Takayuki Hoshino

Room Temperature Operable Autonomously Moving Bio-Microrobot Powered by Insect Dorsal Vessel Tissue

Living muscle tissues and cells have been attracting attention as potential actuator candidates. In particular, insect dorsal vessel tissue (DVT) seems to be well suited for a bio-actuator since it is capable of contracting autonomously and the tissue itself and its cells are more environmentally robust under culturing conditions compared with mammalian tissues and cells. Here we demonstrate an autonomously moving polypod microrobot (PMR) powered by DVT excised from an inchworm. We fabricated a prototype of the PMR by assembling a whole DVT onto an inverted two-row micropillar array. The prototype moved autonomously at a velocity of 3.5×10−2 µm/s, and the contracting force of the whole DVT was calculated as 20 µN. Based on the results obtained by the prototype, we then designed and fabricated an actual PMR. We were able to increase the velocity significantly for the actual PMR which could move autonomously at a velocity of 3.5 µm/s. These results indicate that insect DVT has sufficient potential as the driving force for a bio-microrobot that can be utilized in microspaces.

Improvement of neuronal cell adhesiveness on parylene with oxygen plasma treatment

We improved adhesiveness of a neuron-like cell, PC12, on a Parylene-C surface by O(2) plasma treatment which changes the surface from hydrophobic to hydrophilic. Neural cell adhesiveness on the plasma-treated Parylene-C was more than twenty times better compared to non-treated Parylene-C and it was close to that on a conventional polystyrene tissue-culture dish.

Electron-beam direct processing on living cell membrane

We demonstrated a direct processing on a living Hep G2 cell membrane in conventional cultivation conditions using an electron beam. Electron beam-induced deposition from liquid precursor 3,4-ethylenedioxythiophene and ablation was performed on the living cells. The 2.5-10 keV electron beam which was irradiated through a 100-nm-thick SiN nanomembrane could induce a deposition pattern and a ablation on a living cell membrane. This electron beam direct processing can provide simple in-situ cell surface modification for an analytical method of living cell membrane dynamic.

Closed-looped in situ nano processing on a culturing cell using an inverted electron beam lithography system

The beam profile of an electron beam (EB) can be focused onto less than a nanometer spot and scanned over a wide field with extremely high speed sweeping. Thus, EB is employed for nano scale lithography in applied physics research studies and in fabrication of semiconductors. We applied a scanning EB as a control system for a living cell membrane which is representative of large scale complex systems containing nanometer size components. First, we designed the opposed co-axial dual optics containing inverted electron beam lithography (I-EBL) system and a fluorescent optical microscope. This system could provide in situ nano processing for a culturing living cell on a 100-nm-thick SiN nanomembrane, which was placed between the I-EBL and the fluorescent optical microscope. Then we demonstrated the EB-induced chemical direct nano processing for a culturing cell with hundreds of nanometer resolution and visualized real-time images of the scanning spot of the EB-induced luminescent emission and chemical processing using a high sensitive camera mounted on the optical microscope. We concluded that our closed-loop in situ nano processing would be able to provide a nanometer resolution display of virtual molecule environments to study functional changes of bio-molecule systems.

Neurite outgrowth of PC12 cells on diX (parylene) family materials

We investigated neuronal cell differentiation, particularly neurite outgrowth, on the surface of diX H and diX AM using an in vitro examination of a neuron‐like rat pheochromocytoma cell line, PC12. diX H and diX AM are in the parylene family of diX C (or Parylene‐C), which is widely used as a novel coating material to insulate neural electrodes, and they have been recently commercialized; diX H and diX AM offer different features of biocompatibility. Previously, we found that these new parylene materials have high cell adhesiveness to neuronal cells whereas the adhesiveness of diX C is extremely low. However, their cell differentiation remains unknown although neuronal cell differentiation plays a crucial role in their development and regeneration. This study showed that almost all PC12 cells adhering to the surface of diX AM and diX H were differentiated, but the neurite outgrowth was significantly larger on diX H than that on diX AM and a conventional polystyrene culture dish. The result suggests that diX H may be advantageous as a biocompatible coating material for a scaffold, which can be used on virtually any substrate to get various configurations in neural devices.

Measurement system for biomechanical properties of cell sheet

In this study, we present a new fixture (self-attachable fixture) and tensile test system for measuring mechanical properties of cell sheet. To evaluate strength of cell sheet, it is the most important to measure mechanical properties of tensile mode. However, there has been no study which measured the tensile mechanical properties of cell sheet, since it has been difficult to attach a cell sheet in the tensile test system owing to the structure of the conventional fixture, and there has been no tensile test system which had a measurement range that covered the tension force range of the cell sheets. Therefore, we have addressed these problems by developing a self-attachable fixture and a tensile test system. By using developed system, we measured mechanical properties (tension, stress and initial stiffness) C2C12 of cell sheet cultured in different recipe of culture medium. The initial stiffness of cell sheet cultured in culture medium without FBS had a tendency to become stiffer. This indicates that our new fixture and test system are applicable for evaluating mechanical properties of cell sheets.

Closed-looped nano stimulation microscope for living cell membrane

Electron-beam could stimulate a living cell membrane through a 100-nm-thick SiN nanomembrane. We designed a co-axial dual microscope with an electron-beam lithography system and a fluorescence microscope to investigate bio-molecular dynamic system of living cell membrane. This microscope had a closed-looped controlling system using the fluorescence live cell imaging and the electron beam induced stimulation. The live cell imaging of the fluorescence microscope provides the chemical identity of the target molecule and the response to a following stimulation on the cell membrane, and the electron-beam can induce electro-chemical stimulation in hundreds nanometer resolution. Scanning of the electron-beam could provide high-speed, precise, and large scale stimulation on the target molecules. We attempted that the combination with this stimulation system and fluorescence microscope would provide the closed-loop control of the bio-molecule system on cell membrane. We proposed here the concept of the virtual molecule environmental display to study the functional changes on the target behavior due to our closed-loop control system on this co-axial microscope.

Contracting cardiomyocytes in hydrophobic room-temperature ionic liquid.

Room-temperature ionic liquids (RTILs) are drawing attention as a new class of nonaqueous solvents to replace organic and aqueous solvents for chemical processes in the liquid phase at room temperature. The RTILs are notable for their characteristics of nonvolatility, extremely low vapor pressure, electric conductivity, and incombustibility. These distinguished properties of RTILs have brought attention to them in applications with biological cells and tissue in vacuum environment for scanning electron microscopy, and in microfluidic devices for micro-total analysis system (micro-TAS). Habitable RTILs could increase capability of nonaqueous micro-TAS for living cells. Some RTILs seemed to have the capability to replace water in biological applications. However, these RTILs had been applied to just supplemental additives for biocompatible test, to fixed cells as a substitute for an aqueous solution, and to simple molecules. None of RTILs in which directly soaks a living cell culture. Therefore, we demonstrated the design of RTILs for a living cell culture and a liquid electrolyte to stimulate contracting cardiomyocytes using the RTILs. We assessed the effect of RTILs on the cardiomyocytes using the beating lifetime to compare the applicability of RTILs for biological applications. Frequent spontaneous contractions of cardiomyocytes were confirmed in amino acid anion RTILs [P(8,8,8,8)][Leu] and [P(8,8,8,8)][Ala], phosphoric acid derivatives [P(8,8,8,8)][MeO(H)PO(2)], and [P(8,8,8,8)][C(7)CO(2)]. The anion type of RTILs had influence on applicable characteristics for the contracting cardiomyocyte. This result suggested the possibility for biocompatible design of hydrophobic group RTILs to achieve biological applications with living cells.