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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 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.

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]

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. [ABSTRACT FROM AUTHOR]

There is growing interest in using isochoric freezing and isochoric supercooling for the preservation of biological matter at subfreezing temperatures. Custodiol® is a commonly used intracellular composition type, subnormothermic preservation solution. It is anticipated that Custodiol® will also be used for isochoric freezing and isochoric supercooling preservation of biological matter. The thermodynamic properties of Custodiol® at subfreezing temperatures as well as the metastable behavior of the solution at subfreezing temperatures were not studied in the past. This study was designed to generate the thermodynamic data needed for the use of Custodiol® for the preservation of biological matter in isochoric systems at subfreezing temperatures. The experiments were performed in a specially designed isochoric chamber that can measure simultaneously the temperature and pressure in the isochoric chamber, and thereby correlate pressure and temperature at thermodynamic equilibrium in isochoric systems as well as the nucleation temperature in isochoric supercooling. The primary focus of this study is on determining the temperature at which nucleation is initiated and to identify the temperature threshold for nucleation due to its specific relevance to various applications in medicine. [ABSTRACT FROM AUTHOR]