Cellasys #8: A Microphysiometric Assay for Real-Time Cell Analysis Within 24 Hours.

Traditional biochemical assays present a vital toolbox to identify the effects of a test substance on cells. However, current assays are single-point measurements, only reveal one parameter at a time, and introduce potential interferences with labels and fluorescent lights. We have addressed these limitations by introducing the cellasys #8 test which is a microphysiometric assay for real-time cell analysis. Within 24 h, the cellasys #8 test is able to identify not only the effect of a test substance but also measure recovery effects. Due to the multi-parametric read-out, the test provides insights into metabolic as well as morphological changes in real-time. The following protocol provides a detailed introduction to the materials as well as a step-by-step description to support scientists with protocol adoption. The automated and standardized assay opens up manifold new application areas for scientists to study biological mechanisms, develop new therapeutic approaches, and validate serum-free media formulation.

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

Gunshot wounds to the head: a comparison of postmortem magnetic resonance imaging, computed tomography, and autopsy.

BACKGROUND: Postmortem imaging has become a powerful diagnostic tool in forensics. Postmortem computed tomography (PMCT) is often used currently to complement and sometimes even replace an autopsy. PURPOSE: To compare PMCT, postmortem magnetic resonance imaging (PMMRI), and autopsy findings for gunshot wounds to the head. MATERIAL AND METHODS: Cross-sectional study. We performed a retrospective analysis of 24 cases with gunshot wounds to the head that underwent both PMCT and PMMRI between 2011 and 2018 at the Institute of Forensic Medicine, University of Zurich (Switzerland). RESULTS: Our study confirms that PMCT and, to a slightly lesser degree, PMMRI provide additional information that is valuable when combined with autopsy findings. Air embolism was solely detected in PMCT (67% vs. 0% at autopsy). A retained bullet or projectile and bone fragments were diagnosed more frequently with PMCT (42%, 67%, and 92%) than at autopsy (33%, 42%, and 46%). Soft tissue lesions were more often detected with PMMRI than with PMCT. With regard to autopsy, subdural hemorrhage and ventricular hemorrhage were slightly more frequently diagnosed with PMMRI (63% and 75% vs. 38% and 58% at autopsy). Intracerebral hemorrhage was by far most often diagnosed with PMMRI (92%) compared with both PMCT (38%) and autopsy (14%). CONCLUSION: All three modalities should ideally be considered in cases of craniocerebral gunshot wounds. However, it might be conceivable that depending on the forensic query, PMCT and PMMRI may be an adequate replacement for an autopsy.

Nonequilibrium Floquet Steady States of Time-Periodic Driven Luttinger Liquids.

Time-periodic driving facilitates a wealth of novel quantum states and quantum engineering. The interplay of Floquet states and strong interactions is particularly intriguing, which we study using time-periodic fields in a one-dimensional quantum gas, modeled by a Luttinger liquid with periodically changing interactions. By developing a time-periodic operator algebra, we are able to solve and analyze the complete set of nonequilibrium steady states in terms of a Floquet-Bogoliubov ansatz and known analytic functions. Complex valued Floquet eigenenergies occur when integer multiples of the driving frequency approximately match twice the dispersion energy, which correspond to resonant states. In experimental systems of Lieb-Liniger bosons we predict a change from power-law correlations to dominant collective density wave excitations at the corresponding wave numbers as the frequency is lowered below a characteristic cutoff.

Bosonic Continuum Theory of One-Dimensional Lattice Anyons.

Anyons with arbitrary exchange phases exist on 1D lattices in ultracold gases. Yet, known continuum theories in 1D do not match. We derive the continuum limit of 1D lattice anyons via interacting bosons. The theory maintains the exchange phase periodicity fully analogous to 2D anyons. This provides a mapping between experiments, lattice anyons, and continuum theories, including Kundu anyons with a natural regularization as a special case. We numerically estimate the Luttinger parameter as a function of the exchange angle to characterize long-range signatures of the theory and predict different velocities for left- and right-moving collective excitations.

