In the past few years, microfluidic-based technology has developed microscale models recapitulating key physical and biological cues typical of the native myocardium. However, the application of controlled physiological uniaxial cyclic strains on a defined three-dimension cellular environment is not yet possible. Two-dimension mechanical stimulation was particularly investigated, neglecting the complex three-dimensional cell-cell and cell-matrix interactions. For this purpose, we developed a heart-on-a-chip platform, which recapitulates the physiologic mechanical environment experienced by cells in the native myocardium. The device includes an array of hanging posts to confine cell-laden gels, and a pneumatic actuation system to induce homogeneous uniaxial cyclic strains to the 3D cell constructs during culture. The device was used to generate mature and highly functional micro-engineered cardiac tissues (μECTs), from both neonatal rat and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), strongly suggesting the robustness of our engineered cardiac micro-niche. Our results demonstrated that the cyclic strain was effectively highly uniaxial and uniformly transferred to cells in culture. As compared to control, stimulated μECTs showed superior cardiac differentiation, as well as electrical and mechanical coupling, owing to a remarkable increase in junction complexes. Mechanical stimulation also promoted early spontaneous synchronous beating and better contractile capability in response to electric pacing. Pacing analyses of hiPSC-CM constructs upon controlled administration of isoprenaline showed further promising applications of our platform in drug discovery, delivery and toxicology fields. The proposed heart-on-a-chip device represents a relevant step forward in the field, providing a standard functional three-dimensional cardiac model to possibly predict signs of hypertrophic changes in cardiac phenotype by mechanical and biochemical co-stimulation.

In vitro cardiac models able to mimic the fibrotic process are paramount to develop an effective anti-fibrosis therapy that can regulate fibroblast behaviour upon myocardial injury. In previously developed in vitro models, typical fibrosis features were induced by using scar-like stiffness substrates and/or potent morphogen supplementation in monolayer cultures. In our model, we aimed to mimic in vitro a fibrosis-like environment by applying cyclic stretching of cardiac fibroblasts embedded in three-dimensional fibrin-hydrogels alone. Using a microfluidic device capable of delivering controlled cyclic mechanical stretching (10% strain at 1 Hz), some of the main fibrosis hallmarks were successfully reproduced in 7 days. Cyclic strain indeed increased cell proliferation, extracellular matrix (ECM) deposition (e.g. type-I-collagen, fibronectin) and its stiffness, forming a scar-like tissue with superior quality compared to the supplementation of TGFβ1 alone. Taken together, the observed findings resemble some of the key steps in the formation of a scar: (i) early fibroblast proliferation, (ii) later phenotype switch into myofibroblasts, (iii) ECM deposition and (iv) stiffening. This in vitro scar-on-a-chip model represents a big step forward to investigate the early mechanisms possibly leading later to fibrosis without any possible confounding supplementation of exogenous potent morphogens.