No description available.

No description available.

No description available.

The small intestine represents a strong barrier separating the lumen from blood circulation thereby playing a major role in the absorption and the transport of pharmacological agents prior to their arrival on the respective target site. In order to gain more knowledge about specialized uptake mechanisms and risk assessment for the patient after oral admission of drugs, intestinal in vitro models demonstrating a close similarity to the in vivo situation are needed. In the past, cell line-based in vitro models composed of Caco-2 cells cultured on synthetic cell carriers represented the “gold standard” in the field of intestinal tissue engineering. Expressive advantages of these models are a reproducible, cost-efficient and standardized model set up, but cell function can be negatively influenced by the low porosity or unwanted molecular adhesion effects of the artificial scaffold material. Natural extracellular matrices (ECM) such as the porcine decellularized small intestinal submucosa (SIS) are used as alternative to overcome some common drawbacks; however, the fabrication of these scaffolds is time- and cost-intensive, less well standardized and the 3Rs (replacement, reduction, refinement) principle is not entirely fulfilled. Nowadays, biopolymer-based scaffolds such as the bacterial nanocellulose (BNC) suggest an interesting option of novel intestinal tissue engineered models, as the BNC shows comparable features to the native ECM regarding fiber arrangement and hydrophilic properties. Furthermore, the BNC is of non-animal origin and the manufacturing process is faster as well as well standardized at low costs. In this context, the first part of this thesis analyzed the BNC as alternative scaffold to derive standardized and functional organ models in vitro. Therefore, Caco-2 cells were cultured on two versions of BNC with respect to their surface topography, the unmodified BNC as rather smooth surface and the surface-structured BNC presenting an aligned fiber arrangement. As controls, Caco-2 in vitro models were set up on PET and SIS matrices. In this study, the BNC-based models demonstrated organ-specific properties comprising typical cellular morphologies, a characteristic tight junction protein expression profile, representative ultrastructural features and the formation of a tight epithelial barrier together with a corresponding transport activity. In summary, these results validated the high quality of the BNC-based Caco-2 models under cost-efficient conditions and their suitability for pre-clinical research purposes. However, the full functional diversity of the human intestine cannot be presented by Caco-2 cells due to their tumorigenic background and their exclusive representation of mature enterocytes. Next to the scaffold used for the setup of in vitro models, the cellular unit mainly drives functional performance, which demonstrates the crucial importance of mimicking the cellular diversity of the small intestine in vitro. In this context, intestinal primary organoids are of high interest, as they show a close similarity to the native epithelium regarding their cellular diversity comprising enterocytes, goblet cells, enteroendocrine cells, paneth cells, transit amplifying cells and stem cells. In general, such primary organoids grow in a 3D Matrigel® based environment and a medium formulation supplemented with a variety of growth factors to maintain stemness, to inhibit differentiation and to stimulate cell migration supporting long term in vitro culture. Intestinal primary spheroid/organoid cultures were set up as Transwell®-like models on both BNC variants, which resulted in a fragmentary cell layer and thereby unfavorable properties of these scaffold materials under the applied circumstances. As the BNC manufacturing process is highly flexible, surface properties could be adapted in future studies to enable a good cell adherence and barrier formation for primary