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.

An automatic system for evaluating single cell viscoelasticity is proposed and tested in this paper. The system includes three main operations, and they are the operations of catch, load and launch. In the catch operation, the system is capable of capturing a target cell by high-speed vision and high-frequency flow control. The captured cell is pushed into a narrow constriction for the load operation. Different durations of loading time are applied to cells for evaluating cell viscoelasticity. Finally, the cell is launched out from the constriction, and the recovery response of the cell is monitored for cell characterization. Human red blood cells are experimentally tested by the proposed system. The experimental results show that the system successfully perform the evaluation, and the viscoelastic characteristics of the cells are discussed.

Monitoring of biosignals plays important roles in health management in daily life. Especially, the sensing method of biosignals, which does not require special efforts such as restraining people or wearing the sensors to measure them, is really important to maintain monitoring activities of people. We call such method what a sensing way should be for monitoring of biosignals as casual sensing methods. Previously, we have developed highly sensitive and wide-measurement-range load sensors based on measuring frequency shift of quartz crystal resonator (QCR) as the sensing principal. We have integrated the load sensor into a chair, and measure the load when people just sit on it. Since the load sensor has wide measurement range of 105order, multi-biosignal; heartbeat, respiration, and body motion under the weight-loaded environment can be measured from load information. Thus, people can casually monitor the biosignals by sitting on the chair. However, the previous sensing system required us to measure the frequency shift of one-Hz order in tens-MHz of the resonant frequency of a QCR. In this case, the sensing system requires a expensive frequency counter, and it is not suitable for daily use situation such as in-home sensing. In this paper, we presents the detection of multi-biosignal method using a newly developed load sensing system which utilizes the developed frequency counter unit. In order to measure the frequency shift of one-Hz order in tens-MHz of the resonant frequency, we use a differential method of signal for two QCRs with an electrical signal-mixing circuit. By using the method, we can reduce the required measurement range for frequency from tens-MHz to tens-kHz. The load sensing performances were evaluated, and the results showed that the sensitivity and withstand load were 2.9 [mN] and 300 [N], respectively. Finally, we demonstrated the measurement of multi-biosignal by using constructed system, and succeeded in detecting respiration, heartbeat, and body motion.

We present a super wide-measurement range load sensor with high-durability for multi-biosignal sensing in daily life. We employed quartz crystal resonator (QCR) load sensor with super wide range (from 6 × 10-5to 30 [N]) for sensing of biosignals ranging from mN order (e.g. heartbeat) to 100 N order (e.g. body motion). Furthermore, we implemented an outer case which makes the sensor durable against unexpected loads in practical use. The outer case increased the durability of the sensor 88 times higher than the sensor without outer case. Finally we succeeded in simultaneous sensing of heartbeat (≈ 100 [mN]) and body motion (≈ 25 [N]) with identical sensor.

This paper proposes the method for mechanical characterization of floating cells on a microfluidic chip. We measured the reactive force of cells on the chip by applying mechanical deformation to the cells. Particularly we focused on the method to improve the sensing resolution of the probe position by using the phase detection of moiré fringe. This method realized approximately ten times higher resolution compared to the previous method. The detailed data suggest some unique characteristics of cells. We discussed the phenomenon seen on the data with a mechanical model, which is based on Heltzian contact theory in the light of stiff nucleus.

Cyanobacteria have ion channels which response to osmolarity change in living environment. Since it may lead to the mechanical characteristics changes of the cell itself due to the intracellular pressure changes, we focus on constructing the mechanical characteristics measurement system of Synechocystis sp. PCC 6803, one of unicellular cyanobacteria. There are two important issues in constructing the system. Because of features of the cyanobacteria, which is a kind of floating cell whose diameter is a few micrometer, one is how to transport the cell to the measurement point. The other is how to deform the target cell. In this paper, we propose the system combining optical tweezers for transportation of the target cell with the robot integrated microfluidic chip (robochip). The robochip contains an on-chip probe actuated by an external actuator, and a force sensor. We present the concept of the total system, and demonstrated the results of mechanical characterization of a single cyanobacteria.

We present a novel cell manipulation method based on vibration-induced flow in open-chip environment. By applying circular vibration to a chip having micropillar patterns on its surface, local whirling flow is induced around the micropillars. Therefore, by patterning micropillar array on a chip and applying the circular vibration to the chip, we can achieve cell transportation along the array in an open chip environment. We theoretically analyzed this phenomenon with a single micropillar, and experimentally evaluated behaviours of manipulated microbeads with two micropillars. We confirm there are different manipulation modes according to the pitch of micropillars. We designed the micropillar array for an oocyte transportation based on these evaluations. We succeeded in the transportation of oocytes with up to 35.6 μm/s and confirmed that the velocity can be changed by changing the frequency and amplitude of the applied vibration.

We have developed a highly functional untethered micro-robot that can manipulate cells with high gripping force in a micro-fluidic chip. The robot has gripping mechanism for grasping and transportation of cells with sufficient power. A permanent magnet is attached at the center of the gripping mechanism, and an electrical magnet controls the position of the magnet from the bottom of the micro-fluidic chip. The distance accuracy of the gripper is 3.0 μm, and it can activate about 50 Hz. The robot has four permanent magnets for positioning, and other four permanent magnets control the robot from the bottom of the micro-fluidic chip. The robot is made very thin and small to allow maneuverability in a micro-fluidic chip. We succeeded in high-power handling and cutting of a cell using this micro-robot.

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 discusses the observability of cell stiffness by utilizing a micro-channel whose diameter is slightly smaller than that of a cell. By modeling each cell with distributed springs and dampers, we discuss how many sensing points are necessary and sufficient for evaluating cell stiffness. We define the observability by the velocity of each cell through a micro-channel based upon the experimental observation that the equilibrium velocity of a cell varies corresponding to its stiffness. We conclude that for evaluating cell stiffness, two points are necessary and sufficient while using an infinitely long channel, and three are necessary and sufficient for judging whether cell stiffness is observable or not. This study attempts to provide a practical guidance for developing a simpler, faster, low-cost sensor in the future.

This paper presents a novel measurement system of cellular mechanical properties based on a robot integrated microfluidic chip. In order to achieve the high throughput measurement of cellular mechacal properties, we proposed the robot integrated microfluidic chip (robochip), taking advantages of both of micromechanical manipulator and Lab-on-a-Chip devices. The robochip contained a pair of a magnetically driven on-chip robotic probe and a force sensor. The characterization system based on the robochip performed by the visual feedback control, and the continuous cell measurement was demonstrated. The throughput of our system was 15 to 20 seconds per one oocyte. Moreover, the measurement of the viscoelastic properties were demonstrated as a quality evaluation of oocyte. Experimental results shows that the oocyte has the viscoelastic properties among the same culture condition, and it is important to analyze the mechanical properties of oocyte for the evaluation of the quality. From these results, we concluded that the high through put cellular mechanical characterization was achieved, and our robochip approach was a promising technique for a cellular characterization because the chip part was disposable.

A new concept of μ-cell fatigue test is proposed. By reciprocating a cell across the throat of a micro channel repeatedly, the dynamic deformation behavior of the cell is measured. We define a new index of fatigue characteristics of cells as the number of reciprocatory motion leading to a prescribed recovery ratio. The test system is composed of a piezoelectric (PZT) actuator, a high speed vision sensor and a micro channel with a throat. Preliminary experiments were conducted by using Red Blood Cells (RBCs). The result suggested that the activation level of a cell can be evaluated based on its fatigue characteristics.

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.

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.