Dielectric spectroscopy is a label-free, non-contact, real-time, multi-layer sensing technology, and has been used for identification and quantification of many biological materials. A combination of such sensing features is in demand for monitoring of organ-on-chip systems; however available sensing technologies have yet to address this need. In this work, we explore the possibility of leveraging the inherent features of dielectric spectroscopy for the application in organ-on-chip systems, by investigating three key technological developments using open-ended coaxial probes. Firstly, biocompatible non-contact sensing capabilities are proved by showing similar sensing performance of Parylene C-coated probes and uncoated probes. Secondly, a setup and methodology are developed for highly accurate and non-destructive height positioning of the probe to allow for precise extraction of intermediate sample layers. Finally, non-contact multi-layer sensing performance of the presented technology is successfully demonstrated by means of a biological phantom in a three-layered system. With further integration, dielectric spectroscopy can potentially become a cornerstone sensing technique for organ-on-chip by enabling real-time non-contact tracking of various tissue contents and properties.

Engineered heart tissues (EHTs) formed around flexible pillars are used to measure the contraction force of myocytes. When based on cardiac cells derived from human induced pluripotent stem cells (hiPSCs), EHTs capture human cardiac physiology and drug responses in vitro. However, variability in contractile function often arises due to variation in tissue positioning on the pillar. Here, novel tapered pillars are introduced to achieve spatial confinement of tissues in EHT devices. The devices are fabricated by moulding polydimethylsiloxane (PDMS) into micromachined tapered cavities of a silicon substrate. The symmetrically-tapered geometry, with the minimum cross-section at the pillar mid-height, restricts tissue movement outside of the indented area. This increases sensitivity and accuracy of tissue contractile readout, providing high reproducibility with reduced variability between data points. Design and stiffness of tapered pillars are investigated to determine the optimal mechanical environment, obtain accurate contractile measurements, and achieve long-term culture of EHTs. Results show that tapered pillars provide superior confinement efficiency (over 90%) compared to straight pillars (30%), with tissue confinement directly correlated to pillar geometry rather than stiffness. The optimized precision in force readouts and long-term tissue studies enables higher sensitivity in the detection of contractile responses to drugs or diseases.

In this work, we present a numerical testbench, realized in a circuit simulation environment, enabling a priori uncertainty evaluation of dielectric spectroscopy in the application field of organs-on-chip. This testbench evaluates the impact of noise, ambient temperature variation and impurity of liquid standards on the uncertainty of dielectric spectroscopy measurements. Moreover, the proposed approach allows to account for the impact on measurement sensitivity of system parameters such as probe dimensions and probe coatings. The estimated uncertainty contributions for the considered effects are compared and benchmarked experimentally. Finally, the testbench is employed to project the dielectric spectroscopy accuracy on a relevant biological application, namely monitoring the growth of a 7 μm-thick kidney cell monolayer.

We present a novel silicon-based organ-on-chip (OoC) device featuring integrated microelectrodes to assess barrier function in biological tissue co-cultures. The microfluidic device consists of two vertically-stacked microchannels separated by a submicron-thin, microporous silicon nitride membrane, enabling in vivo-like proximity for co-cultured tissues. The integrated four-probe electrode geometry on slanted microchannel sidewalls ensures unobstructed optical access to the membrane and consistent measurement repeatability. Experimental validation through electrical impedance spectroscopy supported the device's sensitivity to sodium chloride concentration. Fabricated through a scalable, wafer-scale batch process, the device additionally demonstrated biocompatibility and optical transparency, representing a significant advancement for in situ tissue barrier assessments.