Automated 3D Microphysiometry Facilitates High-Content and Highly Reproducible Oxygen Measurements within 3D Cell Culture Models.

Microphysiometry is a powerful technique to study metabolic parameters and detect changes to external stimuli. However, applying this technique for automated label-free and real-time measurements within cell-laden three-dimensional (3D) cell culture constructs remains a challenge. Herein, we present an entirely automated microphysiometry setup that combines needle-type microsensors with motorized sample and sensor positioning systems inside a standard tissue-culture incubator. The setup records dissolved oxygen as a metabolic parameter along the z-direction within cell-laden 3D constructs in a minimally invasive manner. The microphysiometry setup was applied to characterize the spatial oxygen distribution within thick cell-laden 3D constructs, study the time-dependent changes on the oxygen tension within 3D breast cancer models following a chemotherapeutic treatment, and identify kinetics and recovery effects after drug exposure over 5 weeks. Our data suggest that the microphysiometry setup enables highly reproducible measurements without human intervention, due to the high degree of automation and positional accuracy. The results demonstrate the applicability of the setup to provide valuable long-term insights into oxygenation within 3D models using minimally invasive, label-free, and entirely automated analysis methods.

Automated melt electrowritting platform with real-time process monitoring.

Melt electrowriting (MEW) is an additive manufacturing (AM) technology with the ability to fabricate complex designs with high-resolution. The utility of MEW is studied in many fields including tissue engineering and soft robotics. However, current MEW hardware offers only basic functionality and is often designed and built in-house. This affects results replication across different MEW devices and slows down the technological advancement. To address these issues, we present an automated MEW platform with real-time process parameter monitoring and control. We validate the developed platform by demonstrating the ability to accurately print polymer structures and successfully measure and adjust parameters during the printing process. The platform enables the collection of large volumes of data that can be subsequently used for further analysis of the system. Ultimately, the concept will help MEW to become more accessible for both research laboratories and industry and allow advancing the technology by leveraging the process monitoring, control and data collection.

Development and thorough characterization of the processing steps of an ink for 3D printing for bone tissue engineering.

Achieving reproducibility in the 3D printing of biomaterials requires a robust polymer synthesis method to reduce batch-to-batch variation as well as methods to assure a thorough characterization throughout the manufacturing process. Particularly biomaterial inks containing large solid fractions such as ceramic particles, often required for bone tissue engineering applications, are prone to inhomogeneity originating from inadequate mixing or particle aggregation which can lead to inconsistent printing results. The production of such an ink for bone tissue engineering consisting of gellan gum methacrylate (GG-MA), hyaluronic acid methacrylate and hydroxyapatite (HAp) particles was therefore optimized in terms of GG-MA synthesis and ink preparation process, and the ink's printability was thoroughly characterized to assure homogeneous and reproducible printing results. A new buffer mediated synthesis method for GG-MA resulted in consistent degrees of substitution which allowed the creation of large 5 g batches. We found that both the new synthesis as well as cryomilling of the polymer components of the ink resulted in a decrease in viscosity from 113 kPa·s to 11.3 kPa·s at a shear rate of 0.1 s-1 but increased ink homogeneity. The ink homogeneity was assessed through thermogravimetric analysis and a newly developed extrusion force measurement setup. The ink displayed strong inter-layer adhesion between two printed ink layers as well as between a layer of ink with and a layer without HAp. The large polymer batch production along with the characterization of the ink during the manufacturing process allows ink production in the gram scale and could be used in applications such as the printing of osteochondral grafts.

OpenWorkstation: A modular open-source technology for automated in vitro workflows.

