単一運動性微生物の刺激応答計測のためのマイクロロボティックプラットホーム

九州工業大学博士学位論文 学位記番号:生工博甲第355号 学位授与年月日:令和元年9月20日1 Introduction|2 Observation Platform|3 Stimulation Platform|4 Application to Actual Motile Microorganisms|5 Conclusion九州工業大学令和元年

Bioinspired reorientation strategies for application in micro/nanorobotic control

Engineers have recently been inspired by swimming methodologies of microorganisms in creating micro-/nanorobots for biomedical applications. Future medicine may be revolutionized by the application of these small machines in diagnosing, monitoring, and treating diseases. Studies over the past decade have often concentrated on propulsion generation. However, there are many other challenges to address before the practical use of robots at the micro-/nanoscale. The control and reorientation ability of such robots remain as some of these challenges. This paper reviews the strategies of swimming microorganisms for reorientation, including tumbling, reverse and flick, direction control of helical-path swimmers, by speed modulation, using complex flagella, and the help ofmastigonemes. Then, inspired by direction change in microorganisms,methods for orientation control for microrobots and possible directions for future studies are discussed. Further, the effects of solid boundaries on the swimming trajectories of microorganisms and microrobots are examined. In addition to propulsion systems for artificial microswimmers, swimming microorganisms are promising sources of control methodologies at the micro-/nanoscale

MicroBioRobots for Single Cell Manipulation

One of the great challenges in nano and micro scale science and engineering is the independent manipulation of biological cells and small man-made objects with active sensing. For such biomedical applications as single cell manipulation, telemetry, and localized targeted delivery of chemicals, it is important to fabricate microstructures that can be powered and controlled without a tether in fluidic environments. These microstructures can be used to develop microrobots that have the potential to make existing therapeutic and diagnostic procedures less invasive. Actuation can be realized using various different organic and inorganic methods. Previous studies explored different forms of actuation and control with microorganisms. Bacteria, in particular, offer several advantages as controllable micro actuators: they draw chemical energy directly from their environment, they are genetically modifiable, and they are scalable and configurable in the sense that any number of bacteria can be selectively patterned. Additionally, the study of bacteria inspires inorganic schemes of actuation and control. For these reasons, we chose to employ bacteria while controlling their motility using optical and electrical stimuli. In the first part of the thesis, we demonstrate a bio-integrated approach by introducing MicroBioRobots (MBRs). MBRs are negative photosensitive epoxy (SU8) microfabricated structures with typical feature sizes ranging from 1-100 μm coated with a monolayer of the swarming Serratia marcescens. The adherent bacterial cells naturally coordinate to propel the microstructures in fluidic environments, which we call Self-Actuation. First, we demonstrate the control of MBRs using self-actuation, DC electric fields and ultra-violet radiation and develop an experimentally-validated mathematical model for the MBRs. This model allows us to to steer the MBR to any position and orientation in a planar micro channel using visual feedback and an inverted microscope. Examples of sub-micron scale transport and assembly as well as computer-based closed-loop control of MBRs are presented. We demonstrate experimentally that vision-based feedback control allows a four-electrode experimental device to steer MBRs along arbitrary paths with micrometer precision. At each time instant, the system identifies the current location of the robot, a control algorithm determines the power supply voltages that will move the charged robot from its current location toward its next desired position, and the necessary electric field is then created. Second, we develop biosensors for the MBRs. Microscopic devices with sensing capabilities could significantly improve single cell analysis, especially in high-resolution detection of patterns of chemicals released from cells in vitro. Two different types of sensing mechanisms are employed. The first method is based on harnessing bacterial power, and in the second method we use genetically engineered bacteria. The small size of the devices gives them access to individual cells, and their large numbers permit simultaneous monitoring of many cells. In the second part, we describe the construction and operation of truly micron-sized, biocompatible ferromagnetic micro transporters driven by external magnetic fields capable of exerting forces at the pico Newton scale. We develop micro transporters using a simple, single step micro fabrication technique that allows us to produce large numbers in the same step. We also fabricate microgels to deliver drugs. We demonstrate that the micro transporters can be navigated to separate single cells with micron-size precision and localize microgels without disturbing the local environment

\u3ci\u3eVorticella\u3c/i\u3e: A Protozoan for Bio-Inspired Engineering

In this review, we introduce Vorticella as a model biological micromachine for microscale engineering systems. Vorticella has two motile organelles: the oral cilia of the zooid and the contractile spasmoneme in the stalk. The oral cilia beat periodically, generating a water flow that translates food particles toward the animal at speeds in the order of 0.1–1 mm/s. The ciliary flow of Vorticella has been characterized by experimental measurement and theoretical modeling, and tested for flow control and mixing in microfluidic systems. The spasmoneme contracts in a few milliseconds, coiling the stalk and moving the zooid at 15–90 mm/s. Because the spasmoneme generates tension in the order of 10–100 nN, powered by calcium ion binding, it serves as a model system for biomimetic actuators in microscale engineering systems. The spasmonemal contraction of Vorticella has been characterized by experimental measurement of its dynamics and energetics, and both live and extracted Vorticellae have been tested for moving microscale objects. We describe past work to elucidate the contraction mechanism of the spasmoneme, recognizing that past and continuing efforts will increase the possibilities of using the spasmoneme as a microscale actuator as well as leading towards bioinspired actuators mimicking the spasmoneme

