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

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 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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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