JP2009273458A - Cell fusion device and cell fusion method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell fusion device and a cell fusion method which enable such treatment of cell as to simply and rapidly enhance fusion regeneration probability by carrying out substitution of cell fusion liquids, while fixing cells carrying out cell fusion in micropores in a fusion container. <P>SOLUTION: The cell fusion device includes a cell fusion container equipped with a cell fusion liquid inlet and discharge port and consisting of a pair of electrodes composed of electroconductive member and oppositely arranged in a cell fusion region and a flat insulator arranged through a flat spacer between a pair of electrodes and having a plurality of micropores extended through the direction of electrodes oppositely arranged, wherein the insulator is arranged on an electrode surface on a cell fusion region side of either one electrode in the electrodes, an electric power supply having an electric source changeover mechanism and a cell fusion liquid-introducing changeover means of changing over and introducing three or more cell fusion liquids. The cell fusion method comprises using the cell fusion device. <P>COPYRIGHT: (C)2010,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.

  As a conventional general cell fusion technology, a polyethylene glycol method (PEG method) which is a chemical cell fusion method and an electric cell fusion method are known. In the PEG method, (i) polyethylene glycol (PEG) has a strong toxicity to cells. (Ii) It takes time to find optimum conditions such as the degree of polymerization of PEG and the amount of addition in cell fusion. , (Iii) Advanced technology is required for cell fusion, and can only be used by those skilled in specific technology. (Iv) 2 cell contact is accidental, and it is difficult to control cell fusion in a pair of 2 cells. Therefore, there is a problem to be solved such as a very low fusion reproduction probability. Here, the fusion regeneration probability is a value obtained by dividing the number of produced fused cells by the number of spleen cells introduced into the fusion container.

  On the other hand, the electric cell fusion method does not require advanced technology, can easily and efficiently fuse cells, has little toxicity to cells, and can fuse cells with high activity. It is known that the fusion regeneration probability is higher than that of the PEG method because various conditions at the time of cell fusion such as conditions for electric cell fusion can be easily set. 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, the electric conductivity of the cell membrane is instantaneously lowered by applying a DC pulse voltage (hereinafter also referred to as a fusion voltage) in the unit of several μsec to several tens of μsec between the electrodes. Cell membranes are reversibly disrupted and reconstituted, resulting in cell fusion.

  For the electric cell fusion method, a microelectrode method and a parallel electrode method are mainly used. Among these, the microelectrode method is a method of applying a DC pulse voltage by collecting cells with a micromanipulator while observing a cell fusion of a pair of two cells with a microscope, and has a very high fusion probability. Examples have also been reported (see, for example, Patent Document 1). However, this method is a laborious method, and its operation requires skill and is not practical for handling a large number of cells. The parallel electrode method is a method in which a plurality of cells are arranged in a bead shape by dielectrophoresis, and then the cells are fused by applying a DC pulse voltage. Since a plurality of cells are fused, contact of two cells is accidental as in the PEG method, and there is a problem that it is difficult to reliably control cell fusion with a pair of two cells.

  In addition, both the chemical cell fusion method and the electric cell fusion method perform cell fusion by mixing two types of cells nonspecifically in the same component cell fusion solution. It was extremely difficult to control the components of the liquid according to the purpose. Therefore, some attempts to increase the fusion regeneration probability always require a cell pretreatment operation different from cell fusion, and also involve a complicated washing operation after cell fusion, so that the operation is complicated, The treatment time of cells was longer than necessary, resulting in problems such as decreased cell activity and lost cells.

  For example, as a method for increasing the fusion regeneration probability, a case has been reported in which the fusion regeneration probability is improved by increasing the Ca concentration in the cell fusion solution used in the electric cell fusion method (see, for example, Non-Patent Document 1). . In general, it is said that Ca has a cell membrane repairing action, and the fusion regeneration probability may be improved by cell fusion by increasing the Ca concentration. However, in practice, in the conventional electric cell fusion method, cell fusion is performed by mixing two types of cells in a solution of the same component, so that the Ca concentration control is effective in increasing the fusion regeneration probability. However, when Ca concentration is increased, there is an effect of improving the fusion regeneration probability due to Ca's cell membrane repair action, but the cells aggregate before cell fusion and the number of cells that cannot participate in cell fusion increases. As a result, there is a problem that the fusion reproduction probability decreases.

  Another method for increasing the fusion regeneration probability is to treat cells with sialic acid-degrading enzyme before cell fusion to decompose sialic acid on the cell membrane and charge the cells to a positive charge. A case has been reported in which the degree of adhesion is increased and the fusion reproduction probability is improved (see, for example, Non-Patent Document 2). In this case, however, cells are killed during the sialic acid decomposition reaction, and cells that have undergone sialic acid decomposition aggregate to reduce the number of cells contributing to cell fusion, resulting in a decrease in the probability of fusion regeneration. There was a problem of doing it.

  As another method for increasing the probability of fusion regeneration, a case has been reported in which the cell membrane is treated with an enzyme such as a protease before cell fusion to degrade proteins on the cell membrane, thereby improving the fusion regeneration probability (for example, Non-Patent Document 3). However, in this case as well, cell aggregation and death or cell attachment to the container occurs during the enzyme treatment, and the number of cells that can be treated during cell fusion is significantly reduced, resulting in a decrease in the probability of fusion regeneration. There was a problem of ending up. In addition, a complicated cell washing operation for removing these enzyme components before or after cell fusion is necessary.

  As another method to increase the fusion regeneration probability, a case in which the fusion regeneration probability is improved by suspending cells in a sugar solution that is hypotonic than osmotic pressure and applying a fusion voltage has been reported ( For example, refer nonpatent literature 4). In this case, it takes a long time to immerse cells in a hypotonic sugar solution, and the cells must be left in the hypotonic sugar solution for a while after cell fusion. As a result, there is a problem that the fusion reproduction probability decreases as a result.

  Furthermore, as another method for increasing the fusion regeneration probability, a fusion voltage such as polyethylene glycol is added to the cell fusion solution used in the electric cell fusion method and a fusion voltage is applied. Thus, there have been reported cases in which the fusion reproduction probability is improved as compared with the prior art (see, for example, Patent Document 2 and Non-Patent Document 5). In this case, if the added additive is a harmful component for the cells, the cells are killed during the treatment, resulting in a problem that the fusion regeneration probability is lowered. Further, after cell fusion, a complicated cell washing operation for removing components harmful to these cells is required.

  As described above, in the conventional PEG method and electrical cell fusion method, cell pretreatment for increasing the fusion regeneration probability and removal of the fusion accelerator after cell fusion itself cause cell death, respectively. There has been a problem that the number of fused cells produced is reduced, and thus the probability of fusion regeneration is rather deteriorated.

Japanese Patent Publication No. 7-40914 JP 60-9490 A

Ohnishi, K .; , Chiba, J .; Goto, Y .; , Tokunaga, T .; , “Improvement in the basic technology of electrophoretic for generation of antibody-production hybridomas”. Immunol. Methods. , 100, p. 181-189, 1987 Igarashi, M .; , Bando, Y .; "Enhanced efficiency of cell hybridization by neurominidase treatment." Immunol. Methods. 135, p. 91-93, 1990 Ohno-Shosaku, T .; Okada, Y .; “Facility of electrofusion of mouse lymphoma cells by the proteolytic action of proteases.” Biochem. Biophys. Res. Commun. , 120, p. 138-143, 1984 Schmitt, J.M. J. et al. , Zimmermann, U.S. Gessner, P .; "Electrofusion of osmotically treated cells. High and reproducible yields of hybridoma cells." Naturewissenchaften, 76, p. 122-123, 1989 Stoicheva, NG. , Hui, SW. “Electrically Induced Fusion of Mammalian Cells in the Presence of Polyethylene Glycol.” Membr. Biol. 141, p. 177-182, 1994

  The object of the present invention has been proposed in view of the conventional situation, and the cell fusion solution is replaced while the cells to be fused are fixed in the micropores in the fusion vessel, so that the fusion regeneration is simple and quick. Provided are a cell fusion device and a cell fusion method that enable treatment of cells with an increased probability.

