Instrumented cardiac microphysiological devices via multimaterial three-dimensional printing

Biomedical research has relied on animal studies and conventional cell cultures for decades. Recently, microphysiological systems (MPS), also known as organs-on-chips, that recapitulate the structure and function of native tissues in vitro, have emerged as a promising alternative. However, current MPS typically lack integrated sensors and their fabrication requires multi-step lithographic processes. Here, we introduce a facile route for fabricating anewclass of instrumented cardiacmicrophysiological devicesviamultimaterial three-dimensional (3D) printing. Specifically, we designed six functional inks, based on piezo-resistive, high-conductance, and biocompatible soft materials that enable integration of soft strain gauge sensors within micro-architectures that guide the self-assembly of physio-mimetic laminar cardiac tissues. We validated that these embedded sensors provide non-invasive, electronic readouts of tissue contractile stresses inside cell incubator environments. We further applied these devices to study drug responses, as well as the contractile development of human stem cell-derived laminar cardiac tissues over four weeks. Current MPS models of muscle tissue rely on microscopy coupled with optical tracking analysis for assessing tissue contractile stress. For instance, muscular thin-film (MTF) assays track changes in curvature of soft cantilever substrates induced by the contraction of a laminar tissue, and micro-post assays measure the deflection of pillars supporting a micro-tissue. Although these assays have proved valuable for short-term modelling of human disease and small-scale drug screening applications, they are not well suited for higher-throughput or longer-term studies. Moreover, biomimetic microsystems are at present fabricated using soft material lithography-based techniques that require multiple steps, masks and dedicated tools, which hinders rapid prototyping and customization. By contrast,multimaterial 3Dprinting of viscoelastic inks enables a wide range of functional, structural and biological materials to be patterned and integrated in a single programmable manufacturing step. Here, we introduce a fully 3D printed and instrumented microphysiological device that provides continuous electronic readout of the contractile stress ofmultiple laminar cardiacmicro-tissues (Fig. 1a,b). Each device contains three key features: multilayer cantilevers, composed of a base layer, an embedded strain sensor, and a tissue-guiding layer; electrical interconnects for readout; and eight independent wells (Fig. 1c–i). The tissue-guiding layer promotes self-assembly of engineered physio-mimetic laminar tissues from neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). Our microphysiological device facilitates tissue culture and non-invasive analyses of tissue contractile strength over several weeks, and facilitates drug studies inside a controlled incubator environment. To create this integrated device, six materials are patterned sequentially via direct ink writing multimaterial 3D printing. To allow material integration at the microscale, the substrate topology and x–y–z positions of four individually addressable nozzles are determined by an automated calibration process (see SupplementaryMovie 1). After this calibration, the device is printed in a single continuous procedure (Fig. 1c–i and Supplementary Movies 2 and 3). The stress generated by laminar cardiac tissues is limited to the range 1–15 kPa (refs 5,8). Hence, to match this range, both the thickness and stiffness of each cantilever layer must be minimized. Towards this objective, we designed a series of highly dilute polymer-based inks. Their low solids content ensures patterning of thin individual layers (0.5–6.5 μm in thickness). Additionally, by tuning the evaporation rate of the carrier solvent solution, the ink viscosity and corresponding wetting and spreading behaviour is controlled to achieve the desired lateral dimensions (Supplementary Figs 1 and 2). Using these inks, 0.5 μm dextran films are printed first (Fig. 1c). These serve as biocompatible, watersoluble sacrificial release layers that allow the final cantilevers to detach from the substrate and deflect freely. Next, using dilute thermoplastic polyurethane (TPU) inks, 3-μm-thick cantilever bases, 6.5-μm-thick strain gauge wires and 1.5-μm-thick wire covers are printed in steps 2–4, respectively (Fig. 1d–f). The cantilever base and wire covers are printed using an unfilled TPU ink, whereas the strain gauge wires are printed using a TPU ink filled with 25 wt% carbon black nanoparticles (CB:TPU). The printed TPU-based features exhibit elastic mechanical properties with a Young’s modulus of 1.6MPa (Supplementary Fig. 3), whereas CB:TPU features readily cure to form an elastic piezo-resistive material with a Young’s modulus of 8.8MPa and resistivity of 1.19 cm (Supplementary Figs 3–7). Several alternative fillers were investigated, including metals particles and carbon nanotubes. However, we found that carbon black imparts the best combination of ink rheology, low stiffness and sensor hysteresis. The remainder of the microphysiological device is printed using concentrated viscoelastic inks optimized for deposition of self-supporting structures (Supplementary Figs 8 and 9). Using a