Abstract
Blood vessels are composed of endothelial cells (ECs) that form the inner vessel wall and mural cells that cover the ECs to mediate their stabilization. Crosstalk between ECs and VSMCs while the ECs undergo microfluidic flow is vital for the function and integrity of blood vessels. Here, we describe a protocol to generate three-dimensional (3D) engineered vessels-on-chip (VoCs) composed of vascular cells derived from human induced pluripotent stem cells (hiPSCs). We first describe protocols for robust differentiation of vascular smooth muscle cells (hiPSC-VSMCs) from hiPSCs that are effective across multiple hiPSC lines. Second, we describe the fabrication of a simple microfluidic device consisting of a single collagen lumen that can act as a cell scaffold and support fluid flow using the viscous finger patterning (VFP) technique. After the channel is seeded sequentially with hiPSC-derived ECs (hiPSC-ECs) and hiPSC-VSMCs, a stable EC barrier covered by VSMCs lines the collagen lumen. We demonstrate that this 3D VoC model can recapitulate physiological cell-cell interaction and can be perfused under physiological shear stress using a microfluidic pump. The uniform geometry of the vessel lumens allows precise control of flow dynamics. We have thus developed a robust protocol to generate an entirely isogenic hiPSC-derived 3D VoC model, which could be valuable for studying vessel barrier function and physiology in healthy or disease states. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Differentiation of hiPSC-VSMCs Support Protocol 1: Characterization of hiPSC-NCCs and hiPSC-VSMCs Support Protocol 2: Preparation of cryopreserved hiPSC-VSMCs and hiPSC-ECs for VoC culture Basic Protocol 2: Generation of 3D VoC model composed of hiPSC-ECs and hiPSC-VSMCs Support Protocol 3: Structural characterization of 3D VoC model.