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

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

Engineered, functional, human microvasculature in a perfusable fluidic device

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2017.Cataloged from PDF version of thesis.Includes bibliographical references (pages 141-154).Engineered, human tissue models will enable us to study disease more accurately, and develop treatments more economically, than ever before. Functional tissue grown in the laboratory will also provide a much-needed source for the clinical replacement of diseased or damaged tissues. A major hindrance to the development of these technologies has been the inability to vascularize tissue-engineered constructs, resulting in limited size and biological complexity. In this thesis, we report the development of a novel 3D fluidic platform for the generation of functional, human, microvasculature. Using different fabrication methods, we developed both a micro-fluidic system (0.1 - 1 mm tissue dimensions) - used for high throughput disease modeling assays, and a meso-fluidic system (I - 10 mm tissue dimensions) - for generating removable tissue-engineered constructs. These systems were validated by their successful use in a metastasis model - to elucidate the mechanism of cancer cell extravasation, and in the formation of a vascularized, perfusable tissue construct containing pancreatic islets, respectively. Vascularization, in our system, was achieved by encapsulating endothelial cells in a 3D fibrin matrix and relying on their inherent ability to collectively self-assemble into a functional vasculature - as they do during embryonic development. To better understand and characterize this process, we measured the morphological, functional, mechanical, and biological properties of the tissue as they emerged during vascular morphogenesis. We found that juxtacrine interactions between endothelial cells and fibroblasts enhanced the functionality and stability of the newly formed vasculature - as characterized via vascular permeability and gene expression. Under optimal co-culture conditions, the tissue stiffness increased 10- fold, mainly due to organized cellular contraction. Additionally, over the course of 2-weeks, the cells deposited over 50 new extracellular matrix (ECM) proteins, accounting for roughly 1/3 of the total ECM. These results shed light on the mechanisms underlying vascular morphogenesis and will be useful in further developing vascularization strategies for tissue engineering and regenerative medicine applications. Key words: Tissue Engineering, Vascularization, Microfluidics, In Vitro Model.by Jordan Ari Whisler.Ph. D

On-chip human microvasculature assay for visualization and quantification of tumor cell extravasation dynamics

Distant metastasis, which results in >90% of cancer-related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. This requires the completion of a sequence of complex steps including transit, initial arrest, extravasation, survival and proliferation. Increased understanding of the cellular and molecular players enabling each of these steps is key to uncovering new opportunities for therapeutic intervention during early metastatic dissemination. As a protocol extension, this article describes an adaptation to our existing protocol describing a microfluidic platform that offers additional applications. This protocol describes an in vitro model of the human microcirculation with the potential to recapitulate discrete steps of early metastatic seeding, including arrest, transendothelial migration and early micrometastases formation. The microdevice features self-organized human microvascular networks formed over 4-5 d, after which the tumor can be perfused and extravasation events are easily tracked over 72 h via standard confocal microscopy. Contrary to most in vivo and in vitro extravasation assays, robust and rapid scoring of extravascular cells, combined with high-resolution imaging, can be easily achieved because of the confinement of the vascular network to one plane close to the surface of the device. This renders extravascular cells clearly distinct and allows tumor cells of interest to be identified quickly as compared with those in thick tissues. The ability to generate large numbers of devices (∼36) per experiment further allows for highly parametric studies, which are required when testing multiple genetic or pharmacological perturbations. This is coupled with the capability for live tracking of single-cell extravasation events, allowing both tumor and endothelial morphological dynamics to be observed in high detail with a moderate number of data points

Generation of 3D functional microvascular networks with human mesenchymal stem cells in microfluidic systems

The generation of functional microvascular networks is critical for the development of advanced in vitro models to replicate pathophysiological conditions. Mural cells provide structural support to blood vessels and secrete biomolecules contributing to vessel stability and functionality. We investigated the role played by two endothelium-related molecules, angiopoietin (Ang-1) and transforming growth factor (TGF-β1), on bone marrow-derived human mesenchymal stem cell (BM-hMSC) phenotypic transition toward a mural cell lineage, both in monoculture and in direct contact with human endothelial cells (ECs), within 3D fibrin gels in microfluidic devices. We demonstrated that the effect of these molecules is dependent on direct heterotypic cell–cell contact. Moreover, we found a significant increase in the amount of α-smooth muscle actin in microvascular networks with added VEGF and TGF-β1 or VEGF and Ang-1 compared to networks with added VEGF alone. However, the addition of TGF-β1 generated a non-interconnected microvasculature, while Ang-1 promoted functional networks, confirmed by microsphere perfusion and permeability measurements. The presence of mural cell-like BM-hMSCs coupled with the addition of Ang-1 increased the number of network branches and reduced mean vessel diameter compared to EC only vasculature. This system has promising applications in the development of advanced in vitro models to study complex biological phenomena involving functional and perfusable microvascular networks.National Cancer Institute (U.S.) (R33 CA174550-01)National Cancer Institute (U.S.) (R21 CA140096)Italian Ministry of HealthRepligen Corporation (Fellowship in Cancer Research)Charles Stark Draper Laboratory (Fellowship