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.@en

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.@en

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.@en

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.

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Organ-on-chip (OoC) technology is a promising improvement within in vitro cell culture, better mimicking functional units of human organs compared to conventional techniques. Current fabrication of three-Dimensional (3D) components in OoC, such as thin membranes and microfluidic structures, is often achieved via soft lithography, bonding, and punching of access holes of polymers, such as polymethylsiloxane (PDMS). However, these methods often suffer from the need of manual fabrication steps, drastically increasing production time and reducing yield due to handling errors and manual alignment of the layers. Consequently, the scalability is limited, which is a crucial aspect for a more widespread adaptation of OoC technology. In this work, we present a reproducible and scalable process for the direct patterning of various 3D polymer structures. The investigated process employs commercially available systems from IC packaging to mould pillars, membranes, and microfluidic channels with varying dimensions and thicknesses. Our process simultaneously improves the control over the thickness and dimensions of these structures in comparison to conventional fabrication techniques. Furthermore, proof of functionality is presented by adapting this technology to an existing OoC platform which incorporates integrated electrodes used for electrophysiological recording, stimulation, and TEER measurements. We demonstrate a complete process for wafer-scale microfabrication of OoCs, enabling low-cost, high-volume automated production. This is an important next step to large-scale manufacturing of OoCs, enabling more biologists and scientists to integrate OoCs into their workflow. @en

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@en

Microphysiological systems consisting of multiple cell types of the human heart have been shown to recapitulate certain aspects of human physiology better than conventional 2D in vitro models [1]. Engineered heart tissues (EHTs) that self-organise into contractile 3D structures between two flexible pillars are particularly useful to measure contraction against a force. However, conventional EHTs typically require between 50,000 and 2,000,000 cells, which makes creating many EHTs for high throughput screening costly [2]. Here, we show that downscaling EHT size, in our case to include human-induced pluripotent stem cell-derived cardiomyocytes (70%), cardiac fibroblasts (15%) and cardiac endothelial cells (15%), is feasible using as few as 16,000 cells. Tissues of three different sizes formed as expected and consistently, with 47,000, 31,000, and 16,000 cells. Moreover, while keeping the load constant relative to the size of the tissue [3], there was no difference in the viability nor functionality up to 14 days after formation. Electrical pacing of the tissues was conducted within the range of 1 to 3 Hz and with an optimal pacing frequency of 1.4 Hz, which is consistent over the three EHT sizes. Our results indicate that downscaled EHTs might be used as a cost-effective alternative to larger EHTs in drug discovery. @en

Organ-on-Chip (OoC) is a game-changing technology in which human cells are cultured in microfluidic chips to mimic and predict the physiology and pathology of human tissues, as well as to provide insights into drug and disease mechanisms. However, current limitations in manufacturing and technical usability of existing OoC approaches must be overcome in order for industry and regulators to adopt OoCs. Our goal is therefore to develop a Standardized and Modular open-technology OoC platform as a new Approach to Recapitulate human Tissues (SMART OoC), that enables the integration of a novel microfluidic pump. This platform is a further development of the Translational OoC Platform (TOP), which includes a plate with an integrated microfluidic channel network with standardized fluid input/output ports, to which exchangeable chip modules can be connected [1]. To provide fluid pumping to the platform, we are developing a novel micropumping chip module based on magnetic artificial cilia (MAC) [2]. MAC are flexible rod-shaped magnetic micro-actuators inspired by biological cilia, made of polydimethylsiloxane (PDMS) containing magnetic microparticles. MAC can induce microfluidic flow and particle transport when integrated into a microfluidic module and actuated with an external magnet. In this research, we present a novel miniaturized actuation setup for actuating biocompatible MAC integrated in a versatile microfluidic module that generates a variety of fluid flow regimes in the OoC platform. In comparison to other micropumping methods, this module does not require tubings or electrical connections, which opens up a wide range of possibilities for OoC applications. @en

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). @en

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.

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Organ-on-chip (OoC) is emerging as a key technology for improved pre-clinical drug testing. Monitoring tissues and the artificial microenvironment in OoC devices is critical to recapitulate human physiology; however, sensing is often invasive, superficial, and not continuous over time. This work aims to overcome these issues by proposing dielectric spectroscopy as a non-invasive and time-continuous sensing technique capable of extracting information from multi-layer OoC devices, including distinguishable tissue layers. The presented results set the foundations for this goal by proving this technique’s feasibility, showing excellent correspondence between the experimental and modelled data, and providing design guidelines for application-tailored optimization.@en

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.

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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. @en

Organs-on-Chip (OoC) have been a promise of microfluidics since their early days, leading to a widespread use in research. OoC adoption by industry conversely has so far been slow, resulting in a big gap between what is developed by elite pioneers and what can be offered to a broad audience. A way to bridge this gap is the consolidation of standards for design, manufacturing and qualification of OoC, making it easier for experts in different fields to combine their expertise and creating integrated systems with higher value than the sum of the parts. To this end, here we present the Smart Multi-Well Plate (SMWP), a device based on an open technology platform and the standardized 96-well plate format. Within this format, the SMWP integrates 16 OoC devices and piezoelectric micropumps, connected by a microfluidic network and controlled by integrated electronics. Results and discussion: The SMWP allows for high modularity, with design and interfacing rules according to guidelines defined by experts from industry and academia [1]. With the SMWP, OoC devices, sensors and piezoelectric micropumps can be integrated using industrial workflows exploiting automated pick-and-placing and wire-bonding machines. The modules are fluidically connected by means of a polymeric fluidic circuit board and electrically connected by reusable and disposable printed circuits boards. These take care of both control of micropumps and sensors and transfer of power and data to the outside world. Fluidic and electric performance of the plate, including piezoelectric micropumps from Fraunhofer EMFT, were tested in combination with barrier model OoC devices from BEOnChip and Bi/ond and multi electrode array chips from MultiChannel Systems. Preliminary results of the biological validation of the three embodiments of the SMWP will also be presented. Conclusions: An autonomous standardized OoC platform based on an open technology was realized by leveraging industrially upscalable processes and integrating OoC devices. @en

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.

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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. @en

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.@en

Human heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better than standard two-dimensional models in vitro. They typically consist of less than 5000 cells and are used to measure contraction kinetics although not contraction force. By contrast, engineered heart tissues (EHTs) formed around two flexible pillars, can measure contraction force but conventional EHTs often require between 0.5 and 2 million cells. This makes large-scale screening of many EHTs costly. Our goals here were (i) to create a physiologically relevant model that required fewer cells than standard EHTs making them less expensive, and (ii) to ensure that this miniaturized model retained correct functionality. We demonstrated that fully functional EHTs could be generated from physiologically relevant combinations of hiPSC-derived cardiomyocytes (70%), cardiac fibroblasts (15%) and cardiac endothelial cells (15%), using as few as 1.6 × 10⁴ cells. Our results showed that these EHTs were viable and functional up to 14 days after formation. The EHTs could be electrically paced in the frequency range between 0.6 and 3 Hz, with the optimum between 0.6 and 2 Hz. This was consistent across three downscaled EHT sizes tested. These findings suggest that miniaturized EHTs could represent a cost-effective microphysiological system for disease modelling and examining drug responses particularly in secondary screens for drug discovery.

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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.@en