Cell fusion has been widely applied in scientific research for cancer immunotherapy, antibody production, and nuclear reprogramming of somatic cells, and therefore the cell fusion technique that enable us to precisely control the fusion process with high throughput manner has been desired. Here, we present a novel microfluidic method for automatic cell pairing by microdroplets, separation of droplets containing cells, and electrofusion of cells inside a droplet. The proposed microfluidic device mainly composed of three sequential function parts for (i) encapsulation of cells into a droplet by microfluidic droplet generator, (ii) separation of droplets containing cells from empty droplets through a micropillar array, and (iii) electrofusion of cells inside the droplets by applying a voltage during the droplet passing over the pair of electrodes. In the microfluidic device, cell-encapsulated and empty droplets were generated at the upstream cross-junction; they then entered the micropillar array, separating the cell-encapsulated droplets from empty droplets continuously. After separation, they passed over the electrode pairs, and were collected the outside of the microchannel. This continuous process for cell fusion would enable us to observe and isolate the target fused cells for cell analysis.

We have successfully produced mono-dispersed micro-gas bubbles for less than around 10 μm diameter by micro-bubble knife which produce micro-bubble in an electrically induced ultrasonic field. The discharged output power and conductive area of micro-electrode in micro-bubble knife which cuts the cell by micro-bubble were controlled by glass shell insulation around the copper micro-wire. A small space which is called “bubble reservoir” between the wire and glass tip contributes to stabilize the electric discharge and directional bubble generation. The directionally dispensed bubbles can be used for processing of soft materials such as biological cells. For the present study the cell membrane has successfully processed with a few μm order resolution.

This paper presents the high speed microrobot actuation driven by permanent magnets in a microfluidic chip. The riblet surface, which is regularly arrayed V groove reduces the fluid friction and enables stable actuation in high speed. The comprehensive analysis of fluid force, the optimum design and its fabrication were conducted and proved the friction reduction by riblet. The Ni and Si composite fabrication was employed to form the optimum riblet shape on the microrobot surface by wet and dry etching. The evaluation experiments show the microrobot can actuate up to 90 Hs, which is 10 times higher than the original microrobot. In addition, it can be applied to cell manipulation without harm since the microrobot is covered by Si, which is bio-compatible. One of the applications of developed microrobot was demonstrated by assembling cell aggregation in high speed.

In this paper, we introduce an untethered type of magnetically driven microtool (MMT) with a force sensing function to investigate the stimulus characteristics of aquatic microorganisms in a microfluidic chip. The microchip is composed of the proposed MMTs, which have a Si-Ni hybrid structure, a transparent cover made of polydimethylsiloxane (PDMS), a glass substrate, and a microspacer to prevent both friction during the force measurement and damage to the micropattern of the MMT. The fabrication and drive performance of the MMT, which is actuated by permanent magnets from the outside of the microchip, verified in basic experiments. Finally, we succeeded in on-chip manipulation and sensing of microorganisms using this MMT, which we found to be easy to use.

This paper describes an innovative driving method for magnetically driven microtools (MMT). 1.1 μm positioning accuracy of the MMT was obtained by applying a piezoelectric ceramic, which induced nanoscale vibration to the microfluidic chip, and reducing the friction on the MMT. Using this drive method, the enucleation process was conducted by dual-arm MMT. Permanent magnets are used to supply the driving power to the MMT allowing sufficient positioning accuracy to cut oocytes precisely. The nucleus was cut within 10 s, and the removed area was less than 25% of the original oocyte.

The video presents high power, high precision and high speed drive of on-chip robot and its biomedical applications. The magnetically driven microtool (MMT) actuated by permanent magnets was employed as a microrobot because of its strong output force in the order of millinewtons and the ultrasonic vibration was applied to the microfluidic chip in order to reduce the friction on the MMT. We have achieved 1.1 μm positioning accuracy of the MMT and applied this drive method to the enucleation of oocyte process. The dual-arm MMT removed the nucleus part in a few seconds.

In this paper, we newly propose an on-chip force sensing by using a magnetically driven microtool (MMT) equipped with a frame structure for measurement of stimulant property of Pleurosira laevis. The design and fabrication of the force sensing structure with a displacement magnification mechanism based on beam deformation is discussed. Through the basic experiments, the advantages of the proposed layer fabrication technique and the performance of the force sensor are confirmed. The basic characteristics of P. laevis are also confirmed by using the developed MMT.

