JP2007295922A - Cell fusion device and method for cell fusion using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell fusion device for efficiently carrying out a cell fusion with a pair of two types of cells, and to provide a method for the cell fusion using the device. <P>SOLUTION: The cell fusion device includes a pair of electrodes formed by an electrically conducting member and disposed opposite to each other in the cell fusion region in which the cell fusion is carried out, a cell fusion container formed by flat plate-like electrical insulators disposed via a flat plate-like spacer in between the pair of electrodes and provided with one or more fine pores penetrated in the direction of the electrodes disposed opposite to each other, and an electric source for applying an alternating voltage to the pair of electrodes. The method for the cell fusion using the device comprises the following practice: A first cell is introduced into the cell fusion region followed by application of an alternating voltage with the above-mentioned waveform to immobilize the first cell in the fine pore(s). Thereafter, a second cell is introduced into the cell fusion region followed by application of an alternating voltage with the above-mentioned waveform to contact the second cell with the first cell at the position of the fine pore(s) to accomplish the objective cell fusion chemically. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell fusion apparatus for efficiently performing cell fusion and a cell fusion method using the same.

  Conventionally, a chemical fusion method mainly using polyethylene glycol (PEG) has been used as a cell fusion technique in which different cells are fused to form one hybrid cell. In this method, (i) PEG is added to cells. (Ii) It takes time and effort to find the optimum conditions such as the degree of polymerization of PEG and the amount added, and (iii) Advanced technology is required for the fusion. Can only be used by those skilled in the art, (iv) 2 cell contact is accidental, and it is difficult to control cell fusion with a pair of 2 cells, so the cell fusion probability is extremely low, etc. was there.

  In order to solve the above problems, a method has been reported that combines a chemical fusion method using PEG and an electric cell fusion method in which cells are aligned and fused by applying a voltage (for example, Patent Document 1). The electric cell fusion method has the advantage that it does not require advanced technology, can be easily and efficiently fused, has no toxicity to cells, and can fuse cells with high activity. is there.

  The electric cell fusion method was established by Zimmermann in West Germany in 1981, and the principle is as follows. That is, when an alternating voltage is applied between parallel electrodes and cells are introduced therein, the cells are attracted toward the higher current density and form a bead shape. A state in which cells are arranged in a bead shape is generally called a pearl chain. In this state, by applying a pulse voltage of several μsec to several tens of μsec between the electrodes, the electric conductivity of the cell membrane is instantaneously lowered, and the reversible disturbance of the cell membrane constituted by the lipid bilayer and its reconfiguration are performed. Done, and as a result, cell fusion occurs. In the method described in Patent Document 1, an attempt is made to arrange cells in an orderly manner by introducing a solvent of PEG containing cells between the electrodes and applying an alternating voltage between the electrodes to arrange the cells in a bead shape. ing.

  However, although the cells are arranged in a beaded manner, the order of the cells cannot be controlled. Therefore, although the chemical fusion method is improved more than when used alone, the contact between the two cells is still incidental, and the two cells There has been a problem that it is difficult to reliably control cell fusion in a pair, and further improvement has been desired.

JP 60-9490 A

  An object of the present invention has been proposed in view of the conventional situation, and an object of the present invention is to provide a cell fusion device that efficiently performs cell fusion in a pair of two cells and a cell fusion method using the same. To do.

  The present invention, as a solution to the above problems, comprises a pair of electrodes made of conductive members arranged so as to face each other in the cell fusion region, and an insulator formed with fine holes penetrating in the direction of the pair of electrodes, A cell fusion container for chemically fusing cells, wherein the surface of the insulator is hydrophilic, and the insulator is disposed on the electrode surface on the cell fusion region side of one of the electrodes. A planar cell shape of the micropore has a shape having at least one or more corners and an AC power source for applying an AC voltage to the pair of electrodes, and the AC power source is connected between the electrodes. The waveform of the alternating voltage to be applied is a waveform in which charging and discharging of the cells are periodically repeated, and a first cell is introduced into the cell fusion region using the cell fusion device, and the waveform Having sex After fixing the first cell in the micropore by applying a voltage, the second cell is introduced into the cell fusion region, and the first voltage is applied by applying an AC voltage having the waveform. It is found that the above-mentioned problems of the prior art can be solved by using a cell fusion method characterized in that the cell is chemically fused by bringing the second cell into contact with the cell at the position of the micropore. Finally, the present invention has been completed. Hereinafter, the present invention will be described in detail.

  The present invention relates to a pair of electrodes made of conductive members arranged opposite to each other in a cell fusion region, and a plate-like spacer arranged between the pair of electrodes, and the electrodes arranged opposite to each other A cell fusion device comprising a cell fusion container made of a flat insulator having one or more fine holes penetrating in a direction, and a power source for applying an alternating voltage to the pair of electrodes.

  The present invention also provides a cell fusion device in which the micropores formed in the insulator described above have a shape capable of fixing one cell per micropore.

  The present invention is also a cell fusion device in which an AC voltage having a waveform for fixing one cell to one micropore is applied between the electrodes by the power source described above.

  In addition, the present invention is a cell fusion device in which an AC voltage having a waveform in which charging and discharging of the fine particles are periodically repeated is applied between the electrodes by the power source described above.

  Further, the present invention is the cell fusion device, wherein the waveform of the AC voltage described above is a waveform having at least one time in a half cycle in which a voltage having a value other than 0 does not change for a certain period of time.

  In addition, the present invention is a cell fusion device in which the waveform of the AC voltage described above is a rectangular wave, a trapezoidal wave, or a combination of these.

  The present invention also provides a time constant in which the time when the voltage having a value other than 0 does not change for a certain period of time is a product of the capacitance of the cell and the resistance of the cell suspension containing the cell. This is the cell fusion device as described above.

  In the present invention, the diameter of the maximum circle inscribed in the planar shape of the micropore is in the range of 1 to less than twice the diameter of the cell fixed to the micropore, and the depth of the micropore is fixed to the micropore. The cell fusion device according to the above, wherein the cell fusion device has a diameter less than or equal to a cell diameter.

  In addition, the present invention is a cell fusion device in which the insulator described above is disposed on the electrode surface on the cell fusion region side of one of the electrodes.

  Further, the present invention is a cell fusion device in which a plurality of micropores formed in the insulator described above are formed in the same position on the surface of the insulator, the positions of micropores adjacent to any micropore. is there.

  The present invention also provides a cell fusion device in which a plurality of micropores formed in the insulator described above are formed in an array on the surface of the insulator.

  In addition, the present invention is a cell fusion device in which the interval between the micropores described above is in the range of 0.5 to 6 times the diameter of the cells to be inserted into the micropores.

  In addition, the present invention is a cell fusion device in which the spacer described above has a through-hole forming a cell fusion region.

  The present invention also provides a cell fusion device in which the spacer described above has an introduction channel for introducing cells and a discharge channel for discharging cells.

The present invention also provides a cell fusion device characterized in that the insulator described above is hydrophilic.
The present invention also provides a cell fusion device, wherein the planar shape of the micropore described above is a shape having one or more corners.
The present invention also provides a cell fusion device characterized in that the planar shape of the micropore described above is a quadrilateral.

The present invention is also a cell fusion method using the above-described cell fusion device, wherein the first cell is introduced into the cell fusion region and an alternating voltage is applied to the first cell in the micropore. After fixing the cell, the second cell is introduced into the cell fusion region, and an AC voltage is applied to bring the second cell into contact with the first cell at the position of the micropore to chemically fuse the cell. Cell fusion method.
The present invention is also the above cell fusion method, wherein the waveform of the AC voltage is an AC voltage having the waveform described above.

  The present invention is also a cell fusion method in which the cells described above are placed in a cell suspension to which a substance that enhances the fluidity of the cell membrane is added.

  The present invention is also the cell fusion method, wherein the substance that enhances the fluidity of the cell membrane described above is polyethylene glycol.

  Hereinafter, the cell fusion device of the present invention will be described in more detail with reference to the drawings.

