Protocol for fibrin-based endothelial-cardiomyocyte co-culture in a microfluidic device.

Here, we present a protocol for building a vascularized heart-on-a-chip by co-encapsulating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and endothelial cells in vascular endothelial growth factor (VEGF)- and aprotinin-supplemented fibrin. We describe steps for cell preparation, fibrin formulation, chip loading, optional droplet-based pilot or high-throughput setups, and imaging. We then detail procedures for immunofluorescence staining and routine quantitative analysis of vascular and contractile features during culture for up to three weeks.