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 microm 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 micros, 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.

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.

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.

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Study on the discrete dielectrophoresis for particle-cell separationThis paper presents the application of the discrete dielectrophoretic force to separate polystyrene particles from red blood cells. The separation process employs a simple microfluidic device that is composed of interdigitated electrodes and a microchannel. The discrete dielectrophoretic force is generated by adjusting the duty cycle of the applied voltage. The electrodes make a tilt angle with the microchannel to change the moving direction of the red blood cells. By adjusting the voltage magnitude and duty cycle, we investigate the deflection of red blood cells and the variation of cell velocity along electrode edge under positive dielectrophoresis. The experiments with polystyrene particles show that the enrichment of the particles is greater than 150 times. The maximum separation efficiency is 97% for particle-to-cell number ratio equal to 1:2000 in the sample having high cell concentration. Using the appropriate applied voltage magnitude and duty cycle, the discrete dielectrophoretic force can prevent the clogging of microchannel while successfully separating the particles from the cells with high enrichment and efficiency. The proposed principle can be readily applied to dielectrophoresis-based devices for biomedical sample preparation or diagnosis such as the separation of rare or infected cells from a blood sample.

This paper presents a numerical analysis of the membrane voltage induced on biological cells, under the influence of an externally applied field in such processes as electroporation or electrofusion. We focus on the configurations in which an insulator plate with an orifice is used and study the cases of (a) a cell placed on the orifice or (b) two cells in contact at the orifice, when a stepwise voltage is applied across the plate. Formulation based on the boundary element method is made assuming that a biological membrane is an infinitesimally thin insulator. Results of the calculation show that, due to the field constriction created by the orifice plate, almost all the externally applied voltage is imposed on the membrane at the orifice, and the membrane voltage outside the orifice is virtually zero; field tailoring with the use of the orifice plate enables control over the magnitude and localization of the membrane voltage. It is also shown that we can induce breakdown exclusively around the contact point of a pair of cells for high-yield electrofusion with such a geometry.

This article presents an analytical method for calculating electric field and dielectrophoretic force in three-dimensional arrangements of spherical particles. The analytical method is based on the method of images that utilizes the multipole re-expansion and the fundamental solutions for several arrangements of a multipole. It is capable of calculating electric field for various conditions of particles and energization. The method needs much less memory than the already proposed one. The calculation results show that force on a dielectric particle chain in a dielectric fluid depends on the number of particles and the chain direction. However, the maximal attractive and repulsive forces reach their saturation values at about 32 and 12 particles, respectively. When the lower particle of a two-particle chain is in contact with a plate electrode, the dielectrophoretic force on the chain becomes higher on the whole, and it always attracts the chain to the electrode. As a result, the particle chain is stabilized for a wider range of the angle between the chain and the applied field. Neglecting the interaction between the electrodes and particles usually gives adequate accuracy in the force calculation, unless the electrodes are not very close to particles.

SUMMARYThis paper describes a triangular surface charge method (TSCM) called (3,1)-TSCM, which uses curved surface elements for calculating electric fields in composite dielectrics. The boundary element utilizes a cubic shape function with nine degrees of freedom and a linear function for representing the charge density on its surface. Conventional SCMs, including the (3,1)-TSCM, show a very large relative error in the composite dielectrics where the permittivity is much higher in one medium than in the other. A modified method called the E method can suppress such relative errors, which expresses electric fields by surface charges without subtraction causing large relative errors. We have applied the E method to the (3,1)-TSCM and calculated electric fields for a spherical dielectric under a uniform field. The calculated results show that the (3,1)-TSCM improves the accuracy of the electric field by more than one order compared with the method using flat surface elements with constant charge density on each element. Furthermore, the E method completely suppresses the divergence of relative errors even when the ratio of the permittivity of two media reaches 10