  The cell fusion device of the present invention includes a pair of electrodes made of a conductive member arranged to face each other in a cell fusion region, a flat spacer disposed between the pair of electrodes, and the face to face A cell fusion device comprising: a cell fusion container made of a flat insulator having one or more fine holes penetrating in the direction of the electrodes formed; and a power source for applying an AC voltage to the pair of electrodes. Preferably, the cell fusion container is a cell fusion device provided with a container for chemically fusing cells.

  Here, chemically fusing cells means that cells are fused in a suspension added with a chemical substance that increases the fluidity of the cell membrane, such as polyethylene glycol, and generally the fluidity of the cell membrane is low. This is one type of cell fusion utilizing the fact that membrane fusion occurs easily when cells come into contact in an elevated state.

  The present invention is also the cell fusion method, wherein the substance that enhances the fluidity of the cell membrane described above is polyethylene glycol.

  FIG. 1 shows a conceptual diagram of the cell fusion device of the present invention. The cell fusion device of the present invention is roughly divided into a cell fusion container (13) and a power source (4). As shown in FIG. 1, in the cell fusion container, the insulator (8) in which the spacer (16) is arranged between the upper electrode (14) and the lower electrode (15) and the micropore (9) is formed is used as the lower electrode. It has a structure arranged on the cell fusion region side.

  The material of the upper electrode and the lower electrode is not particularly limited as long as it is a conductive member and is a chemically stable member. An alloy such as platinum, gold, copper or an alloy such as stainless steel or ITO (Indium Tin Oxide) Although a glass substrate on which a transparent conductive material such as tin) is formed may be used, in order to observe cell fusion, it is preferable to use a glass substrate on which a transparent conductive material such as ITO is formed as an electrode. The area of the upper electrode and the lower electrode is not particularly limited, but as a size that is easy to handle, for example, a size of about 70 mm long × 40 mm wide × 1 mm thick is preferable.

  The spacer is provided so that the upper electrode and the lower electrode are not in direct contact with each other, and has a through-hole that forms a cell fusion region for securing a space for storing the cell suspension in the cell fusion container The material may be any insulating material, such as glass, ceramic, resin, and the like. In addition, in the spacer, in order to introduce and discharge cells to and from the cell fusion container, an introduction flow path for introducing cells, an introduction port (19) communicating with the cells, a discharge flow path for discharging cells and a discharge port (20) communicating therewith ) May be provided. The size of the spacer is not particularly limited as long as the upper electrode and the lower electrode are not in contact with each other, but a size matched to the electrode is preferable. For example, if the electrode size is about 70 mm long × 40 mm wide, the spacer size is preferably about 40 mm long × 40 mm wide, for example. The space and thickness inside the spacer forming the cell fusion region (1) are not particularly limited, but it is sufficient that the cell suspension has a capacity for storing several μL to several mL, for example, the spacer size is 40 mm in length. X In the case of about 40 mm in width, the space inside the spacer may be about 20 mm in length x about 20 mm in width, and the thickness of the spacer may be about 0.5 to 2.0 mm.

  A fine hole (9) is formed in the insulator (8). The material of the insulator (8) is not particularly limited as long as it is an insulating material such as glass, ceramic, resin, etc. However, since it is necessary to form through holes, it is relatively easy to process the resin or the like. Material is preferred.

  In addition, the material of the insulator is preferably a material having affinity with the cell because the cell is attracted and fixed to the micropores formed in the insulator, and generally the surface of the cell is hydrophilic. It is preferable that the surface of the insulator is hydrophilic. Here, hydrophilic means that the affinity of the cell suspension or pure water used for cell fusion is high, and generally the suspension or pure water is applied to the surface of the insulator. Indicated by the contact angle between the droplet formed when dropped and the surface of the insulator (the smaller the contact angle, the higher the affinity between the surface of the insulator and the cell suspension or pure water, that is, the cell High affinity). The cell suspension is an aqueous solution of saccharides such as mannitol, glucose and sucrose, an electrolyte such as calcium chloride and magnesium chloride, a protein such as BSA (bovine serum albumin), and a cell membrane such as polyethylene glycol. This means a suspension containing cells to be fused with an aqueous solution containing a chemical substance that enhances the fluidity of the cells. Examples of the insulating film having relatively high hydrophilicity include glass and titanium oxide, and these materials may be used as an insulator. Alternatively, when a low hydrophilic material such as a resin is used for the insulator, the surface of the insulator may be modified to be hydrophilic by performing a hydrophilic treatment. Here, as a method for hydrophilizing the surface of the insulator, plasma treatment, chemical modification, modification by protein physical adsorption, or any combination of these methods may be used.

Here, the plasma treatment of the surface of the insulator means that the surface of the insulator is irradiated with an electrically neutral ionized gas (plasma) in which active species such as electrons, ions, and radicals are present. This is a process for modifying the surface of the insulator by removing organic contaminants on the surface and changing the chemical bonding state. Plasma treatment includes plasma surface treatment using a non-polymerizable gas (Ar, O _2, etc.) and plasma polymerization in which an insulator surface is coated with a polymer thin film using an organic monomer. Plasma surface treatment includes formation of a surface cross-linked layer with a non-reactive gas such as Ar, introduction of a functional group with a reactive gas such as O _2, etc., and —COOH or —CO is introduced by oxygen plasma treatment, and an insulator It is possible to increase the affinity with cells by improving the hydrophilicity of the surface.

  Further, the chemical modification of the surface of the insulator is a method of making the surface hydrophilic by bonding a derivative having a hydrophilic group such as a hydroxyl group, a carboxyl group, an amino group or a sulfone group or a silane coupling agent to the surface of the insulator. is there. A silane coupling agent is a compound composed of an organic substance and silicon, and has a reactive group (hydroxyl group, carboxyl group, amino group, sulfone group, etc.) having hydrophilicity in the molecule and a reactive group (vinyl group, (Methyl group, ethyl group, propyl group, etc.) having two or more different reactive groups. Therefore, if a hydrophobic insulator is immersed in a dilute solution of a silane coupling agent, the reactive group exhibiting hydrophobicity is chemically bonded to the surface of the hydrophobic insulator, and the reactive group exhibiting hydrophilicity is the insulator. Since the surface of the insulator is covered, the surface of the insulator can be uniformly hydrophilized.

  Furthermore, the surface of the insulator can be hydrophilized by physically adsorbing the protein by immersing the insulator in a protein-containing solution such as BSA (bovine serum albumin) for several minutes to several hours.

  Moreover, as a hydrophilicity evaluation method, the general method described below was used. That is, pure water was dropped on the surface of the insulator, and the hydrophilicity of the surface of the insulator was evaluated by measuring the contact angle between the droplet formed on the surface of the insulator and the surface of the insulator. Here, although there is generally no strict definition of hydrophilicity, the hydrophilicity in the present invention is defined as that the contact angle is 45 ° or less, preferably 30 ° or less.

  As a means for forming fine holes penetrating the resin, a method of irradiating a laser at the position of the fine hole to be formed, or a method of molding using a mold having a pin for forming the through hole at the position of the fine hole A known method such as the above may be used. In addition, when UV curable resin or the like is used for the insulator, a fine hole penetrating through general photolithography (exposure) and etching (development) using an exposure photomask on which a pattern corresponding to the fine hole is drawn is formed. Can be formed. When forming a plurality of fine holes in the insulator, it is preferable to form the fine holes by a general photolithography and etching method using a UV curable resin for the insulator.

  FIG. 2 is a schematic view showing a cross-sectional view of the cell fusion container of FIG. As a means for bonding the upper electrode (14), the spacer (16), the insulator (8), and the lower electrode (15) as shown in FIG. 2, each of them is bonded with an adhesive or heated in a pressurized state. A known method may be used such as a method of fusing or a method in which a spacer is bonded using a surface sticky PDMS (poly-dimethylsiloxane) or a resin such as a silicon sheet. In this way, the cell fusion region (1) shown in FIG. 2 can be formed.

  A power source (4) is connected to the upper and lower electrodes of the cell container via a conductive wire (3). The power source (4) is composed of an AC power source for applying an AC voltage waveform between the upper electrode (14) and the lower electrode (15).

