Engineered, perfusable, human microvascular networks on a microfluidic chip

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (pages 61-64).In this thesis, we developed a reliable platform for engineering perfusable, microvascular networks on-demand using state of the art microfluidics technology. We have demonstrated the utility of this platform for studying cancer metastasis and as a test bed for drug discovery and analysis. In parallel, this platform enabled us to study, in a highly controlled environment, the physiologic processes of angiogenesis and vasculogenesis to further elucidate their underlying mechanisms. In addition to using our platform for real-time observation of physiological processes, we also took advantage of the ability to influence these processes through precise control of the extracellular environment. By manipulating the mechanical and bio-chemical inputs to our system, we controlled the dynamics of microvascular network formation as well as key properties of the network morphology. These findings will aid in the design and engineering of organ specific constructs for tissue engineering and regenerative medicine applications. Finally, we explored the potential use of stem cells for engineering microvascular networks in our system. We found that human mesenchymal stem cells can act as secondary, support cells during microvascular network formation.by Jordan Ari Whisler.S.M

Control of Perfusable Microvascular Network Morphology Using a Multiculture Microfluidic System

The mechanical and biochemical microenvironment influences the morphological characteristics of microvascular networks (MVNs) formed by endothelial cells (ECs) undergoing the process of vasculogenesis. The objective of this study was to quantify the role of individual factors in determining key network parameters in an effort to construct a set of design principles for engineering vascular networks with prescribed morphologies. To achieve this goal, we developed a multiculture microfluidic platform enabling precise control over paracrine signaling, cell-seeding densities, and hydrogel mechanical properties. Human umbilical vein endothelial cells (HUVECs) were seeded in fibrin gels and cultured alongside human lung fibroblasts (HLFs). The engineered vessels formed in our device contained patent, perfusable lumens. Communication between the two cell types was found to be critical in avoiding network regression and maintaining stable morphology beyond 4 days. The number of branches, average branch length, percent vascularized area, and average vessel diameter were found to depend uniquely on several input parameters. Importantly, multiple inputs were found to control any given output network parameter. For example, the vessel diameter can be decreased either by applying angiogenic growth factors—vascular endothelial growth factor (VEGF) and sphingosine-1-phsophate (S1P)—or by increasing the fibrinogen concentration in the hydrogel. These findings introduce control into the design of MVNs with specified morphological properties for tissue-specific engineering applications.National Science Foundation (U.S.). Science and Technology Center Emergent Behaviors of Interated Cellular Systems (EBICS) (Grant CBET-0939511)National Science Foundation (U.S.) (Fellowship

Mechanisms of tumor cell extravasation in an in vitro microvascular network platform

A deeper understanding of the mechanisms of tumor cell extravasation is essential in creating therapies that target this crucial step in cancer metastasis. Here, we use a microfluidic platform to study tumor cell extravasation from in vitro microvascular networks formed via vasculogenesis. We demonstrate tight endothelial cell–cell junctions, basement membrane deposition and physiological values of vessel permeability. Employing our assay, we demonstrate impaired endothelial barrier function and increased extravasation efficiency with inflammatory cytokine stimulation, as well as positive correlations between the metastatic potentials of MDA-MB-231, HT-1080, MCF-10A and their extravasation capabilities. High-resolution time-lapse microscopy reveals the highly dynamic nature of extravasation events, beginning with thin tumor cell protrusions across the endothelium followed by extrusion of the remainder of the cell body through the formation of small (~1 μm) openings in the endothelial barrier which grows in size (~8 μm) to allow for nuclear transmigration. No disruption to endothelial cell–cell junctions is discernible at 60×, or by changes in local barrier function after completion of transmigration. Tumor transendothelial migration efficiency is significantly higher in trapped cells compared to non-trapped adhered cells, and in cell clusters versus single tumor cells.National Cancer Institute (U.S.) (R33 CA174550-01)National Science Foundation (U.S.). Graduate Research FellowshipNational Science Foundation (U.S.). Science and Technology Center Emergent Behaviors of Interated Cellular Systems (EBICS) (CBET-0939511

Cell Invasion Dynamics into a Three Dimensional Extracellular Matrix Fibre Network