intestinal cells, too. However, the application of these organoid cultures in pre-clinical research represents an enormous challenge, as the in vitro culture is complex and additionally time- and cost-intensive. With regard to the high potential of primary intestinal spheroids/organoids and the necessity of a simplified but predictive model in pre-clinical research purposes, the second part of this thesis addressed the establishment of a primary-derived immortalized intestinal cell line, which enables a standardized and cost-efficient culture (including in 2D), while maintaining the cellular diversity of the organoid in vitro cultures. In this study, immortalization of murine and human intestinal primary organoids was induced by ectopic expression of a 10- (murine) or 12 component (human) pool of genes regulating stemness and the cell cycle, which was performed in cooperation with the InSCREENeX GmbH in a 2D- and 3D-based transduction strategy. In first line, the established cell lines (cell clones) were investigated for their cell culture prerequisites to grow under simplified and cost-efficient conditions. While murine cell clones grew on uncoated plastic in a medium formulation supplemented with EGF, Noggin, Y-27632 and 10% FCS, the human cell clones demonstrated the necessity of a Col I pre coating together with the need for a medium composition commonly used for primary human spheroid/organoid cultures. Furthermore, the preceding analyses resulted in only one human cell clone and three murine cell clones for ongoing characterization. Studies regarding the proliferative properties and the specific gene as well as protein expression profile of the remaining cell clones have shown, that it is likely that transient amplifying cells (TACs) were immortalized instead of the differentiated cell types localized in primary organoids, as 2D, 3D or Transwell®-based cultures resulted in slightly different gene expression profiles and in a dramatically reduced mRNA transcript level for the analyzed marker genes representative for the differentiated cell types of the native epithelium. Further, 3D cultures demonstrated the formation of spheroid-like structures; however without forming organoid-like structures due to prolonged culture, indicating that these cell populations have lost their ability to differentiate into specific intestinal cell types. The Transwell®-based models set up of each clone exhibit organ-specific properties comprising an epithelial-like morphology, a characteristic protein expression profile with an apical mucus-layer covering the villin-1 positive cell layer, thereby representing goblet cells and enterocytes, together with representative tight junction complexes indicating an integer epithelial barrier. The proof of a functional as well as tight epithelial barrier in TEER measurements and in vivo-like transport activities qualified the established cell clones as alternative cell sources for tissue engineered models representing the small intestine to some extent. Additionally, the easy handling and cell expansion under more cost-efficient conditions compared to primary organoid cultures favors the use of these newly generated cell clones in bioavailability studies. Altogether, this work demonstrated new components, structural and cellular, for the establishment of alternative in vitro models of the small intestinal epithelium, which could be used in pre-clinical screenings for reproducible drug delivery studies. / Der Dünndarm bildet eine starke Barriere aus, welche das Lumen vom Blutkreislauf trennt, und dadurch maßgeblich an der Absorption und dem Transport von pharmakologischen Wirkstoffen beteiligt ist, bevor diese ihren Wirkort erreichen. Um ein detaillierteres Wissen über die speziellen Aufnahmemechanismen zu erlangen und zur Risikoabschätzung für den Patienten nach oraler Aufnahme dieser Medikamente, sind intestinale in vitro Modelle erforderlich, die eine große Ähnlichkeit mit der Situation in vivo aufweisen. In der Vergangenheit