京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21453号 / 医博第4420号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 浅野 雅秀, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Automated and continuous processes are the future trends in downstream protein purification. A functionalized nanometer-scale membrane electrode system, mimicking the function of cell wall transporters, can selectively capture genetically modified proteins and subsequently pump them through the system under programmed voltage pulses. Numerical study of the two-step pulse pumping cycles coupled with experimental His-GFP releasing study reveals the optimal 14s/1s pumping/repel pulse pumping condition at 10 mM bulk imidazole concentration in the permeate side. A separation factor for GFP: BSA of 9.7 was achieved with observed GFP electrophoretic mobility of 3.1×10-6 cm2 s-1 V-1 at 10 mM bulk imidazole concentration and 14 s/1 s pumping/repel duration. The purification of His6-OleD Loki variant directly from crude E. coli extracts expression broth was demonstrated using the pulse pumping process, simplifying the separation process as well as reducing biopharmaceutical production costs. The enzymatic reactions showed that His6-OleD Loki was still active after purification. A nanoporous membrane/electrode system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzymes-complex system was demonstrated. The substrates residence time on the immobilized enzyme can be precisely controlled by changing the pumping rate and thereby prevent a secondary hydrolysis reaction. Immobilized enzyme showed long term storage longevity with activity half-life of 50 days at 4℃ and the ability to be regenerated. One-step immobilization and purification of His-tagged OleD Loki variant directly from expression broth, yielded 98% Uridine Diphosphate glycosylation and 80% 4-methylumbelliferone glycosylation conversion efficiency for the sequential reaction. A flow-through electroporation system, based on a novel membrane/electrode design, for the delivery of membrane-impermeant molecules into Model Leukocyte cells was demonstrated. The ability to apply low voltage between two short distance electrodes contributes to high cell viability. The flow-through system can be easily scaled-up by varying the micro-fluidic channel geometry and/or the applied voltage pulse frequency. More importantly, the system allows the electrophoretical pumping of molecules from the reservoir across the membrane/electrode system to the micro-fluidic channel for transfection, which reduces large amount of reagents used.

Microfluidics, the manipulation of fluids at nanoliter scale, has emerged to offer an ideal platform for biological analysis of a low number of cells. The technological advances in microfluidics have allowed both forming of valves, mixers and pumps and integrating of optic and electronic components into microfluidic devices to construct complete and functional systems. In this dissertation, I present novel microfluidic techniques and their applications in cellular probes delivery, cell separation and epigenetic study. In the first part of the dissertation, electroporation is implemented on microfluidic platform to generate uniform delivery of "exposed" nanoparticle or protein into cells. In contrast to endocytosis, electroporation is a physical method to breach cell membrane and does not involve vesicle encapsulation of delivered probes, which means these probes have exposed surface in the cytosol. Such trait enables the use of delivered nanoparticle and protein for intracellular targeting of native biomolecules. Laser-induced fluorescent microscopy was used for single particle illuminating to track single molecules in cells. Microfluidic device provide integrated platform for conducting electroporation, cell culture and imaging. In the second part, microfluidic immunomagnetic cell separation is introduced. I showed two new approaches to enhance immunomagnetic cell separation based on (1) uniquely microfabricated paramagnetic patterns inside separation channels; and (2) using combination of nonmagnetic beads and magnetic beads for selection of tumor initiating cells based on two markers of opposite preference in one step. Enhancement in cell isolation (high capture efficiency or high selection purity) is experimentally observed and the former is explained by computational model. In the final part of the dissertation, microfluidic device incorporating valves and mixers for sensitive study of chromosome conformation is presented. This device has small reaction chamber minimizing sample requirement, and allows multiple steps of biological analysis in a single chip avoiding sample loss during sample transfer. Several orders of magnitude improved detection sensitivity is achieved with our microfluidics based method. I envision all novel techniques discussed in this dissertation have great potential in application of disease prognosis, diagnosis and treatment. / Ph. D.

Microfluidic devices are ideal for analysis of biological systems. The small dimensions result to controlled handling of the flow profile and the cells in suspension. Implementation of additional forces in the system, such as an electric field, promote further manipulation of the cells. In this dissertation, I show novel, unique microfluidic approaches for manipulation and analysis of mammalian cells by the aid of electrical methods or the architecture of the device. Specifically, for the first time, it is shown, that adoption of electrical methods, using surface electrodes, promotes cell concentration in a microchamber due to isoelectric focusing (IEF). In contrast to conventional IEF techniques for protein separation, a matrix is not required in our system, the presence of which would even block the movement of the bulky cells. Electric field is, also, used to breach the cell membrane and gain access to the cell interior by electroporation (irreversible and reversible). Irreversible electroporation is used in a unique, integrated microfluidic device for cell lysis and reagentless extraction of DNA. The genomic material is subsequently analyzed by on-chip PCR, demonstrating the possible elimination of the purification step. On the other hand, reversible electroporation is used for the delivery of exogenous molecules to cells. For the first time, the effect of shear stress on the electroporation efficiency of both attached and suspended cells is examined. On the second part of my dissertation, I explore the capabilities of the architecture of microfluidic devices for cell analysis. A simple, unique method for compartmentalization of a microchamber in an array of picochambers is presented. The main idea of the device lies on the fabrication of solid supports on the main layer of the device. These features may even hold a dual nature (e.g. for cell trapping, and chamber support), in which case, single cell analysis is possible (such as single cell PCR). On the final chapter of my dissertation, a computational analysis of the flow and concentration profiles of a device with hydrodynamic focusing is conducted. I anticipate, that all these novel techniques will be used on integrated microfluidic systems for cell analysis, towards point-of-care diagnostics. / Ph. D.

Microfluidics has revolutionized how molecular biology studies are conducted. It permits profiling of genomic and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. These assays dramatically improved the sensitivities of conventional approaches over 1000 times without compromising genomic coverages. We applied these assays to examine the neuronal/glial nuclei isolated from mouse brain tissues. We successfully identified the distinctive epigenomic signatures from neurons and glia. Another focus of this thesis is applying electrical field to investigate the intracellular contents. We report two projects, drug delivery to encapsulated bacteria and mRNA extraction under ultra-high electrical field intensity. We envision rapid growth in these directions, driven by the needs for testing scarce primary cells samples from patients in the context of precision medicine. / Ph. D. / Microfluidics is a technology that manipulates solution with extremely small volume. It is an emerging platform that has revolutionized how molecular biology studies are conducted. It permits profiling of genome wide DNA changes or DNA-related changes (e.g. epigenomics) for a wide range of applications. One of the major contribution of microfluidics is to improve the next generation sequencing (NGS) technologies with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. DNA methylation and histone modification have been proved to affect a lot of biological processes, such as disease development. These developed technologies would benefit the development of precision medicine (a medical model that proposes the customization of healthcare) and treatment to various diseases. We applied these technologies to study the epigenomic differences between several cell types in the mouse brain.