Engineered heart tissues (EHTs) have shown great potential in recapitulating tissue organization, functions, and cell-cell interactions of the human heart in vitro. Currently, multiple EHT platforms are used by both industry and academia for different applications, such as drug discovery, disease modelling, and fundamental research. The tissues’ contractile force, one of the main hallmarks of tissue function and maturation level of cardiomyocytes, can be read out from EHT platforms by optically tracking the movement of elastic pillars induced by the contractile tissues. However, existing optical tracking algorithms which focus on calculating the contractile force are customized and platform-specific, often not available to the broad research community, and thus hamper head-to-head comparison of the model output. Therefore, there is the need for robust, standardized and platform-independent software for tissues’ force assessment. To meet this need, we developed ForceTracker: a standalone and computationally efficient software for analyzing contractile properties of tissues in different EHT platforms. The software uses a shape-detection algorithm to single out and track the movement of pillars’ tips for the most common shapes of EHT platforms. In this way, we can obtain information about tissues’ contractile performance. ForceTracker is coded in Python and uses a multi-threading approach for time-efficient analysis of large data sets in multiple formats. The software efficiency to analyze circular and rectangular pillar shapes is successfully tested by analyzing different format videos from two EHT platforms, developed by different research groups. We demonstrate robust and reproducible performance of the software in the analysis of tissues over time and in various conditions. ForceTracker’s detection and tracking shows low sensitivity to common incidental defects, such as alteration of tissue shape or air bubbles. Detection accuracy is determined via comparison with manual measurements using the software ImageJ. We developed ForceTracker as a tool for standardized analysis of contractile performance in EHT platforms to facilitate research on disease modeling and drug discovery in academia and industry.

We investigated the evaporative crystallization of aqueous glycine sessile droplets on hydrophilic glass, hydrophobic Teflon surfaces, and hydrophobic Teflon surfaces, where the contact angle is manipulated dynamically with electrowetting. Microscopy experiments and analytical characterization revealed that the size, morphology, and polymorphic form (α, β, and γ) of the glycine crystals are influenced by the surface wettability as well as the amplitude and frequency of electrowetting. On a hydrophilic glass surface, a coffee-stain-shaped residue composed of a mixture of bipyramidal α and needle-like β crystals was observed. On a hydrophobic Teflon surface, the droplets evaporated with minimum contact line pinning, producing hemispherical residue shapes, and bipyramidal α crystals smaller than 100 μm were formed. On a Teflon surface with electrowetting, glycine could be manipulated to crystallize into distinct polymorphic forms (β and γ) and residue shapes not observed on hydrophilic glass and hydrophobic Teflon surfaces. The frequency and amplitude of electrowetting were optimized to produce single large crystals. We observed the highest chance of producing single-millimeter-scale crystals at a frequency of 1 kHz and a voltage amplitude of 80 Vrms. We attribute this observation to a combination of nucleation at lower bulk supersaturation compared to the experiment on Teflon surfaces and electrowetting-induced mixing most prominent at 1 kHz. Our results highlight the opportunities arising from the dynamic manipulation of surface wettability

It is estimated that 99 % of the world population is exposed to air pollution above air quality guidelines and this is responsible for 6.7 million premature deaths annually. Lung and skin are the first organs exposed to air pollution, and this is associated with carcinogenesis, inflammation and atopic disease. Proposed mechanisms of adverse health effects in lung and skin include oxidative stress, inflammation, and loss of epithelial barrier integrity. Most knowledge has been gained using simple 2D or more complex culture models, however these cultures have important limitations, such as a lack of perfusion and stretching and lack of cell-cell crosstalk. Organ-on-chip (OoC) technology may be used to overcome limitations of the in vitro models currently used in air pollution research and opens possibilities for studying the pathways underlying adverse health effects of air pollution on immune-mediated diseases of the lung and skin using more physiologically relevant exposure experiments. In this review we discuss currently used in vitro models to study the effect of air pollution on epithelial barrier integrity and development of immune-mediated diseases and identify gaps in current knowledge on adverse health effects of air pollution. We then focus on how OoC technology can enhance mechanistic studies of the skin and lung's response to air pollution.

Real-time pH and oxygen concentration sensing is critical for monitoring tissue damage and organ health; however, there is no report to date in such context of a single device that can simultaneously detect both pH and oxygen changes. This paper presents the development of a single optical sensor device that can simultaneously and reversibly respond to changes in both pH and oxygen concentration. The proposed optical sensor integrates both pH- and oxygen-sensitive probes, and is optimized to achieve minimal cross-sensitivity during simultaneous measurements. This approach enables high accuracy in early detection of chemical correlates of tissue or organ damage, improving screening and efficacy in organ transplants.

