Shigehiro Hashimoto

Locomotion of medical micro robot with spiral ribs using mucus

An intracorporeal microrobot will not become practical unless an efficient and low invasive driving device is developed. This paper presents a new technology to drive medical microrobots and experimental results which confirm the new principle. Thrust force up to 1 N was measured when gap width was small and sliding speed was high. While it is pointed out that rupture of fluid film may limit the magnitude of thrust force. The experimental study also confirms that as thrust force arises only due to hydrodynamic action and direct contact does not arise, low invasive operation is possible if good lubrication conditions are kept.

Control of myotube contraction using electrical pulse stimulation for bio-actuator

The contractility of tissue-engineered muscle on the application of electrical signals is required for the development of bio-actuators and for muscle tissue regeneration. Investigations have already reported on the contraction of myotubes differentiated from myoblasts and the construction of tissue-engineered skeletal muscle using electrical pulses. However, the relationship between myotube contraction and electrical pulses has not been quantitatively evaluated. We quantitatively investigated the effect of electrical pulse frequency on the excitability of myotubes and developed bio-actuators made of tissue-engineered skeletal muscle. C2C12 cells were seeded on a collagen-coated dish and in collagen gel and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and antibiotics. When the cells reached confluence or after 2 days in culture, the medium was shifted to DMEM containing 7% horse serum to allow them to differentiate to C2C12 myotubes. We electrically stimulated the myotubes and tissue-engineered skeletal muscle, and contractions were observed under a microscope. The myotubes contracted synchronously with electrical pulses between 0.5 and 5 Hz and unfused tetanus was generated at 10 Hz. The contractile performance of tissue-engineered skeletal muscle made of collagen gel and C2C12 was similar to that of the myotubes. Both the rheobase and chronaxie of the myotubes were lowest when the electric field was applied parallel to the myotube axis, and the values were 8.33 ± 2.78 mA and 1.19 ± 0.38 ms, respectively. The motion of C2C12 myotube contraction depended on the pulse frequency and showed anisotropy in the electric field. These results suggest that a tissue-engineered bio-actuator may be controlled using electrical signals.

The effect of pulsatile shear flow on thrombus formation and hemolysis

The hydrodynamic effects of periodically fluctuating shear rate on clot growth and on erythrocyte destruction have been investigated in vitro to study quantitatively thrombus formation and hemolysis in pulsatile flow. Sinusoidally fluctuated uniform shear rates in the Couette flow were applied to the blood, which filled the space in the concave-convex cones or in the parallel-discs-type of rheoscope systems. The results show that clot growth is controlled so as not to occupy a large space when the time of application of lower (<100 s/sup -1/) shear rates are modified by the wash-out effect of intermittent application of higher (>500 s/sup -1/) shear rates, and that erythrocyte destruction including sub-lethal damage decreases when the exposure time of larger (>500 s/sup -1/) shear rates is interspersed with smaller (<300 s/sup -1/) shear rates. This study demonstrates that pulsatile flow plays an important role in controlling thrombus formation and hemolysis.

Clot growth under periodically fluctuating shear rate

To control the morphology of a clot formed on an artificial flow path in pulsatile blood flow, the hydrodynamic effect of periodically fluctuating shear rate on clot growth has been quantitatively investigated in vitro. Uniform shear rates were applied to a sample of beagle blood in the concave-convex cones system. These shear rates were sinusoidally fluctuated between a maximum and a minimum in one direction at frequencies between 0.1 and 0.6 Hz. Evaluation of clot growth was derived from a clot ratio, which was experimentally determined from the rate of increase of frictional torque between the two cones. The results show that clot growth is controlled so as not to occupy a large space when the minimum shear rate is higher than 100 s-1, or when the time of application of lower (< 100 s-1) shear rates is modified by the intermittent application of higher (> 500 s-1) shear rates as long as the frequency is less than 0.6 Hz.

Environmental Design for Muscle Cell Culture with Magnetic Field

Control methodology on cell culture has been studied for regenerative medicine and micro-actuator in an interdisciplinary research field. Environment has been designed around cultured muscle cells with a magnetic field, and an effect of the magnetic field at an extremely low frequency on proliferation and orientation of cultured skeletal muscle cells has been studied in vitro. Skeletal muscle cells were isolated from a hind-limb quadriceps, and cultured on a dish coated with collagen. The culture dishes were placed parallel to the uniform alternating magnetic field, which was generated with a solenoid coil. The result shows that the cultured skeletal muscle cells tend to align to the direction of the magnetic field at the extremely low frequency.