Automation liberates scientific staff from repetitive tasks, decreases the probability of human error and consequently enhances the reproducibility of lab experiments. However, the use of laboratory automation in academic laboratories is limited due to high acquisition costs and the inability to customize off-the-shelf hardware. To address these challenges, we present an Open Source Hardware concept, referred to as OpenWorkstation, to build an assembly line-inspired platform consisting of ready-to-use and customizable modules. In contrast to current standalone solutions, the OpenWorkstation concept enables the combination of single hardware modules - each with a specific set of functionalities - to a modular workstation to provide a fully automated setup. The base setup consists of a pipetting and transport module and is designed to execute basic protocol steps for in vitro research applications, including pipetting operations for liquids and viscous substances and transportation of cell culture vessels between the modules. We demonstrate the successful application of this concept within a case study by the development of a storage module to facilitate high-throughput studies and a photo-crosslinker module to initiate photo-induced polymerization of hydrogel solutions. We present a Systems Engineering framework for customized module development, guidance for the design and assembly of the presented modules, and operational instructions on the usage of the workstation. By combining capabilities from various open source instrumentations into a modular technology platform, the OpenWorkstation concept will facilitate efficient and reliable experimentation for in vitro research. Ultimately, this concept will allow academic groups to improve replicability and reproducibility in cell culture process operations towards more economical and innovative research in the future.

Dirac Spin Liquid on the Spin-1/2 Triangular Heisenberg Antiferromagnet.

We study the spin liquid candidate of the spin-1/2 J_{1}-J_{2} Heisenberg antiferromagnet on the triangular lattice by means of density matrix renormalization group (DMRG) simulations. By applying an external Aharonov-Bohm flux insertion in an infinitely long cylinder, we find unambiguous evidence for gapless U(1) Dirac spin liquid behavior. The flux insertion overcomes the finite size restriction for energy gaps and clearly shows gapless behavior at the expected wave vectors. Using the DMRG transfer matrix, the low-lying excitation spectrum can be extracted, which shows characteristic Dirac cone structures of both spinon-bilinear and monopole excitations. Finally, we confirm that the entanglement entropy follows the predicted universal response under the flux insertion.

In vitro disease models 4.0 via automation and high-throughput processing.

While much progress has been accomplished in the development of physiologically relevant in vitro disease models, current manufacturing and characterisation workflows still rely on manual, time-consuming, and low-throughput processes, which are not efficient and prone to human errors. For these reasons adoption and, more importantly, reproducibility and validation of 3D in vitro disease models is rather low for fundamental and applied research concepts. This article argues in form of a perspective view that automation and high-throughput methodologies will play a vital role to act as a catalyst to accelerate the development and characterisation process for generations to come. Innovative engineering concepts are required to overcome current limitations of in vitro disease models and to foster the scientific rigour as well as the applied research potential.

Fatal bilateral pneumothorax and generalized emphysema following contraindicated speaking-valve application.

We report a case of a contraindicated attachment of a speaking valve to a tracheal tube with an inflated cuff, which rapidly resulted in the patient's death. The attached one-way valve allowed unrestrained inspiration through the tracheal tube but prevented physiological expiration. The increased pulmonary pressure resulted in alveolar rupture and replaced expiration with a steady release of air into the peribronchial sheaths and the mediastinum, resulting in what is commonly known as the Macklin effect. From the mediastinum, air inflated both pleural cavities, the peritoneum, and the subcutaneous tissue of the entire body. No gas was found in the blood vessels, the brain, the bones, or in the inner organs. The entire air volume was estimated by radiological segmentation to be more than 25 l. This implies continuous inspiration, while expiration turned into an aberrant pulmonary decompression by whole-body gas-enclosure. Death ultimately resulted from asphyxia following bilateral (tension) pneumothorax.

Printomics: the high-throughput analysis of printing parameters applied to melt electrowriting.

