Human Neurons Form Axon-Mediated Functional Connections with Human Cardiomyocytes in Compartmentalized Microfluidic Chip

The cardiac autonomic nervous system (cANS) regulates cardiac function by innervating cardiac tissue with axons, and cardiomyocytes (CMs) and neurons undergo comaturation during the heart innervation in embryogenesis. As cANS is essential for cardiac function, its dysfunctions might be fatal; therefore, cardiac innervation models for studying embryogenesis, cardiac diseases, and drug screening are needed. However, previously reported neuron-cardiomyocyte (CM) coculture chips lack studies of functional neuron–CM interactions with completely human-based cell models. Here, we present a novel completely human cell-based and electrophysiologically functional cardiac innervation on a chip in which a compartmentalized microfluidic device, a 3D3C chip, was used to coculture human induced pluripotent stem cell (hiPSC)-derived neurons and CMs. The 3D3C chip enabled the coculture of both cell types with their respective culture media in their own compartments while allowing the neuronal axons to traverse between the compartments via microtunnels connecting the compartments. Furthermore, the 3D3C chip allowed the use of diverse analysis methods, including immunocytochemistry, RT-qPCR and video microscopy. This system resembled the in vivo axon-mediated neuron–CM interaction. In this study, the evaluation of the CM beating response during chemical stimulation of neurons showed that hiPSC-neurons and hiPSC-CMs formed electrophysiologically functional axon-mediated interactions.