Are Micro Back Markers on Thin Film of Scaffold Effective to Evaluate Contractive Movement of Myotube

The scaffold of the transparent film with micro markers has been designed to evaluate the contractive movement of myotube under the electric pulse stimulations in vitro. The scaffold of a thin film (6 μm of thickness) of polydimethylsiloxane was made with ten million micro markers: 4 μm of diameter, 2 μm of height, and 30 μm of interval. Ten million markers were made on the backside of the scaffold by the photolithography technique. C2C12 (mouse myoblast cell line) was seeded on the film at the counter surface to the markers at the density of 5000 cells/cm2. The cells were cultured for 12 days in the medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 310 K with 5% of CO2 content. The electric pulses (1 s of pulse cycle, 1 ms of pulse width) were applied between platinum electrodes dipped in the medium. The myoblasts were able to be cultured on the film to be differentiated into myotubes. The thin film with micro markers was successfully made of polydimethylsiloxane, and had enough transparency for observation of myotubes by the microscope. The myotube was contracted synchronously with the stimulation of electric pulses. The contractile movement was able to be analyzed by the repetitive displacement of the length between backside micro markers of the film. The micro backside markers on thin film of scaffold are effective to evaluate contractive movement of the myotube.

How Does Cell Deform Through Micro Slit Made by Photolithography Technique

Several slits sort cell according to the deformability in vivo. A micro slit (0.87 mm of width, 0.010 mm height) was newly designed between a micro ridge on a transparent polydimethylsiloxane plate and micro ridges on a borosilicate glass plate. These ridges made by photolithography technique make contact each other in the perpendicular position to make the slit between the ridges. A one-way flow system was designed to observe each cell passing through the slit in vitro. Four kinds of cells were used in the test: C2C12 (mouse myoblast cell), HUVEC (human umbilical vein endothelial cell), Hepa1-6 (mouse hepatoma cell), and Neuro-2a (mouse neural crest-derived cell). The suspension of each kind of cells was injected to the slits. The deformation of each cell passing through the slit was observed with an inverted phase-contrast microscope. At the microscopic images, the outline of each cell was traced, and the area (S) was calculated. The deformation ratio was calculated as the ratio (S2/S1) of the projected area of each cell before the slit (S1) and that in the slit (S2). The velocity of the cell passing through the slit was calculated by the trace at the microscopic movie. The experimental results show that each cell deforms to the flat circular disk and passes through the micro slit. Hepa1-6 is flattened with the increase of the passing velocity, and HUVEC is elongated along the flow. The designed slit between micro ridges is effective to evaluate the deformability of cells.

Accelerated differentiation of myoblast with high gravitational force in vitro

The accelerated differentiation of myoblast with high gravitational force has been studied in an experimental system with centrifugal force in vitro. Mouse myoblasts were seeded on a culture dish of 35 mm diameter, and cultured in the Dulbeccos Modified Eagles Medium until the sub-confluent condition. To apply the excess gravitational force on the cultured cells, the dish was set in a conventional centrifugal machine. Constant gravitational force was applied to the cultured cells for three hours. Variation was made on the gravitational force (<800 G) with control of the rotational speed of the rotator in the centrifugal machine. Morphology of the cells was observed with a phase-contrast microscope for eight days. The experimental results show that the myoblast thickens day by day after the exposure to the high gravitational force field.

Accelerated differentiation of myoblast with electric pulses in vitro

An effect of repetitive electric pulses on differentiation of myoblasts has been studied in vitro. C2C12 (Mouse myoblast cell line originated with cross-striated muscle of C3H mouse) was exposed to repetitive electric pulses. The adhered cells were exposed to the electric field between two electrodes made of a platinum wire dipped in the medium at 37 degrees Celsius. The repetitive electric pulses at a period of one second were generated with a function generator. Variation was made on the amplitude of the pulse. To find the limit of amplitude of the electric pulse, the number of adhered cells was counted after exposure to the continuous electric stimulation for 72 hours. The cells were microscopically observed during cultivation, after electric stimulation with repetitive pulses for 72 hours. The responsive movement of myoblast was observed with the electric stimulation to confirm differentiation. The results show that the repetitive electric pulses accelerate differentiation of myoblast.

Application of laser to measurement of cyclic contractile movement of cultured myotubes

Laser technique has been applied to measure cyclic contractile movement of cultured myotubes in vitro. The designed measurement system includes light source (helium neon, 632.8 nm wave length), charge-coupled devise cameras and detectors. Cyclic contraction of myotubes cultured from C2C12 (mouse myoblast) was generated by cyclic electric pulses (amplitude < 60 V, 1 ms width) through electrodes of platinum wire dipped in the medium. The spectrum of fluctuating intensity of the transmitted laser beam through the myotubes, which periodically repeated contraction and relaxation, was analyzed. The results show that the designed laser system is effective to detect frequency of cyclic contractile movement of myotubes between 0.5 Hz and 5 Hz.