The ability to identify individual protein molecules using Surface-Enhanced Raman Scattering (SERS) spectroscopy, without the need for labelling, is a significant advancement in biomedical diagnostics. However, the inherently small Raman scattering cross-section of most (bio) molecules necessitates significant signal amplification for successful detection, particularly at the single-molecule level. A novel approach is introduced for fabricating plasmonic nanopores suitable for sequential Raman readout, namely the recording of Raman spectra from portions of molecules, which are progressively flowing into plasmonic hot spots. The method is based on Capillary-Assisted Particle Assembly (CAPA)of gold nanoparticles (Au NPs), thus ensuring high stability and cost-effectiveness. By electrophoretically driving polypeptides through these nanopores, real-time Raman detection is achieved with a Single-Photon Avalanche Diode (SPAD) camera, attaining single-molecule detection at 1 nm concentration with 100 microsecond resolution. Statistical analysis of translocation times, photon scattering rates, and spectral data confirms a linear correlation between dwell time, molecular length, and Raman signal intensity. On average, a photon scattering of 6 photons/amino acid and a translocation time of 7 µs per amino acid is recorded. These results demonstrate the feasibility of sequential Raman readout, overcoming key limitations of SERS. This method represents a significant step toward label-free, high-resolution molecular identification, with future potential applications in protein identification.

Poor stimulus-response correlation, caused by acoustic reflections from conventional culture substrates, poses a significant challenge in cellular mechanistic studies of ultrasound neuromodulation. Existing specialized setups that mitigate this interference have limited recording capabilities. In this study, we propose an anti-reflective microengineered substrate (ARMS) that can be incorporated into a standard in vitro platform. The substrate's dimensions and material composition were optimized in simulation. The optimized simulated platform exhibited an 86.3% reduction in reflection amplitude on the substrate surface compared to the conventional glass substrate. Furthermore, the ARMS reduced stimulation signal distortion to a 19.2% deviation from the expected amplitude, a substantial improvement compared to the 76.4% deviation observed with glass.

This abstract describes the design, simulation and experimental characterization of a thin film thermal flow sensor fabricated using flat panel display technology. Patterned microelectrodes were successfully applied as a thermal flow sensor, showing good correlation between experimental and simulated data. The use of flat panel display technology could provide an interesting option for cost-effective integration of flow sensing in Organ-on-Chip technologies.

Climate and justice are interconnected. However, simply raising ethical issues associated with the links between climate change, technology, and health is insufficient. Rather, policies and practices need to consider ethics ahead of time. If it is only added “after the fact,” policy will be less efficient and opportunities for carbon minimization will be lost. This will require the cooperation of people at many levels and can be guided by two essential ethical principles: distributive justice and environmental sustainability.

Microphysiological systems (MPSs) are cellular models that replicate aspects of organ and tissue functions in vitro. In contrast with conventional cell cultures, MPSs often provide physiological mechanical cues to cells, include fluid flow and can be interlinked (hence, they are often referred to as microfluidic tissue chips or organs-on-chips). Here, by means of examples of MPSs of the vascular system, intestine, brain and heart, we advocate for the development of standards that allow for comparisons of quantitative physiological features in MPSs and humans. Such standards should ensure that the in vivo relevance and predictive value of MPSs can be properly assessed as fit-for-purpose in specific applications, such as the assessment of drug toxicity, the identification of therapeutics or the understanding of human physiology or disease. Specifically, we distinguish designed features, which can be controlled via the design of the MPS, from emergent features, which describe cellular function, and propose methods for improving MPSs with readouts and sensors for the quantitative monitoring of complex physiology towards enabling wider end-user adoption and regulatory acceptance.