stellten Caco-2 Zelllinien-basierte in vitro Modelle, die auf synthetischen Trägerstrukturen aufgebaut sind, den „Goldstandard“ auf dem Gebiet der intestinalen Geweberekonstruktion dar. Bedeutende Vorteile dieser Modelle sind der reproduzierbare, kosteneffiziente und standardisierte Modellaufbau, jedoch können die zellulären Funktionen durch die geringe Porosität oder die unerwünschten molekularen Adhäsionseffekte des künstlichen Trägermaterials negativ beeinflusst werden. Um einige häufige Nachteile zu überwinden werden natürliche extrazelluläre Matrizen (ECM) wie die porzine dezellularisierte Dünndarm-submukosa (SIS) verwendet, jedoch ist die Herstellung dieser Trägerstrukturen zeit- und kostenintensiv, weniger gut standardisiert und entspricht nicht ganzheitlich dem 3R-Prinzip (Replace = Vermeiden, Reduce = Verringern, Refine = Verbessern). Heutzutage ermöglichen biopolymer-basierte Trägerstrukturen wie die bakterielle Nanozellulose (BNC) die Entwicklung von neuartigen intestinalen Gewebemodellen, da die BNC eine große Ähnlichkeit hinsichtlich der Faseranordnung und der hydrophilen Eigenschaften mit der nativen ECM aufweist. Darüber hinaus ist die BNC nicht tierischen Ursprungs und der Herstellungsprozess schneller, gut standardisiert als auch kostengünstig. In diesem Zusammenhang wurde im ersten Teil dieser Arbeit nachgewiesen, dass die BNC als alternative Trägerstruktur für standardisierte und funktionelle Organmodelle in vitro geeignet ist. Dafür wurden Caco-2 Zellen auf zwei Varianten der BNC kultiviert, die sich in ihrer Oberflächentopographie unterscheiden, wobei die nicht-modifizierte BNC eine glatte Oberfläche und die oberflächen-strukturierte BNC eine ausgerichtete Faseranordnung aufweist. Als Kontrollen dienten Caco 2 zellbasierte in vitro Modelle, die auf PET- oder SIS Matrizes aufgebaut wurden. In dieser Studie wiesen die BNC-basierten Modelle die wichtigsten organ-spezifischen Eigenschaften auf, darunter eine typische zelluläre Morphologie, ein charakteristisches Expressionsprofil der Tight Junction Proteine, repräsentative ultrastrukturelle Merkmale und die Bildung einer dichten epithelialen Barriere verbunden mit einer entsprechenden Transportaktivität. Zusammenfassend bestätigten diese Ergebnisse die hohe Qualität der BNC-basierten Caco-2 Modelle unter kosteneffizienten Herstellbedingungen und ihre Eignung für präklinische Forschungszwecke. Allerdings kann die volle Funktionsvielfalt des menschlichen Darms durch Caco-2 Zellen aufgrund ihres kanzerogenen Ursprungs und der exklusiven Repräsentanz von Enterozyten nicht abgebildet werden. Neben der Trägerstruktur die für den Aufbau der in vitro Modelle verwendet wird, trägt auch die zelluläre Einheit zur Etablierung von funktionalen Modellen bei, weshalb es von großer Bedeutung ist, die zelluläre Vielfalt des Dünndarms in diesen Modellen in vitro nachzuahmen. In diesem Zusammenhang sind die primären intestinalen Organoide, die sich hauptsächlich aus Enterozyten, Becherzellen, enteroendokrinen Zellen, Paneth Zellen, Vorläuferzellen und Stammzellen zusammensetzen, von großem Interesse, da die zelluläre Komponente eine große Ähnlichkeit zum nativen Epithel aufweist. Derartige primäre Organoide werden üblicherweise in einer 3D-Matrigel® Umgebung und einer speziellen Formulierung des Mediums, die mit einer Vielzahl an Wachstumsfaktoren ergänzt wird, um das Stammzellpotenzial zu erhalten, die Differenzierung zu hemmen, die Zellmigration zu stimulieren und somit eine langfristige in vitro-Kultivierung zu unterstützt. Intestinale primäre Sphäroid-/Organoidkulturen wurden auf beiden BNC Varianten als Transwell®-ähnliche Modelle aufgebaut. Dabei zeigte sich eine fragmentierte Zellschicht was darauf schließen lässt, dass die Matrix unter diesen Bedingungen für den Modellaufbau ungeeignet ist. Da der BNC-Herstellungsprozess sehr flexibel ist, könnten die Oberflächen-eigenschaften in zukünftigen Studien angepasst werden, um so eine gute Zelladhäsion auch für primäre Darmzellen zu ermöglichen. Die Anwendung