Microvascular networks support metabolic activity and define microenvironmental conditions within tissues in health and pathology. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of complex vascular phenomena, including angiogenesis and thrombosis. We have engineered living microvascular networks in three-dimensional tissue scaffolds and demonstrated their biofunctionality in vitro. We describe the lithographic technique used to form endothelialized microfluidic vessels within a native collagen matrix; we characterize the morphology, mass transfer processes, and long-term stability of the endothelium; we elucidate the angiogenic activities of the endothelia and differential interactions with perivascular cells seeded in the collagen bulk; and we demonstrate the nonthrombotic nature of the vascular endothelium and its transition to a prothrombotic state during an inflammatory response. The success of these microvascular networks in recapitulating these phenomena points to the broad potential of this platform for the study of cardiovascular biology and pathophysiology.tissue engineering | regenerative medicine | microfluidics | cancer | blood T he microvasculature is an extensive organ that mediates the interaction between blood and tissues. It defines the biological and physical characteristics of the microenvironment within tissues and plays a role in the initiation and progression of many pathologies, including cancer (1) and cardiovascular diseases (2, 3). Conventional planar cultures fail to recreate the in vivo physiology of the microvasculature with respect to three-dimensional (3D) geometry (lumens and axial branching points), and interactions of endothelium with perivascular cells, extracellular tissue and blood flow (4). Studies of the microvasculature in vivo allow only limited control of physical, chemical, and biological parameters influencing the microvasculature and present challenges with respect to observation (5). In vitro cultures that produce tubular vessels within 3D matrices will aid in elucidation of the roles of the microvasculature in health and disease. Important progress has been made toward this goal: Biologically derived or synthetic materials have been used to generate macrovessel tubes (6) and endothelialized microtubes (7); cellular self-assembly has been used to generate random microvasculature (8); microfabrication has been used to define complex geometries in hydrogels at the micro-scale (9); and distributions of cells and biochemical factors within 3D scaffolds (10). Of particular note, the group of Tien has pioneered the use of collagen to template the growth of vascular endothelium (7, 11) and demonstrated appropriate permeability (7), response to cyclic AMP (12), and differential properties as a function of the luminal shear stress and composition of the medium (13). Nonetheless, prior methodologies have been unable to produce endothelialized networks that can undergo substantial remodeling via angiogenesis; elucidate the ...

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This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. These microvascular networks (μVNs) allow for multiweek cultures of endothelial cells or cocultures with parenchymal or tissue cells in the extra-lumen space. The platform enables real-time fluorescence imaging of living engineered tissues, in situ confocal fluorescence of fixed cultures and transmission electron microscopy (TEM) imaging of histological sections. This protocol enables studies of basic vascular and blood biology, provides a model for diseases such as tumor angiogenesis or thrombosis and serves as a starting point for constructing prevascularized tissues for regenerative medicine. After one-time microfabrication steps, the system can be assembled in less than 1 d and experiments can run for weeks.