Skeletal muscle spatial analyses have revealed unexpected regionalized gene expression patterns challenging the understanding of muscle as a homogeneous tissue. Here, we present a protocol for the spatial analysis of transcript and protein levels in murine skeletal muscle. We describe steps for tibialis anterior dissection, formaldehyde fixation, tissue chopper cutting, and hybridization chain reaction (HCR) detection and amplification. We then detail procedures for immunostaining, tissue clearing, and imaging. This protocol is easily adaptable to other tissues.

Micro-physiological systems (MPS) hold the potential for advancing drug research by emulating realistic in vitro human (patho)physiology models. These systems replicate organ microenvironments, delivering stimuli similar to those experienced by organs in vivo. Active biomechanical cues, like shear force and tensile stress, profoundly influence cell activity and can alter biochemical responses. The critical factors encompass the nature, duration, magnitude, and frequency of these cues [1]. A currently popular solution to provide biomechanical stimulation employs pneumatic actuation, which is powerful and multifunctional, but bulky, expensive, and inconvenient to use. Electroactive polymers (EAP), also known as artificial muscles, are gaining attention as a potential alternative for improved versatility [2]. Ionic polymer-metal composites (IPMCs) are a class of ionic EAPs which operate at low voltage and exhibit large displacement [3]. They are intrinsically suitable for cell culture media as their operating principle is based on electrolytes (Fig. 1A). However, IPMCs employ metallic electrodes, which eliminate the optical transparency essential in fundamental biological experiments.
In this study, we present the design, fabrication, and characterization of a selectively transparent IPMC for utilization in MPS to apply controllable mechanical stimuli to tissues. A multiphysics-based finite-element model was constructed and validated basing on literature data [4] to estimate the maximum tip displacement of IPMC cantilevers. The model was used to study several cantilever configurations to determine the best electrode patterning topology for the transparency, stiffness, and tip displacement trade-off. The optimized designs were implemented in wafer-scale cleanroom-compatible fabrication (Fig. 1B-C). The novel fabrication process involved sequential patterning of planar Au electrodes on polydimethylsiloxane (PDMS) substrates, and covalent bonding of a pair of such Au-patterned PDMS substrates to an ionomer (Nafion) through silanization (Fig 1B). Preliminary electro-mechanical characterization of the performance of the selectively transparent IPMC cantilevers (Fig. 1D) and biocompatibility tests indicate a potential for integration and use in MPS and organ-on-chip platforms.

Bulk piezoelectric ultrasound transducers on integrated circuits offer unique properties for therapeutic applications of ultrasound neuromodulation. However, current implementations of such transducers are not optimized for the high transmit efficiency required to stimulate neurons. This is mainly due to the challenge of implementing a metal layer on top of the piezoelectric film using microfabrication techniques. Here, we propose a micromachined capping structure providing an electrical connection on top of the piezoelectric film with minimal acoustic losses. The structure can potentially be used as a common ground connection in phased-array ultrasound transducers.

We present a novel design of elastic micropillars for tissue self-assembly in engineered heart tissue (EHT) platforms. The innovative tapered profile confines reproducibly the tissue position along the main micropillar axis, increasing the accuracy of tissue contraction force measurement. Polydimethylsiloxane-based pillars were designed and fabricated by wafer-level molding in an hourglass shape, with symmetric tapering producing a restriction for tissue movement in the middle of the pillars’ length. Confinement efficacy of the new geometry was validated by comparing the tissue performance in straight versus tapered (75° or 80° tapering angle) micropillars. While in all three cases compact tissues formed successfully, for both tapered designs the functionality assays evidenced yield increase from 15% to 100%, higher spatial tissue confinement, and correspondingly higher accuracy and smaller dispersion in measurements of tissue contraction force.