  In the cell fusion device of the present invention, the planar shape of the micropore is a shape having at least one or more corners, and further, the planar shape of the micropore is The cell fusion device is preferably a quadrilateral. Further, the diameter of the maximum circle inscribed in the planar shape of the micropore is less than the diameter of the cell fixed in the micropore, or the diameter of the maximum circle inscribed in the planar shape of the micropore is the micropore. A cell fusion device characterized in that it is in the range of 1 or more and less than 2 times the diameter of the cell fixed to the cell, and the depth of the micropore is less than or equal to the diameter of the cell fixed to the micropore, The cell fusion device is preferably such that the interval between adjacent micropores is in the range of 0.5 to 6 times the diameter of the cell to be fixed. In addition, a plurality of micro holes formed in the insulator are formed in the same position on the surface of the insulator, and the positions of the micro holes adjacent to any one of the micro holes are, for example, arrayed on the surface of the insulator. It is preferable to be formed.

  FIG. 3 shows a schematic diagram of the cell fusion device of the present invention when a plurality of micropores (9) are formed in an array in the insulator (8). FIG. 4 is a schematic view showing a B-B ′ cross-sectional view of the cell fusion container of FIG. 3. Here, the array shape means that the vertical and horizontal intervals of the fine holes are arranged at substantially equal intervals. By arranging the fine holes in an array, an electric field generated by a voltage applied between the electrodes is generated almost uniformly in all the fine holes.

  In addition, in order to fix one cell in one micropore, it is inappropriate that the interval between micropores formed in an array is too narrow or too wide. When the interval between the micropores is too narrow, the probability that a plurality of cells are fixed in one micropore is increased, and as a result, the probability that a micropore that does not contain cells is increased. In addition, when the interval between the micropores is too wide, cells are left between the micropores, and the probability that micropores that do not contain cells are increased. Therefore, more specifically, the interval between adjacent micropores is preferably in the range of 0.5 to 6 times the diameter of the cells to be fixed in the micropores, and further, the interval between the micropores is fixed. More preferably, it is about 1 to less than 2 times the diameter of the cell.

  Hereinafter, the waveform of the alternating voltage and the shape of the micropores for fixing one cell per micropore will be described.

  Since the cell fusion device of the present invention fixes one cell per micropore, the waveform of the AC voltage applied between the electrodes by the AC power source periodically charges and discharges the cell by the power source. A cell fusion device having a repeated waveform, preferably, the waveform of the alternating voltage is a waveform having at least one time in a half cycle in which a voltage having a value other than 0 does not change for a certain period of time, for example, The waveform of the AC voltage is a waveform characterized by being a rectangular wave, a trapezoidal wave, or a combination thereof, and more preferably, the waveform of the AC voltage has a voltage other than 0 for a certain period of time. A cell fusion device comprising a power source that applies a waveform alternating voltage, characterized in that the time during which it does not change is equal to or greater than the time constant consisting of the product of the capacitance of the cell and the resistance of the cell suspension containing the cell A.

  In the cell fusion device of the present invention, the diameter of the maximum circle inscribed in the planar shape of the micropore is in the range of 1 to less than twice the diameter of the cell fixed to the micropore in the previous period, and There is a cell fusion device having a depth equal to or less than the diameter of the cell fixed in the micropore, and the interval between adjacent micropores is in the range of 0.5 to 6 times the diameter of the fixed cell. It is a cell fusion device.

  By using the waveform of the alternating voltage as described above and forming a micropore shape, one cell can be fixed per one micropore, and another cell is fixed from above the fixed cell, By bringing a pair of two cells into contact with each other through a plurality of micropores, cell fusion with a pair of two cells can be performed in a plurality of micropores at a time.

  The waveform of the AC voltage used in the cell fusion device of the present invention is not particularly limited as long as the charging and discharging of the cells can be periodically repeated. FIG. 5 shows an example of the waveform of the AC voltage in this mode. . The charging and discharging of the cells are repeated every T / 2, which is a half cycle of the AC voltage waveform in FIG. In the case of FIG. 5, since the polarity of the voltage is inverted every half cycle, the polarity of the charge is inverted between positive and negative when the cell is charged every half cycle. In addition, it is preferable that the waveform of the alternating voltage used for the cell fusion apparatus of this invention does not have a direct-current component. This is because the cells move by receiving a biased force in a specific direction due to the electrostatic force generated by the DC component, and it becomes difficult to fix the cells in the micropores due to the dielectrophoretic force. The ions contained in the suspension generate an electric reaction on the electrode surface, which generates heat, causing the cells to undergo thermal motion, and the cell movement cannot be controlled by the dielectrophoretic force, attracting the cells to the micropores. This is because it becomes difficult.

  More specifically, the waveform of the AC voltage used in the cell fusion device of the present invention is a waveform having at least one or more times in a half cycle in which a voltage having a value other than 0 does not change for a certain period of time. S [s] in FIG. 5 is a time during which the voltage does not change for a certain time. In the present invention, the time S [s] during which a voltage having a value other than 0 does not change for a certain period of time is the product of the capacitance C [F] of the cell and the resistance R [Ω] of the cell suspension containing the cell. Since it is preferable that the time constant be equal to or greater than τ [s], a relationship of S> τ (= C × R) is preferable. Note that the waveform of the AC voltage in the present invention is not limited to the waveform shown in FIG. 5 and can be arbitrarily changed without departing from the gist of the invention. For example, the waveform of the AC voltage may be a rectangular wave (FIG. 6), a trapezoidal wave (FIG. 7), or a combination of these (FIG. 8).

  The voltage value and frequency to be applied may be set appropriately depending on the distance between the electrodes of the cell fusion container, the type and size of cells to be fused, and the type of cell suspension containing cells. For example, when the area of the cell fusion container is about 2 cm × 2 cm, the distance between the electrodes is about 1 mm, the cell to be fused is a cell having a diameter of about 10 μm, and the suspension cage component is a 300 mM mannitol aqueous solution, the cell having a diameter of about 10 μm The capacitance is generally about 1 pF, the area is about 2 cm × 2 cm, and the resistance value when a 300 mM mannitol aqueous solution is put into a cell fusion container having a distance between electrodes of about 1 mm is about 5 kΩ. The time constant τ [s] consisting of the product of F] and the resistance R [Ω] of the cell suspension containing the cells is 5 ns. Therefore, it is preferable that the waveform of the AC voltage applied between the electrodes is an AC voltage waveform having at least one or more time within a half cycle in which a voltage having a value other than 0 does not change by at least 5 ns. For example, when the rectangular wave AC voltage waveform shown in FIG. 6 is used, the S time is preferably longer than 5 ns, that is, the frequency is preferably less than 100 MHz (= 1 / (2 × 5 ns), Considering the ease of electrical handling and the ease of handling with a commercially available signal generator, a rectangular wave AC voltage waveform with a frequency of about 1 to 3 MHz is preferable. In this case, the voltage of the rectangular AC voltage waveform is preferably about 10 to 20 Vpp in order to generate a dielectrophoretic force sufficient to attract the cells to the micropores. In the case of the conditions of this example, the time for attracting cells to the micropores is about 1 to 5 seconds, and the cells can be fixed in the micropores instantaneously.

  Next, the reason why one cell is fixed per one minute hole when the waveform of the alternating voltage of the above aspect and the minute hole of the above aspect is used will be described with reference to FIGS.

  9 to 11 show conceptual diagrams of processes in which cells enter micropores in the cell fusion device of the present invention. The thickness of the insulator (8) is substantially equal to the diameters of the cells A (10) and B (11), and the inner diameter of the micropores is in the range of 1 to less than 2 times the diameter of the cells A and B. FIG. It is assumed that after the cell A enters the micropore A (17) as shown in FIG. 11, the cell B enters the micropore B (21) as shown in FIG. FIGS. 12 to 14 show diagrams in which FIGS. 9 to 11 are expressed by electrical equivalent circuits, respectively. A cell suspension containing cells can be expressed by resistance (resistance value: 5 kΩ), and a cell can be expressed by a capacitor (capacity: 1 pF).

