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

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

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

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

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 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