In this paper, on-chip particle loading and dispensing modules are presented with their results for the automation of a single particle retrieving from a microfluidic channel. Our proposed microfluidic chip has several modules. Each one of them has important functions as (a) loading micro-particles singly to main microfluidic flow by the aid of magnetically driven microtools (MMT); (b) finding particle position in a microfluidic channel by micro-capacitance sensors; (c) adjusting micro-channel height locally by pneumatic pressure valve; (d) dispensing particles out from the microfluidic chip to incubation environment. Novelty of this paper is summarized as follows: (1) Multi-photoresist combination technique for the pneumatic pressure valve; (2) Automatic on-chip particle dispensing with micro-capacitance sensors. We showed feasibility of automatic dispensing of a single polystyrene bead (about 100 μm) from the chip to atmosphere. The performances of each module (hybrid structure, sensor and dispensing parts) were evaluated individually. We succeeded in determination of the movement of micro-particles (about 50-100 μm) with the velocity of over 6 mm/sec. by the micro-capacitance sensors. The advantages of the proposed system are that composed of the reusable drive system such as xy motorized stage, pumps and a disposable microfluidic chip.

In this paper, we discuss the design and fabrication approach to increase the success rate of single particle dispensing. Two pairs of capacitance sensors are placed in a biochip to detect the flow velocity of particles, and the air pressure is applied to eject particles by synchronizing the timing. Comprehensive design theory, which is taken into account of the back pressure caused by air pressure, the response time of the system, sensor property, and the delay of the dispensing from the air pressure, is developed in order to minimize the disturbance of the system and maximize the throughput of the ejection system. Then, the system has capability to eject 3 particles/sec and maximum flow velocity is 10 mm/s. The novelty of the system is that the biochip is disposable which is unlike the conventional mechanical inkjet system and it can prevent contamination. Therefore, the fabricated disposable biochip based on photolithography is low cost and the drive system is reusable. Finally, we succeed in automatic dispensing of a single particle (=100 µm) from a biochip to culture well atmosphere using developed cell ejection system with the success rate of 50 %.

Cell stiffness could be an index for evaluating its activity. Although various systems measuring cell stiffness have been proposed so far, they are slow for adaptively connecting to cell sorters capable of handling more than 1000 [cells/sec]. This paper proposes a new approach that can indirectly evaluate the cell stiffness by measuring the passing time for a narrow channel. When a cell passes through the channel, it receives a viscous force depending upon how much deformation is exerted on the cell. We show that the stiffness is a function of both the passing time and the initial diameter of cell. We also show that the stiffness is proportional to the passing time and inversely proportional to the initial diameter, under the assumption that the thickness of fluid film is inversely proportional to the normal force. The experimental validation is given together with the basic working principle.

We have successfully produced emulsion droplets on a chip with size-control and on-demand droplet generation by using magnetically driven microtool (MMT) which has a parallel plate structure to be constrained in translational motion. With a lateral motion of MMT in microchannels, the continuous phase can be cut into different size of droplets and the dispersed phase flow can inflow into the microchannels by the movement of MMT to obtain both of size-controlled and on-demand droplets actively. The size range of the produced droplet was three times as large as the previous system by using novel hydraulic design of the chip, and also the dispersing the fluid was successfully prevented from leakage when it is chopped by using MMT.

This paper presents two innovative driving methodologies using magnetically driven microtools for precise cell manipulations and its automation systems. First, the magnetic analysis has been conducted to show the current MMT problem. Then, the new driving methodologies are introduced and backed up with FEM analysis and the experimental results. The positioning accuracy improves 3–10 times and the following response become 10 times higher against the driving linear stage. Using this methodology, the enucleation of oocytes is demonstrated to show the effectiveness of the method.

We succeeded in powerful noncontact actuation of magnetically driven microtool (MMT) by magnetizing it and focusing magnetic field in a microfluidic chip. Novelty of this paper is summarized as follows. (1) We employed neodium powder as the main component of MMT. The density of magnetic flux was improved about 100 times larger after magnetization. (2) We fabricated a pair of magnetic sharp needles in the chip by electroplating. MMT was placed between the needles and the density of magnetic flux was improved about 3 times larger. As a result, we succeeded in powerful actuation of MMT in a chip. Drive frequency was improved about 10 times faster (up to 180 Hz). We applied it for sorting of copolymer beads in a chip. MMT is put in the microchannel. The size of the bead was measured by image processing (15 Hz). Moving frequency of the MMT was made higher than the sampling frequency of measurement and secure sorting was achieved.