dieser Organoid-basierten Kulturen stellt jedoch für die präklinische Forschung eine enorme Herausforderung dar, da die Kultivierung komplex und zudem sehr zeit- und kosten-intensiv ist. Im Hinblick auf das hohe Potenzial der primären intestinalen Sphäroide/Organoide und der Notwendigkeit eines vereinfachten aber prädiktiven Modells für präklinische Forschungs-zwecke, befasste sich der zweite Teil der Arbeit mit der Etablierung einer primären immortalisierten intestinalen Zelllinie, die eine standardisierte und kosteneffiziente Kultur ermöglicht, wobei die zelluläre Vielfalt der in vitro Organoid-Kulturen erhalten bleibt. In dieser Studie wurden primäre Organoide aus dem murinen und dem menschlichen Dünndarm durch die ektopische Expression eines 10- (murin) bzw. 12 Komponenten (human) Pools von Genen, welche im Hinblick auf die Regulation der Stammzellen und dem Zellzyklus bekannt sind, in Zusammenarbeit mit der InSCREENeX GmbH in einer 2D- und 3D-basierten Transduktionsstrategie immortalisiert. In erster Linie wurden die etablierten Zelllinien (Zellklone) auf ihren Bedarf an Wachstumsfaktoren für die Kultivierung unter vereinfachten und kosteneffizienten Bedingungen hin untersucht. Während die murinen Zellklone auf unbeschichteten Kunststoff in einer Mediumformulierung mit hEGF, mNoggin, Y-27632 und 10% FCS wuchsen, zeigten die humanen Zellklone eine Notwendigkeit für eine Col I-Vorbeschichtung zusammen mit einer Zusammensetzung des Mediums, wie sie üblicherweise für primäre humane Sphäroide/Organoide verwendet wird. Darüber hinaus führten diese vorangegangenen Analysen dazu, dass nur ein humaner Zellklon und drei murine Zellklone umfänglich charakterisiert wurden. Studien zu proliferativen Eigenschaften und spezifischen Gen- sowie Proteinexpressionsprofilen dieser Klone haben gezeigt, dass vermutlich Vorläuferzellen (TACs) anstelle der differenzierten Zelltypen der primären Organoide immortalisiert wurden, da die Kultivierung in 2D, 3D oder in Transwell®-basierten Modellen zu einem geringfügig veränderten Genexpressionsprofil im Vergleich untereinander und zudem zu einem stark reduzierten mRNA-Transkriptionswert für die analysierten Markergene, welche die differenzierten Zelltypen des nativen Epithels repräsentieren, die Folge war. Weiterhin zeigte die 3D-Kultivierung die Bildung von Sphäroid-ähnlichen Strukturen, jedoch keine Organoid-ähnlichen Strukturen unter verlängerten Kultur-bedingungen, was darauf hinweist, dass diese Zellpopulationen ihre Eigenschaft zur Differenzierung hin zu spezifischen intestinalen Zelltypen eingebüßt haben. Die Transwell®-basierten Modelle, welche für jeden Klon etabliert wurden, weisen zudem Organ-spezifische Eigenschaften auf, wie eine epitheliale Morphologie, ein charakteristisches Protein-expressionsprofil mit einer apikalen Schleimschicht, welche den Villin-1 positiven Zelllayer bedeckt und somit den Nachweis erbringt, dass die entstandenen immortalisierten Zellpopulationen zu einem gewissen Anteil aus Becherzellen und Enterozyten bestehen. Zudem konnten repräsentative Tight-Junction Komplexe, die auf eine dichte epitheliale Barriere hinweisen, in entsprechenden Proteinexpressionsprofilanalysen nachgewiesen werden. Der Nachweis einer sowohl dichten als auch funktionellen epithelialen Barriere konnte weitergehend durch TEER-Messungen und in vivo-ähnliche Transportmechanismen für die etablierten Zellklone qualifiziert werden, wodurch diese Zellen als alternative Zellquelle für in vitro Modelle des Dünndarms verwendet werden können. Darüber hinaus begünstigt die einfache Handhabung und Zellexpansion unter kostengünstigeren Bedingungen im Vergleich zu primären Organoidkulturen den Einsatz dieser neu-generierten Zellklone für Bioverfügbarkeits-Studien. Zusammenfassend zeigte diese Arbeit neue Komponenten, strukturelle und zelluläre, für die Etablierung alternativer in vitro-Modelle des Dünndarmepithels, die in präklinischen Screenings für reproduzierbare Studien hinsichtlich der Medikamententestung verwendet werden können.

