Taisuke Masuda

On-chip magnetically actuated robot with ultrasonic vibration for single cell manipulations

This paper presents an innovative driving method for an on-chip robot actuated by permanent magnets in a microfluidic chip. A piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the high-frequency vibration reduces the effective friction on the MMT significantly. As a result, we achieved 1.1 micrometre positioning accuracy of the microrobot, which is 100 times higher accuracy than without vibration. The response speed is also improved and the microrobot can be actuated with a speed of 5.5 mm s(-1) in 3 degrees of freedom. The novelty of the ultrasonic vibration appears in the output force as well. Contrary to the reduction of friction on the microrobot, the output force increased twice as much by the ultrasonic vibration. Using this high accuracy, high speed, and high power microrobot, swine oocyte manipulations are presented in a microfluidic chip.

Development of a new rapid isolation device for circulating tumor cells (CTCs) using 3D palladium filter and its application for genetic analysis.

Circulating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

A microfabricated platform to form three-dimensional toroidal multicellular aggregate

Techniques that allow cells to self-assemble into three-dimensional (3D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular in fields such as stem cell research, tissue engineering, and cancer biology. Appropriate simulation of the 3D environment in which tissues normally develop and function is crucial for the engineering of in vitro models that can be used for the formation of complex tissues. We have developed a unique multicellular aggregate formation platform that utilizes a maskless gray-scale photolithography. The cellular aggregate formed using this platform has a toroidal-like geometry and includes a micro lumen that facilitates the supply of oxygen and growth factors and the expulsion of waste products. As a result, this platform was capable of rapidly producing hundreds of multicellular aggregates at a time, and of regulating the diameter of aggregates with complex design. These toroidal multicellular aggregates can grow as long-term culture. In addition, the micro lumen can be used as a continuous channel and for the insertion of a vascular system or a nerve system into the assembled tissue. These platform characteristics highlight its potential to be used in a wide variety of applications, e.g. as a bioactuator, as a micro-machine component or in drug screening and tissue engineering.

Changes in stress distribution of orthodontic miniscrews and surrounding bone evaluated by 3-dimensional finite element analysis.

INTRODUCTION Miniscrews can be used to provide absolute anchorage during orthodontic treatment. If we could obtain the optimum design or shape of the miniscrew, we might be able to reduce its size and lessen the chance of root contact. In addition, miniscrews are placed at several angles, and orthodontic forces are applied in various directions for clinical requirements. In this study, we used finite element analysis to investigate changes in stress distribution at the supporting bone and miniscrew by changing the angle and the shape of the miniscrew and the direction of force. METHODS Three types of miniscrews (cylindrical pin, helical thread, and nonhelical thread) were designed and placed in 2 types of supporting bone (cancellous and cortical). The miniscrews were inclined at 30°, 40°, 45°, 50°, 60°, 70°, 80°, and 90° to the surface of the supporting bone. A force of 2N was applied in 3 directions. RESULTS Significantly lower maximum stress was observed in the cancellous bone compared with the cortical bone. By changing the implantation angle, the ranges of the maximum stress distribution at the supporting bone were 9.46 to 14.8 MPa in the pin type, and 17.8 to 75.2 MPa in the helical thread type. On the other hand, the ranges of the maximum stress distribution at the titanium element were 26.8 to 92.8 MPa in the pin type, and 121 to 382 MPa in the helical thread type. According to the migration length of the threads in the nonhelical type, the maximum stresses were 19.9 to 113 MPa at the bone, and 151 to 313 MPa at the titanium element. By changing the angle of rotation in the helical thread type, the maximum stress distributions were 25.4 to 125 MPa at the bone, and 149 to 426 MPa at the titanium element. Furthermore, the maximum stress varied at each angle according to the direction of the applied load. CONCLUSIONS From our results, the maximum stresses observed in all analyzed types and shapes of miniscrews were under the yield stress of pure titanium and cortical bone. This indicates that the miniscrews in this study have enough strength to resist most orthodontic loads.

Compressive force-produced CCN2 induces osteocyte apoptosis through ERK1/2 pathway.

Osteocytes produce various factors that mediate the onset of bone formation and resorption and play roles in maintaining bone homeostasis and remodeling in response to mechanical stimuli. One such factor, CCN2, is thought to play a significant role in osteocyte responses to mechanical stimuli, but its function in osteocytes is not well understood. Here, we showed that CCN2 induces apoptosis in osteocytes under compressive force loading. Compressive force increased CCN2 gene expression and production, and induced apoptosis in osteocytes. Application of exogenous CCN2 protein induced apoptosis, and a neutralizing CCN2 antibody blocked loading‐induced apoptosis. We further examined how CCN2 induces loaded osteocyte apoptosis. In loaded osteocytes, extracellular signal‐regulated kinase 1/2 (ERK1/2) was activated, and an ERK1/2 inhibitor blocked loading‐induced apoptosis. Furthermore, application of exogenous CCN2 protein caused ERK1/2 activation, and the neutralizing CCN2 antibody inhibited loading‐induced ERK1/2 activation. Therefore, this study demonstrated for the first time to our knowledge that enhanced production of CCN2 in osteocytes under compressive force loading induces apoptosis through activation of ERK1/2 pathway.