Attachment of Therapeutic and Imaging Agents to Magnetotactic Bacteria Acting as Self-Propelled Bio-Carriers for Cancer Treatment

RÉSUMÉ Malgré les progrès de la médecine moderne, les traitements anticancéreux actuels n’arrivent toujours pas à vaincre le cancer. Seulement une fraction des doses de médicaments administrées parvient à la tumeur en raison d’un ciblage non spécifique, de barrières physiologiques au niveau du système vasculaire ainsi que de l’élimination immédiate de médicaments par le système immunitaire. Des dosages fréquents de médicaments deviennent nécessaires afin de surmonter ces obstacles, entraînant une toxicité systémique, des effets secondaires et un échec thérapeutique. De plus, les systèmes actuels d’imagerie médicale sont incapables de produire des images de haute qualité des structures tumorales pour les diagnostiques et les traitements. Ceci est dû aux restrictions de la résolution spatiale et de l’incapacité des agents de contraste à pénétrer dans les zones tumorales afin de générer un signal suffisamment intense. Le développement de nouveaux agents thérapeutiques ainsi que de nouvelles techniques de ciblage thérapeutique sont donc requis afin d’améliorer l’efficacité des traitements actuels. Pour ce projet de recherche doctorale, l'attachement de charges utiles à la surface de bactéries magnétotactiques flagellées Magnetococcus Marinus MC-1 (BMT) a été mise en place pour transporter de façon ciblée une quantité optimale de médicaments profondément dans les zones tumorales. Ces bio-robots autopropulsés de dimensions adéquates sont équipés d’un système de propulsion dirigeable, d’un système de navigation, et de capacités sensorielles. Divers types de complexes BMT ont été fabriquées en attachant aux BMT (i) des liposomes vides (BMT-LP), (ii) des liposomes contenant un agent anticancéreux SN38 (BMT-LSC), et (iii) des nanoparticules superparamagnétiques de magnétite (BMT-S200). L’efficacité de l'attachement des charges et du comportement des bactéries soumises à un champ magnétique directionnel ont été étudiés. Par la suite, la capacité des complexes BMT à naviguer le long d’une trajectoire prédéterminée, à infiltrer profondément l'espace interstitiel, et à cibler des zones tumorales inaccessibles, ont été étudiés dans un modèle animal soumis à un champ magnétique externe. Pour parvenir à des complexes BMT aptes à transporter suffisamment de produits pharmaceutiques et de s’accumuler préférentiellement dans les régions affectées, il faut assurer un attachement solide et stable qui ne compromet pas la motilité des BMT.----------ABSTRACT Despite the substantial achievements of modern medicine, current medical therapies cannot eradicate cancer. Due to nonspecific targeting, the multiple physiological barriers that blood-borne agents must encounter, and the rapid sequestration of drugs by the immune system, a suboptimal fraction of the total injected dose reaches the intended target. These obstacles necessitate frequent dosing to compensate therapeutic effects, resulting in systemic toxicity, undesirable side effects, and treatment failure. In addition, existing medical imaging modalities struggle to provide high quality clinical images of tumor structures for treatment purposes due to limitations in spatial resolution and lack of penetration of contrast agents into tumoral regions to induce sufficient signal intensity. To address these issues, the development of new therapeutic agents alongside improved strategies for targeting therapy with the ability to control their fate is required. The attachment of payloads to the flagellated Magnetococcus Marinus MC-1 magnetotactic bacteria (MTB) to directly transport optimal quantities of pharmaceutical agents to regions located deep in tumors is what has been proposed during the accomplishment of this PhD project. These engineered self-propelled bio-robots with an appropriate dimension are equipped with steerable propulsion, navigation system, and onboard sensory capabilities. MTB complexes were fabricated by attaching the MTB to (i) empty liposomes (MTB-LP), (ii) SN38 anticancer drug encapsulated in liposomes (MTB-LSC), and (iii) 200 nm superparamagnetic magnetite nanoparticles (MTB-S200). The attachment efficacy and magnetic response behavior from the influence of a directional magnetic field of loaded bacteria with therapeutic or imaging agents were studied. Subsequently, results showed that the attachment method was suitable to allow MC-1 MTB to transport therapeutic and imaging agents along a planned trajectory prior to penetrate deep through the interstitial space in order to reach the hypoxic regions of a tumor in an animal model. To achieve MTB complexes capable of carrying sufficient pharmaceutical agents and accumulating preferentially at disease sites, the attachment must be strong and stable without compromising the natural motility of MTB. The MTB-LP were prepared by direct covalent attachment of functionalized liposomes to the amine groups naturally presented on the surface of MTB using carbodiimide (EDC/NHS) chemistry