The dynamics of filopodia interacting with the surrounding extracellular matrix (ECM) play a key role in various cell-ECM interactions, but their mechanisms of interaction with the ECM in 3D environment remain poorly understood. Based on first principles, here we construct an individual-based, force-based computational model integrating four modules of 1) filopodia penetration dynamics; 2) intracellular mechanics of cellular and nuclear membranes, contractile actin stress fibers, and focal adhesion dynamics; 3) structural mechanics of ECM fiber networks; and 4) reaction-diffusion mass transfers of seven biochemical concentrations in related with chemotaxis, proteolysis, haptotaxis, and degradation in ECM to predict dynamic behaviors of filopodia that penetrate into a 3D ECM fiber network. The tip of each filopodium crawls along ECM fibers, tugs the surrounding fibers, and contracts or retracts depending on the strength of the binding and the ECM stiffness and pore size. This filopodium-ECM interaction is modeled as a stochastic process based on binding kinetics between integrins along the filopodial shaft and the ligands on the surrounding ECM fibers. This filopodia stochastic model is integrated into migratory dynamics of a whole cell in order to predict the cell invasion into 3D ECM in response to chemotaxis, haptotaxis, and durotaxis cues. Predicted average filopodia speed and that of the cell membrane advance agreed with experiments of 3D HUVEC migration at r[superscript 2] > 0.95 for diverse ECMs with different pore sizes and stiffness.Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology)National Science Foundation (U.S.). Science and Technology Center and Emergent Behaviors of Integrated Cellular Systems (Grant EFRI-0735997)National Science Foundation (U.S.). Science and Technology Center and Emergent Behaviors of Integrated Cellular Systems (Grant STC-0902396)National Science Foundation (U.S.). Science and Technology Center and Emergent Behaviors of Integrated Cellular Systems (Grant CBET-0939511

Engineering of In Vitro 3D Capillary Beds by Self-Directed Angiogenic Sprouting

In recent years, microfluidic systems have been used to study fundamental aspects of angiogenesis through the patterning of single-layered, linear or geometric vascular channels. In vivo, however, capillaries exist in complex, three-dimensional (3D) networks, and angiogenic sprouting occurs with a degree of unpredictability in all x,y,z planes. The ability to generate capillary beds in vitro that can support thick, biological tissues remains a key challenge to the regeneration of vital organs. Here, we report the engineering of 3D capillary beds in an in vitro microfluidic platform that is comprised of a biocompatible collagen I gel supported by a mechanical framework of alginate beads. The engineered vessels have patent lumens, form robust ~1.5 mm capillary networks across the devices, and support the perfusion of 1 µm fluorescent beads through them. In addition, the alginate beads offer a modular method to encapsulate and co-culture cells that either promote angiogenesis or require perfusion for cell viability in engineered tissue constructs. This laboratory-constructed vascular supply may be clinically significant for the engineering of capillary beds and higher order biological tissues in a scalable and modular manner.Singapore-MIT Alliance for Research and Technolog

Confidence Intervals for Concentration and Brightness from Fluorescence Fluctuation Measurements

AbstractThe theory of photon count histogram (PCH) analysis describes the distribution of fluorescence fluctuation amplitudes due to populations of fluorophores diffusing through a focused laser beam and provides a rigorous framework through which the brightnesses and concentrations of the fluorophores can be determined. In practice, however, the brightnesses and concentrations of only a few components can be identified. Brightnesses and concentrations are determined by a nonlinear least-squares fit of a theoretical model to the experimental PCH derived from a record of fluorescence intensity fluctuations. The χ2 hypersurface in the neighborhood of the optimum parameter set can have varying degrees of curvature, due to the intrinsic curvature of the model, the specific parameter values of the system under study, and the relative noise in the data. Because of this varying curvature, parameters estimated from the least-squares analysis have varying degrees of uncertainty associated with them. There are several methods for assigning confidence intervals to the parameters, but these methods have different efficacies for PCH data. Here, we evaluate several approaches to confidence interval estimation for PCH data, including asymptotic standard error, likelihood joint-confidence region, likelihood confidence intervals, skew-corrected and accelerated bootstrap (BCa), and Monte Carlo residual resampling methods. We study these with a model two-dimensional membrane system for simplicity, but the principles are applicable as well to fluorophores diffusing in three-dimensional solution. Using simulated fluorescence fluctuation data, we find the BCa method to be particularly well-suited for estimating confidence intervals in PCH analysis, and several other methods to be less so. Using the BCa method and additional simulated fluctuation data, we find that confidence intervals can be reduced dramatically for a specific non-Gaussian beam profile

Human Vascular Tissue Models Formed from Human Induced Pluripotent Stem Cell Derived Endothelial Cells

Here we describe a strategy to model blood vessel development using a well-defined induced pluripotent stem cell-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats.National Institutes of Health (U.S.) (NIH 1UH2 TR000506-01)National Institutes of Health (U.S.) (3UH2 TR000506-02S1)National Institutes of Health (U.S.) (T32 HL007936-12)National Institutes of Health (U.S.) (RO1 HL093282)National Institutes of Health (U.S.) (R21 EB016381-01

Emergent mechanical control of vascular morphogenesis

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine

Emergent mechanical control of vascular morphogenesis

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine

Characterization of Protein-Lipid Nanodomains using FCS (Fluorescence Correlation Spectroscopy)

Mentor: Guy Genin From the Washington University Undergraduate Research Digest: WUURD, Volume 4, Issue 1, Fall 2008. Published by the Office of Undergraduate Research. Henry Biggs, Director of Undergraduate Research and Associate Dean in the College of Arts & Sciences; Joy Zalis Kiefer, Undergraduate Research Coordinator, Co-editor, and Assistant Dean in the College of Arts & Sciences; Kristin Sobotka, Editor