Virus purification and enrichment by hydroxyapatite chromatography on a chip

Abstract The spread of infectious diseases has become a global health concern. In order to diagnose infectious diseases quickly and accurately, next-generation DNA sequencing techniques for genetic analysis of infectious viruses have been developed rapidly. However, it takes a very long time to pretreat clinical samples for genetic analysis using next-generation sequencers. We have therefore developed a microfluidic chromatography chip that can purify and enrich viruses in a sample using hydroxyapatite particles packed in a micro-column. We demonstrated the purification of virus from a mixture of virus and FBS protein, and enrichment of the virus using this novel microfluidic chip.

Virus enrichment for single virus manipulation by using 3D insulator based dielectrophoresis

We developed an active virus filter (AVF) that enables virus enrichment for single virus infection, by using insulator-based dielectrophoresis (iDEP). A 3D-constricted flow channel design enabled the production of an iDEP force in the microfluidic chip. iDEP using a chip with multiple active virus filters (AVFs) was more accurate and faster than using a chip with a single AVF, and improved the efficiency of virus trapping. We utilized maskless photolithography to achieve the precise 3D gray-scale exposure required for fabrication of constricted flow channel. Influenza virus (A PR/8) was enriched by a negative DEP force when sinusoidal wave was applied to the electrodes within an amplitude range of 20 Vp-p and a frequency of 10 MHz. AVF-mediated virus enrichment can be repeated simply by turning the current ON or OFF. Furthermore, the negative AVF can inhibit virus adhesion onto the glass substrate. We then trapped and transported one of the enriched viruses by using optical tweezers. This microfluidic chip facilitated the effective transport of a single virus from AVFs towards the cell-containing chamber without crossing an electrode. We successfully transported the virus to the cell chamber (v = 10 µm/s) and brought it infected with a selected single H292 cell.

Microfluidic perfusion culture system for multilayer artery tissue models

We described an assembly technique and perfusion culture system for constructing artery tissue models. This technique differed from previous studies in that it does not require a solid biodegradable scaffold; therefore, using sheet-like tissues, this technique allowed the facile fabrication of tubular tissues can be used as model. The fabricated artery tissue models had a multilayer structure. The assembly technique and perfusion culture system were applicable to many different sizes of fabricated arteries. The shape of the fabricated artery tissue models was maintained by the perfusion culture system; furthermore, the system reproduced the in vivo environment and allowed mechanical stimulation of the arteries. The multilayer structure of the artery tissue model was observed using fluorescent dyes. The equivalent Youngs modulus was measured by applying internal pressure to the multilayer tubular tissues. The aim of this study was to determine whether fabricated artery tissue models maintained their mechanical properties with developing. We demonstrated both the rapid fabrication of multilayer tubular tissues that can be used as model arteries and the measurement of their equivalent Youngs modulus in a suitable perfusion culture environment.

Selective injection and laser manipulation of nanotool inside a specific cell using Optical pH regulation and optical tweezers

We developed Optical pH regulation using functional nanotool impregnated with photo-responsive chemical for selective cell injection of nanotool. The nanotool was modified by fluorescent dye for intracellular measurement. The nanotool was included in the fusogenic liposome. Membrane fusion of the liposome to the cell membrane was used for invasive cell injection of the nanotool. The liposome fuses to the cell in weak acidic condition. Local pH regulation inside the liposome was developed using photochromic chemical for selective cell injection of the nanotool. The nanotool was modified by Leuco crystal violet (LCV). LCV emits the proton by ultraviolet (UV) illumination. The emitted proton decreases the pH value in the liposome. This pH regulation is reversible by UV/VIS illumination. The liposome was manipulated by optical tweezers. After contact of the liposome to the cell, the liposome was adhered to the cell by UV induced membrane fusion. Injected nanotool was manipulated by optical tweezers. Intracellular temperature was detected by measuring the fluorescence intensity from the nanotool. We demonstrated optical pH regulation, selective cell injection of the nanotool, and manipulation of the nanotool in the cell.

3D fabrication and manipulation of hybrid nanorobots by laser

We developed fabrication and manipulation of hybrid nanorobots by 3D nano exposure and optical tweezers. Hybrid nanorobot is composed of photoresist and silicon nanowire. The robot is fabricated by femtosecond laser exposure and is connected to the glass substrate by a micropillar. The processing resolutions of the femtosecond laser exposure are 270 nm (line width) and 600 nm (thickness), respectively. The robot is released by cutting of the pillar by ablation with femtosecond laser. Release time is within four minutes. The released robot is manipulated by holographic optical tweezers (HOT). We succeeded in high-speed manipulation of the robot using HOT (transport speed: 100 μm/s, rotation speed: 1140 deg/s). The robot can be fabricated by incorporating temperature indicator to the robot body and inserting a silicon nanowire to the probe. The robot can be used as a thermal sensor by measuring temperature change from the probe to the robot body because silicon nanowire has high thermal conductivity (168 W/m·K). In this paper, we demonstrated fabrication, on-demand release, and manipulation of the robot in a solution. We also demonstrated temperature calibration and measurement to confirm the effectiveness of the temperature sensor.