  As a means for solving the above-described problems, the present invention includes a pair of electrodes each including a cell fusion solution inlet and a cell fusion solution discharge port and arranged to face the cell fusion region, and between the pair of electrodes. It consists of a flat insulator having a plurality of fine holes penetrating in the direction of the opposed electrodes arranged via a flat spacer, and the insulator is one of the electrodes A cell fusion device comprising a cell fusion container disposed on an electrode surface on the cell fusion region side of the electrode, a power source, and cell fusion solution introduction switching means for switching and introducing three or more cell fusion solutions Using the cell fusion device, wherein the power source has a power source switching mechanism for switching and connecting an AC power source for applying an AC voltage to the pair of electrodes and a DC pulse power source for applying a DC pulse voltage; Dress When the first cell and the second cell are fused in the cell fusion region using the cell fusion solution, the cell fusion solution containing the first cell is introduced into the cell fusion region, and the alternating voltage is applied to the cell fusion region. After fixing the first cell in the micropore, a cell fusion solution containing the second cell is introduced into the cell fusion region, and the AC voltage is applied to the second cell to the second cell. In which the cells are contacted in the micropores, and a DC pulse voltage is applied to the micropores to fuse the first cells with the second cells, the cell fusion solution introduction switching means The cell fusion solution containing the cell fusion treatment solution is introduced at least one time before introduction of the second cell, before the cell fusion, and after the cell fusion. By using the method, the above-mentioned problems of the prior art are solved. Found that it is possible to attain, it has led to the completion of the finally the present invention. Hereinafter, the present invention will be described in detail.

  The cell fusion device of the present invention includes a cell fusion solution inlet and a cell fusion solution discharge port, a pair of electrodes made of a conductive member arranged to face the cell fusion region, and a flat plate shape between the pair of electrodes. It is composed of a flat insulator having a plurality of fine holes that are arranged through a spacer and penetrated in the direction of the electrodes arranged to face each other, and the insulator is one of the electrodes. A cell fusion device comprising a cell fusion container disposed on an electrode surface on the cell fusion region side, a power source, and cell fusion solution introduction switching means for switching and introducing three or more cell fusion solutions, The power supply has a power supply switching mechanism for switching and connecting an AC power supply for applying an AC voltage to the pair of electrodes and a DC pulse power supply for applying a DC pulse voltage.

  In the cell fusion method of the present invention, when the first cell and the second cell are fused in the cell fusion region using the cell fusion device, a cell fusion solution containing the first cell in the cell fusion region is obtained. After the introduction and application of an alternating voltage to fix the first cells in the micropores, a cell fusion solution containing the second cells in the cell fusion region is introduced, and the alternating voltage is applied to the first cells. In which the second cell is brought into contact with the micropore, a DC pulse voltage is applied to the micropore, and the first cell and the second cell are fused, using cell fusion solution introduction switching means. In this cell fusion method, the cell fusion solution containing the cell fusion treatment solution is introduced at least one time before introduction of the second cell, before cell fusion, and after cell fusion.

  In the cell fusion method of the present invention, the cell fusion solution containing the cell fusion treatment solution is 2 or more, and the cell fusion solutions containing the two or more cell fusion treatment solutions are all the same, partly different, or all different. The cell fusion method is a cell fusion solution containing any of the cell fusion treatment solutions.

  The cell fusion method of the present invention is a cell fusion method in which the specific component is an enzyme, and the enzyme is a sialic acid degrading enzyme or a protease.

  The cell fusion method of the present invention is a cell fusion method in which the specific component is calcium ion.

  The cell fusion method of the present invention is a cell fusion method in which the above additive is an additive that increases the fusion regeneration probability. Examples of these additives include calcium salt, magnesium salt, amino acid, bovine serum albumin (BSA). , Sugar, ficoll, calmodulin, sericin, albumin, insulin, transferrin, cytokine, lipopolysaccharide, polyethylene glycol, cholesterol, catechin, serum, hormone, containing at least one of components derived from animal cell culture media It is a cell fusion method which is an additive.

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

  The cell fusion device of the present invention includes a cell fusion solution inlet and a cell fusion solution discharge port, a pair of electrodes made of a conductive member disposed to face the cell fusion region, and a flat plate between the pair of electrodes. It is composed of a flat insulator having a plurality of fine holes that are arranged through a spacer and penetrated in the direction of the electrodes arranged to face each other, and the insulator is one of the electrodes. A cell fusion device comprising a cell fusion container disposed on an electrode surface on the cell fusion region side, a power source, and cell fusion solution introduction switching means for switching and introducing three or more cell fusion solutions, The power supply has a power supply switching mechanism for switching and connecting an AC power supply for applying an AC voltage to the pair of electrodes and a DC pulse power supply for applying a DC pulse voltage.

  First, in order to explain the characteristics of the present invention, an outline of a basic cell fusion method using the cell fusion device of the present invention, that is, a method for cell fusion by introducing two cells into a cell fusion region is shown in FIGS. This will be described with reference to FIG. In the description, in order to briefly explain the basic operation of the present invention, the step of introducing the cell fusion solution containing the cell fusion treatment solution is omitted, and the cell fusion solution is a cell fusion method unique to the present invention. The method of introducing the cell fusion solution containing the cell fusion treatment solution using the introduction switching means is at least one time before the introduction of the second cell, before the cell fusion, and after the cell fusion. The description will be described later.

  The procedure of the cell fusion method of the present invention is shown in the order of FIG. 1, FIG. 2, and FIG.

  As shown in FIG. 1, the cell fusion solution containing the first cells (18) is first placed in the cell fusion region (1), and the power supply switching mechanism (7) is connected to the AC power supply (5). At this time, the first cells move toward the micropores (9) formed in the insulator (8) and are fixed. The force acting when the first cell moves toward the micropore is referred to as dielectrophoretic force (10). As shown in FIG. 1, the dielectrophoretic force means that when an alternating voltage of a specific frequency is applied between the electrodes, like the upper electrode (14) and the lower electrode (15) covered with the fine holes (9), When there is a concentrated part of the electric field lines (12), it is a force that moves dielectric particles such as cells toward the direction of the concentrated part (12) of the electric field lines (that is, the direction of the micropores). In general, the dielectrophoretic force is proportional to the volume of the dielectric particles, the difference between the dielectric constant of the dielectric particles and the dielectric constant of the solution, and the square of the applied voltage.

  Next, the cell fusion solution containing the second cells (22) is put into the cell fusion region while the first cells are fixed in the micropores while the dielectrophoretic force is continuously applied with the alternating voltage applied. At this time, as shown in FIG. 2, the second cell is fixed on the first cell by the dielectrophoretic force. Next, as shown in FIG. 3, when the power source switching mechanism is switched to the direct current pulse power source (6) and a direct current pulse voltage is applied, the cell membrane changes at the contact point of the first cell and the second cell (reversible destruction and Presumed) occurs, and cell fusion between the first cell and the second cell occurs to produce a fused cell (32). By doing in this way, a 1st cell and a 2nd cell can be made to contact in the position of a micropore, and cell fusion can be carried out with the fusion regeneration probability higher than the conventional electrical cell fusion method. In this case, ideally, a higher fusion regeneration probability can be obtained when one second cell is brought into contact with one first cell and fused. That is, it is possible to obtain a higher fusion regeneration probability when one first cell is fixed in one micropore and one second cell is further fixed thereon.

