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

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

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

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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The high death toll of cardiovascular diseases worldwide and the lack of effective treatments for them are the main motivation for developing alternative and more efficient models for cardiac drug development and disease research. The missing link between current laboratory research on static in vitro and animal models and the clinical stage research on human patients could be created using the rapidly emerging Organ-on-Chip (OoC) technology. Themicrophysiological models developed within OoC research combine devices made of biocompatible, soft materials and human-origin organ-specific cell types, which are then exposed to flow, chemical, electrical or biomechanical stimuli. Modeling a human cardiac in vivo environment in an artificial model represents quite a challenge from several aspects. First, cardiac tissue in vivo is exposed to a strong coupling between different biomechanical and electrical stimuli that need to be faithfully captured by an in vitro model. Furthermore, such an in vitro model should recapitulate the complexity of cell-cell and cell-extracellular matrix (ECM) interactions between different cardiac cell types, while obtaining physiologically relevant responses. This thesis addresses the first challenge, in an attempt to engineer a dynamic, artificial microenvironment, suitable for the growth, monitoring, and stimulation of hiPSC-based engineered cardiac tissues (EHTs).....@en

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

Stemming from the convergence of tissue engineering and microfluidics, organ-on-chip (OoC) technology can reproduce in vivo-like dynamic microphysiological environments for tissues in vitro. The possibility afforded by OoC devices of realistic recapitulation of tissue and organ (patho)physiology may hold the key to bridge the current translational gap in drug development, and possibly foster personalized medicine. Here we underline the biotechnological convergence at the root of OoC technology, and outline research tracks under development in our group at TU Delft along two main directions: fabrication of innovative microelectromechanical OoC devices, integrating stimulation and sensing of tissue activity, and their embedding within advanced platforms for pre-clinical research. We conclude with remarks on the role of open technology platforms for the broader establishment of OoC technology in pre-clinical research and drug development.@en

We present a wafer-scale fabricated, PDMS-based platform for culturing miniaturized engineered heart tissues (EHTs) which allows highly accurate measurements of the contractile properties of these tissues. The design of the platform is an anisometrically downscaled version of the Heart-Dyno system, consisting of two elastic micropillars inside an elliptic microwell with volume ranging from 3 down to 1μL which supports EHT formation. Size downscaling facilitates fabrication of the platform and makes it compatible with accurate and highly reproducible batch wafer-scale processing; furthermore, downscaling reduces the cost of cell cultures and increases assay throughput. After fabrication, the devices were characterized by nanoindentation to assess the mechanical properties of the pillars and transferred to 96-well plates for cell seeding. Regardless the size of the platform, cell seeding resulted in successful formation of EHTs and all tissues were functionally active (i.e. showed cyclic contractions). The precise characterization of the stiffness of the micropillars enabled accurate measurements of the contractile forces exerted by the cardiac tissues through optical tracking of micropillar displacement. The miniature EHT platforms described in this paper represent a proper microenvironment for culturing and studying EHTs.@en

We present an extremely compact field effect transistor (FET)-based electrochemical sensor for in situ real-time and label-free measurement of ion concentrations in the cell culture area of organs-on-chip (OoCs) devices. This sensor replaces the functionality of an external reference electrode, crucial in standard electrochemical sensing, by controlling the FET threshold voltage via a capacitive control gate. The silicon- and polymer-based charge sensor can be integrated in OoC platforms by means of a wafer-scale and CMOS-compatible microfabrication process. This fabrication approach inherently allows a superior level of accuracy, repeatability and scalability compared to common OoC manufacturing methods. The sensor combines in a single device the complementary benefits of silicon-based electronics and of flexible polymer membranes with integrated microelectrodes – congenial substrates to sustain dynamic stimuli and mimic physiological tissue microenvironments. The integration of the polymer membrane in the sensing region makes this miniature sensor a preferable option for high sensitivity biochemical measurements in OoC applications, including monitoring the pH of cell culture media and of tissue culturing microenvironments, quantification of ion displacement in cells, and complementary research on disease modeling.@en

Monitoring cell conditions and microenvironment in real time is crucial for Organ-on-Chip (OoC) functionality. In particular, biological cues such as ions, including metals and metabolites, play a critical role in physiology and homeostasis in the human body. • Real-time monitoring of ions without optical systems is an unmet need for OOCs [1]. • Electrochemical sensors, such as organic electrochemical [2] and thin-film transistors [3], may address this need. Most of these sensors however rely on reference electrodes.@en

We presented the smallest and best characterised EHT devices to date. The devices were fabricated by wafer-scale silicon and polymer processing, characterised by nanoindentation and finite-element simulations, and transferred to 96-well plates for cell seeding and optical tracking of bundle contraction. Cell bundles remained functional for at least 18 days. Pacing electrodes and strain sensors will be added for improved bundle control.@en