Biological building blocks for 3D printed cellular systems

Advancements in the fields of tissue engineering, biomaterials, additive manufacturing, synthetic and systems biology, data acquisition, and nanotechnology have provided 21st-century biomedical engineers with an extensive toolbox of techniques, materials, and resources. These “building blocks” could include biological materials (such as cells, tissues, and proteins), biomaterials (bio-inert, -instructive, -compatible, or -degradable), soluble factors (growth factors or small molecules), and external signals (electrical, chemical, or mechanical). “Forward engineering” attempts to integrate these building blocks in different ways to yield novel systems and machines that, by promoting new relationships and interactions among their individual components, are greater than the sum of their parts. Drawing from an extensive reserve of parts and specifications, these bio-integrated forward-engineered cellular machines and systems could acquire the ability to sense, process signals, and produce force, and could also contain a countless array of applications in drug screening and delivery, programmable tissue engineering, and biomimetic machine design. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. In contrast to traditional machines that use external energy to produce an output, muscle cells can be fueled by glucose and other biomolecules. While cardiac cell driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel “bio-bots” powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot upon applied electrical stimulation. The muscle strips were composed of differentiated skeletal myofibers in a matrix of natural proteins, including fibrin, that provide physical support and cues to the cells as an engineered basement membrane. The hierarchical organization, modularity, and scalable nature of mature skeletal muscle fibers (which can be combined in parallel to increase force production, for example), lends itself to “building with biology.” Few systems have shown net movement from an autonomous, freestanding biological machine composed of skeletal muscle, and even fewer have attempted to incorporate multiple cell types for greater functionality. Modular and flexible platforms for fabrication of such multi-cellular modules and their characterization have been lacking. We also present a modular heterotypic cellular system, made up of multi-layered tissue rings containing integrated skeletal muscle and motor neurons embedded in an extracellular matrix. Site-specific innervation of a group of muscle fibers in the multi-layered tissue rings allowed for muscle contraction via chemical stimulation of motor neurons with glutamate, a major excitatory mammalian neurotransmitter, with the frequency of contraction increasing with glutamate concentration. The addition of the nicotinic receptor antagonist tubocurarine chloride halted the contractions, indicating that muscle contraction was motor neuron-induced. We also present a thorough characterization and optimization of a co-culture system that harnesses the potential of engineered skeletal muscle tissue as the actuating component in a biological machine through the incorporation of motor neurons, and creates an environment that is amenable to both cell types and prime for functional neuromuscular formation. With a bio-fabricated system permitting controllable mechanical and geometric attributes on a range of length scales, our novel engineered cellular system can be utilized for easier integration of other modular “building blocks” in living cellular and biological machines. We are poised to design the next generation of complex biological machines with controllable function, specific life expectancy, and greater consistency. In the future, we envision that this system can be used for applications beyond bio-robotics and muscular actuators; as a functioning heterotypic co-culture, the muscle- neuron arrangement is also a highly relevant machine for the study of neuromuscular diseases and related drug toxicity studies. These results could prove useful for the study of disease-specific models, treatments of myopathies such as muscular dystrophy, and tissue engineering applications

Engineering derivatives from biological systems for advanced aerospace applications

The present study consisted of a literature survey, a survey of researchers, and a workshop on bionics. These tasks produced an extensive annotated bibliography of bionics research (282 citations), a directory of bionics researchers, and a workshop report on specific bionics research topics applicable to space technology. These deliverables are included as Appendix A, Appendix B, and Section 5.0, respectively. To provide organization to this highly interdisciplinary field and to serve as a guide for interested researchers, we have also prepared a taxonomy or classification of the various subelements of natural engineering systems. Finally, we have synthesized the results of the various components of this study into a discussion of the most promising opportunities for accelerated research, seeking solutions which apply engineering principles from natural systems to advanced aerospace problems. A discussion of opportunities within the areas of materials, structures, sensors, information processing, robotics, autonomous systems, life support systems, and aeronautics is given. Following the conclusions are six discipline summaries that highlight the potential benefits of research in these areas for NASA's space technology programs

Climbing and Walking Robots

Nowadays robotics is one of the most dynamic fields of scientific researches. The shift of robotics researches from manufacturing to services applications is clear. During the last decades interest in studying climbing and walking robots has been increased. This increasing interest has been in many areas that most important ones of them are: mechanics, electronics, medical engineering, cybernetics, controls, and computers. Today’s climbing and walking robots are a combination of manipulative, perceptive, communicative, and cognitive abilities and they are capable of performing many tasks in industrial and non- industrial environments. Surveillance, planetary exploration, emergence rescue operations, reconnaissance, petrochemical applications, construction, entertainment, personal services, intervention in severe environments, transportation, medical and etc are some applications from a very diverse application fields of climbing and walking robots. By great progress in this area of robotics it is anticipated that next generation climbing and walking robots will enhance lives and will change the way the human works, thinks and makes decisions. This book presents the state of the art achievments, recent developments, applications and future challenges of climbing and walking robots. These are presented in 24 chapters by authors throughtot the world The book serves as a reference especially for the researchers who are interested in mobile robots. It also is useful for industrial engineers and graduate students in advanced study