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

  FIG. 4 is a conceptual diagram of the cell fusion device of the present invention. The cell fusion device of the present invention comprises a cell fusion container (13), a power source (4), and a cell fusion solution introduction switching means (17). The power source (4) is constituted by a power source switching mechanism (7) for switching and connecting an AC power source (5) for applying an AC voltage to the pair of electrodes and a DC pulse power source (6) for applying a DC pulse voltage. Has been.

  As shown in FIG. 4, the cell fusion container secures the cell fusion region (1) by arranging the spacer (16) between the upper electrode (14) and the lower electrode (15), and the micropore (9). Insulator (8) having a structure is arranged on the cell fusion region side of the lower electrode.

  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, such as platinum, gold, copper, or an alloy such as stainless steel, and 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.

  There are no particular limitations on the dimensions of the upper electrode and the lower electrode, but a size that is easy to handle is preferably, for example, a size of about 70 mm long × 40 mm wide × 1 mm thick. A power source (4) is connected to the upper electrode and the lower electrode of the cell fusion container via a conductive wire (3). The power source (4) includes an AC power source (5) for applying an AC voltage between the upper electrode and the lower electrode, and a DC pulse power source for applying a DC pulse voltage for cell fusion between the upper electrode and the lower electrode ( 6), and the AC power source and the DC pulse power source can be used by appropriately switching them by the power source switching mechanism (7).

  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 fusion solution in the cell fusion container. The material may be an insulating material, and examples thereof include glass, ceramic, resin, and the like. In addition, the spacer includes an introduction channel (29) for introducing cells and an introduction port (19) communicating therewith, a discharge channel (30) for discharging cells, and a cell for introducing and discharging cells to and from the cell fusion container. A communicating outlet (20) is provided.

  The cell fusion liquid introduction switching means (17) is connected to the introduction port of the cell fusion container shown in FIG. 4 via the cell fusion liquid introduction flow path (2), and the syringe A (34) is connected to the cell fusion liquid introduction switching means. ), Syringe B (35), syringe C (36), syringe D (37), and syringe E (38). The cell fusion solution introduction switching means only needs to select one of syringe A, syringe B, and syringe C and connect the syringe to the cell fusion solution introduction channel. It consists of A valve may be attached to each syringe as shown in FIG. In the example shown in FIG. 4, for example, a cell fusion solution containing the first cells in syringe A, a cell fusion solution containing the second cells in syringe B, and a cell fusion solution containing the cell fusion treatment solution in syringe C are added. be able to. With such a configuration, it becomes possible to switch and introduce three or more cell fusion solutions, and when the cell fusion solution containing the cell fusion treatment solution is introduced using the cell fusion solution introduction switching means, At least one period of time before introduction of the second cell, before cell fusion, and after cell fusion can be achieved. The number of syringes is not particularly limited as long as one of the syringes can be selected and the syringe can be connected to the cell fusion solution introduction flow path. The number of cells to be used and the cell fusion solution containing the cell fusion treatment solution are not limited. What is necessary is just to install suitably according to a number.

  As two types of cells to be fused, for example, a combination of antibody-producing cells and myeloma cells used for producing a monoclonal antibody may be used.

  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. 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.

  The shape and size of the micropores are not particularly limited, but when the cell fusion device of the present invention is used, it is possible to obtain a higher fusion regeneration probability by fixing one cell in one micropore. Therefore, the diameter of the maximum circle inscribed in the planar shape of the micropore is smaller than the diameter of the cell fixed to the micropore (depending on the cell, about 1 μm to several tens of μm), or 1 of the cell diameter. It is preferable that it is in the range of about ˜2 times and the depth of the micropore is not more than the diameter of the cell fixed in the micropore. The reason for this will be described in more detail with reference to the drawings.

  As shown in FIG. 6, when the diameter of the largest circle inscribed in the planar shape of the micropore is larger than the diameter of two cells fixed to the micropore, a plurality of first cells and second cells are contained in the micropore. It becomes impossible to perform cell fusion in a one-to-one relationship between the first cell and the second cell, and the fusion regeneration probability is lowered. However, as shown in FIG. 7, when the diameter of the maximum circle inscribed in the planar shape of the micropore is smaller than the diameter of the two cells fixed in the micropore, a pair of the first cell and the second cell 1 cell fusion is possible and the fusion regeneration probability is high. Moreover, as shown in FIG. 8, the diameter of the largest circle inscribed in the planar shape of the micropore is about 1 to 2 times larger than that of the first cell, and the depth of the micropore is fixed to the micropore. If it is larger than the diameter, the second cell cannot contact the first cell fixed in the micropore and cannot be fused. However, as shown in FIG. 9, the diameter of the maximum circle inscribed in the planar shape of the micropore is about 1 to 2 times larger than that of the first cell, and the depth of the micropore is fixed to the micropore. When the diameter is less than or equal to the diameter, one second cell and one first cell fixed in the micropore are surely in contact with each other, so that a high fusion regeneration probability can be obtained.

  In a more preferred embodiment, the diameter of the second cell is preferably larger than the diameter of the first cell. When introducing a cell, a cell having a smaller diameter is first introduced as the first cell, Large cells are then preferably introduced as second cells, in which case the micropore diameter is preferably smaller than the diameter of the second cell and larger than the diameter of the first cell. The reason for this will be described below.

  Since electric field lines are concentrated in the fine holes, the electric field strength in the vicinity of the fine holes is the highest on the electrode surface in the fine holes as shown in FIG. 10, and is directed from the insulating film surface to the other electrode. The electric field strength gradually decreases. FIG. 10 shows a finite element method for calculating the electric field strength when an arbitrary voltage is applied between electrodes by arranging one fine hole having an arbitrary diameter and depth in an insulating film of an arbitrary film thickness on one electrode. Used to calculate. The vertical axis is the value obtained by normalizing the electric field intensity with the maximum electric field intensity, and the horizontal axis is the position between the electrodes. There is an electrode in which an insulating film is arranged at the origin of the horizontal axis. The insulating film surface corresponds to the position indicated by the dotted line in the figure, and the range from the origin of the horizontal axis to the dotted line corresponds to the insulating film thickness. According to the calculation, the electric field strength of the electrode surface in the microhole is less than the electric field strength of the insulating film surface as shown in FIG. 10 without much depending on the material and thickness of the insulating film and the size and depth of the microhole. The result is about 20% higher.

  In general, as described above, in the electric cell fusion method, by applying a DC pulse voltage for cell fusion, the electric conductivity of the cell membrane is instantaneously reduced, and the reversible disturbance of the cell membrane composed of lipid bilayers. And the cell is fused by reconfiguration. Here, in general, the smaller the cell diameter, the higher the DC pulse voltage that causes reversible disturbance of the cell membrane. Therefore, if a cell having a small diameter is put in the micropore as a first cell and a cell having a large diameter is fixed as a second cell from above the first cell fixed in the micropore, the DC pulse voltage to be applied is Even in the same case, as shown in FIG. 10, since the electric field strength in the micropore is higher than the electric field strength on the surface of the micropore, a higher voltage is applied to the first cell having a small diameter fixed in the micropore. Then, a voltage about 20% lower than the voltage applied to the first cell is applied to the second cell having a large diameter fixed to the surface of the micropore. By doing so, it becomes possible to cancel to some extent the voltage difference that causes reversible disturbance of the cell membrane when cells of different diameters are fused.