Melt electrowriting (MEW) combines the fundamental principles of electrospinning, a fibre forming technology, and 3D printing. The process, however, is highly complex and the quality of the fabricated structures strongly depends on the interplay of key printing parameter settings including processing temperature, applied voltage, collection speed, and applied pressure. These parameters act in unison, comprising the principal forces on the electrified jet: pushing the viscous polymer out of the nozzle and mechanically and electrostatically dragging it for deposition towards the collector. Although previous studies interpreted the underlying mechanism of electrospinning with polymer melts in a direct writing mode, contemporary devices used in laboratory environments lack the capability to collect large data reproducibly. Yet, a validated large data set is a condition sine qua non to design an in-process control system which allows to computer control the complexity of the MEW process. For this reason, we engineered an advanced automated MEW system with monitoring capabilities to specifically generate large, reproducible data volumes which allows the interpretation of complex process parameters. Additionally, the design of an innovative real-time MEW monitoring system identifies the main effects of the system parameters on the geometry of the fibre flight path. This enables, for the first time, the establishment of a comprehensive correlation between the input parameters and the geometry of a MEW jet. The study verifies the most stable process parameters for the highly reproducible fabrication of a medical-grade poly(ε-caprolactone) fibres and demonstrates how Printomics can be performed for the high throughput analysis of processing parameters for MEW.

Continuous Easy-Plane Deconfined Phase Transition on the Kagome Lattice.

We use large scale quantum Monte Carlo simulations to study an extended Hubbard model of hard core bosons on the kagome lattice. In the limit of strong nearest-neighbor interactions at 1/3 filling, the interplay between frustration and quantum fluctuations leads to a valence bond solid ground state. The system undergoes a quantum phase transition to a superfluid phase as the interaction strength is decreased. It is still under debate whether the transition is weakly first order or represents an unconventional continuous phase transition. We present a theory in terms of an easy plane noncompact CP^{1} gauge theory describing the phase transition at 1/3 filling. Utilizing large scale quantum Monte Carlo simulations with parallel tempering in the canonical ensemble up to 15552 spins, we provide evidence that the phase transition is continuous at exactly 1/3 filling. A careful finite size scaling analysis reveals an unconventional scaling behavior hinting at deconfined quantum criticality.

Skin-on-a-Chip: Transepithelial Electrical Resistance and Extracellular Acidification Measurements through an Automated Air-Liquid Interface.

Skin is a critical organ that plays a crucial role in defending the internal organs of the body. For this reason, extensive work has gone into creating artificial models of the epidermis for in vitro skin toxicity tests. These tissue models, called reconstructed human epidermis (RhE), are used by researchers in the pharmaceutical, cosmetic, and environmental arenas to evaluate skin toxicity upon exposure to xenobiotics. Here, we present a label-free solution that leverages the use of the intelligent mobile lab for in vitro diagnostics (IMOLA-IVD), a noninvasive, sensor-based platform, to monitor the transepithelial electrical resistance (TEER) of RhE models and adherent cells cultured on porous membrane inserts. Murine fibroblasts cultured on polycarbonate membranes were first used as a test model to optimize procedures using a custom BioChip encapsulation design, as well as dual fluidic configurations, for continuous and automated perfusion of membrane-bound cultures. Extracellular acidification rate (EAR) and TEER of membrane-bound L929 cells were monitored. The developed protocol was then used to monitor the TEER of MatTek EpiDermTM RhE models over a period of 48 hours. TEER and EAR measurements demonstrated that the designed system is capable of maintaining stable cultures on the chip, monitoring metabolic parameters, and revealing tissue breakdown over time.

A novel lab-on-a-chip platform for spheroid metabolism monitoring.