Towards increased throughput and automated workflows for organs- on-a-chip, a novel high-definition electrophysiology multiwell plate is developed in the Moore4Medical project [1]. It consists of an advanced CMOS microelectrode array (MEA) chip with 16 sampling areas, each featuring 1024 electrodes [2], and of a polymeric fluidic cartridge with 16 corresponding microchambers, each connected to an integrated pneumatic peristaltic pump [3]. It can perform 16-well assays with single-cell-resolution electrical recording and integrated microfluidics. Building on an earlier prototype, we present recent developments showing a scalable integration route and excellent fluidic and electrical functionalities of the plate in an easy-to-use system. Direct bonding of the MEA chip and the cartridge was achieved using accurate glue deposition and a high-precision pick-&-place procedure, resulting in 16 well-aligned, sealed microchambers on top of the MEA. This method is developed for high-volume production. To run the plate, a user-friendly toolbox, which can be placed inside an incubator, is made together with a data acquisition unit, a pneumatic control unit, and custom software. The plate can be clamped directly inside the toolbox, ensuring communication of the MEA with the data acquisition unit by a pogo-pin connection. The pneumatic control unit actuates the 16 integrated pumps simultaneously via three pressure lines. Hence, 16 assays can run in parallel, with simple pipetting steps for cell seeding and media perfusion. To demonstrate the plate’s functions, all the 16 microchambers were primed and filled with water and 1% PBS buffer sequentially. No liquid leakage or bubble entrapment was observed. The voltage scan of the 16 MEA areas showed uniform signals and a clear difference between the two liquids (0.08 mVrms for water; 0.02 mVrms for PBS). Plate biological-validation is currently on-going to study the effect of drugs on electrical and contractile properties of human stem-cell derived cardiomyocytes and to demonstrate the plate potential for OoC standardization and workflow streamlining.

Engineered heart tissues (EHTs) showed great potential in recapitulating tissue organization and function of the human heart in vitro [1]. Contractile kinetics is one key hallmark of cardiac tissue function and maturation level of cardiomyocytes, and a critical readout from EHT platforms. Typically-used optical methods to track elastic micropillar displacement upon tissue contraction are laborious and in most cases not conducted in real-time. This hampers automation and precise control of the EHT microenvironment. We address these unmet needs by developing a co-planar capacitive displacement sensor for tissue contraction force measurement integrated within an EHT platform. The working principle of the displacement sensor relies on the deformation of the substrate wherein the sensors are integrated. Bending of each micropillar, caused by tissue contraction, results in local anti-symmetric out-of-plane deformation of the substrate. Two spiral capacitors are integrated below each micropillar of a previously developed EHT platform [2] to exploit the maximum substrate deformation. The capacitive sensors were fabricated using a combination of wafer-level micromachining and polymer processing. The mould for the micropillars and elliptic well was fabricated by deep reactive ion etching of a Si wafer. Another Si wafer was covered with an 80 μm-thick polydimethylsiloxane (PDMS) layer, whereupon sputtered Al was photolithographically patterned into sensor designs. De-moulded micropillars and wells were aligned and bonded to the wafer with sensors. Single 10 x 10 mm2 PDMS chips with integrated sensors were wire-bonded to custom-designed printed circuit boards. Analog Device AD7746 was selected to readout the expected aF-range change in base capacitance. Static characterization of the sensors showed good agreement between measured and FEM-simulated values of base capacitance. The dynamic behavior was tested using a nanoindentation setup by applying specific force at different positions along the micropillars length while measuring the electrical response. Responsivity of 0.35 ± 0.07 fF/μN was measured. Preliminary experiments with EHTs proved the biocompatibility of the new platform with integrated sensors, as tissues were functional and in culture for at least 14 days.

We present a novel capacitive displacement sensor integrated in an engineered heart tissue (EHT) platform to measure tissue contractile properties in-situ. Co-planar spiral capacitors were integrated into the elastomeric substrate underneath the two micropillars of a previously developed EHT platform. The capacitor plates are displaced by the tension and compression that occurs in the substrate when the micropillars bend under contractile tissue force. For a contraction force of ~200 µN, applied in the middle of pillar length, the expected change in base capacitance is in the aF range. Readout of such low capacitance changes was achieved using a commercial low-noise high-sensitivity device. Characterization of static and dynamic sensor behavior agreed with numerical simulations, demonstrating a responsivity of 0.35 ± 0.07 fF/µN. Preliminary tests with cardiac tissues proved biocompatibility of the platform, as EHTs successfully formed and remained functional for at least 14 days