  As described above, in the cell fusion method using the cell fusion device of the present invention, it is preferable that the diameter of the first cell is smaller than the diameter of the second cell, and further, the diameter of the micropore is cell-fused. Of the two cells having different diameters, the diameter is preferably equal to or smaller than the diameter of the large diameter cell and equal to or larger than the diameter of the small diameter cell. By doing so, it becomes possible to securely fix cells having a diameter smaller than the micropores to the micropores, and then introduce cells having a diameter larger than the micropores, thereby making contact and cells in pairs of two cells. It becomes possible to perform fusion efficiently. However, in essence, the diameter of the first cell may be equal to or greater than the diameter of the second cell.

  In the cell fusion method of the present invention, the first cell and the second cell are preferably fused in the vicinity of the surface of the micropore. Essentially, the first cell and the second cell are in the micropore. These cells may be cell-fused, and can be arbitrarily changed without departing from the gist of the invention.

  Also, as will be described later, in order to increase the contact probability between the first cells fixed in the micropores and the second cells, the number of the second cells is made larger than the number of the first cells and excessively introduced into the cell fusion region. Even in this case, as shown in FIG. 10, the electric field strength is the highest in the vicinity of the fine hole, and becomes weaker as the distance from the fine hole becomes smaller. The cell membranes of only one cell and the second cell cause reversible disturbance and cell fusion. Therefore, it is possible to selectively fuse only the first cell and the second cell that are in contact with each other in the vicinity of the micropore.

  Since the cell fusion device of the present invention can obtain a higher fusion regeneration probability by fixing one cell in one micropore, the plurality of micropores formed in the insulator described above, On the surface of the insulator, the positions of the adjacent micro holes from any one of the micro holes are formed at the same position, that is, as shown in FIG. 4, a plurality of micro holes are formed in an array on the surface of the insulator. It is preferable that Here, the array shape means that the vertical and horizontal intervals of the fine holes are arranged at substantially equal intervals. By arranging the micropores in an array, the electric field generated by the voltage applied between the electrodes is generated almost uniformly in all the micropores, so that the probability that the cells are fixed in the micropores is the same in each micropore. The probability that one cell can be fixed in one micropore increases.

  In addition, in order to fix one cell in one micropore, it may be inappropriate if the interval between micropores formed in an array is too narrow or too wide. When the interval between the micropores is too narrow, there is a high probability that a plurality of cells are fixed in one micropore, and as a result, there is a high probability that micropores that do not contain cells are generated. Moreover, when the space | interval of a micropore is too wide, a cell will remain between a micropore and a micropore which a cell does not enter may become high. Therefore, more specifically, it is preferable that the interval between adjacent micropores is in the range of 0.5 to 6 times the diameter of the cells fixed in the micropores, and further the cell diameter in which the interval between the micropores is fixed. More preferably, it is about 1 to 2 times.

  The shape of the micropore in the present invention is not limited to a circular shape, and may be a polygonal shape such as a triangular shape or a square shape. In the case of a polygon such as a triangle or a quadrangle, the concentration of the electric field lines is increased at the corners, so the dielectrophoretic force is stronger than the circular micropores, and the probability that the cells are fixed in the micropores is increased. There is a merit that it becomes higher. However, when the micropores are arranged in an array, the probability that one cell can be fixed to each micropore is higher when the dielectrophoretic force from the front, rear, left and right micropores acts equally. The shape of is preferably point-symmetric and more preferably square. FIG. 5 is a schematic view showing an XX ′ cross-sectional view of the cell fusion container of FIG. 4. As a means for bonding the upper electrode (14), the spacer (16), the insulator (8), and the lower electrode (15) as shown in FIG. For example, a known method may be used, such as a method in which the spacers are fused together or a method in which the spacers are bonded using pressure-sensitive adhesives made by using a resin such as PDMS (poly-dimethylsiloxane) having surface adhesiveness or a silicon sheet. . In this way, the cell fusion region (1) shown in FIG. 5 can be formed.

  When putting the second cell, it becomes harder to be fixed to the micropore than the first cell entering the micropore, so by adding a larger number of the second cells than the number of the first cells, One cell and the second cell can be reliably brought into contact with each other. Here, when the number of the first cells is larger than the number of the micropores, there are cells that are not fixed in the micropores, and as a result, the ratio of the cells involved in the cell fusion decreases. The number is preferably equal to or less than the number of. If the number of the second cells is smaller than the number of the first cells, there may be a first cell that cannot be contacted with the second cell, and as a result, the combination of one set of two cells for cell fusion may be reduced. On the other hand, if the number of the second cells is too large, it may be impossible to introduce cells practically. Therefore, the number of the second cells is preferably about the same number to four times as the number of the first cells. .

  The AC power supply used in the cell fusion device of the present invention is, for example, an AC power supply that can output an AC voltage such as a sine wave, a rectangular wave, a triangular wave, or a trapezoidal wave having a peak voltage of about 1 V to 20 V and a frequency of about 100 kHz to 3 MHz. There is no particular limitation, and the DC pulse power supply is not particularly limited as long as it is a DC pulse power supply that can output a DC pulse voltage of about 50 V to 1000 V and a pulse width of about 10 μsec to 50 μsec.

  When the cell fusion device of the present invention is used, it is possible to obtain a higher fusion regeneration probability when one cell is fixed in one micropore, but one cell is fixed per one micropore. The waveform of the AC voltage is preferably a rectangular wave. The reason for this is that, as shown in FIGS. 12 to 15, the waveform of the alternating voltage is instantaneous compared to the sine wave (FIG. 12), the triangular wave (FIG. 13), and the trapezoidal wave (FIG. 14). Since the cell quickly moves into the micropores because the peak voltage (31) set in is reached, the probability that the cells overlap and enter the micropores is low, and thus the probability of fixing one cell per micropore Becomes higher. In addition, cells can be regarded as capacitors electrically, and while the peak voltage of the rectangular wave does not change, it is difficult for current to flow into the cells that have entered the micropores. Since the dielectrophoretic force is less likely to be generated in the micropores, once a cell enters the micropore, the probability that another cell enters the micropore is reduced, and electric lines of force are generated to generate dielectrophoretic force. This is because cells sequentially enter the empty micropores.

  The waveform of the AC voltage used in the cell fusion device of the present invention preferably has no DC 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. Ions contained in the suspension cause an electrical reaction on the electrode surface, generating 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.

  Next, the cell fusion method of the present invention will be described.

  The cell fusion method of the present invention is a cell fusion method using the cell fusion device, wherein the cell fusion solution containing the first cell is introduced into the cell fusion region using the cell fusion solution introduction switching means. Then, after the first cell is fixed in the micropore by applying the AC voltage, a cell fusion solution containing the second cell is introduced into the cell fusion region, and the AC voltage is applied. A cell fusion method in which the second cell is brought into contact with the first cell at the position of the micropore, and the cell switching is performed by applying the DC pulse voltage using the power supply switching mechanism, The time when the cell fusion solution containing the cell fusion treatment solution is introduced using the cell fusion solution introduction switching means is at least one time before the introduction of the second cell, before the cell fusion, and after the cell fusion. Cell fusion method. In the cell fusion method of the present invention, the cell fusion solution containing the cell fusion treatment solution is 2 or more, and the cell fusion solutions containing the two or more cell fusion treatment solutions are all the same or partially different or all It may be a cell fusion solution containing any one of the different cell fusion treatment solutions.