  When an AC voltage having a rectangular waveform with a frequency f [Hz] shown in FIG. 15 is applied between the upper electrode (14) and the lower electrode (15) in FIG. 9, in the fine hole in FIG. ), A concentration of electric lines of force is generated, so that a dielectrophoretic force is generated on the cell, and the cell A (10) is attracted to the micropore A (17) as shown in FIG. The cells A are fixed in the holes A, and the cells A block the micropores A. In addition to the dielectrophoretic force, the cells are induced into the micropores by gravity and electrostatic force from the electrodes. The portion of the micropore A closed by the cell A is electrically equivalent to the capacitor A (29) as shown in FIG.

  When the voltage waveform shown in FIG. 15 is applied, the voltage waveform in the capacitor A of FIG. 13 is shown in FIG. 16, and the current waveform is shown in FIG. As shown in FIG. 16, the capacitor A has a time constant τ [s] (= C × R) determined by the product of the capacity C [F] of the cell A and the resistance value R [Ω] of the cell suspension containing the cell. Need to be charged. Since the time during which the voltage having a value other than 0 does not change for a certain period of time is preferably equal to or less than the time constant formed by the product of the capacitance of the cell and the resistance of the cell suspension containing the cell, τ <1 / 2f is set. It is preferable to use a rectangular wave AC voltage having a frequency to satisfy. Since the current stops flowing when the capacitor A is charged, as shown in FIG. 17, the current flowing in the capacitor A flows in a pulse shape only for the time of the time constant τ. It becomes electrically equivalent. For this reason, in the micropore A containing the cells A, the concentration of the electric lines of force does not occur, and the probability that the micropores A attract new cells becomes low.

  On the other hand, since the electric lines of force are concentrated in the micropore B, the cell B is attracted by the dielectrophoretic force, the cell B is fixed to the micropore B, and the cell B closes the micropore B. By repeating this, one cell can be fixed to one minute hole by successively entering cells into empty minute holes. If the inner diameter of the micropore is larger than the diameter of the cell, the cell cannot sufficiently close the micropore, the electric field lines are concentrated, and the cell is attracted by the dielectrophoretic force. The probability that cells will enter increases. When cell fusion is performed in a state where two or more cells are fixed in the micropores, the diameter of the micropores may be larger than the diameter of the cells fixed in the micropores. However, when cell fusion is performed with one cell fixed in one micropore, the diameter of the maximum circle inscribed in the planar shape of the micropore is the diameter of the maximum circle inscribed in the planar shape of the micropore. The diameter of the cells fixed in the micropores is preferably in the range of 1 to less than 2 times, and the depth of the micropores is preferably less than or equal to the diameter of the cells fixed in the micropores. It may be less than the diameter of the cell to be fixed.

  The cell fusion device of the present invention is a cell fusion device characterized in that the planar shape of the micropore (9) is a shape having at least one corner, and further, The cell fusion device is characterized in that the planar shape is a quadrilateral. Here, the corner is a portion where two sides constituting the shape of the microhole intersect at an acute angle or an obtuse angle, and includes a shape in which the tip of the corner is slightly rounded. FIG. 26 shows a typical shape in which the planar shape of the micropore has at least one corner. In addition, the quadrilateral includes the shape of the fine hole having the four corners, and the four corners include a shape in which the tip of the corner is slightly rounded. Further, the four sides may be straight lines, or any of the four sides or any of the four sides may be slightly curved toward the center or outside of the microhole. In FIG. 27, the typical example of the shape of the quadrilateral micropore in this invention was shown.

  As described above, if corners are present in a part of the planar shape of the micropores, the electric force lines concentrate at the corners and the dielectrophoretic force becomes strong, and the cells are attracted by the stronger dielectrophoretic force. As a result, the probability that the cells are fixed in the micropores is improved. It is sufficient that at least one corner exists in the micropore, but it is more preferable that a plurality of corners exist. However, since the corner shape is more acute than the obtuse angle, electric lines of force are more likely to be concentrated, and the dielectrophoretic force is stronger, and therefore, a polygon less than a quadrangle is more preferable than a polygon more than a pentagon. In addition, there is no particular limitation as long as it is a quadrangle. For example, as shown in FIG. 27, there are trapezoids, rhombuses, parallelograms, and the like. If all four lines are substantially equal and point-symmetrical at an angle of 90 degrees at the center of the micropore, all four dielectrophoretic forces generated on the four sides of the quadrangular micropore are equal, and the dielectrophoretic forces generated on the four corners are also equal. Since all four are equal and the distribution of dielectrophoretic force of the micropores is point-symmetric regardless of the direction of the micropores, it is possible to apply a dielectrophoretic force with little bias regardless of the position of the cells with respect to the micropores. Therefore, the shape of the fine holes is more preferably a square as shown in FIGS. 27A to 27D or a shape close to a square. FIG. 28 shows an example of the cell fusion device in the present invention when the shape of the micropores is square.

  Furthermore, in order to fix one cell in one micropore, it is inappropriate that the interval between micropores is too narrow or too wide. When the interval between the micropores is too narrow, the probability that a plurality of cells are fixed in one micropore is increased, and as a result, the probability that a micropore that does not contain cells is increased. In addition, when the interval between the micropores is too wide, cells are left between the micropores, and the probability that micropores that do not contain cells are increased. Therefore, specifically, the interval between the micropores is preferably in the range of about 0.5 to 6 times the diameter of the cells to be fixed, and further, the interval between the micropores is 1 or more than the diameter of the cells to be fixed. More preferably, it is less than about twice.

  When the time during which the voltage having a value other than 0 does not change for a certain time is equal to or less than the time constant formed by the product of the capacitance of the cell and the resistance of the cell suspension containing the cell, the capacitor A in FIG. Since the battery is not charged, the current continues to flow through the capacitor A, and the current continues to flow in the micropore A containing the cells A, causing the electric field lines to concentrate. Therefore, since the cell B may be attracted to the micropore A containing the cell A, the probability that two or more cells are fixed in one micropore increases.

  Next, when an AC voltage having a sinusoidal waveform with a frequency f [Hz] shown in FIG. 18 is applied, the electric force lines concentrate in the micropores as in the case shown in FIG. As shown in FIG. 10, the cells are attracted to the micropores by the dielectrophoretic force, and the cells A block the micropores A. The portion of the micropore A closed by the cell A is equivalent to the capacitor A as shown in FIG. When the voltage waveform shown in FIG. 18 is applied, the voltage waveform and current waveform in the capacitor A are shown in FIG. As shown in FIG. 19, when the waveform of the applied AC voltage is a sine wave, the phase of the sine wave only advances 90 degrees and the waveform of the sine wave does not change. Flows and concentration of electric lines of force occurs. For this reason, since there exists a possibility that the cell B may be attracted | sucked to the micropore A containing the cell A, the probability that two or more cells will be fixed to one micropore becomes high. Therefore, in the waveform of the AC voltage in which the voltage continuously changes like a sine wave or a triangular wave, there are fine holes in which a plurality of cells are concentrated and fixed, and fine holes in which the cells are not fixed at all. It is difficult to fix one cell per hole. When the applied voltage is direct current, the ions contained in the cell suspension containing the cells generate an electric reaction on the electrode surface, which generates heat, causing the cells to undergo thermal motion. This makes it impossible to control the movement of the cells, making it difficult to draw the cells into the micropores.

  For the above reasons, in the cell fusion device of the present invention, the waveform of the AC voltage applied between the electrodes by the AC power source is a waveform characterized by periodically repeating charging and discharging of the cells, More specifically, the waveform of the AC voltage is a waveform having at least one time in a half cycle in which a voltage having a value other than 0 does not change for a certain period of time, such as a rectangular wave, a trapezoidal wave, or the like More preferably, the time when the voltage having a value other than 0 does not change for a certain period of time is equal to or more than the time constant formed by the product of the capacitance of the cell and the resistance of the cell suspension containing the cell. The diameter of the maximum circle inscribed in the planar shape of the micropore penetrating the insulator is less than the diameter of the cell fixed to the micropore, or fixed to the micropore. Cells The micropore depth is in the range of 1 to less than 2 times the diameter and the micropore depth is equal to the cell diameter, and the interval between adjacent micropores is 0.5 to 6 times the diameter of the cell to be fixed. By being in the following range, it becomes possible to fix one cell per minute hole in the plurality of minute holes formed in an array on the insulator.

