Binding of Plasmodium falciparum-Infected Red Blood Cells to Engineered 3D Microvessels.

P. falciparum-infected red blood cell (iRBC) sequestration in the microvasculature is a pivotal event in severe malaria pathogenesis. In vitro binding assays using endothelial cell monolayers under static and flow conditions have revealed key ligand-receptor interactions for iRBC sequestration. However, mechanisms remain elusive for iRBC sequestration in specific vascular locations, which prevents further development of effective therapies. New models are needed to better recapitulate the complex geometry of blood flow in human blood vessels and organ-specific vascular signatures. Recent advances in engineering 3D microvessels in vitro have emerged as promising technologies to not only model complex human vascular structures but also allow for precise and step-wise control of individual biological and biomechanical parameters. By designing networks with different branching structures and change of vessel diameter along the flow path, these models recapitulate pressure and flow changes occurring in vivo. Here, we describe the methodology employed to build 3D microvessels using soft lithography and injection molding techniques, as well as the protocol to fabricate capillary-size vessels through collagen photoablation. Furthermore, we describe the methodology of using these models to study malaria and narrate necessary steps for perfusion of P. falciparum through 3D microvessels and different options to quantify P. falciparum-iRBC binding.