  By such a cell fusion method, each cell to be fused is sequentially introduced in a state of being suspended in a component solution according to the purpose to perform a two-cell paired cell fusion, and further each cell is introduced. It becomes possible for the first time to introduce a cell fusion solution containing a cell fusion treatment solution for removing components unnecessary for cell fusion or before or after cell fusion. In addition, since each cell is fixed to the micropores by an alternating voltage, it is possible to replace the cell fusion solution, and it is also possible for the first time to suppress the loss of cells accompanying the replacement of the cell fusion solution to an extremely low level. It becomes possible.

  The cell fusion method filed by the inventors of the present invention as Japanese Patent Application No. 2006-160744 is the following process (1). Moreover, as for the specific order of introduction of the cell fusion solution or cell fusion solution and application of the fusion voltage when carrying out the present invention, for example, the following processes (2) to (8) may be mentioned. Needless to say, the present invention is not limited to the process examples (2) to (8), and can be arbitrarily changed without departing from the gist of the invention. Further, the components of the first cell fusion solution and the second cell fusion solution may be the same component or different components.

  Process (1): The first cells suspended in the first cell fusion solution are introduced into the cell fusion region, and then the second cells suspended in the second cell fusion solution are introduced into the cell fusion region. Then, after making two cells contact with each other through the micropores, a fusion voltage is applied to fuse the cells.

  Process (2): The first cell suspended in the first cell fusion solution is introduced into the cell fusion region, and then the second cell suspended in the second cell fusion solution is introduced into the cell fusion region. After contacting two cells in a micropore, a cell fusion solution containing a cell fusion treatment solution containing a specific component is introduced into the cell fusion region to replace the cell fusion solution, and a fusion voltage is applied. Cell fusion.

  Process (3): introducing the first cell suspended in the first cell fusion solution into the cell fusion region, and introducing the cell fusion solution containing the cell fusion treatment solution containing a specific component into the cell fusion region. After replacing the cell fusion solution in step 2, the second cells suspended in the second cell fusion solution are introduced into the cell fusion region and contacted by a pair of two cells through the micropores. Apply to fuse cells.

  Process (4): introducing the first cell suspended in the first cell fusion solution into the cell fusion region, and introducing the cell fusion solution containing the cell fusion treatment solution containing a specific component into the cell fusion region. Then, the cell fusion solution is replaced with, and then the second cells suspended in the second cell fusion solution are introduced into the cell fusion region and contacted by a pair of two cells through the micropores. The cell fusion solution containing the cell fusion treatment solution is introduced into the cell fusion region to replace the cell fusion solution, and a cell is fused by applying a fusion voltage.

  Process (5): The first cells suspended in the first cell fusion solution are introduced into the cell fusion region, and then the second cells suspended in the second cell fusion solution are introduced into the cell fusion region. The cell fusion solution is introduced by introducing a cell fusion solution containing a cell fusion treatment solution containing a specific component into the cell fusion region after bringing the cell fusion into contact with a pair of two cells through a micropore and applying a fusion voltage. Replace.

  Process (6): The first cell suspended in the first cell fusion solution is introduced into the cell fusion region, and then the second cell suspended in the second cell fusion solution is introduced into the cell fusion region. After contacting two cells in a micropore, a cell fusion solution containing a cell fusion treatment solution containing a specific component is introduced into the cell fusion region to replace the cell fusion solution, and a fusion voltage is applied. The cell fusion solution is replaced by introducing a cell fusion solution containing a cell fusion treatment solution containing a specific component into the cell fusion region.

  Process (7): introducing the first cell suspended in the first cell fusion solution into the cell fusion region, and introducing the cell fusion solution containing the cell fusion treatment solution containing a specific component into the cell fusion region. After replacing the cell fusion solution in step 2, the second cells suspended in the second cell fusion solution are introduced into the cell fusion region and contacted by a pair of two cells through the micropores. The cell fusion solution is replaced by introducing a cell fusion solution containing a cell fusion treatment solution containing a specific component into the cell fusion region.

  Process (8): Introducing the first cell suspended in the first cell fusion solution into the cell fusion region, and introducing the cell fusion solution containing the cell fusion treatment solution containing a specific component into the cell fusion region. After the cell fusion solution is replaced with the above, the second cells suspended in the second cell fusion solution are introduced into the cell fusion region, brought into contact with a pair of two cells through the micropores, and then again identified. A cell fusion solution containing a component-containing cell fusion treatment solution is introduced into the cell fusion region to replace the cell fusion solution, and then a cell fusion is performed by applying a fusion voltage, and then a cell fusion treatment solution containing a specific component again. The cell fusion solution is replaced by introducing the cell fusion solution containing the cell fusion region into the cell fusion region.

  As described above, the process (1) is the most basic process of the cell fusion method already filed in the Japanese Patent Application No. 2006-160744 by the inventors of the present invention. It is a standard form that can be implemented more efficiently and reliably.

  The above processes (2) to (8) are obtained by arbitrarily adding a cell fusion solution replacement step in the cell fusion region to the process (1). Specifically, process (2) is after introduction of two types of cells, process (3) is after introduction of the first cell, process (4) is after introduction of the first cell and introduction of the second cell. Later, a cell fusion solution replacement step is added. Further, the process (5) is performed after applying the fusion voltage of the process (1), the process (6) is applied after applying the fusion voltage of the process (2), and the process (7) is applied after applying the fusion voltage of the process (3). ) Is obtained by adding a cell fusion solution replacement step after application of the fusion voltage in process (4). Here, the number of replacements of the cell fusion solution can be repeated any number of times according to the purpose. When the replacement is repeated a plurality of times, a cell fusion solution containing a cell fusion treatment solution containing a different component can be used each time. Is possible. In addition, when replacing with a specific cell fusion solution, it is desirable to fix the cells in the micropores with an AC voltage, but the cell fusion solution is slowly fed even when no AC voltage is applied. Thus, the cell fusion solution in the cell fusion region can be replaced without detaching the cells from the micropores.

  Further, by replacing the cell fusion solution as described above, the cell fusion method of the present invention removes specific components in the cell fusion solution in the cell fusion region with the cell fusion solution containing the cell fusion treatment solution. May be.

  In the cell fusion method of the present invention, the cell fusion solution containing the cell fusion treatment solution is used as a cell fusion solution containing a specific component, and the cell fusion solution is replaced with the cell fusion solution in the cell fusion region. The specific component may be introduced into Here, the specific component may be, for example, an enzyme such as a sialic acid-degrading enzyme or protease that modifies the cell surface, may be a calcium ion having a cell membrane repairing action, etc. Depending on the type of cells to be performed, an additive that increases the fusion regeneration probability may be used.

  In general, additives that increase the probability of fusion regeneration include inorganic salts such as calcium and magnesium salts, amino acids, bovine serum albumin (BSA), sugar, ficoll, calmodulin, sericin, albumin, insulin, transferrin, cytokines, and other proteins. , Lipopolysaccharide, polyethylene glycol, cholesterol, catechin, serum, hormones, components derived from animal cell culture media, and the like. The above-described process of the present invention can be applied by using any combination of these components in any concentration.

  Hereinafter, examples of applying the cell fusion method of the present invention and effects thereof will be described in more detail.

  As a first example, in the conventional electric cell fusion method, two types of cells are mixed in a mixed solution in the same component solution, so that the Ca concentration control is effective in increasing the fusion regeneration probability. Is difficult (see, for example, Non-Patent Document 1 above). In other words, when the Ca concentration is increased, there is an effect of improving the fusion regeneration probability due to the cell membrane repair action of Ca. However, cells aggregate before cell fusion, and the number of cells that cannot participate in cell fusion increases. There was a problem that the fusion reproduction probability decreased.