  By the way, in the cell fusion method of the present invention, after fixing the first cell in one micropore, the second cell is fixed further from above the fixed first cell. Dielectric migration force, gravity, and electrostatic force of the first cell act on the second cell and come into contact with the first cell. However, for the reasons described above, the first cells block the micropores, making it difficult for the current to flow, thereby suppressing the generation of electric lines of force and weakening the dielectrophoretic force acting on the second cells. Therefore, the probability of bringing the second cells into contact with the first cells fixed in the micropores one by one is reduced. However, it is possible to increase the contact probability between the first cell and the second cell by making the concentration of the first cell higher than the concentration of the second cell and introducing it excessively into the cell fusion region. .

  Next, the cell fusion method in the present invention will be described in more detail with reference to the drawings.

  The cell fusion method of the present invention is a cell fusion method using the cell fusion device, wherein the first cell is introduced into the cell fusion region, and an alternating voltage is applied to the first cell in the micropore. After fixing one cell, a second cell is introduced into the cell fusion region, and an alternating voltage is applied to bring the second cell into contact with the first cell at the position of the micropore. Cell fusion method in which cells are fused together. The AC voltage waveform is more preferably an AC voltage having the waveform described above. Here, schematic views of the cell fusion method of the present invention are shown in FIGS.

  20 to 22 show examples in which the diameter of the first cell is smaller than the diameter of the second cell. In the cell fusion method of the present invention, the diameter of the first cell is preferably smaller than the diameter of the second cell, but the diameter of the first cell is essentially equal to or larger than the diameter of the second cell. In the cell fusion method of the present invention, it is preferable that the first cell and the second cell are fused in the vicinity of the surface of the micropore. The cells and the second cells may be fused, and any modifications other than those shown in FIGS. 20 to 22 can be arbitrarily made without departing from the scope of the invention.

  FIG. 20 shows a case where the diameter and depth of the micropore (9) are approximately equal to the diameter of the first cell (18), and the first cell just enters the micropore, and FIG. FIG. 22 shows the case where the diameter and depth of the micropore (9) are larger than the diameter of the first cell (18), and FIG. 22 shows the case where the diameter and depth of the micropore (9) are the first cell (18). Is smaller than the diameter of

  The reason why the fusion method of the present invention is preferred will be described below. The best mode of the present invention is the cell fusion method shown in FIG. First, as the first cell (18), a cell having a small diameter is introduced into the cell fusion region (1) together with the cell suspension (6), and the AC voltage (34) having the above-described waveform is applied to obtain one cell. Fix one first cell per micropore (9). In this case, the first cell is guided to the micropore mainly by dielectrophoretic force, gravity, and electrostatic force from the electrode. The diameter and depth of the micropore is approximately equal to the diameter of the first cell, and the first cell just enters the micropore. By doing so, electrostatic force is generated on the electrode surface of the bottom surface of the micropore and the first cell, and the first cell is reliably fixed to the micropore. Although there is no restriction | limiting in particular in the number of 1st cells, When considering using the 1st cell effectively, it is preferable that it is equivalent to the number of micropores. Next, as the second cell (22), a cell having a large diameter is introduced into the cell fusion region (1) together with the cell suspension (6). At this time, since the first cell (18) is fixed by electrostatic force with the electrode and is surrounded by the micropore (9), the first cell (18) is fed by feeding the second cell. Cells rarely detach from the micropores.

  The introduced second cell is contacted and fixed from above the first cell fixed in the micropore by applying the AC voltage (34) having the waveform described above. In this case, the dielectrophoretic force in the micropores also acts on the second cells, but is induced by the first cells fixed in the micropores mainly by gravity and electrostatic force from the first cells. The number of the second cells is not particularly limited, but considering the effective use of the second cells, it is preferably equal to the number of micropores, but introduced in excess of the number of micropores. Thus, it is possible to increase the contact probability between the first cell and the second cell.

  The cell fusion method of the present invention is a cell fusion method characterized in that, in the cell fusion method, the cells are placed in a cell suspension added with a substance that enhances the fluidity of the cell membrane. Membrane fusion occurs easily when cells come into contact with the fluidity of the cell membrane being increased. Therefore, as described above, when two cell pairs come into contact with each other in a micropore, cell fusion occurs in a pair of two cells. Here, the substance that enhances the fluidity of the cell membrane is not particularly limited as long as it is a substance that causes membrane fusion between the contacted cells. Examples thereof include polyethylene glycol and lysotium, and polyethylene glycol is particularly preferable. . Furthermore, polyethylene glycol having an average molecular weight of about 1000 to 6000 is preferable.

According to the present invention, the following effects can be obtained.
(1) In the cell fusion device of the present invention, by disposing the insulator having the micropores on the electrode surface on the cell fusion region side, the cells are reliably fixed in the micropores, and 2 located near the micropores. A pair of cells can be selectively fused, and chemical cell fusion between two pairs of cells can be efficiently performed.
(2) In the cell fusion device of the present invention, the diameter of the maximum circle inscribed in the planar shape of the micropore is in the range of 1 to 2 times the diameter of the cell fixed to the micropore, and the depth of the micropore Is a cell fusion device having a diameter equal to or less than the diameter of the cells to be fixed in the micropores, and in this way, the cells can be reliably fixed in the micropores.
(3) In the cell fusion device of the present invention, a plurality of micropores are formed in an array on the insulator, and in this way, a pair of two cells fixed in the plurality of micropores can be obtained. Cell fusion can be performed simultaneously, and chemical cell fusion using a pair of two cells can be efficiently performed.
(4) In the cell fusion device of the present invention, the cell fusion device is characterized in that the interval between adjacent micropores is in the range of 0.5 to 6 times the diameter of the cells to be inserted into the micropores. By doing so, it is possible to increase the probability of fixing one cell in one micropore, and it is possible to increase the probability of making a pair of two cells contact in the micropore.
(5) In the cell fusion device of the present invention, it is possible to fix one cell in one minute hole by controlling the waveform of the AC voltage applied between the electrodes by the AC power source.
(6) In the cell fusion device of the present invention, one cell is fixed to one micropore by hydrophilizing the surface of the insulator formed with the micropore arranged on the electrode surface on the cell fusion region side. It is possible to further increase the probability that the two cells come into contact with each other in the micropore.
(7) In the cell fusion device of the present invention, since the planar shape of the micropores has corners, electric field lines are concentrated at the corners, and the dielectrophoretic force is increased, so that one micropore has one. The probability of fixing the cells can be further increased, and the probability of bringing two cells into contact with each other in the micropores can be increased.
(8) The cell fusion method of the present invention is a cell fusion method using the cell fusion device described above, wherein the first cell is introduced into the cell fusion region, and an alternating voltage is applied to the inside of the micropore. After fixing the first cell to the cell, the cell is introduced, and an AC voltage having the waveform described above is applied to bring the second cell into contact with the first cell at the position of the micropore, thereby chemically More preferably, the AC voltage waveform is an AC voltage having the waveform described above, and the cells such as polyethylene glycol are added to a cell suspension added with a substance that enhances the fluidity of the cell membrane. This is a cell fusion method. By doing in this way, two cell pairs in the vicinity of the micropores can be selectively fused, and cell fusion with two cell pairs can be performed efficiently. The AC voltage is used to fix a pair of two cells in a micropore. Even after fixing a pair of two cells in a micropore, the two cells in contact with each other can be dielectrophoresised by continuing to apply the AC voltage. Since it is pressed by force, electrostatic force, and gravity, a pair of two cells is more easily fused.