Cardiovascular diseases (CVD) are the leading cause of death around the world, being responsible for 31.8% of all deaths in 2017. The leading cause of CVD is Ischemic heart disease (IHD), which caused 8.1 million deaths in 2013. IHD occurs when coronary arteries in the heart are narrowed or blocked, preventing the flow of oxygen and blood into the cardiac muscle, which could provoke acute myocardial infarction (AMI) and ultimately lead to heart failure and death. Cardiac regenerative therapy aims to repair and refunctionalize damaged heart tissue through the application of (1) intramyocardial cell delivery, (2) epicardial cardiac patch, and (3) acellular biomaterials. These approaches have provided benefit of cell localization and tissue structure respectively. However, to improve cell retention and integration, there is a need for the intramyocardial delivery of functional tissues while preserving anisotropic muscle alignment. Here, we developed a biodegradable z-wire scaffold that supports the scalable gel-free production of an array of functional cardiac tissues in a 384-well plate format. The z-wire scaffold design supports cellular alignment, provides tunable mechanical support, and allows for hallmark tissue contraction. When the scaffold is imparted with magnetic properties, individual tissues can be assembled with macroscopic alignment under magnetic guidance. When used in combination with a customized surgical delivery tool, z-wire tissues can be injected directly into the myocardial wall, with controlled tissue orientation according to the injection path. This modular tissue engineering approach, in combination with the use of smart scaffolds, could expand opportunity in functional tissue delivery. / Thesis / Master of Science in Chemical Engineering (MSChE)

Osteoarthritis is the most prevalent musculoskeletal disease in humans, severely reducing the standard of living of millions of people. Osteoarthritis is characterized by degeneration and loss of articular cartilage which leads to pain, and loss of joint motility and function. Individuals suffering from severe osteoarthritis are commonly treated with full knee replacements. The procedure does eliminate the problem of degrading cartilage tissue; however, it does not fully restore function and its lifetime can be limited. To overcome the disadvantages of current treatments, tissue engineering has become a focus of research to regenerate cartilage. Tissue engineering attempts to repair or replace damaged tissue with cells, biomaterials, and/or molecular signals. Biodegradable scaffolds serve as a temporary replacement for the tissue until it has regenerated. Two types of scaffolds that have been used in tissue engineering are electrospun scaffolds and hydrogels. We have proposed and fabricated a scaffold for cartilage tissue engineering that incorporates an electrospun cylinder and a thermosetting hydrogel in order to provide improved properties compared to either individual material. Electrospun cylinders were created by sintering electrospun mats that include salt pores. The addition of salt pores decreased the mechanical properties of the electrospun materials while also improving the capability of cells to infiltrate into the scaffold. The sintering process involved the connecting of one electrospun mat to an adjacent one. Specifically, poly(d,l-lactide) was capable of sintering to an adjacent electrospun mat when exposed to either heat (near the glass transition temperature) or tetrahydrofuran vapor. The sintering process did not deteriorate the structure or function of the electrospun material. Sintering allowed the creation of unique structures of electrospun material that previously could not be produced. A thermosetting hydrogel was added to the scaffold to replicate the function of proteoglycans present in articular cartilage. A composite scaffold of electrospun polymer and hydrogel showed improved mechanical properties and better integration of the scaffold in vivo compared to an electrospun scaffold with no hydrogel. In conclusion, the composite electrospun and hydrogel scaffold could become an excellent tissue engineering scaffold to treat patients suffering from osteoarthritis. / Ph. D.