  On the other hand, in the cell fusion method of the present invention, by applying the above-described process (1), process (5) or process (6), each cell to be fused is suspended in a cell fusion solution of separate components. Since it becomes cloudy and can be individually introduced into the cell fusion region, such a problem can be overcome for the first time.

  A specific example in which the process (1) is applied in the first example will be described below. In the process (1), the first cell to be introduced into the cell fusion region first uses a suspension of cells in a cell fusion solution with a low Ca concentration to prevent cell aggregation and apply an alternating voltage. Thus, one cell can be fixed in one micropore. Next, the second cells to be introduced into the cell fusion region are suspended in a cell fusion solution having a high Ca concentration within a range that does not cause a voltage drop when the fusion voltage is applied. The second cell suspended in the cell fusion solution of this high Ca concentration component is introduced into the cell fusion region, an alternating voltage is applied to bring the first cell and the second cell into contact with each other through the micropore, and fusion is performed. Apply voltage to fuse cells. Thus, only the cell fusion solution in which the second cells are suspended uses a cell fusion solution having a high Ca concentration, thereby preventing the aggregation of the first cells and enhancing the effect of repairing the cell membrane after cell fusion. , The fusion reproduction probability can be increased. In addition, in order to efficiently form a two-cell pair with respect to the first cells fixed in the micropores, the second cells can be used excessively, and aggregation of the second cells themselves is not a problem.

  Next, a specific example in which the process (5) is applied in the first example will be described. In the process (5), the first cell to be first introduced into the cell fusion region is one in which cells are suspended in a cell fusion solution having a low Ca concentration to prevent cell aggregation and to apply an AC voltage. Thus, one cell can be fixed in one micropore. Next, the second cells to be introduced into the cell fusion region are suspended in a cell fusion solution having a high Ca concentration within a range that does not cause a voltage drop when the fusion voltage is applied. The second cell suspended in the cell fusion solution of this high Ca concentration component is introduced into the cell fusion region, an alternating voltage is applied to bring the first cell and the second cell into contact with each other through the micropore, and fusion is performed. Apply voltage to fuse cells. Further, after cell fusion, a medium for culturing the fused cells is rapidly introduced into the cell fusion region, and the cell fusion solution in the cell fusion region is replaced with the medium. Thus, only the cell fusion solution in which the second cells are suspended uses a cell fusion solution having a high Ca concentration, thereby preventing the aggregation of the first cells and enhancing the effect of repairing the cell membrane after cell fusion. , The fusion reproduction probability can be increased. In addition, after cell fusion, the cell fusion solution in the cell fusion region can be promptly replaced with a medium for culturing the fused cells, thereby preventing a decrease in the activity of the fused cells and further increasing the fusion regeneration probability. .

  A specific example in which the process (6) is applied in the first example will be described below. In this example, when significant aggregation occurs when the second cells are suspended in the cell fusion solution having a high Ca concentration, the aggregation can be prevented. In the process (6), the first cell to be introduced into the cell fusion region first uses a suspension of cells in a cell fusion solution having a low Ca concentration to prevent cell aggregation and apply an alternating voltage. Thus, one cell can be fixed in one micropore. Next, the second cell to be introduced into the cell fusion region is similarly suspended in a cell fusion solution having a low Ca concentration component, the second cell is introduced into the cell fusion region, and an alternating voltage is applied to the first cell. The cells and the second cells are brought into contact with each other through micropores. Thereafter, before applying the fusion voltage, a cell fusion solution having a high Ca concentration is introduced into the cell fusion region to replace the cell fusion solution. Next, cell fusion is performed by applying a fusion voltage. Finally, a medium for culturing the fused cells is rapidly introduced into the cell fusion region, and the cell fusion solution in the cell fusion region is replaced with the medium. Thus, the first cells and the second cells can be prevented from aggregating by suspending in the cell fusion solution having a low Ca concentration, and the first cells and the second cells are microporous. Then, by introducing a cell fusion solution having a high Ca concentration component and replacing the cell fusion solution, the effect of repairing the cell membrane after cell fusion is increased, and the fusion regeneration probability can be increased. In addition, after cell fusion, the cell fusion solution in the cell fusion region can be promptly replaced with a medium for culturing the fused cells, thereby preventing a decrease in the activity of the fused cells and further increasing the fusion regeneration probability. .

  As a second example, sialic acid on the cell membrane is decomposed by treating the cells with sialic acid degrading enzyme before cell fusion, and the cells are fused with each other by charging the cells with a positive charge. An example of improving the fusion reproduction probability has been reported (see, for example, Non-Patent Document 2). However, in general, in the conventional electric cell fusion method, it is necessary to perform an enzyme treatment with a sialic acid-degrading enzyme before introducing the cell into the cell fusion region. In addition to the decrease in activity and cell loss, the cells that have undergone sialic acid degradation aggregate to reduce the number of cells that contribute to cell fusion, resulting in a decrease in fusion regeneration probability. there were. However, in the cell fusion method of the present invention, since the replacement of the cell fusion solution can be sequentially performed, by applying the above-described process (3) or process (4), cell fusion is performed before cell fusion in the cell fusion region. It is possible to simply perform sialic acid decomposition treatment and continue cell fusion as it is.

A specific example in which the process (3) is applied in the second example will be shown below. In the process (3), first, the first cell is mixed with a cell fusion solution of a normal electric cell fusion method (for example, 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL in 300 mM mannitol aqueous solution). In a cell fusion solution supplemented with BSA (bovine serum albumin), the suspension is introduced, and the first cells are fixed in the micropores using an alternating voltage, and then sialic acid-degrading enzyme is contained in the state where the alternating voltage is applied. Substitution is performed with a normal cell fusion solution for electric cell fusion, and the sialic acid decomposition treatment of the first cell can be performed. In this case, since the first cells are fixed in the micropores, cell aggregation does not occur during the treatment with the sialic acid-degrading enzyme, and therefore, loss of the cells before cell fusion due to the sialic acid-degrading treatment should be avoided as much as possible. Can do. Next, the second cell is introduced into the cell fusion region. In this case as well, the cell fusion solution for suspending the second cell is a cell fusion solution for normal electric cell fusion containing sialic acid degrading enzyme, so that the sialic acid of the second cell in the cell fusion region is used. It is possible to perform a decomposing enzyme treatment. Next, by bringing the second cell into contact with the first cell with an alternating voltage through a micropore and then applying a fusion voltage, the two cells can be rapidly fused.

A specific example in which the process (4) is applied in the second example will be described below. In the process (4), first, the first cell is mixed with a cell fusion solution of a normal electric cell fusion method (for example, 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL in 300 mM mannitol aqueous solution). In a cell fusion solution to which BSA has been added, the suspension is introduced and the first cells are fixed in micropores using an alternating voltage, and then a serum-free medium containing sialic acid-degrading enzyme is introduced to carry out sialic acid degradation treatment. Do. In this case, since the first cells are fixed in the micropores, cell aggregation does not occur during the treatment with the sialic acid-degrading enzyme, and therefore, loss of the cells before cell fusion due to the sialic acid-degrading treatment should be avoided as much as possible. Can do. Next, the second cell is introduced into the cell fusion region. In this case as well, a serum-free medium containing sialic acid-degrading enzyme can be used as a cell fusion solution for suspending the second cell, so that the second cell can be treated with sialic acid-degrading enzyme in the cell fusion region. It is. However, although the serum-free medium used in this specific example is more advantageous for maintaining the cell activity than a normal cell fusion solution for electric cell fusion, components such as salt contained in the medium are advantageous. As a result, the voltage drop at the time of applying the fusion voltage becomes remarkable, and the fusion reproduction probability decreases. Therefore, after bringing the first cell and the second cell into contact with each other through the micropores with an alternating voltage, a normal cell fusion solution for electric cell fusion is applied to the cell fusion region 2 to 3 times before applying the fusion voltage. After the introduction and replacement of the cell fusion solution, cell fusion can be performed by applying a fusion voltage. By doing this, it is possible to remove components such as salt contained in the medium, prevent voltage drop when applying fusion voltage, and maintain cell activity, and cell fusion with high fusion regeneration probability Can be performed.