  Hereinafter, the present invention will be described in detail with reference to examples. Needless to say, the present invention is not limited to these examples, and can be arbitrarily changed without departing from the scope of the invention.

Example 1
FIG. 3 shows a conceptual diagram of the cell fusion device used in Example 1. The cell fusion device is roughly divided into a cell fusion container (13) and a power source (4). As shown in FIG. 3, the cell fusion container has an insulator (8) in which a spacer (16) is arranged between an upper electrode (14) and a lower electrode (15), and a plurality of micropores are formed in an array. It has a structure sandwiched between a spacer and a lower electrode.

  In this embodiment, as will be described later, an insulator-integrated lower part with a fine hole in which a lower electrode (15) and an insulator in which a plurality of fine holes are formed in an array are integrally formed by general photolithography and etching. An electrode (28) was used.

  The upper electrode and the lower electrode were formed by depositing ITO (film thickness 150 nm) on a Pyrex (registered trademark) substrate 70 mm long × 40 mm wide × 1 mm thick. The spacer was used by hollowing out the center of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm into a length of 20 mm and a width of 20 mm.

  Further, as shown in FIG. 3, an introduction port (19) and a discharge port (20) for introducing and discharging a cell suspension containing cells were provided. The insulator (8) having a plurality of fine holes was produced by integrally forming on the lower electrode by a photolithography and etching method shown in FIG.

  First, a resist (25) was applied to the ITO film-forming surface of Pyrex (registered trademark) glass (24) on which ITO (23) was formed using a spin coater so as to have a film thickness of 2.5 μm. After drying, pre-baking (80 ° C., 15 minutes) was performed using a hot plate. A xylene negative resist was used as the resist. Next, an exposure depicting a microhole pattern having a diameter of 7 μm arranged in an array of 1000 vertical x 1000 horizontal in an area of 30 mm vertical by 30 mm horizontal and the vertical and horizontal spacing of the micropores is 30 μm. The resist was exposed (27) with a UV exposure machine using a photomask (26), and developed with a developer (7). The exposure time and development time were adjusted so that the depth of the micropores was 2.5 μm, which is equal to the film thickness of the resist, so that the ITO was exposed on the bottom surfaces of the micropores. After development, the resist was hardened by post-baking (115 ° C., 30 minutes) using a hot plate. The upper electrode (14), the spacer (16), and the insulator-integrated lower electrode (28) with fine holes were laminated and pressure-bonded as shown in FIG. 4 is a B-B ′ cross-sectional view of the cell fusion container shown in FIG. 3. The surface of the silicon sheet was sticky, and the parts were brought into close contact with each other by pressure bonding, and the cell suspension containing the cells could be put into the cell fusion container without leakage. Since the area where the spacer is hollowed is 20 mm long × 20 mm wide, the number of micropores existing in this space is about 400,000. Moreover, the power supply which applies a voltage between electrodes connected the signal generator (NF circuit design block make, WF1966) as an alternating current power supply via the conductive wire (3).

As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Remove both cells from the medium, separate the cells from the medium by centrifugation, and suspend the removed cells in a 50% aqueous polyethylene glycol solution with a molecular weight of 4000 to a density of 0.7 × 10 ⁶ cells / mL. A cell suspension was prepared.

  First, 600 μL of the mouse antibody-producing cell suspension (number of mouse antibody-producing cells: about 400,000) was injected from the introduction port of the spacer using a syringe, and a rectangular wave with a voltage of 10 Vpp and a frequency of 3 MHz was supplied from an AC power source. When an alternating voltage is applied between the electrodes, one or two mouse antibody-producing cells can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. A plurality of cells could be arranged in an array. At this time, the probability of one mouse antibody-producing cell entering one micropore was 35%. Here, the cell fixation rate means that 225 fine pores of 15 vertical x 15 horizontal are visible in the field of view of the microscope, and the fine cells containing 1-2 cells when cells are introduced and fixed. It was defined as a value obtained by dividing the number of holes by the number of fine holes of 225. In addition, the cell fixation rate in the following Example 2 and a comparative example is also the same definition.

  Subsequently, 600 μL of the cell suspension of mouse myeloma cells (number of mouse myeloma cells: about 400,000) was applied to the spacer inlet while a rectangular wave AC voltage of 10 Vpp and frequency 3 MHz was applied between the electrodes by an AC power source. When injected with a syringe, one mouse myeloma cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. It was possible to arrange in the shape. At this time, the probability that one or two mouse myeloma cells enter one micropore was 55%. When the mouse myeloma cells were introduced, it was hardly observed that the mouse antibody-producing cells put in the micropores were detached from the micropores. Therefore, the mouse antibody-producing cells and the mouse myeloma cells were paired in pairs in the micropores. Estimated contact is estimated to be about 20% (= 35% × 55%).

Next, after allowing to stand for 1 minute in this state, the cell suspension in the cell fusion container is taken out and diluted with a medium. Then, the cells and the cell suspension are separated by centrifugation, and the removed cells are put into HAT medium (H : Hypoxanthine, A: medium containing aminopterin, T: medium containing thymidine as components, and culturing the fused cells. The HAT medium is a medium for selectively growing only fused cells. The HAT medium containing the cell suspension was placed in a CO ₂ incubator, and the cells were cultured. After 6 days, the number of fused cells was counted. As a result, 20 fused cells could be confirmed. A fusion probability of 0.5 / 10000 was obtained. This is a fusion probability that is about four times the fusion probability of 0.12 / 10000 in normal cell fusion using polyethylene glycol shown in Comparative Example 1, and it is possible to confirm efficient fusion in a pair of two cells. did it.

(Example 2)
In order to evaluate the hydrophilicity of the fine holed insulator used in Example 1, pure water was dropped on the surface of the insulator, and the droplet formed on the surface of the insulator at that time and the surface of the insulator When the contact angle was measured, the contact angle was about 47 °. Therefore, in order to make the insulator with micropores hydrophilic, the insulator-integrated lower electrode with micropores is immersed in a 300 mM mannitol aqueous solution containing BSA (1 mg / mL) for about 1 hour, and the BSA is physically applied to the insulator surface. Adsorbed. Similarly, after BSA is physically adsorbed, pure water is dropped on the surface of the insulator, and the contact angle between the droplet formed on the surface of the insulator and the surface of the insulator is measured. Was about 27 °, confirming that the hydrophilicity was improved.

  Cell fusion was performed as described below using the cell fusion apparatus assembled in the same manner as in Example 1 using the insulator-integrated lower electrode with micropores thus subjected to hydrophilic treatment.

As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Remove both cells from the medium, separate the cells from the medium by centrifugation, and suspend the removed cells in a 50% aqueous solution of polyethylene glycol having a molecular weight of 4000 to a density of 0.7 × 10 ⁶ cells / mL. A cell suspension was prepared.

  First, 600 μL of the mouse antibody-producing cell suspension (number of mouse antibody-producing cells: about 400,000) was injected from the introduction port of the spacer using a syringe, and a rectangular wave with a voltage of 10 Vpp and a frequency of 3 MHz was supplied from an AC power source. When an alternating voltage is applied between the electrodes, one mouse antibody-producing cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. Could be arranged in an array. At this time, the probability that one or two mouse antibody-producing cells enter one micropore was 70%. From this result, it was found that the cell fixing probability to the micropores was improved as compared with Example 1 by making the insulating film with micropores hydrophilic.

  Subsequently, 600 μL of the cell suspension of mouse myeloma cells (number of mouse myeloma cells: about 400,000) was applied to the spacer inlet while a rectangular wave AC voltage of 10 Vpp and frequency 3 MHz was applied between the electrodes by an AC power source. When injected with a syringe, one mouse myeloma cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. It was possible to arrange in the shape. At this time, the probability of one mouse myeloma cell entering one micropore was 65%. From this result, it was found that the cell fixing probability to the micropores was improved as compared with Example 1 by making the insulating film with micropores hydrophilic.

  When the mouse myeloma cells were introduced, it was hardly observed that the mouse antibody-producing cells put in the micropores were detached from the micropores. Therefore, the mouse antibody-producing cells and the mouse myeloma cells were paired in pairs in the micropores. Estimated contact is estimated to be about 46% (= 70% × 65%).

