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 ...

Most methods to culture cells in three dimensions depend on a cell-seedable biomaterial to define the global structure of the culture and the microenvironment of the cells. Efforts to tailor these scaffolds have focused on the chemical and mechanical properties of the biomaterial itself. Here, we present a strategy to control the distributions of soluble chemicals within the scaffold with convective mass transfer via microfluidic networks embedded directly within the cell-seeded biomaterial. Our presentation of this strategy includes: a lithographic technique to build functional microfluidic structures within a calcium alginate hydrogel seeded with cells; characterization of this process with respect to microstructural fidelity and cell viability; characterization of convective and diffusive mass transfer of small and large solutes within this microfluidic scaffold; and demonstration of temporal and spatial control of the distribution of non-reactive solutes and reactive solutes (that is, metabolites) within the bulk of the scaffold. This approach to control the chemical environment on a micrometre scale within a macroscopic scaffold could aid in engineering complex tissues.

Type I collagen is a favorable substrate for cell adhesion and growth and is remodelable by many tissue cells; these characteristics make it an attractive material for the study of dynamic cellular processes. Low mass fraction (1.0-3.0 mg/ml), hydrated collagen matrices used for threedimensional cell culture permit cellular movement and remodeling, but their microstructure and mechanics fail to mimic characteristics of many extracellular matrices in vivo and limit the definition of fine-scale geometrical features (< 1 mm) within scaffolds. In this study, we worked with hydrated type I collagen at mass fractions between 3.0 and 20 mg/ml to define the range of densities over which the matrices support both microfabrication and cellular remodeling. We present pore and fiber dimensions based on confocal microscopy and longitudinal modulus and hydraulic permeability based on confined compression. We demonstrate faithful reproduction of simple pores of 50 µm-diameter over the entire range and formation of functional microfluidic networks for mass fractions greater than 10.0 mg/ml. We present quantitative characterization of the rate and extent of cellular remodelability using human umbilical vein endothelial cells. Finally, we present a co-culture with tumor cells and discuss the implications of integrating microfluidic control within scaffolds as a tool to study spatial and temporal signaling during tumor angiogenesis and vascularization of tissueengineered constructs.

We report on the incorporation of microfluidic structure within a high-water-content hydrogel [4% (w/v) calcium alginate]. We used the microfluidic network to control the chemical environment within the hydrogel and demonstrated higher rates of delivery and extraction of solutes than was achievable by diffusion alone.

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.