  As a third example, a case has been reported in which cells on a cell membrane are decomposed by treating the cells with an enzyme such as a protease before cell fusion to improve the fusion regeneration probability (for example, the above-mentioned non-patent document). 3). However, in general, the conventional electric cell fusion method requires an enzyme treatment with a protease before introducing the cell into the cell fusion region, and the cell is killed during the enzyme treatment or the activity of the cell. There has been a problem that cells are lost due to decrease in cell adhesion, cell adhesion to the container, or aggregation between cells, resulting in a decrease in the probability of fusion regeneration. However, in the cell fusion method of the present invention, by applying the above-mentioned process (8), the cell protease treatment is simply performed before cell fusion in the cell fusion region, followed by cell fusion, and further removal of the protease. Is possible.

  A specific example in which the process (8) is applied in the third example will be shown below. In the process (8), first cells are suspended in a normal cell fusion solution for electric cell fusion and introduced into the cell fusion region, and the first cells are fixed in the micropores using an alternating voltage. Thereafter, the cell fusion solution can be replaced with a normal cell fusion solution for electric cell fusion containing protease in a state where an AC voltage is applied, and the protease treatment of the first cell can be performed. In this case, since the first cells are fixed in the micropores, aggregation between the cells does not occur during the protease treatment, and loss of the cells before cell fusion can be avoided. Next, with the alternating voltage applied, the second cells are suspended in a normal cell fusion solution for electric cell fusion and introduced into the cell fusion region. Next, after the second cell is brought into contact with the first cell by an AC voltage, the cell fusion solution is replaced with a normal cell fusion solution for electric cell fusion containing protease in a state where the AC voltage is applied. The protease treatment of the second cell can be performed. However, since protease is cytotoxic, it has been necessary to remove it by repeating a complicated cell washing operation before or after fusion in the conventional electric cell fusion method, but in the cell fusion method of the present invention, it is necessary to remove the cell fusion region. The protease can be easily removed by substituting the cell fusion solution. That is, after the protease treatment of the first cell and the second cell, and before the fusion voltage is applied, a normal cell fusion solution for electric cell fusion is introduced into the cell fusion region 2 to 3 times. By replacing the fusion solution, the protease can be removed, and then a fusion voltage is applied to fuse the two cells. By doing in this way, the protease which has cytotoxicity at the time of cell fusion can be prevented from entering the fused cell, and the activity of the fused cell can be maintained. Furthermore, after cell fusion, the cell fusion solution in the cell fusion region can be quickly replaced with a medium for culturing the fused cells, so that the decrease in the activity of the fused cells can be prevented. Since it can be propagated, the fusion regeneration probability can be further increased.

  As a fourth example, in the case of cell fusion of two types of cells, a case was reported in which the fusion regeneration probability was improved by suspending the cells in a sugar solution that is less than osmotic pressure and applying a fusion voltage. (For example, refer nonpatent literature 4). In this case, in the conventional electric cell fusion method, the cells are treated with a hypotonic sugar solution for a long time, and after fusion, the cells must be left in the hypotonic sugar solution for a while. Alternatively, after cell fusion, the number of cells that rupture and die increases, resulting in a problem that the fusion regeneration probability is lowered. However, in the cell fusion method of the present invention, by applying the above-described process (6) or process (7), the cell fusion solution in the cell fusion region can be sequentially replaced. The cell can be treated with a hypotonic solution to prevent cell loss.

A specific example in which the process (6) is applied in the fourth example will be described below. In the process (6), the first cell was added to a sugar solution that is isotonic with the cell (for example, 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL BSA was added to a 300 mM mannitol aqueous solution). The cells are introduced into the cell fusion region in a suspended state in a cell fusion solution, and the first cells are fixed in the micropores with an alternating voltage, and then the second cells are made isotonic with the cells (for example, 300 mM mannitol). A cell fusion solution in which 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL BSA is added) is suspended in an aqueous solution and introduced into the cell fusion region. after contacting the second cell with micropores, hypotonic sugar solutions (e.g., in 200mM mannitol solution, CaCl ₂ 0.1 mM, of 0.2mM of MgCl _2, 1 mg / mL B Performs substitution cell fusion solution by introducing several cell fusion solution) was added A, then, by applying a fusion voltage, an effect is obtained that processing hypotonic solution before fusion cells. In this case, unlike the conventional electric cell fusion method, since it is not necessary to immerse the cells in a hypotonic sugar solution from the beginning, it is possible to minimize damage to the cells due to a decrease in osmotic pressure. . Furthermore, by replacing the cell fusion region with an isotonic sugar solution again immediately after application of the fusion voltage, loss due to rupture of the fused cells during the stationary treatment after fusion can be prevented as much as possible. In addition, it cannot be overemphasized that arbitrary composition suitable for the kind of cell which should be united can be used for the sugar concentration of the solution which performs substitution.

A specific example in which the process (7) is applied in the fourth example will be described below. In the process (7), the first cell was added to a sugar solution that is isotonic with the cell (for example, 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL BSA was added to a 300 mM mannitol aqueous solution). The cells are introduced into the cell fusion region in a state suspended in a cell fusion solution, and the first cells are fixed in the micropores by an alternating voltage. Next, a hypotonic sugar solution (for example, a cell fusion solution in which 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL BSA is added to a 200 mM mannitol aqueous solution) is introduced several times to perform cell fusion. Replace the liquid. Next, the second cells are made into an isotonic sugar solution with the cells (for example, a cell fusion solution in which 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL BSA is added to a 300 mM mannitol aqueous solution) The cell is introduced into the cell fusion region in a suspended state, and the first cell and the second cell are brought into contact with each other through a micropore with an alternating voltage, and then the fusion voltage is applied. Thus, it is possible to treat only the first cells with the hypotonic sugar solution by replacing with the hypotonic sugar solution after the introduction of the first cells. In this case, the fusion reproduction probability can be increased by adjusting the size of the first cell having a small radius so that it can be easily fused with the second cell. Furthermore, by replacing the cell fusion region with an isotonic sugar solution again immediately after application of the fusion voltage, loss due to rupture of the fused cells during the stationary treatment after fusion can be prevented as much as possible. In addition, it cannot be overemphasized that arbitrary composition suitable for the kind of cell which should be united can be used for the sugar concentration of the solution which performs substitution.

  As a fifth example, in the cell fusion of two types of cells, the fusion regeneration probability can be increased by adding a fusion voltage additive such as polyethylene glycol in the cell fusion solution and applying a fusion voltage. Improved cases have been reported (see, for example, Patent Document 2 and Non-Patent Document 5). In this case, in the conventional electric cell fusion method, if the added additive is a harmful component for cells such as polyethylene glycol, the cell is killed during the cell fusion treatment, and these components are removed after the fusion. For this reason, there is a problem in that complicated cell washing operations are required for this purpose, resulting in cell death, cell activity reduction, and cell loss, resulting in a decrease in fusion regeneration probability. However, since the cell fusion solution can be introduced sequentially in the cell fusion method of the present invention, the cell fusion solution containing these additives is given to the cells by applying the process (2) or the process (6) described above. It is possible to minimize damage and quickly remove these components before and after cell fusion.