In the field of tissue engineering, a major area of study is developing bone scaffolds that will provide support for osteoblasts. Despite many advances in recent years there is still a significant need for new bio-based 3-D porous scaffolds that possess sufficient initial mechanical properties to prevent immediate failure upon implantation. Ovalbumin (OVA), a glycoprotein from chicken egg whites, has been use to fabricate biodegradable, porous hydrogel bone scaffolds that promote osteoblast attachment and proliferation. Although ovalbumin scaffolds encourage bioactivity and are naturally resorbed into the body after bone regeneration, they are also very fragile. Extremely stiff cellulose nanocrystals (CNCs), derived from wood pulp, can be utilized to reinforce these scaffolds while improving biocompatibility. When chemically modified to incorporate surface amine groups, cellulose nanocrystals become capable of covalently crosslinking with the OVA matrix for improved mechanical resilience. Three concentrations (2, 5, 10 wt. %) of CNCs were incorporated and crosslinked to form nanocomposite scaffolds then were compared to pure OVA scaffolds. After fabrication, pore size morphology was compared between each CNC loading using SEM. The images revealed that the 10 wt. % CNC concentration doubled the pore compared to pure OVA scaffolds. Under high magnification, the CNCs were incorporated into the pore walls, providing a contoured surface. AFM was applied to analyze the topography of OVA with CNCs present. The surfaces laden with CNCs had a higher mean surface roughness, but was insufficient to impact cell behavior. Compression testing was carried out on both Instron and DMA machines to demonstrate any reinforcing effect provided by the CNCs. While the compressive modulus remained constant, the elastic limit and strain increased with CNC loading, indicating a change in the resilience of the reinforced scaffolds. With a MTT Assay, it was shown that MC3T3-E1 preosteoblasts significantly increase in metabolic activity on 2 wt. % films and scaffolds, an indication of proliferation. All scaffolds had a net increase in metabolic activity suggesting overall biocompatibility for OVA scaffolds and those incorporating CNCs. Overall, the 5 wt. % scaffolds had the highest mechanical strength and had a positive cell response. / Master of Science

Skeletal loss and bone deficiencies are major worldwide problem that is only expected to increase due to increase in aging population. As current standards in treatment autografts and allografts are not without drawbacks, there is a need for alternative bone grafts substitutes. The goal of this project was to utilize electrospinning and heat sintering techniques to create biodegradable full thickness three dimensional biomimetic polymeric scaffolds with macro and nano architecture similar to natural bone for bone tissue engineering. First we have investigated pretreatment with 0.1M NaOH and electrospinning gelatin/PLLA blends as means to increase overall mineral precipitation and distribution throughout the scaffolds when incubated in concentrated simulated body fluid (SBF)10XSBF. Mixture of 10% gelatin and PLLA resulted in the significantly higher degree of mineralization, increased mechanical properties, and scaffolds that supported cellular adhesion and proliferation. In the next step we applied heat sintering technique to fabricate 3D electrospun scaffolds that were used to evaluate effects of mineralization and fiber orientation on scaffold strength. Fiber orientation can make a slight difference in nanofibrous scaffold compressive mechanical properties, but this difference is not as profound as the difference seen with increased mineralization. We also developed a technique to fabricate scaffolds that mimic the organization of an osteon, the structural unit of cortical bone. Resulting scaffolds consisted of concentric layers of electrospun gelatin/PLLA nanofibers wrapped around microfiber core with diameters that ranged from 200-600µm. Individual osteon-like scaffolds were heat sintered to fabricate three dimensional scaffolds contained a system of channels running parallel to the length of the scaffolds, as found naturally in bone tissue. Finally we combined two previously fabricated structures, sintered electrospun sheets and individual osteon-like scaffolds, to create novel scaffolds that mimic dual structural organization of natural bone with cortical and trabecular regions. Mineralization for 24 hr significantly increased mechanical properties of the scaffolds, both yield stress and compressive modulus under physiological conditions. Both nonminerlized and mineralized scaffolds were found to support cellular attachment and proliferation over 28 days in culture, but scaffolds mineralized for 24hr were found to better support osteoblastic differentiation and mineral deposition. / Ph. D.

