Hidehiro Oana

Dielectrophoretic cell trapping and parallel one-to-one fusion based on field constriction created by a micro-orifice array

Micro-orifice based cell fusion assures high-yield fusion without compromising the cell viability. This paper examines feasibility of a dielectrophoresis (DEP) assisted cell trapping method for parallel fusion with a micro-orifice array. The goal is to create viable fusants for studying postfusion cell behavior. We fabricated a microfluidic chip that contained a chamber and partition. The partition divided the chamber into two compartments and it had a number of embedded micro-orifices. The voltage applied to the electrodes located at each compartment generated an electric field distribution concentrating in micro-orifices. Cells introduced into each compartment moved toward the micro-orifice array by manipulation of hydrostatic pressure. DEP assisted trapping was used to keep the cells in micro-orifice and to establish cell to cell contact through orifice. By applying a pulse, cell fusion was initiated to form a neck between cells. The neck passing through the orifice resulted in immobilization of the fused cell pair at micro-orifice. After washing away the unfused cells, the chip was loaded to a microscope with stage top incubator for time lapse imaging of the selected fusants. The viable fusants were successfully generated by fusion of mouse fibroblast cells (L929). Time lapse observation of the fusants showed that fused cell pairs escaping from micro-orifice became one tetraploid cell. The generated tetraploid cells divided into three daughter cells. The fusants generated with a smaller micro-orifice (diameter approximately 2 mum) were kept immobilized at micro-orifice until cell division phase. After observation of two synchronized cell divisions, the fusant divided into four daughter cells. We conclude that the presented method of cell pairing and fusion is suitable for high-yield generation of viable fusants and furthermore, subsequent study of postfusion phenomena.

Microorifice-Based High-Yield Cell Fusion on Microfluidic Chip: Electrofusion of Selected Pairs and Fusant Viability

Microorifice-based fusion makes use of electric field constriction to assure high-yield one-to-one fusion of selected cell pairs. The aim of this paper is to verify feasibility of high-yield cell fusion on a microfluidic chip. This paper also examines viability of the fusant created on the chip. We fabricated a microfluidic chip to fuse selected cell pairs and to study postfusion behavior. We used a self-forming meniscus-based fabrication process to create microorifice with a diameter of 2-10 ¿m on the vertical walls in a microfluidic channel. When 1 MHz was applied to electrodes located on both sides of the microorifice, dielectrophoretic force attracted the cells toward microorifice to form a cell pair. Once the cells get into contact, fusion pulse was applied. Real time imaging of cells during fusion and cytoplasmic dye transfer between cells indicated success of cell fusion. We found that when high frequency voltage for dielectrophoresis was swept from 1 MHz to 10 kHz in 100 ¿s, cell fusion was initiated. The effective electric field strength was 0.1-0.2 kV/cm. We analyzed viability by imaging fusant going into cell division phase after 48 h of incubation. We conclude that fabricated microfluidic chip is suitable for high-yield one-to-one fusion and creation of viable fusants. This technology should be a useful tool to study fusion phenomena and viability of fusants, as it allows imaging of the cells during and after the fusion.

Spontaneous Formation of Giant Unilamellar Vesicles from Microdroplets of a Polyion Complex by Thermally Induced Phase Separation

Water pump: Polyion complex (PIC) vesicles are spontaneously formed from PIC microdroplets, which are formed by mixing cationic and anionic polymers (see picture). The formation process can be reversibly controlled by local heating with a focused infrared laser that triggers microphase separation and subsequent water influx. The size of the resulting giant unilamellar vesicles is determined by the initial size of the PIC droplets.

Electroporation through a micro-fabricated orifice and its application to the measurement of cell response to external stimuli

A device is developed for low-voltage electroporation using field constriction at a micro-orifice, and the application to the real-time measurement of single cell response is demonstrated. The device consists of a pair of electrodes separated by an insulator film having a regularly arranged array of micro-fabricated orifices with a typical diameter of 1?2 ?m. Cells are immobilized at the orifices by aspiration, and a pulse voltage is applied. The field lines, being unable to penetrate the insulator, go into the orifices and create a field constriction. This means that most voltage drop occurs in the vicinity of the orifice, and is imposed locally on the membrane in contact with the orifice. Hence, electroporation can be achieved regardless of the cell size, shape or orientation. The experimental verification is made with human monocytes, and uptake of a fluorescence dye is observed with pulses as low as 1 V, and almost 100% yield is achieved at 1.5 V. Then the dynamic response of a myocyte to external stimuli is measured. When the substrates for the metabolic cycle are fed by the method, a clear increase in fluorescence emission from the resultant NADH is observed.

