancies between results of different clinical studies and should be taken into consideration in the design of optimal electroporation ablation protocols.

This study is the first experimental evidence showing that a living multicellular organism, the nematode Caenorhabditis elegans, can survive subfreezing temperatures in an isochoric (constant volume) thermodynamic system, while immersed in a simple isotonic solution, without the addition of cryoprotectants. Some of the test conditions were more extreme than those found at the ice/water interface of the Antarctic subglacial Vostok lake. On earth, life takes place in an isobaric (constant pressure) environment. In isobaric systems, subfreezing temperature survival of organisms in nature and subfreezing temperature preservation of living material for biotechnology and medicine, is made possible by use of cryoprotective chemicals additives. Our theoretical thermodynamic studies suggested that in an isochoric system, living biological material could survive subfreezing temperatures, without any cryoprotective chemicals. By confirming the theoretical predictions, this paper suggests a new technology for subfreezing preservation of cells, organs and organisms of possible value for biotechnology and medicine as well as new possible mechanisms of living organism survival in nature.

A new minimally invasive tissue ablation technique, that combines the biophysical processes of electroporation and electrolysis, is introduced. The technology employs electrode needles inserted in the target tissue to deliver an Electrolytic Electroporation Waveform (EEW) in the form of an exponential decay voltage, several tens of microsecond long. A case study in a large animal model demonstrates that clinically significant size lesions can be achieved with a single, milliseconds long EEW. Ablation with EEW has major advantages over the comparable tissue ablation techniques of electrolysis, reversible electroporation and irreversible electroporation. EEW is orders of magnitude faster than conventional electrolytic ablation. EEW does not require the use of muscle relaxant as irreversible electroporation and EEW does not require injection of drugs as reversible electroporation. This new technology is simple to use and may become an important addition to the minimally invasive surgery armamentarium.