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京都大学 / 新制・課程博士 / 博士(工学) / 甲第23885号 / 工博第4972号 / 新制||工||1776(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 横川 隆司, 教授 安達 泰治, 教授 井上 康博 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Therapeutic viruses are a class of biotherapeutic which have enabled new treatments and medical advances in the areas of vaccines, cancer treatment, gene therapy, and more. In the production and purification of these products, the sterile filtration unit operation is known to have poor yields and contribute to the high cost of the final product, significantly hampering the large-scale production of some therapeutic viruses. Thus, this thesis seeks to explore various aspects of process development and fundamental understanding in the sterile filtration of therapeutic viruses. This thesis explores the mechanisms and membrane properties which govern how bacteria are retained during filtration, and applies these insights to improve the sterile filtration recovery of a therapeutic virus through proper membrane selection. To better understand the factors which cause membrane fouling and loss of virus during sterile filtration, the effect of host cell impurities on filtration performance was investigated. This revealed that small amounts of host cell protein are a major factor in both membrane fouling and reduced virus yield, and that there is a synergistic effect between the virus and the host cell protein adsorbing to the membrane surface. Recognizing that conventional polymeric membranes have many limitations, a novel ultrathin, isoporous, microfabricated silicon nitride membrane was tested for suitability as a sterile filter. Finally, the application of nanoparticles as model virus particles in filtration testing was examined, and a process was developed through which nanoparticles could be fused together to create controlled amounts of particle aggregates, similar to how viruses can be prone to aggregation. The work described here will help enable the development of next generation sterile filtration membranes and provides both insights and methodologies for improving sterile filtration performance. / Thesis / Doctor of Philosophy (PhD) / While many people are aware that viruses can be used in medicine as vaccines, there are even more new and developing ways they can be used, such as in fighting cancer or treating previously uncurable diseases. However, testing of and patient access to these new treatments is often limited due to the challenges in producing and purifying enough of the virus. Viruses are highly complex and large relative to other products, and so many of the common methods and manufacturing processes which are standard in the industry need to be significantly adapted or improved to suit the production of viruses. This study investigates one step of the purification process, sterile filtration, and considers how a variety of factors from the materials used to the properties of the virus solution can be optimized to improve performance. With a deeper understanding of the sterile filtration process, recommendations can be made to help improve the production of future virus-based therapies.

Cancer metastasis is the leading cause of death related to cancer diseases. Once the cancer cells depart the primary tumor site and enter the blood circulation, they spread through the body and will likely initiate a new tumor site. Therefore, understanding the cell migration and stopping the spread in the initial stage is the utmost of importance. In this dissertation, we have proposed a 3-D microenvironment that (partially) mimics the structures, complexity and circulation of human organs for cell migration studies. We have developed the tools to fabricate 3-D complex geometries in PDMS from our previously developed single-mask, single-etch technology in silicon. In this work, 3-D patterns are transferred from silicon structures to glass following anodic bonding and high temperature glass re-flow processes. Silicon is etched back thoroughly via wet etching and the glass is used as master device to create 3-D PDMS structures for use in dielectrophoresis cell sorting applications. Furthermore, this work has been modified to fabricate 3-D master devices in PDMS to create 3-D structures in collagen hydrogels to mimic native tissue structures. We have studied the interaction of endothelial cells with model geometries of blood vessels in collagen hydrogel at different concentrations to mimic the biomechanical properties of tissues varying from normal to tumor under the growth factor stimulation. Finally, we have designed and fabricated a silicon-based transmigration well with a 30um-thick membrane and 8um pores. This platform includes a deep microfluidic channel on the back-side sealed with a glass wafer. The migratory behavior of highly metastatic breast cancer cells, MDA-MB-231, is tested under different drug treatment conditions. This versatile platform will enable the application of more complex fluidic circulation profile, enhanced integration with other technologies, and running multiple assays simultaneously. / Ph. D.

L’objectif de cette thèse est de développer de nouveaux robots qui combinent dextérité, compacité et précision afin de réaliser des tâches de micromanipulation complexes dans des environnements confinés. Ainsi, deux architectures robotiques parallèles ont été développées. La première est un poignet à 4 degrés de liberté (DDL) en rotation et la seconde est un robot redondant à 7 DDL. Les deux structures intègrent la fonction de préhension grâce à une plateforme configurable et un actionnement déporté. L’étude géométrique et cinématique des deux robots ainsi que des résultats expérimentaux validant les deux architectures sont présentés. Pour miniaturiser le robot à 7 DDL, les liaisons mécaniques (rotules) ont été remplacées par des liaisons en élastomère (PDMS). Cette solution permet, entre autres, d’éliminer les jeux mécaniques au niveau des articulations tout en gardant une grande plage de déplacement. Cependant, comme le comportement de telles articulations ne correspond pas parfaitement à des liaisons rotules, un modèle de robot prenant en compte le comportement élastique de ces articulations a été développé. Afin de réaliser la structure à l’échelle désirée (jambes et liaisons à 400 µm de côté), un nouveau processus de micro-fabrication en salle blanche a été développé. Contrairement aux méthodes existantes, le nouveau processus permet de réduire le nombre d’étapes de gravure et d’intégrer différents types d’élastomères à des microstructures robotiques en silicium. Enfin, le micro-robot a été réalisé et les capacités de déplacement dans les 6 DDL en plus de la préhension ont été validées. Les applications visées des robots développées dans cette thèse sont le micro/nano-assemblage, la manipulation de cellules biologiques et la chirurgie mini-invasive, notamment en neurochirurgie. / The objective of this thesis is the development of new robots that combine dexterity, compactness and precision to perform complex micromanipulation tasks in confined environments. Thus, two parallel robotic structures have been developed. The first is a wrist that can insure 4 degrees of freedom (DOF) in rotation and the second is a redundant robot with 7 DOF. Both structures integrate the grasping function thanks to a configurable platform and a deported actuation. The kinematic study of the two robots and the experimental results validating the two architectures are presented. To miniaturize the 7 DOF robot, the mechanical joints (spherical) have been replaced by elastomeric articulations (PDMS). This solution allows, among others, to eliminate the mechanical backlash in the joints while keeping a large range of movements. However, as the behavior of such joints does not correspond perfectly to spherical joints, a model for the robot taking into account the elastic behavior of these joints has been developed. In order to made the structure on the desired scale (the cross sectional side of its legs and connections are 400 µm), a new microfabrication process in the clean room has been developed. Unlike the existing methods, the new process reduces the number of etching steps and allow the integration of different types of elastomers into silicon robotic microstructures. Finally, the micro-robot was realized and the displacement capacities in the 6 DOF with the grasping were validated. The targeted applications by the developed robots in this thesis are micro / nano-assembly, manipulation of biological cells and minimally invasive surgery, particularly in neurosurgery.

