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

Techaumnat, Boonchai; Tsuda, Kazuhiko; Kurosawa, Osamu; Murat, Gaël; Oana, Hidehiro; Washizu, Masao

doi:10.1049/iet-nbt:20080008

Cited by 26 publications

(20 citation statements)

References 8 publications

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“…Owing to a short distance between two counter microelectrodes, a low electric voltage is sufficient to generate a high electric field for cell fusion, which could decrease the manufacturing difficulty and the cost of the power supply and also improve the safety and reliability of the electrofusion. However, one or several electrode pairs were commonly integrated in these devices in order to avoid cumbersome fabrication and obtain accurate control ability [11][12][13][14][15][16]. Thus, the electrofusion efficiencies in these devices were too low for practical applications.…”

Section: Introductionmentioning

confidence: 99%

A high‐throughput dielectrophoresis‐based cell electrofusion microfluidic device

Yang

Yin

et al. 2011

Electrophoresis

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A high-throughput cell electrofusion microfluidic chip has been designed, fabricated on a silicon-on-insulator wafer and tested for in vitro cell fusion under a low applied voltage. The developed chip consists of six individual straight microchannels with a 40-μm thickness conductive highly doped Si layer as the microchannel wall. In each microchannel, there are 75 pairs of counter protruding microelectrodes, between which the cell electrofusion is performed. The entire highly doped Si layer is covered by a 2-μm thickness aluminum film to maintain a consistent electric field between different protruding microelectrode pairs. A 150-nm thickness SiO₂ film is subsequently deposited on the top face of each protruding microelectrode for better biocompatibility. Owing to the short distance between two counter protruding microelectrodes, a high electric field can be generated for cell electrofusion with a low voltage imposed across the electrodes. Both mammalian cells and plant protoplasts were used to test the cell electrofusion. About 42-68% cells were aligned to form cell-cell pairs by the dielectrophoretic force. After cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroporation and electrofusion signals. The averaged fusion efficiency in the paired cells is above 40% (the highest was about 60%), which is much higher than the traditional polyethylene glycol method (<5%) and traditional electrofusion methods (∼12%). An individual cell electrofusion process could be completed within 10 min, indicating a capability of high throughput.

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Section: Introductionmentioning

confidence: 99%

A high‐throughput dielectrophoresis‐based cell electrofusion microfluidic device

Yang

Yin

et al. 2011

Electrophoresis

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“…To solve the problem, our group has developed a highyield electrofusion method based on field constriction at a micro-orifice [12][13][14], as schematically depicted in Fig. 1B.…”

Section: -10mentioning

confidence: 99%

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

et al. 2011

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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.

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“…16,[20][21][22][23][24] The last two methods are very efficient for cell pairing and electrofusion but are both coupled with mechanical handling (fluidic and mechanic in the case of Ref. 9, electric and mechanic in the case of Refs.…”

Section: Introductionmentioning

confidence: 99%

Microarray of non-connected gold pads used as high density electric traps for parallelized pairing and fusion of cells

et al. 2013

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Cell fusion consists of inducing the formation of a hybridoma cell containing the genetic properties of the progenitor cells. Such an operation is usually performed chemically or electrically. The latter method, named electrofusion, is considered as having a strong potential, due to its efficiency and non-toxicity, but deserves further investigations prior to being applicable for key applications like antibody production and cancer immunotherapy. Indeed, to envision such applications, a high amount of hybrid cells is needed. In this context, we present in this paper a device for massive cell pairing and electrofusion, using a microarray of nonconnected conductive pads. The electrofusion chamber--or channel--exposes cells to an inhomogeneous electric field, caused by the pads array, enabling the trapping and pairing of cells with dielectrophoresis (DEP) forces prior to electrofusion. Compared to a mechanical trapping, such electric trapping is fully reversible (on/ off handling). The DEP force is contactless and thus eases the release of the produced hybridoma. Moreover, the absence of wire connections on the pads permits the high density trapping and electrofusion of cells. In this paper, the electric field mapping, the effect of metallic pads thickness, and the transmembrane potential of cells are studied based on a numerical model to optimize the device. Electric calculations and experiments were conducted to evaluate the trapping force. The structure was finally validated for cell pairing and electrofusion of arrays of cells. We believe that our approach of fully electric trapping with a simple structure is a promising method for massive production of electrofused hybridoma. V C 2013 AIP Publishing LLC. [http://dx

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

Cited by 26 publications

References 8 publications

A high‐throughput dielectrophoresis‐based cell electrofusion microfluidic device

A high‐throughput dielectrophoresis‐based cell electrofusion microfluidic device

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

Microarray of non-connected gold pads used as high density electric traps for parallelized pairing and fusion of cells

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