Next, after allowing to stand for 1 minute in this state, the cell suspension in the cell fusion container is taken out and diluted with a medium. Then, the cells and the cell suspension are separated by centrifugation, and the removed cells are put into HAT medium (H : Hypoxanthine, A: medium containing aminopterin, T: medium containing thymidine as components, and culturing the fused cells. The HAT medium is a medium for selectively growing only fused cells. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured, and after 6 days, the number of fused cells was counted. As a result, 30 fused cells could be confirmed and 400,000 mouse-producing cells were produced. A fusion probability of 0.75 / 10000 was obtained. This is a fusion probability that is about 6 times the fusion probability of 0.12 / 10000 in normal cell fusion using the polyethylene glycol shown in Comparative Example 1, and it is possible to confirm efficient fusion in a pair of two cells. did it.

(Example 3)
FIG. 28 shows the cell fusion device used in Example 3. The shape of the micropores is the same as that of the cell fusion device used in Example 2 except that the shape of the micropores is a square micropore pattern with a side of 10 μm.

As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Remove both cells from the medium, separate the cells from the medium by centrifugation, and suspend the removed cells in a 50% aqueous polyethylene glycol solution with a molecular weight of 4000 to a density of 0.7 × 10 ⁶ cells / mL. A cell suspension was prepared.

  First, 600 μL of the mouse antibody-producing cell suspension (number of mouse antibody-producing cells: about 400,000) was injected from the introduction port of the spacer using a syringe, and a rectangular wave with a voltage of 10 Vpp and a frequency of 3 MHz was supplied from an AC power source. When an alternating voltage is applied between the electrodes, one mouse antibody-producing cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. Could be arranged in an array. At this time, the probability that one mouse antibody-producing cell enters one micropore was 75%. From this result, the plane shape of the micropores has corners, and the lines of electric force are concentrated at the corners, the dielectrophoretic force is increased, and the insulating film with micropores is made hydrophilic, so that It was found that the cell fixation probability was further improved as compared with Example 1 and Example 2.

  Subsequently, 600 μL of the cell suspension of mouse myeloma cells (number of mouse myeloma cells: about 400,000) was applied to the spacer inlet while a rectangular wave AC voltage of 10 Vpp and frequency 3 MHz was applied between the electrodes by an AC power source. When injected with a syringe, one mouse myeloma cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. It was possible to arrange in the shape. At this time, the probability of one mouse myeloma cell entering one micropore was 70%. From this result, the plane shape of the micropores has corners, and the lines of electric force are concentrated at the corners, the dielectrophoretic force is increased, and the insulating film with micropores is made hydrophilic, so that It was found that the cell fixation probability was further improved as compared with Example 1 and Example 2.

  When the mouse myeloma cells were introduced, it was hardly observed that the mouse antibody-producing cells put in the micropores were detached from the micropores. Therefore, the mouse antibody-producing cells and the mouse myeloma cells were paired in pairs in the micropores. Estimated contact is estimated to be about 53% (= 75% × 70%).

Next, after allowing to stand for 1 minute in this state, the cell suspension in the cell fusion container is taken out and diluted with a medium. Then, the cells and the cell suspension are separated by centrifugation, and the removed cells are put into HAT medium (H : Hypoxanthine, A: medium containing aminopterin, T: medium containing thymidine as components, and culturing the fused cells. The HAT medium is a medium for selectively growing only fused cells. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured, and after 6 days, the number of fused cells was counted. As a result, 30 fused cells could be confirmed and 400,000 mouse-producing cells were produced. A fusion probability of 0.9 / 10000 was obtained. This is a fusion probability that is about 7.5 times the fusion probability of 0.12 / 10000 in normal cell fusion using the polyethylene glycol shown in Comparative Example 1, and confirms efficient two-cell fusion. I was able to.

(Comparative Example 1)
As Comparative Example 1, normal cell fusion using polyethylene glycol was performed. As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Mouse antibody-producing cells and mouse myeloma cells are mixed at a ratio of 10: 1, both cells are removed from the medium, and the cells and the medium are separated by centrifugation, and the removed cells are each suspended in a 50% aqueous solution of polyethylene glycol having a molecular weight of 4000. The cell suspension was adjusted to a density of 1.7 × 10 ⁷ cells / mL.

40 μL of the above cell suspension (the number of mouse antibody-producing cells: about 600,000, the number of mouse myeloma cells: 60,000) was placed in a test tube, and the bottom of the test tube was tapped for 1 minute. Then, after leaving still for about 1 minute, the cell suspension in a test tube was put into HAT culture medium. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured. After 6 days, the number of fused cells was counted. As a result, 7 fused cells could be confirmed, and 600,000 total mouse antibody-producing cells were produced. A fusion probability of 0.12 / 10000 was obtained.

(Comparative Example 2)
Using the same cell fusion device as in the example, 600 μL of the same cell suspension of mouse antibody-producing cells as in the example (number of antibody-producing cells: about 400,000) was injected from the inlet of the spacer using a syringe, and alternating current When a sine wave AC voltage with a voltage of 10 Vpp and a frequency of 3 MHz is applied between the electrodes by the power source, a plurality of mouse antibody-producing cells gather in a bead shape and a micropore in which the cells are not fixed at all is generated, and the mouse antibody-producing cells are arrayed. It was not possible to arrange in the shape. In this state, 600 μL of the same mouse myeloma cell suspension as in the example (number of mouse myeloma cells: about 400,000) was applied while applying a sinusoidal AC voltage having a voltage of 10 Vpp and a frequency of 3 MHz between the electrodes by an AC power source. The syringe was injected from the introduction port of the spacer.

Next, after leaving in this state for 1 minute, the cell suspension in the cell fusion container is taken out and diluted with a medium, and then the cells and the cell suspension are separated by centrifugation, and the removed cells are put into a HAT medium. The fused cells were cultured. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cell culture was performed. After 6 days, the number of fused cells was counted. As a result, the fused cells could not be confirmed.

(Comparative Example 3)
FIG. 24 shows a conceptual diagram of the cell fusion device used in Comparative Example 3. The cell fusion device is roughly divided into a cell fusion container (13) and a power source (4). As shown in FIG. 24, the cell fusion container has an insulator (8) in which two spacers (16) are arranged between the upper electrode (14) and the lower electrode (15) and a plurality of micropores are formed in an array. ) Is sandwiched between two spacers.

  The upper electrode and the lower electrode were formed by depositing ITO (film thickness 150 nm) on a Pyrex (registered trademark) substrate 70 mm long × 40 mm wide × 1 mm thick. The spacer was used by hollowing out the center of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1 mm into a length of 20 mm and a width of 20 mm. Moreover, as shown in FIG. 24, the introduction port (19) and the discharge port (20) for introducing and discharging the cell suspension containing the cells were provided. Insulator (8) having a plurality of fine holes is formed in a polyimide film having a length of 40 mm, a width of 40 mm, and a film thickness of 25 μm, by a method using photolithography and etching. Fine holes having a diameter of 20 μm arranged in an array of 300 vertical × 300 horizontal were formed with a space between the vertical and horizontal holes of 50 μm.

  The upper electrode (14), spacer (16), fine-holed insulator (36), and lower electrode (15) thus fabricated were stacked and pressure bonded as shown in FIG. FIG. 25 is a CC ′ sectional view of the cell fusion container shown in FIG. The surface of the silicon sheet was sticky, and the parts were brought into close contact with each other by pressure bonding, and the cell suspension containing the cells could be put into the cell fusion container without leakage. Since the area formed by hollowing out the spacer is 20 mm long × 20 mm wide, the number of micropores existing in this space is about 40,000. The power source for applying a voltage between the electrodes is a signal generator (WF1966, manufactured by NF circuit design block) connected as an AC power source via a conductive wire (3). The connection of can be switched.

