Replicating in vitro the complex in vivo tissue microenvironment has the potential to transform our approach to medicine and also our understanding of biology. In order to accurately model the 3D arrangement and interaction of cells and extracellular matrix, new microphysiological systems must include a vascular supply. The vasculature not only provides the necessary convective transport of oxygen, nutrients, and waste in 3D culture, but also couples and integrates the responses of organ systems. Here we combine tissue engineering and microfluidic technology to create an in vitro 3D metabolically active stroma (*1 mm 3 ) that, for the first time, contains a perfused, living, dynamic, interconnected human capillary network. The range of flow rate (mm/s) and shear rate (s -1 ) within the network was 0-4000 and 0-1000, respectively, and thus included the normal physiological range. Infusion of FITC dextran demonstrated microvessels (15-50 mm) to be largely impermeable to 70 kDa. Our high-throughput biology-directed platform has the potential to impact a broad range of fields that intersect with the microcirculation, including tumor metastasis, drug discovery, vascular disease, and environmental chemical toxicity.

Disturbances in the microcirculation can lead to secondary lymphedema, a common pathological condition that, despite its frequency, still lacks a cure. Lymphedema is clinically well described, but while the genetic underpinnings that cause lymphatic malformations and primary lymphedema are being discovered, the pathophysiology and pathobiology of secondary lymphedema remain poorly understood, partly due to the lack of well-described experimental models. Here, we provide a detailed characterization of secondary lymphedema in the mouse tail and correlate the evolution of tissue swelling to changes in tissue architecture, infiltration of immune cells, deposition of lipids, and proliferation and morphology of the lymphatic vessels. We show that sustained swelling leads to lymphatic hyperplasia and upregulation of vascular endothelial growth factor (VEGF)-C, which may exacerbate the edema because the hyperplastic vessels are poorly functional. The onset of lymphatic hyperplasia occurred prior to the onset of lipid accumulation and peak VEGF-C expression. Langerhans dendritic cells were seen in the dermis migrating from the epidermis to the lymphatic capillaries in edematous tissue. Furthermore, these results were consistent between two different normal mouse strains, but swelling was significantly greater in a matrix metalloproteinase (MMP)-9 null strain. Thus, by characterizing this highly reproducible model of secondary lymphedema, we conclude that VEGF-C upregulation and lymphatic hyperplasia resulting from dermal lymphatic ligation and lymphedema leads to decreased drainage function and that MMP-9 may be important in counteracting tissue swelling.

We report the first demonstration of a microfluidic platform that captures the full physiological range of mass transport in 3-D tissue culture. The basis of our method used long microfluidic channels connected to both sides of a central microtissue chamber at different downstream positions to control the mass transport distribution within the chamber. Precise control of the Péclet number (Pe), defined as the ratio of convective to diffusive transport, over nearly five orders of magnitude (0.0056 to 160) was achieved. The platform was used to systematically investigate the role of physiological mass transport on vasculogenesis. We demonstrate, for the first time, that vasculogenesis can be independently stimulated by interstitial flow (Pe>10) or hypoxic conditions (Pe<0.1), and not by the intermediate state (normal living tissue). This simple platform can be applied to physiological and biological studies of 3D living tissue followed by pathological disease studies, such as cancer research and drug screening.

This paper reports a polydimethylsiloxane microfluidic model system that can develop an array of nearly identical human microtissues with interconnected vascular networks. The microfluidic system design is based on an analogy with an electric circuit, applying resistive circuit concepts to design pressure dividers in serially-connected microtissue chambers. A long microchannel (550, 620 and 775 mm) creates a resistive circuit with a large hydraulic resistance. Two media reservoirs with a large cross-sectional area and of different heights are connected to the entrance and exit of the long microchannel to serve as a pressure source, and create a near constant pressure drop along the long microchannel. Microtissue chambers (0.12 μl) serve as a two-terminal resistive component with an input impedance > 50-fold larger than the long microchannel. Connecting each microtissue chamber to two different positions along the long microchannel creates a series of pressure dividers. Each microtissue chamber enables a controlled pressure drop of a segment of the microchannel without altering the hydrodynamic behaviour of the microchannel. The result is a controlled and predictable microphysiological environment within the microchamber. Interstitial flow, a mechanical cue for stimulating vasculogenesis, was verified by finite element simulation and experiments. The simplicity of this design enabled the development of multiple microtissue arrays (5, 12, and 30 microtissues) by co-culturing endothelial cells, stromal cells, and fibrin within the microchambers over two and three week periods. This methodology enables the culturing of a large array of microtissues with interconnected vascular networks for biological studies and applications such as drug development.

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We are currently performing in vivo experiments to determine the ability of angiogenic proteins released from this outer layer to induce neovascularization around the grafts in the omentum pouch. We will subsequently examine the effect of co-encapsulation of islets with angiogenic protein on blood glucose control in diabetic animals. It is hoped that addition of tissue engineering to encapsulated islet transplantation will result in long-term survival of the islets and their ability to control blood glucose in type 1 diabetes without the necessity to use risky immunosuppressive drugs to prevent transplant rejection.

Alginate microcapsules coated with a permselective poly-L-ornithine (PLO) membrane have been investigated for the encapsulation and transplantation of islets as a treatment for type 1 diabetes. The therapeutic potential of this approach could be improved through local stimulation of microvascular networks in order to meet mass transport demands of the encapsulated cells. Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor with optimal effect occurring when it is delivered in a sustained manner. In this paper, a technique is described for the generation of multilayered alginate microcapsules with an outer alginate layer that can be used for the delivery of FGF-1. The influence of alginate concentration and composition (high mannuronic acid (M) or guluronic acid (G) content) on outer layer size and stability, protein encapsulation efficiency, and release kinetics was investigated. The technique results in a stable outer layer of alginate with a mean thickness between 113-164 µm, increasing with alginate concentration and G-content. The outer layer was able to encapsulate and release FGF-1 for up to thirty days, with 1.25% of high G alginate displaying the most sustained release. The released FGF-1 retained its biologic activity in the presence of heparin, and the addition of the outer layer did not alter the permselectivity of the PLO coat. This technique could be used to generate encapsulation systems that deliver proteins to stimulate local neovascularization around encapsulated islets.