Digital microfluidics (DMF) has emerged in the past decade as a novel microfluidic paradigm. As a liquid handling technology, DMF facilitates the electrostatic manipulation of discrete nano- and micro- litre droplets across open electrode arrays providing the advantages of single sample addressability, automation, and parallelization. This thesis presents DMF advances toward improved functionality and compatibility for automated miniaturized cell culture in two and three dimensions. Through the development and integration of surface patterning techniques we demonstrate a virtual microwell method for high precision on-device reagent dispensing in one and two plate DMF geometries. These methods are shown to be compatible with two-dimensional culture of immortalized cell lines on ITO, primary cells on coated surfaces, and for co-culture assays. We further extrapolate this method for the formation of microgels on-demand where form micro scale hydrogel structures through passive dispensing in a wide array of geometries. With this system we interrogate three-dimensional cell culture models, specifically for the recapitulation of kidney epthelialization and the analysis of functional cardiac microgels.

Digital microfluidics (DMF) has emerged in the past decade as a novel microfluidic paradigm. As a liquid handling technology, DMF facilitates the electrostatic manipulation of discrete nano- and micro- litre droplets across open electrode arrays providing the advantages of single sample addressability, automation, and parallelization. This thesis presents DMF advances toward improved functionality and compatibility for automated miniaturized cell culture in two and three dimensions. Through the development and integration of surface patterning techniques we demonstrate a virtual microwell method for high precision on-device reagent dispensing in one and two plate DMF geometries. These methods are shown to be compatible with two-dimensional culture of immortalized cell lines on ITO, primary cells on coated surfaces, and for co-culture assays. We further extrapolate this method for the formation of microgels on-demand where form micro scale hydrogel structures through passive dispensing in a wide array of geometries. With this system we interrogate three-dimensional cell culture models, specifically for the recapitulation of kidney epthelialization and the analysis of functional cardiac microgels.

Although asthma is primarily thought to be an inflammatory disease of the airways, it has recently been hypothesized that the altered mechanical environment of an asthmatic airway may contribute to the development of the disease through changes in cellular phenotype. In regards to this hypothesis, the effects of stretch on airway smooth muscle (ASM) have previously been investigated using 2D cell culture. However, over the last few years there has been an increasing appreciation to the importance of the role of the 3D extracellular matrix in the regulation of cellular response. For this reason, the work presented in this thesis covers the development of a device capable of high-throughput investigations into the effects of acute or chronic, uniaxial, oscillatory mechanical strain on an array of miniature, 3D, multi-cell, tissue-engineered constructs.

The garlic extract ajoene is considered to have antimicrobial and antitumor effects against a variety of cell types, and it is suggested to have the potential to be used as an antifungal or antitumor drug clinically. The underlying mechanism of its inhibitory effects is still uncertain. In this project, the effects of ajoene on the growth of fungal and oomycete cells were studied on Candida albicans, Neurospora crassa and Achlya bisexualis. Endometrial cancer is the most common gynecologic cancer. A 3D spheroid model of endometrial cancer cells were for the first time used to investigate the antitumor effects of ajoene and selected antitumor agents. Ajoene was extracted from fresh garlic by chromatographic methods and the outcome of the extractions was verified with Mass spectrometry and NMR spectroscopy. Ajoene was then tested on the yeast form or germ tubes of C. albicans, and the cell division and germ tube formation was analyzed. N. crassa and A. bisexualis were treated with ajoene on plates or on glass slides to measure the hyphae radial extension or individual hyphal extension. 3D endometrial adenocarcinoma cell (Ishikawa) spheroids were treated with ajoene, paclitaxel, targeted drugs everolimus, sorafenib, gefitinib and canertinib alone or in combinations. The growth activity, metabolic activity, cell proliferation, apoptotic activity and the cytoskeletons were analyzed after the treatments. Cell division of C.albicans was inhibited by ajoene at 5µg/ml or higher concentrations. The length of C.albicans germ tubes was significantly shorter in ajoene treated groups than the untreated ones. Radial extension and individual hyphal extension of N. crassa and A. bisexualis were both inhibited by ajoene. Ajoene did not show any antitumor effects on the 3D cell model of Ishikawa cells. No synergistic effect was detected between ajoene and paclitaxel or ajoene and everolimus. The targeted drugs Canertinib and everolimus showed an inhibitory effect on growth activity of the spheroids, but no synergy with paclitaxel. In conclusion, ajoene was able to inhibit various forms of fungal and oomycete growth, but any antitumor activity of ajoene did not show on 3D culture of endometrial cancer cells.

