JP2009178105A - Electric cell fusion apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high-yield electric cell fusion apparatus that enables a massively parallel cell fusion. <P>SOLUTION: The electric cell fusion apparatus includes an insulating thin film (hereinafter orifice sheet) having a small hole (orifice) smaller than any diameter of two kinds of cells to be fused, a detachable insulating plate having two through-holes and placed so as to hold the orifice sheet therebetween, electrodes arranged in the through-holes, respectively, and a power source to excite the electrodes, and has a constitution that each cell suspension to be fused is introduced into the inside of each through-hole, the cells are drawn near the orifice by dielectrophoretic force by utilizing electric field concentration when a high-frequency voltage is applied to the electrodes, the two kinds of the cells are opposed on the orifice, a pulse voltage or a high-frequency modulation pulse voltage is applied to the electrodes, the cells are fused, respectively, then, the insulating plate is removed and a fused cell having a shape of two balls connected through the orifice is removed together with the orifice plate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a high-yield electric cell fusion device that enables mass parallelism.

  Conventional cell fusion technologies include those that use viruses and those that use drugs such as PEG, but these have problems with the toxicity of viruses and drugs to cells. Unlike this, electric cell fusion is purely by physical means and has no problems such as cytotoxicity, but its yield has been lower than before (for example, 1 / 10,000). This is because a conventional electric cell fusion method applies a high-frequency electric field to a cell suspension to form a cell array, and a pulse voltage is applied to the cell suspension to fuse the cell membrane at the cell contact point. This is because there was no means for applying an appropriately controlled voltage for fusion to each cell contact point in the cell suspension.

On the other hand, Non-Patent Document 1 and Non-Patent Document 2 below show that the electric field generated by the orifice is obtained by performing an electrofusion operation by combining two cells with an orifice having a size less than or equal to the cell diameter. Theoretically, it is shown that a controlled membrane voltage can be applied only in the vicinity of the contact point of two cell membranes by concentration, and the following Non-Patent Document 3 describes the inside of a fluid circuit fabricated using a microfabrication technique. It has been experimentally demonstrated that fusion operation can be performed with a yield of 90% or more if a pulse voltage is applied after placing cells with the orifice placed in between the orifices using dielectrophoresis. . In addition, it can be easily assumed that if such orifices are arranged in an array, a large number of parallel cell fusions can be performed simultaneously.
However, this method has the following problems.

  1. Since the range of dielectrophoretic force is about the diameter of the orifice, only cells in the immediate vicinity of the orifice can be attracted. Therefore, how to arrange cells with high yield in the orifice array becomes a big problem.

  2. Since the cells settle in normal culture, it is easy to bring them to the upper side of the horizontally placed orifice sheet. However, the cells placed under the orifice sheet move away from the orifice sheet due to sedimentation. Filling the lower side of the orifice sheet with a solution with a high specific gravity and floating the cells brings it to the orifice sheet, but Ficoll and other molecules used to adjust the density of the solution have problems with cytotoxicity and high viscosity.

  3. When the fluid circuit is routed, the dead volume is large, that is, not all the inserted cells can be used effectively.

4. It is not easy to sort out the fusion products obtained in the fluid circuit into those that have not been fused.
[1] Boonchai Techaumnat and Masao Washizu: "Analysis of the effects of an orifice plate on the membrane potential in electroporation and electrofusion of cells", J. Phys. D: Appl. Phys. Vol.40, p.1831-1837 ( 2007) [2] Masao Washizu and Boonchai Techaumnat: "Cell Membrane Voltage During Electrical Cell Fusion Calculated by Re-expansion Method", J. Electrostatics vol.65, p.555-561 (2007) [3] Kinya Tsuda, Murat Gel, Hidehiro Oana, Boonchai Techaumnat, Hidetoshi Kotera and Masao Washizu: "Very high yield electro cell-fusion based on field constriction at an microorifice", μ-TAS 2007, p.1375-1377 (2007 )

An object of the present invention is to provide a high-yield electric cell fusion device that enables mass parallelism.

In the present invention, the fusion device itself has a structure that can be turned upside down, and by assisting by gravity sedimentation, the cell can be easily sucked into the orifice by dielectrophoresis, and the orifice smaller than the cell diameter can be used. Only the fused cells are obtained by geometrically constraining the fused cells and taking out the orifice sheet.
"Action"
According to the present invention, it is possible to fix a large amount of cells to be fused on both surfaces of an orifice array formed of silicon rubber or the like, and to easily take out only the fused cells after the fusion operation.

According to the present invention, it is possible to realize an apparatus that makes effective use of a valuable cell sample and performs high-yield electric cell fusion in large quantities in parallel.

An insulating thin film (orifice sheet) having a small hole (orifice) smaller than the diameter of any of the two types of cells to be fused, and two removable through-holes sandwiched between them A cell-containing solution comprising an insulating plate having a portion for forming a chamber for containing a cell suspension, electrodes installed in the through-holes, and a power source for exciting the electrodes. Preferably, the upper and lower through-holes are held by surface tension or the like, and are not spilled even if they are turned upside down. A chip-formable cell fusion device that electrically fuses cells can be realized.