Sensor-based cellular microphysiometry is a technique that allows non-invasive, label-free, real-time monitoring of living cells that can greatly improve the predictability of toxicology testing by removing the influence of biochemical labels. In this work, the Intelligent Mobile Lab for In Vitro Diagnostics (IMOLA-IVD) was utilized to perform cellular microphysiometry on 3D multicellular spheroids. Using a commercial 3D printer, 3 × 3 microwell arrays were fabricated to maintain nine previously cultured HepG2 spheroids on a single BioChip. Integrated layers above and under the spheroids allowed fluidic contact between spheroids in microwells and BioChip sensors while preventing wash out from medium perfusion. Spheroid culturing protocols were optimized to grow spheroids to a diameter of around 620 µm prior to transfer onto BioChips. An ON/OFF pump cycling protocol was developed to optimize spheroid culture within the designed microwells, intermittently perfuse spheroids with fresh culture medium, and measure the extracellular acidification rate (EAR) and oxygen uptake rate (OUR) with the BioChips of the IMOLA-IVD platform. In a proof-of-concept experiment, spheroids were perfused for 36 h with cell culture medium before being exposed to medium with 1% sodium dodecyl sulphate (SDS) to lyse cells as a positive control. These microphysiometry studies revealed a repeatable pattern of extracellular acidification throughout the experiment, indicating the ability to monitor real-time metabolic activity of spheroids embedded in the newly designed tissue encapsulation. After perfusion for 36 h with medium, SDS exposure resulted in an instant decrease in EAR and OUR signals from 37 mV/h (± 5) to 8 mV/h (± 8) and from 308 mV/h (± 21) to -2 mV/h (± 13), respectively. The presented spheroid monitoring system holds great potential as a method to automate screening and analysis of pharmaceutical agents using 3D multicellular spheroid models.

Unexpected brain finding in pre-autopsy postmortem CT.

A case is presented in which pre-autopsy postmortem computed tomography (PMCT) revealed an unexpected brain abscess with a related frontal sinusitis and an erosion of the posterior wall of the frontal sinus. PMCT findings enabled the forensic pathologists to adapt protective measures during autopsy and protect their health from infection. Pre-autopsy PMCT has been also useful in the early differential diagnosis procedure. The complementary use of postmortem imaging and autopsy can improve the quality of forensic death investigations.

Movement of steel-jacketed projectiles in biological tissue in the magnetic field of a 3-T magnetic resonance unit.

PURPOSE: The fact that ferromagnetic bullets can move in air or gelatine when subjected to magnetic resonance (MR) units is well known. A previous study showed that the movement of 7.5-mm GP 11 Suisse bullets also depends on their orientation toward the gantry. In order to compare the movement in gelatine to that in real tissue, we decided to measure the movement of these bullets, as well as 9-mm Luger bullets, in the brain and liver. METHODS: The GP 11 and 9-mm Luger bullets were inserted into the fresh calf brain or pig liver either vertically or horizontally in the x- or z-axis to the gantry. Before and after exposure to a 3-T MR unit, their position was documented by CT. RESULTS: GP 11 bullets rotated more readily and in general proved to be more mobile than the 9-mm Luger. All GP 11 bullets and a large amount of the 9-mm Luger bullets exited the brain. Sliding toward the gantry was easier for 9-mm Luger bullets in the brain than in the liver. CONCLUSIONS: The orientation of a ferromagnetic object influences its mobility in a strong magnetic field. Tipping is easier than sliding for longish ferromagnetic projectiles, probably due to the lesser tissue resistance. The bullets moved more readily in biological tissue, especially brain tissue, compared to gelatine, thus implying that gelatine is not a suitable substitute for soft tissues when examining the movement of ferromagnetic objects in MR units.

Perfect Spin Filter by Periodic Drive of a Ferromagnetic Quantum Barrier.

We consider the problem of particle tunneling through a periodically driven ferromagnetic quantum barrier connected to two leads. The barrier is modeled by an impurity site representing a ferromagnetic layer or a quantum dot in a tight-binding Hamiltonian with a local magnetic field and an ac-driven potential, which is solved using the Floquet formalism. The repulsive interactions in the quantum barrier are also taken into account. Our results show that the time-periodic potential causes sharp resonances of perfect transmission and reflection, which can be tuned by the frequency, the driving strength, and the magnetic field. We demonstrate that a device based on this configuration could act as a highly tunable spin valve for spintronic applications.