In pharmaceutical development cell cultures are used as in vitro models to evaluate the function of drug candidates. In such research it is vital to have models that resemble the in vivo environment to get reliable results. In 3D models with hydrogels ECM like scaffolds are supporting the cells in a more in vivo like environment than flat 2D cultures. In this project PEG-peptide based hydrogels with cell binding RGD incorporated on one PEG-peptide type has been evaluated for culturing of HepG2 cells. Structure and viscoelastic properties were evaluated with techniques like circular dichroism spectroscopy, dynamic light scattering and rheology. Sterilisation impact was also evaluated for PEG-peptides. For cell culturing, observations in light microscope and evaluation with Live/Dead assay and albumin assay were performed. A few companies were interviewed regarding 3D culturing and interest in mechanically tuneable hydrogels. The HepG2 cells grows and forms spherical clusters in the 3D environment with hydrogels, percentage of RGD seems to not impact cell adhesion, growth or albumin secretion. UV irradiation was the most suitable sterilisation method for gel components. The most rigid gel combination formed had storage modulus of around 230 Pa. Mechanically tuneable hydrogels is interesting for the industry. The PEG-peptide based gels are suitable tor growing cells but too soft to closely resemble the in vivo rigidity of hepatocytes.

La Protéine Prion (PrP) est une glycoprotéine ubiquitaire attachée au feuillet externe de la membrane plasmique par une ancre glycosylphosphatidylinositole (GPI). Cette dernière est l’agent infectieux responsable de la maladie Creutzfeld-Jacob ou « maladie de la vache folle ». Cette protéine existe sous sa forme cellulaire mais également sous sa forme infectieuse, nommée PrPSc (Scrapie). Alors que la fonction de PrPSc est établie au cours de la pathogenèse, la fonction de la protéine cellulaire est beaucoup plus énigmatique notamment chez les mammifères. Il est clairement admis que la forme infectieuse découle d’un changement de conformation de la forme cellulaire. Ainsi afin de mieux appréhender le rôle de la protéine prion dans les cellules saines mais également lors de la pathogenèse il apparaît essentiel d’étudier le trafic de cette protéine. La protéine prion est exprimée dans les cellules neuronales qui sont comme les cellules épithéliales des cellules polarisées. J’ai au cours de ma thèse étudié le trafic de la protéine prion dans les cellules polarisées MDCK. MDCK est la lignée épithéliale sur laquelle nous avons la plus grande connaissance. Dans mon travail j’ai utilisé des cellules MDCK polarisées classiquement en culture bidimensionnelle (2D) mais également en culture tridimensionnelle (3D) où les cellules forment des kystes, structures hautement polarisées, physiologiquement proches de l’épithélium in vivo. Il apparaît que dans les cellules MDCK polarisées sur filtre (en 2D) la localisation de la PrP est controversée. Nous avons trouvé que, contrairement à la majorité des protéines à ancre GPI, la PrP suit la voie de transcytose. La PrP qui se retrouve à la membrane basolatérale est transcytosée vers la membrane apicale. De plus la PrP envoyée à la surface apicale est clivée (clivage alpha) générant deux fragments distincts : le fragment C1, pourvu de l’ancre GPI qui reste associé à la surface apicale et le fragment soluble N1 qui est sécrété dans le milieu de culture des cellules MDCK cultivées en 2D ou dans le lumen des cellules MDCK cultivées en 3D. Mon travail permet de mieux comprendre les études réalisées auparavant mais surtout révèle l’existence d’un mécanisme de transcytose de la protéine prion dans les cellules épithéliales. Cette information est essentielle et nous permet de supposer que ce mécanisme pourrait être également utilisé par les cellules neuronales. / The Prion Protein (PrP) is a ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of “prion diseases” the cellular isoform (PrPC) is an enigmatic protein with unclear function. Prion protein has received considerable attention due to its central role in the development of Transmissible Spongiform Encephalopathies (TSEs) known as “prion diseases”, in animals and humans. Understanding the trafficking, the processing and degradation of PrP is of fundamental importance in order to unravel the mechanism of PrPSc mediated pathogenesis, its spreading and cytotoxicity. The available data regarding PrP trafficking are contradictory. To investigate PrP trafficking and sorting we used polarized MDCK cells (two-dimensional and tree-dimensional cultures) where the intracellular traffic of GPI-anchored proteins (GPI-APs) is well characterized. GPI-APs that are sorted in the Trans Golgi Network follow a direct route from the Golgi apparatus to the apical plasma membrane. The exception to direct apical sorting of native GPI-APs in MDCK cells is represented by the Prion Protein. Of interest, PrP localization in polarized MDCK cells is highly controversial and its mechanism of trafficking is not clear. We found that full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures and that the C1/PrP full-length ratio increases upon MDCK polarization. We revealed that differently from other GPI-APs, PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells and is α-cleaved during its transport to the apical surface. This study not only reconciles and explains the different findings in the previous literature but also provides a better picture of PrP trafficking and processing, which has been shown to have major implications for its role in prion disease.