Microfabricated fuel cells have been designed and constructed on silicon integrated circuit wafers using many processes common in integrated circuit fabrication, including sputtering, polymer spin coating, reactive ion etching, and photolithography. Fuel delivery microchannels were made through the use of sacrificial polymers. The characteristics of different sacrificial polymers were studied to find the most suitable for this work. A polypropylene carbonate solution containing a photo-acid generator could be directly patterned with ultraviolet exposure and thermal decomposition. The material that would serve as the fuel cells proton exchange membrane (PEM) encapsulated the microchannels. Silicon dioxide deposited by plasma enhanced chemical vapor deposition (PECVD) at relatively low temperatures exhibited material properties that made it suitable as a thin-film PEM in these devices. By adding phosphorous to the silicon dioxide recipe during deposition, a phosphosilicate glass was formed that had an increased ionic conductivity. Various polymers were tested for use as the PEM or in combination with oxide to form a composite PEM. While it did not work well alone, using Nafion on top of the glass layer to form a dual-layer PEM greatly enhanced the fuel cell performance, including yield and long-term reliability. Platinum and platinum/ruthenium catalyst layers were sputter deposited. Experiments were performed to find a range of thicknesses that resulted in porous layers allowing contact between reactants, catalyst, and the PEM. When using the deposited glasses, multiple layers of catalyst could be deposited between thin layers of the electrolyte, resulting in higher catalyst loading while maintaining porosity. The current and power output were greatly improved with these additional catalyst layers.

Le projet de thèse a pour but de synthétiser de nouveaux matériaux siliciques à microstructure contrôlée en utilisant des procédés de polycondensation à basse température, et de développer de nouvelles approches de microfabrication 3D à haute résolution par excitation bi-photonique d’une formulation sol-gel. La synthèse de matériaux microstructurés est ici centrée sur le procédé sol-gel, impliquant des réactions d’hydrolyse-polycondensation. Les mtériaux les plus étudiés par ces approches en particulier dans le cadre d’un contrôle de la porosité sont préparés à partir de tetraéthoxide de silicium (TEOS)/ Le matériau est structuré généralement par des agent moléculaires structurants (« templates ») tels que les composés tensioactifs. Ces derniers sont en général éliminés, après condensation des silanols, par traitement thermique ou lavage afin de libérer la porosité et générer la structuration. Cette étape d’élimination des agents structurants est souvent problématique, en particulier au niveau industriel, car elle utilise soit des hautes températures, soit des quantités importantes de solvants. Il y a un enjeu important à développer des approches de microstructuration s’affranchissant de ces agents moléculaires structurants, cela représentant le principal objectif de cette thèse.Deux stratégies sont abordées aux cours de ce travail de recherche. La première implique l’utilisation de précurseurs organosilylés donc la structure moléculaire permet une microstructuration du matériau synthétisé. Cette partie explore l’utilisation de précurseurs de la famille des silsesquioxanes et leur transformation vers les matériaux. La deuxième approche propose d’évaluer pour la première fois l’utilisation de l’excitation biphotonique pour contrôler l’étape de condensation sol-gel par modification de pH au point focal d’un laser et ainsi apporter une résolution micrométrique à la structuration du matériau / The main goal of this research was the synthesis of new siliceous materials with controlled microstructures, using low-temperature polycondensation process, and develop the new approach towards 3D microfabrication under two-photon excitation of a sol-gel formulation. Synthesis of microstructured materials is based on the sol-gel process, i.e. hydrolytic polycondensation. Most known siliceous materials with controlled microstructure are synthesized from tetraethoxysilane (TEOS). The materials are generally structured by structuring molecular agents ("templates") such as surfactant compounds, which are then removed by heat treatment or washing with appropriate solvents. This stage of removal of structuring agents is often problematic, particularly at the industrial level, because it uses either high temperatures or large amounts of solvents. Therefore, new methods to obtain materials with controlled porosity without the need to use templates are still being developed. Two strategies are discussed during this research work. First one involved the use of organosilicon precursors with a define structure that at molecular level modify the structure of the final materials. The use of well-defined precursors (silsesquioxanes), in appropriate proportions, allows the control of the porosity of the obtained silica materials. The second approach investigated in this work is the local photo-induced structuration of silicon based pre-hydrolyzed precursors under two-photon excitation allowing high 3D resolution