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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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 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