In this study, we successfully produced a functional microtool made of gel microbeads using size-dependent microparticle classification. Gel microbeads are made by salting-out hydrophilic photo-crosslinkable resin ENT-3400. These gel microbeads were separated according to their size using microfilters made of polydimethylsiloxane (PDMS). The first filter is a row of fluidic microchannels that block microbeads with a size greater than the channel's width. Another filtration method has been examined using a magnetically driven microtool (MMT) to separate the beads using the centrifugal force created by this MMT actuated with a DC motor. Separated gel microbeads were recovered after filtration and used to fabricate functional microtools, for example, a tether-shaped gel tool, by contact with other gel microbeads under UV illumination. The produced gel tool is manipulated using optical tweezers in a microchip. We successfully achieved size-dependent separation of gel microbeads and production of a tether-shaped gel tool.

We have investigated an active size controlled droplet generation system by using magnetically driven microtool (MMT). With a lateral motion of the MMT in microchannels, the continuous phase can be pinched off by the movement of MMT to obtain size-controlled droplets actively. With this method, particle-enclosed droplet can be produced on demand to fit the size of each enclosed particle, and which is difficult to carry out only by the fluid dynamic force. For the current study, the system has been evaluated in terms of the frequency of the actuation of MMT and the size of the produced droplets, and found out the response time to change the droplet size by MMT actuation is one third of that only by the fluid dynamic force. This system contributes to the effective transportation of cells in microchannel.

We have developed magnetically modified polymeric soft microactuators in microchip as replacement of conventional manual cell manipulations such as pipetting and centrifugation. In the past papers in Yamanishi, Y., et al, (2007) we have reported novel magnetically driven polymeric microtools for non-intrusive and no contamination experiments on a chip. The main features of them were 1. fabrication of any shape, 2. soft (harmless to cellls) 3. no stiction and 4. mass production at low cost enabling disposability. For the current research, we have demonstrated manipulation of oocyte with produced novel soft microactuators. These magnetic microtools provide a number of functions such as microvalve and microrotor. The potential impact of this technology includes sample preparations, selection and separation, loading and immobilization, genetic operation, tracking, mixing and reaction techniques into portable microfluidic labs-on-a-chip, culture systems.

In this paper we describe a novel fabrication method of three dimensional polymeric magnetically driven microtools (MMT) for non-intrusive and no contamination experiments on a chip. In order to obtain precise and complicated three dimensional patterns of magnetically driven 3D microtools, a grayscale photolithography technique has been applied by making good use of thick nega-photoresist as sacrificed mold. By controlling an amount of ultraviolet light with a gradation of gray tone mask, we have fabricated smoothly curved (100 mum gap) object without steps which tend to be appeared in case of conventional layer by layer photolithography techniques. A wide range of on-chip application of microactuators can be proposed by using a softness of polymer-based 3D MMT. For example, microrotor was operated as miromixer and micropump, also microloader was actuated by the magnetic and fluidic force. The surface of the 3D MMT was fabricated with a group of spiked pattern to reduce the contact area between PDMS (poly dimethyl siloxane) microchannel and PDMS-based 3D MMT. The produced 3D MMT can be applied to complicated on-chip manipulations of sensitive materials such as cells.

The paper describes a novel non-contact manipulation system using an on-chip, magnetically driven micro-tool (MMT) rather than conventional biomanipulation by hand, which has a higher risk of contamination and lower success rate and repeatability. MMTs can sort particles individually, and have the unique feature that they can be installed directly in a microchannel (width = 150 mum), unlike conventional cell sorting systems. The drive unit was significantly downsized by amplifying the magnetic power using a permanent magnet. Lower power consumption could also be realized, because no energy is required to keep MMTs stationary at a particular position. Sorting was performed using real-time sensing images of microbeads, where the system successfully sorted beads of different sizes at switching speeds up to 18 Hz. The MMT developed in this study is unique with respect to its flexibility and biocompatibility; in addition, since the PDMS (polydimethylsiloxane) microchip is disposable, it can be applied to cell sorting without any risk of contamination. Potential fields of application of this technology include cloning techniques, which require sorting of oocytes with and without nuclei.

In this paper we describe novel magnetically driven polymeric microtool for non-intrusive and no contamination experiments on a chip. The composite is formed by suspending magnetite particles (Fe3O4) in polydimethylsiloxane (PDMS). In order to obtain precise and complicated pattern of micromagnetic tools, a photolithography techniques has been applied by making good use of thick KMPR-1050 photoresist as sacrificed mould. The surface of the produced micromagnetic tools is specially coated in order to suppress stiction in the biochip. The novelties of these tools are (1) fabrication of any 2D shape, (2) softness (harmless to cells), (3) no contact actuation (no stiction), 4. mass production with low cost. Here we have demonstrated that the mass-produced versatile micromagnetic tools such as stirrer and valve. The potential impact of this technology includes sample selection and separation, cell immobilization, genetic operation, tracking, mixing and reaction techniques into portable microfluidic labs-on-a-chip, culture systems and cell loading system.