Controlled cell assembly is essential for fabricating in vitro models that mimic native tissue architecture. Conventional tissue engineering techniques are time-consuming and offer limited control over the spatial organization of cells within the assembled cell aggregates. We describe a label-free, scaffold-free, rapid cell manipulation technique to assemble cells into layered aggregates. Suspensions of cells in a culture medium with higher magnetic susceptibility are seeded into wells of a 96-well plate placed on a quartet magnet array. An FDA-approved paramagnetic agent is added to the regular cell culture medium to enhance the magnetic susceptibility. The inhomogeneous magnetic field and the susceptibility difference drive cells toward the lowest magnetic field region on the well surface. Two cell types are sequentially added to the wells to form layer-on-layer aggregates within 6 h. Next, the label-free technique is extended to develop a cell migration assay. Besides being time-consuming, the traditional scratch-based cell migration assay is not reproducible, whereas the alternate physical barrier-based method is expensive. Annular aggregates of human bronchial epithelial cells (HBEC3 KT) are formed within 3 h using a coaxially arranged ring-cylinder magnet array. The effects of the paramagnetic agent on cell viability, metabolism, and transcriptional profiles are investigated. The closures of the circular cell-free areas enclosed by HBEC3 KT are analyzed at different times in response to various signaling molecules and surface conditions. Further, we demonstrate the formation of the annular aggregates on human lung fibroblast-laden collagen hydrogel surfaces. The cell-free area closures on hydrogel surfaces in response to signaling molecules are analyzed. The high reproducibility and scalability of the label-free method make it amenable for preclinical research. / Thesis / Doctor of Philosophy (PhD) / Cell cultures are essential tools for studying cell functions under controlled conditions. A better understanding of cell behavior in tissues is required to develop effective treatments for diseases. The organized arrangement of cells in tissues controls tissue functions. The existing culture techniques are time-consuming and have limited control over the cellular arrangement. We describe a simple, rapid, and inexpensive bioprinting technique to arrange cells in layers, which resembles the cellular organization in tissues, such as the skin. The layered structures are formed in standard well plates within 6 h. Cell movement is an essential cell function in various biological processes, such as wound healing. Using the bioprinting method, we form ring-shaped cellular structures within 3 h to study cell movements in response to various signals. The ring structures enclose cell-free areas, which are populated over time as the cells move from the ring into the cell-free regions. The bioprinting method is easy to use and can rapidly form organized cellular structures for drug testing.

To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

No description available.

In this work, a hydrogel made from hyaluronic acid is synthesized and utilized to study neural stem cell behavior within a custom tailored three dimensional microenvironment. The physical properties of the hydrogel have been optimized to create an environment conducive for neural stem cell differentiation by mimicking the native brain extracellular matrix (ECM) environment. The physical properties characterized include degree of methacrylation, swelling ratios, enzymatic degradation rates, and viscoelastic moduli. One dimensional proton nuclear magnetic resonance (1HNMR) confirms modification of the hyaluronic acid polymers, and is used to quantify the degree of methacrylation. Rheological measurements are made to quantify the viscoelastic moduli. Further post-processing by lyophilization leads to generation of large voids to facilitate re-swelling and cell infiltration. ReNcell VM (RVM), and adult human neural stem cell line derived from the ventral mesencephalon, are cultured and differentiated inside the hydrogel for up to 2 weeks. Differentiation is characterized by immunocytochemistry (ICC) and real time quantitative polymerase chain reaction (qRT-PCR). / Bioengineering