Abstract
The complex network of blood vessels plays a key role in transporting oxygen and nutrients and maintaining homeostasis in the human body. The inner walls of all blood and lymphatic vessels are lined by the endothelium, a monolayer of endothelial cells (ECs) oriented along the direction of blood flow. ECs play a pivotal role in vascular homeostasis, including regulating vascular tone, delivering oxygen and nutrients, modulating pro-inflammatory molecules and pro-inflammatory immune responses, and performing other vital functions. Therefore, the study of EC biology and vascular responses is key for a deeper understanding of vascular biology and the development of new therapeutics. Most studies in vivo and in vitro present technical challenges, either complexity or oversimplification, respectively, which slow down advances in the field. Therefore, 3D models and microfluidics offer a complementary alternative that integrates shapes similar to those observed in vivo, with the advantages of an in vitro system. Here, we present a robust and reproducible vessel-on-a-chip (VOC) composed of an EC monolayer and a microvascular microenvironment maintained by a peristaltic pump to ensure continuous media circulation and physiological levels of shear stress. In addition, we validated this model for in vitro studies of vascular inflammation by monitoring EC status. We observed cellular alignment after shear stress exposure, increased E-selectin expression, and TNF-induced morphological changes in ECs. This new VOC is a promising approach to studying EC mechanobiology and inflammation and opens new avenues for its versatile use in vascular biology, inflammation, and immune and cancer cell migration in a controlled, scalable manner. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: 3D Vessel Formation within a microfluidic organ-on-a-chip system Basic Protocol 2: Evaluation of shear stress Basic Protocol 3: Evaluation of inflammation.