Dielectrophoresis‐assisted massively parallel cell pairing and fusion based on field constriction created by a micro‐orifice array sheet

In this paper, we present a novel electrofusion device that enables massive parallelism, using an electrically insulating sheet having a two‐dimensional micro‐orifice array. The sheet is sandwiched by a pair of micro‐chambers with immersed electrodes, and each chamber is filled with the suspensions of the two types of cells to be fused. Dielectrophoresis, assisted by sedimentation, is used to position the cells in the upper chamber down onto the orifices, then the device is flipped over to position the cells on the other side, so that cell pairs making contact in the orifice are formed. When a pulse voltage is applied to the electrodes, most voltage drop occurs around the orifice and impressed on the cell membrane in the orifice. This makes possible the application of size‐independent voltage to fuse two cells in contact at all orifices exclusively in 1:1 manner. In the experiment, cytoplasm of one of the cells is stained with a fluorescence dye, and the transfer of the fluorescence to the other cell is used as the indication of fusion events. The two‐dimensional orifice arrangement at the pitch of 50 μm realizes simultaneous fusion of 6×103 cells on a 4 mm diameter chip, and the fusion yield of 78–90% is achieved for various sizes and types of cells.

On-site manipulation of single whole-genome DNA molecules using optical tweezers

In this letter, we describe a noninvasive methodology for manipulating single Mb-size whole-genome DNA molecules. Cells were subjected to osmotic shock and the genome DNA released from the burst cells was transferred to a region of higher salt concentration using optical tweezers. The transferred genome DNA exhibits a conformational transition from a compact state into an elongated state, accompanied by the change in its environment. The applicability of optical tweezers to the on-site manipulation of giant genome DNA is suggested, i.e., lab-on-a-plate.

Folding transition of large DNA completely inhibits the action of a restriction endonuclease as revealed by single-chain observation

The biochemical characteristics of lambda DNA chains in folded/unfolded states upon cleavage by the restriction enzyme ApaLI were investigated in the presence of spermine. These characteristics of DNA chains depending on their higher‐order structure were studied at the single‐molecule level using fluorescence microscopy. With a low concentration of spermine, lambda DNA takes a random coiled conformation and allows digestion by the enzyme, while under a high concentration of spermine, lambda DNA takes a compact folded structure and inhibits such attack. Together with comparative experiments on short oligomeric DNA, our results suggest that the transition in the higher‐order structure causes on/off‐type switching of sensitivity to the enzyme.

Self-oscillating polymer chain

Abstract We report a novel rhythmic phenomenon of a single polymer chain between the elongated coil state and the folded compact state under thermodynamically open conditions. It is shown that a T4 DNA molecule (166 kbp) in poly(ethylene glycol) (PEG) solution exhibits rhythmic conformational change at the focus of a continuous wave (CW) Nd:YAG laser beam (1064 nm), where the laser beam plays dual roles: trapping a molecular chain and making a temperature gradient around the focus. The conformational oscillation is discussed in terms of the limit-cycle oscillation of a single polymer chain driven through the dissipation of the photon energy.

High-yield electrofusion of biological cells based on field tailoring by microfabricated structures

The authors present the use of electric-field constriction created by a microfabricated structure to realise high-yield electrofusion of biological cells. The method uses an orifice on an electrically insulating wall (orifice plate) whose diameter is as small as that of the cells. Owing to the field constriction created by the orifice, we can induce the controlled magnitude of membrane voltage selectively around the contact point, regardless of the cell size. The field constriction also ensures 1:1 fusion even when more than two cells are forming a chain at the orifice. A device for electrofusion has been made with a standard SU-8 lithography and PDMS molding, and real-time observation of the electrofusion process is made. Experiments using plant protoplasts or mammalian cells show that the process is highly reproducible, and the yield higher than 90% is achieved.

Dynamics of a DNA molecule hanging over an obstacle in gel electrophoresis

Direct observation of a DNA molecule in gel electrophoresis indicates that the DNA repeats a stretch-contraction cycle every time when it is hooked by a gel fiber. The dynamics of this process is analyzed for a simple situation that a DNA is hooked by a single obstacle. It is shown that in the weak field limit, the characteristic time of the stretch-contraction process is independent of the electric field, while in the strong field limit, it is proportional to L / v 0 , where L is the contour length of DNA and v 0 is the migration velocity of DNA in free space. The experimental result of Oana e t a l . (submitted to Macromolecules) is interpreted by this result.