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.

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.

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.

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 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

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.

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.

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.

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.

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.

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.

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.

Continuous monitoring of tissue microphysiology is a key enabling feature of the organ-on-chip (OoC) approach for in vitro drug screening and disease modeling. Integrated sensing units are particularly convenient for microenvironmental monitoring. However, sensitive in vitro and real-time measurements are challenging due to the inherently small size of OoC devices, the characteristics of commonly used materials, and external hardware setups required to support the sensing units. Here we propose a silicon-polymer hybrid OoC device that encompasses transparency and biocompatibility of polymers at the sensing area, and has the inherently superior electrical characteristics and ability to house active electronics of silicon. This multi-modal device includes two sensing units. The first unit consists of a floating-gate field-effect transistor (FG-FET), which is used to monitor changes in pH in the sensing area. The threshold voltage of the FG-FET is regulated by a capacitively-coupled gate and by the changes in charge concentration in close proximity to the extension of the floating gate, which functions as the sensing electrode. The second unit uses the extension of the FG as microelectrode, in order to monitor the action potential of electrically active cells. The layout of the chip and its packaging are compatible with multi-electrode array measurement setups, which are commonly used in electrophysiology labs. The multi-functional sensing is demonstrated by monitoring the growth of induced pluripotent stem cell-derived cortical neurons. Our multi-modal sensor is a milestone in combined monitoring of different, physiologically-relevant parameters on the same device for future OoC platforms.

The high rate of drug withdrawal from the market due to cardiovascular toxicity or lack of efficacy, the economic burden, and extremely long time before a compound reaches the market, have increased the relevance of human in vitro models like human (patient-derived) pluripotent stem cell (hPSC)-derived engineered heart tissues (EHTs) for the evaluation of the efficacy and toxicity of compounds at the early phase in the drug development pipeline. Consequently, the EHT contractile properties are highly relevant parameters for the analysis of cardiotoxicity, disease phenotype, and longitudinal measurements of cardiac function over time. In this study, we developed and validated the software HAARTA (Highly Accurate, Automatic and Robust Tracking Algorithm), which automatically analyzes contractile properties of EHTs by segmenting and tracking brightfield videos, using deep learning and template matching with sub-pixel precision. We demonstrate the robustness, accuracy, and computational efficiency of the software by comparing it to the state-of-the-art method (MUSCLEMOTION), and by testing it with a data set of EHTs from three different hPSC lines. HAARTA will facilitate standardized analysis of contractile properties of EHTs, which will be beneficial for in vitro drug screening and longitudinal measurements of cardiac function.

Ionic polymer metal composites (IPMCs) are a class of materials with a rising appeal in biological micro-electromechanical systems (bio-MEMS) due to their unique properties (low voltage output, bio-compatibility, affinity with ionic medium). While tailoring and improving actuation capabilities of IPMCs is a key motivator in almost all IPMC manufacturing reports, very little efforts have been dedicated to sensing using IPMC thinner than 100 µm. Most reports on IPMC manufacturing and utilization rely on 180 µm-thick Nafion with platinum electrodes, too stiff for bio-MEMS applications. The same fabrication process on thinner membranes does yield in very poor electrodes and performance, and needs to be studied to increase flexibility and sensitivity in the microscale range. This study demonstrates an electroless Pt deposition method for fabricating bio-MEMS-suitable 50 µm-thick IPMC samples. First, we perform a comparative study on the platinum distribution within the Nafion backbone as well as on the surface for the standard electroless deposition recipe for thin (50 µm) and thick (180 µm) Nafion. We report strong differences in platinum distribution for thick and thin IPMC that experienced the same manufacturing process. By varying chemical concentrations from the standard recipe we obtain convenient platinum distribution on thin Nafion, with platinum mainly localized in proximity of surface, as well as electrodes with lower sheet resistance. We could measure the flexural rigidity as 3.43 × 10 − 8 N·m², 46 times lower than standard 180 µm-thick IPMC. The calculated sensitivity is 0.476 ± 0.02 mV mm⁻¹ and the limit of detection for our sensor is 500 ± 20 µm. This procedure sets a milestone for manufacturing 50 µm-thick IPMC for transducers and sensors in bio-MEMS applications.

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

The Smart Multi-Well Plate (SMWP), an open technology platform for Organ-on-Chip (OoC) technology developed as part of the Moore4Medical (M4M) consortium, aims to showcase the advantages of standardization in design, manufacturing and assembly for OoC [1]. In previously presented work [2], we showed integration and characterization of piezoelectric micropumps for in-line perfusion of OoC devices. Here we present the integration and preliminary biological evaluation of three OoC devices in a SMWP prototype. This prototype, a downscaled version of the full SMWP, is constructed using stacked, predefined layers. The reservoirs of a 96-well plate are fluidically connected to integrated OoC devices and micropumps through a fluidic circuit board (FCB). A printed circuit board, assembled below the FCB, enables the electrical interfacing. The following devices were integrated in the prototype: OoC devices from Bi/ond and BEOnChip, a microelectrode array (MEA) device from MultiChannel Systems, and piezoelectric micropumps from Fraunhofer EMFT. In the OoC devices, cell culture was performed on integrated on-chip membranes. On the MEA, neuronal cells were cultured directly on the surface of the chip. For initial experiments, static cultures were performed to investigate the biocompatibility of all included materials inside the prototype. BEOnChip performed a static cell culture with skin cells (Ha- CaT) in their device. Normal cell viability and morphology was observed after 96 h and 21 days of culture using Calcein-AM/PI live/ dead staining. MCS performed static cell culture using hiPSCs-derived neurons directly cultured on the PLO/laminin-coated MEA chips. After 14 days of culture, eGFP staining showed normal cell morphology and network formation. Bi/ond performed an endothelial cell culture (HMEC-1), showing proper cell adhesion and viability in their devices. The biological experiments under static conditions showed normal cell morphology and viability in all integrated devices. In the next phase of the project, the full SMWP platform with integrated perfusion will be used for biological experimentation to generate an air-liquid interface with skin cells (BEOnChip), a perfusable MEA (MCS) and endothelial tube formation under unidirectional flow (Bi/ond).