A novel high-throughput assay to produce ring-shaped 3D cardiac tissues from human induced pluripotent stem cells

Abstract

Cardiac tissue engineering aims at creating contractile structures that can optimally reproduce the features of human cardiac tissue. These constructs, associated to the induced pluripotent stem cell (iPSC) technology, are becoming valuable tools to model some of the cardiac functions, to set preclinical platforms for drug testing, or to model cardiac pathologies. Nevertheless, the techniques used to generate engineered tissues still require expertise and produce a limited number of tissues per batch. To develop a novel assay which allows a fast, reproducible and high-throughput generation of 3D cardiac rings in a 96-well plate. A specific mold was designed to generate ring-shaped tissues around a central PEG pillar mounted on glass coverslips (Fig. 1). Cardiomyocytes derived from wild-type human iPSCs (hiPSC-CMs) were mixed with human dermal fibroblasts (HDF) and then seeded in the wells. An inhouse Matlab code was used to measure the deformation of the central pillar in time, deriving the force exerted by the tissue, and several contraction parameters. At day 14, the tissues were fixed and stained to evaluate their organization by confocal imaging. Finally, drug response of the tissues was assessed with different drugs. After seeding, the cells mix fell at the bottom of the PEG microwells and compacted around the pillars, forming 21 beating cardiac tissues per well. An iPSC-CM/fibroblast ratio of 3:1 was optimal to generate the highest number of stable tissues in time. The staining of vimentin and troponin T showed that the fibroblasts form a layer, stabilizing the iPSC-CMs which form a ring-shaped muscular fiber above. Tissue motion was monitored by real-time video-microscopy. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25 ± 0.55%, that maintained up to 14 days. The average stress developed was of 1,43 ± 0,37 μN/mm2. The cardiac constructs showed a positive inotropic response to increasing extracellular calcium concentrations and isoproterenol, while verapamil induced a strong negative inotropic response. Dofetilide induced arrhythmias at low concentrations and cardiotoxic effects. We report on the development on a novel, easy-to-use, reproducible and high-throughput platform to generate cardiac tissues using hiPSC-CMs. The platform can serve for disease modeling and pharmacological testing.