As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Remove both cells from the medium, separate the cells from the medium by centrifugation, and suspend the removed cells in a 50% aqueous polyethylene glycol solution with a molecular weight of 4000 to a density of 0.7 × 10 ⁶ cells / mL. A cell suspension was prepared.

  First, 600 μL of the mouse antibody-producing cell suspension (the number of mouse antibody-producing cells: about 400,000) was injected from the introduction port of the spacer using a syringe, and the voltage was 15 Vpp and the frequency was 3 MHz with a rectangular wave AC voltage. When a rectangular wave AC voltage was applied between the electrodes, mouse antibody-producing cells could be fixed in the micropores formed in an array in an extremely short time of about 2 to 3 seconds. However, there are micropores in which a plurality of mouse antibody-producing cells are fixed in one micropore and micropores in which no mouse antibody-producing cells are fixed at all, and one mouse antibody is produced for each of the plurality of micropores. Micropores in which cells were fixed could hardly be confirmed.

  Subsequently, 600 μL of the mouse myeloma cell suspension (number of mouse myeloma cells: about 400,000) was applied to the spacer inlet while a rectangular wave AC voltage having a voltage of 15 Vpp and a frequency of 3 MHz was applied between the electrodes by an AC power supply. When injected with a syringe more, mouse antibody-producing cells previously fixed in the micropores are almost detached from the micropores, and the mouse antibody-producing cells and mouse myeloma cells cannot be fused in the micropores. There wasn't.

It is a conceptual diagram which shows an example of the cell fusion apparatus of this invention. FIG. 6 is an AA ′ cross-sectional view of the cell fusion container shown in FIG. 5. It is a conceptual diagram of an example of the cell fusion apparatus of this invention, and the cell fusion apparatus used in the Example. FIG. 8 is a BB ′ sectional view of the cell fusion container shown in FIG. 7. It is an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the rectangular wave as an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the trapezoid wave as an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the waveform which combined the square wave and the trapezoid wave as an example of the waveform of the alternating voltage used for this invention. It is a 1st figure explaining the cell operation method of this invention. It is a 2nd figure explaining the cell operation method of this invention. It is a 3rd figure explaining the cell operation method of this invention. FIG. 10 is a diagram illustrating FIG. 9 with an electrical equivalent circuit. It is the figure which represented FIG. 10 with the electrical equivalent circuit. FIG. 12 is a diagram illustrating FIG. 11 as an electrical equivalent circuit. It is a figure which shows the waveform of the rectangular wave alternating voltage of frequency f [Hz]. It is a figure which shows the voltage waveform in the capacitor | condenser A of FIG. 13 at the time of applying the waveform of the rectangular wave alternating voltage of the frequency f [Hz] shown in FIG. 15 between electrodes. It is a figure which shows the electric current waveform in the capacitor | condenser A of FIG. 13 at the time of applying the waveform of the rectangular wave alternating voltage of the frequency f [Hz] shown in FIG. 15 between electrodes. It is a figure which shows the waveform of the sine wave alternating voltage of frequency f [Hz]. It is a figure which shows the voltage waveform or current waveform in the capacitor | condenser A of FIG. 13 at the time of applying the waveform of the sine wave alternating voltage of the frequency f [Hz] shown in FIG. 18 between electrodes. It is a figure which shows the 1st example of the cell fusion method of this invention. It is a figure which shows the 2nd example of the cell fusion method of this invention. It is a figure which shows the 3rd example of the cell fusion method of this invention. It is the schematic of general photolithography and the etching method. It is a conceptual diagram of the cell fusion apparatus used in the comparative example 3 of this invention. It is CC 'sectional drawing of the cell fusion container shown in FIG. It is a 1st figure of the fine hole shape example in this invention. It is a 2nd figure of the micropore shape example in this invention. It is a figure which shows an example of the cell fusion apparatus in case the shape of the micropore in this invention is a square.

Explanation of symbols

1: Cell fusion region 2: Electrode 3: Conductive wire 4: Power supply 5: AC power supply 6: Cell suspension 7: Developer 8: Insulator 9: Micropore 10: Cell A
11: Cell B
12: Electric lines of force 13: Cell fusion container 14: Upper electrode 15: Lower electrode 16: Spacer 17: Micropore A
18: first cell 19: inlet 20: outlet 21: micropore B
22: Second cell 23: ITO
24: Pyrex (registered trademark) glass 25: Resist 26: Photomask for exposure 27: Exposure 28: Insulator-integrated lower electrode with fine holes 29: Capacitor A
30: Capacitor B
31: Resistance 32: Fusion cell

Claims (21)

A pair of electrodes made of conductive members arranged opposite to each other in the cell fusion region, and a flat spacer disposed between the pair of electrodes and penetrating in the direction of the oppositely arranged electrodes A cell fusion device comprising: a cell fusion container made of a flat insulator having one or a plurality of micropores; and a power source for applying an alternating voltage to the pair of electrodes. The cell fusion device according to claim 1, wherein the micropore formed in the insulator has a shape capable of fixing one cell per micropore. The cell fusion device according to claim 1 or 2, wherein an AC voltage having a waveform for fixing one cell to each micropore is applied between the electrodes by a power source. The cell fusion device according to any one of claims 1 to 3, wherein an AC voltage having a waveform in which charging and discharging of the fine particles are periodically repeated is applied between the electrodes by a power source. The cell fusion according to any one of claims 1 to 4, wherein the waveform of the AC voltage is a waveform having at least one or more times in a half cycle in which a voltage having a value other than 0 does not change for a certain period of time. apparatus. The cell fusion device according to any one of claims 1 to 5, wherein the waveform of the AC voltage is a rectangular wave, a trapezoidal wave, or a combination of these. The AC voltage waveform has a time during which a voltage having a value other than 0 does not change for a certain period of time is equal to or more than a time constant consisting of the product of the capacitance of the cell and the resistance of the cell suspension containing the cell. The cell fusion device according to any one of claims 1 to 6. The diameter of the maximum circle inscribed in the planar shape of the micropore is in the range of 1 to less than twice the diameter of the cell fixed in the micropore, and the depth of the micropore is equal to or less than the diameter of the cell fixed in the micropore. The cell fusion device according to claim 1, wherein the device is a cell fusion device. The cell fusion device according to any one of claims 1 to 8, wherein the insulator is disposed on the electrode surface on the cell fusion region side of one of the electrodes. The plurality of micro holes formed in the insulator are formed in the same position on the surface of the insulator, the positions of the micro holes adjacent to each other from any micro hole. A cell fusion device according to claim 1. The cell fusion device according to any one of claims 1 to 10, wherein a plurality of micropores formed in the insulator are formed in an array on the surface of the insulator. The cell fusion device according to any one of claims 1 to 6, wherein the interval between adjacent micropores is in the range of 0.5 to 6 times the diameter of the cells to be inserted into the micropores. The cell fusion device according to any one of claims 1 to 12, wherein the spacer has a through-hole forming a cell fusion region. The cell fusion device according to any one of claims 1 to 13, wherein the spacer has an introduction channel for introducing cells and a discharge channel for discharging cells. The cell fusion device according to claim 1, wherein the insulator is hydrophilic. The cell fusion device according to any one of claims 1 to 15, wherein the planar shape of the micropore is a shape having one or more corners. The cell fusion device according to claim 16, wherein the planar shape of the micropore is a quadrilateral. A cell fusion method using the cell fusion device according to any one of claims 1 to 17, wherein a first cell is introduced into the cell fusion region, and an alternating voltage is applied to the inside of the micropore. After fixing the first cell, the second cell is introduced into the cell fusion region, and the second cell is brought into contact with the first cell at the position of the micropore by applying an alternating voltage. A cell fusion method characterized by chemically fusing cells. The cell fusion method according to claim 18, wherein the AC voltage according to claim 18 is an AC voltage having the waveform according to any one of claims 3 to 7. 20. The cell fusion method according to claim 18 or 19, wherein the cells are placed in a cell suspension added with a substance that enhances the fluidity of the cell membrane. 21. The cell fusion method according to claim 18, wherein the substance that enhances the fluidity of the cell membrane is polyethylene glycol.

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