FIG. 1 is a schematic diagram of cell fusion according to the present invention immediately before the fusion operation. As described in Non-Patent Documents [1] to [3], this arrangement enables cell fusion with high yield using electric field concentration.
An embodiment of the present invention for realizing this will be described below.

1. Two insulating plates (upper plate 1 and lower plate 2) with through holes on both sides of orifice sheet 3 made of soft silicon rubber with a thickness of about 2μm are clamped and fixed so that they can be removed later. To do. The through-hole 4 has a function as a chamber for loading cells. The upper surface of the upper plate 1 and the lower plate 2 having the through holes 4 is made hydrophobic, and the inner surface and the orifice sheet 3 are made hydrophilic. This can be obtained, for example, by processing a through hole in a glass plate and applying a hydrophobic coating on the upper surface thereof. Electrodes 5 and 6 are respectively provided at arbitrary positions on the inner side surface of the through hole 4.

  2. First, a suspension 12 of cells 14 is filled in an upper chamber formed with the bottom surface surrounded by the orifice sheet 3 and surrounded by the upper plate 1. Since the orifice sheet 3 is hydrophilic, the solution enters the orifice without any bubbles. Since the upper surface of the plate having the through holes 4 is hydrophobic, the suspension does not ooze out here.

3. Flip the device upside down. If the chamber diameter is about 2 to 3 mm, the solution will not spill due to surface tension, but if necessary, it may be covered with a cover glass from above.
4. The suspension 13 of the cells 15 is placed from above the lower chamber formed with the bottom surface of the orifice sheet 3 and the periphery surrounded by the lower plate 2.
5. Since the cell 14 in the upper chamber sinks on the orifice plate 3, the switch 9 is switched and an AC voltage is applied from the AC voltage generator 7 to the electrodes 5 and 6 through the electrical leads 10 and 11 to generate dielectric. Pull onto the orifice by electrophoresis. If the density of the cells is sufficiently high, the cells are fixed to the through holes 4 of almost all the orifice plates 3. If the cell diameter is 5 μm and the specific gravity is 1.2, the sedimentation rate is about 10 μm / sec. Therefore, if the chamber depth is 1 mm, the time required for sedimentation is about 100 seconds.

6. Flip the device upside down again. Since the dielectrophoretic force is sufficiently strong, the cells 15 fixed to the orifice plate 3 in the previous stage are not detached from the orifice plate 3. The cells 15 in the upper chamber sink and are fixed to the orifice plate 3 by the dielectrophoretic force. The cells 14 and the already fixed cells 14 are paired with the orifice plate 3 interposed therebetween.

7. When the switch 9 is switched and a pulse is applied from the pulse generator 8, the cells in contact with the orifice plate 3 are fused at 1: 1. The surplus cells existing outside the orifice plate 3 do not fuse because the electric field becomes extremely weak when they are far from the orifice.
8. Remove the chamber, take out the orifice plate 3 and gently wash it. Since the fused cells cannot pass through the orifice, they are collected together with the orifice plate 3. Since the orifice plate 3 is made of thin silicon rubber, if the cell undergoes a movement such as division, it can be removed from the orifice sheet by deformation or destruction of the sheet.

9. By the above, only cells fused at 1: 1 can be taken out.
10. As is clear from the above process, the cell suspension of the sample only needs to have a volume sufficient to fill the through-hole, and the cells contained therein are effectively used for fusion.

Needless to say, the distance and pitch between the through-holes of the orifice need only be equal to or larger than the cell diameter, and by arranging them in a two-dimensional manner on the orifice sheet, it is needless to say that mass parallel fusion is possible.

  INDUSTRIAL APPLICABILITY The present invention is used as a high-yield electric cell fusion device that enables mass parallel fusion in cell fusion used in gene manipulation and the like.

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Explanation of symbols

1 Upper plate 2 Lower plate 3 Orifice sheet 4 Through hole

Claims (4)

  An insulating thin film (orifice sheet) having a small hole (orifice) smaller than the diameter of any of the two types of cells to be fused, and two removable through-holes sandwiched between them. Each of the cell suspensions to be fused into each through-hole, and the electrode is placed in each through-hole, and an electrode installed in each through-hole and a power source for exciting the electrode. The cell is attracted to the orifice by the dielectrophoretic force using the electric field concentration on the orifice when a high frequency voltage is applied, and after combining two types of cells on the orifice, a pulse voltage or a high frequency modulation pulse voltage is applied. Apply and fuse each cell, then remove the insulating plate and remove the fused cells with the orifice plate together with the orifice plate. An electric cell fusion device characterized by 2. An electric cell fusion device characterized in that the insulating plate having a through hole according to claim 1 is characterized in that the inner surface of the through hole is hydrophilic and the other part is hydrophobic. In the fusion device according to claim 1, after the cells in the upper through-hole are settled by gravity and adsorbed to the orifice by dielectrophoresis, the cells on the opposite side are reversed by gravity. An electric cell fusion device with a structure that can be turned upside down, with the aim of aligning cells with the orifice part by adsorbing to the orifice by dielectrophoresis with the help of sedimentation. The orifice sheet according to claim 1 is formed of a deformable soft rubber having a thickness of 10 μm or less, and the orifice sheet is deformed or broken when the cells are divided and proliferated after fusion. An electric cell fusion device characterized in that the proliferated cells can escape from the orifice sheet.

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