Abstract: Cardiac autonomic nervous system regulates the heart function through innervation. In Stroke-Heart Syndrome (SHS) an acute stroke leads to elevated serum levels of cardiac markers and arrhythmias, impacting heart function, leading to cardiovascular complications that are the second leading cause of post-stroke mortality. Completely human cell-based in vitro models studying functional neuron-cardiomyocyte connections with central nervous system (CNS), peripheral nervous system (PNS) and cardiac tissue are missing from the field. Our aim is to model SHS in vitro by combining three cell types; CNS type neurons (CNs), PNS type neurons (PNs) and cardiomyocytes (CMs) all derived from human induced pluripotent stem cells (hiPSC), in compartmentalized microfluidic devices, so called 3D3C chips. The structure of 3D3C chip enables the culturing of each cell type separately while allowing axonal growth through microtunnels to the adjacent cardiac compartment. Integrated in-house produced microelectrode arrays (MEA) enable the electrophysiological functionality measurements. By modifying the oxygen conditions in different compartments of the chip, such as subjecting CNs to oxygen deprivation, local stroke-like simulations can be generated for studying aspects of SHS on-a-chip. Here, hiPSC-derived CNs, PNs and CMs were successfully cocultured in the 3D3C chip up to three weeks. The physical axonal interactions were investigated with microscopy and immunocytochemistry and the functionality of the cells with MEAs. The axonal elongations between CNs to PNs and PNs to CMs were detectable, indicating successful innervation. All cell types developed cell-specific electrophysiological functionality. Oxygen deprivation was induced chemically solely to CN compartment and cell response measured morphologically, metabolically and functionally. Stroke-like conditions altered the function of all cell types, mimicking SHS on-a-chip. As a proof of concept, the compartmentalized microfluidic chip with integrated MEA allowed formation of physiologically relevant and functional connections with neurons and cardiomyocytes. This advanced, completely human cell-based cardiac innervation on-a-chip presents a powerful platform not only for the study of SHS but any disease where brain-heart axis is essential.