The vascular system, responsible for the transport of nutrients, oxygen, and waste removal, overcomes the limitations of oxygen diffusion in solid tissues through blood perfusion, thereby preventing necrosis. The mechanical stimuli from blood flow are pivotal for vascular development and engineering, influencing endothelial cell morphology and vessel remodeling via mechanosensing. Current organ-on-chip systems, while successful in applying dynamic flow to endothelial cells, have limitations, including dependency on pumps and confinement to closed microfluidic channels. Additionally, the interaction between immune cells and these systems under long-term recirculating flow conditions has not been adequately demonstrated. This thesis introduces a novel biofabrication and device manufacturing technique that utilizes a flexible, patternable sacrificial material on a 2D surface. This material morphs in response to an aqueous hydrogel and then degrades, forming perfusable vascular networks within a natural hydrogel matrix. We achieved perfusion using a rocker mechanism that periodically changes tilt direction, while the open-well design facilitates the visualization of perfusable tubular tissues via clinical ultrasound imaging and the construction of complex, vascularized hepatic tissues embedded in gel matrices (Chapter 2). To mimic the unidirectional recirculating flow characteristic of blood vessels, we created the UniPlate platform, combining injection molding with 3D printing (Chapter 3). This innovation allows for the perfusion and recirculation of monocytes through vascular channels without compromising cell viability or eliciting an inflammatory response. Furthermore, by integrating cancer spheroids into the vascular tissues on UniPlate, we developed a vascularized cancer spheroid model that exhibited temporally dependent and tissue-specific macrophage recruitment toward tumor sites with continuous monocyte recirculation (Chapter 4). Collectively, this series of research work introduces a versatile and robust platform capable of replicating vascular functions and immune responses, offering a substantial advancement in the investigation of vascular biology and the mechanism of disease progression. / Thesis / Doctor of Philosophy (PhD) / Vascular networks of the circulatory system are crucial organs in the body, determining the life and death of tissues and organisms by distributing nutrients and oxygen throughout the body. Dysfunction in blood vessel systems is closely related to clinical diseases such as stroke, atherosclerosis, tumor angiogenesis, and cancer metastasis. Mechanical stimuli in blood vessels play a crucial role in regulating the structure and function of endothelial cells during in vivo embryonic development and in vitro vascular tissue formation. Understanding and mimicking the complex environment of blood vessels is vital for studying diseases related to endothelial dysfunction. In this thesis, we introduce a novel subtractive manufacturing method to create three-dimensional (3D) perfusable tubular tissues within a hydrogel. Unidirectional recirculating flow, stromal cells and spheroids, as well as circulating immune cells, were then introduced to the engineered vascular tissues to develop more complex tissue models. These models reproduce the cell diversity, 3D structure, mechanical stimuli, and immune components found in the native tissue microenvironment, providing a valuable tool for the study of vascular diseases and the development of potential treatments.

The structural and functional abnormalities of the tumor vasculature generate regions of elevated interstitial fluid pressure and aberrant flow shear stress within the tumor microenvironment. While research has shown that the hydrodynamics of the tumor vasculature reduce transport and uptake of therapeutic agents, the underlying mechanisms by which fluid forces regulate vascular organization are not well known. Understanding the reciprocal interaction between tumor and endothelial cells to mediate angiogenesis, and the role of flow shear stress on this process, may offer insight into the design of improved therapeutic strategies to control vascularized tumors. Instrumental to this is the development of physiologically relevant models that enable tumor-endothelial co-culture under dynamic conditions. By integrating tissue-engineering strategies with cancer biology, micro-scale fluid mechanics, and optical flow diagnostics, the goal of this research was to develop a 3D in vitro microfluidic culture model to investigate tumor-endothelial cross talk under physiologically relevant flow shear stress. This objective was motivated by early findings demonstrating a contact-independent, paracrine-mediated mechanism by which endothelial cells enhance tumor-expressed angiogenic factors during 2D, static co-culture. The 3D tumor vascular model consists of a central microchannel embedded within a type I collagen hydrogel, through which a range of normal (4 dyn/cm^2), low (1 dyn/cm^2) and high (10 dyn/cm^2) microvascular wall shear stresses (WSS) were introduced. Endothelial cells lining the microchannel lumen form a confluent endothelium across which soluble growth factors are exchanged with tumor cells in the gel. Microscopic particle image velocimetry ("-PIV) was integrated within the model to enable noninvasive optical measurement of velocity profiles and quantification of WSS, which were then correlated with angiogenic potential. Results demonstrate that endothelial permeability decreases as a function of increasing WSS, while co-culture with tumor cells increases permeability. This response is likely due to shear stress-mediated endothelial cell alignment and tumor-VEGF-induced permeability. In addition, high WSS (10 dyn/cm^2) significantly down-regulates tumor-expressed angiogenic factors, suggesting flow shear stress-mediates endothelial cross talk with surrounding tumor cells. Collectively, this research demonstrates the utility of the 3D in vitro microfluidic culture model as a versatile platform for elucidating the role of tumor-relevant hydrodynamic stress on cellular response. / Ph. D.