A specific example in which the process (2) is applied in the fifth example will be described below. In the process (2), first, the first cell is mixed with a cell fusion solution of a normal electric cell fusion method (for example, 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL in 300 mM mannitol aqueous solution). The suspension is introduced into a cell fusion solution supplemented with BSA, and the first cells are fixed in the micropores using an alternating voltage. Next, the second cell is a cell fusion solution of a normal electric cell fusion method (for example, a cell obtained by adding 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL BSA to a 300 mM mannitol aqueous solution). The suspension is introduced into the fusion solution), and the first cell and the second cell are brought into contact with each other through a micropore using an alternating voltage. Then, the cell fusion solution of the usual electric cell fusion method containing an additive (for example, polyethylene glycol) having a fusion promoting action is introduced several times to replace the cell fusion solution, and the fusion voltage is applied, thereby fusing Cell fusion can be achieved by adding an additive having a promoting action. In this case, even if the additive is harmful to the cells, since these components can act on the cells in a short time just before the fusion, damage to the cells can be minimized.

A specific example in which the process (6) is applied in the fifth example will be described below. In the process (6), first, the first cell is mixed with a cell fusion solution of a normal electric cell fusion method (eg, 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL in 300 mM mannitol aqueous solution). The suspension is introduced into a cell fusion solution supplemented with BSA, and the first cells are fixed in the micropores using an alternating voltage. Next, the second cell is a cell fusion solution of a normal electric cell fusion method (for example, a cell obtained by adding 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL BSA to a 300 mM mannitol aqueous solution). The suspension is introduced into the fusion solution), and the first cell and the second cell are brought into contact with each other through a micropore using an alternating voltage. Then, the cell fusion solution of the usual electric cell fusion method containing an additive (for example, polyethylene glycol) having a fusion promoting action is introduced several times to replace the cell fusion solution, and the fusion voltage is applied, thereby fusing Cell fusion can be achieved by adding an additive having a promoting action. In this case, even if the additive is harmful to the cells, since these components can act on the cells in a short time just before the fusion, damage to the cells can be minimized. In addition, after applying the fusion voltage, a normal cell fusion solution for electric cell fusion is introduced into the cell fusion region 2 to 3 times to replace the cell fusion solution, thereby quickly removing the components of the additive. The fused cells can be directly transferred to a medium for growth. In this way, in the conventional electric cell fusion method, when a cytotoxic substance is used as an additive, it was necessary to remove these by repeating a complicated cell washing operation after cell fusion. In the cell fusion method of the invention, it is possible to easily remove these components by replacing the cell fusion solution in the cell fusion region.

  Further, in the cell fusion method of the present invention, a cell fusion solution containing a plurality of cell treatment solutions having different compositions containing a specific component is introduced before the second cell introduction, before cell fusion by application of a fusion voltage, and Since it can be replaced as many times as desired at least at one time after cell fusion, for example, as described above, the effects of the first to fifth examples can be arbitrarily combined. . Below, the specific example which applied the process (2) combining the cell processing which has a mutually different effect is shown. In this example, after the first cell and the second cell are treated with protease, the cells are suspended in a sugar solution having a hypotonicity lower than the osmotic pressure, and a fusion voltage is applied, whereby the third and fourth examples are applied. It is possible to increase the fusion reproduction probability more effectively by combining the effects shown in.

Specific example of applying a plurality of cell treatment effects to process (2): First, the first cell is suspended in a normal cell fusion solution for electric cell fusion, introduced into the cell fusion region, and finely divided using an AC voltage. After fixing the first cells in the pores, the second cells are suspended in a normal cell fusion solution for electric cell fusion and introduced into the cell fusion region with an alternating voltage applied. Next, after the second cell is brought into contact with the first cell with an alternating voltage, the cell fusion solution is replaced with a normal cell fusion solution for electric cell fusion containing a protease. Protease treatment of two cells can be performed simultaneously. In this case, since the first cells are fixed in the micropores, aggregation between the cells does not occur during the protease treatment, and loss of the cells before cell fusion can be avoided. Moreover, in order to efficiently form a pair of two cells with respect to the first cells fixed in the micropores, the second cells can be used excessively, and the aggregation of the second cells themselves is not a problem. Next, by introducing the cell fusion solution for normal electric cell fusion into the cell fusion region 2 to 3 times and replacing the cell fusion solution, the protease having cytotoxicity can be removed. Activity can be maintained. Still further, a hypotonic sugar solution (for example, a cell fusion solution obtained by adding 0.1 mM CaCl ₂ , 0.2 mM MgCl ₂ , 1 mg / mL BSA to a 200 mM mannitol aqueous solution) was introduced several times. By replacing the cell fusion solution and then applying a fusion voltage, the effect of treating the cells with a hypotonic solution before fusion can be obtained. In this case, unlike the conventional electric cell fusion method, since it is not necessary to immerse the cells in a hypotonic sugar solution from the beginning, it is possible to minimize damage to the cells due to a decrease in osmotic pressure. .

  As described above, in the cell fusion method of the present invention, the cell fusion solution in the cell fusion region can be replaced as many times as desired by using a cell fusion solution having a different composition, so that the fusion regeneration probability is increased. Multiple cell treatments can be performed simultaneously in a short time. In this case, it is naturally possible to combine three or more cell treatment steps, and a plurality of cell treatments can be applied in any of the processes (2) to (8) described above.

According to the present invention, the following effects can be obtained.
(1) According to the cell fusion device of the present invention, cell fusion can be reliably performed with a pair of two cells in a micropore, and in addition to a cell fusion solution containing two types of cells, cell fusion treatment It is possible to introduce at least one kind of cell fusion solution containing liquid, put the cells in the cell fusion region, leave the cells fixed in the micropores, change the components of the cell fusion solution in the cell fusion region, It is possible to change the cell fusion conditions to obtain a higher fusion regeneration probability.
(2) According to the cell fusion method of the present invention, the components of the cell fusion solution can be changed at any timing of the cell fusion step, and the cell fusion conditions are changed to obtain a higher fusion regeneration probability. Things will be possible.
(3) According to the cell fusion method of the present invention, although there is an effect of increasing the fusion regeneration probability, if the cell is in contact with the cell for a long time, the cell is killed, the cell activity is decreased, or the cell is aggregated. Such a component can be removed easily and quickly, and loss of cells can be prevented, and a higher fusion regeneration probability can be obtained.
(4) According to the cell fusion method of the present invention, it is possible to easily and quickly introduce a component having an effect of increasing the fusion regeneration probability at an arbitrary timing of the cell fusion step, and a higher fusion regeneration probability. Obtainable.

It is a 1st figure which shows the concept of the basic cell fusion method in this invention. It is a 2nd figure which shows the concept of the basic cell fusion method in this invention. It is a 3rd figure which shows the concept of the basic cell fusion method in this invention. 1 is a conceptual diagram of a cell fusion device in the present invention and a conceptual diagram of a cell fusion device used in Example 1. FIG. FIG. 5 is a sectional view taken along line XX ′ shown in FIG. 4. It is a conceptual diagram which shows the case where the diameter of the largest circle inscribed in the planar shape of a micropore is larger than the diameter of two cells fixed to a micropore. It is a conceptual diagram which shows the case where the diameter of the largest circle inscribed in the planar shape of a micropore is smaller than the diameter of two cells fixed to a micropore. It is a conceptual diagram showing a case where the diameter of the maximum circle inscribed in the planar shape of the micropore is about 1 to 2 times larger than the first cell and the depth of the micropore is larger than the diameter of the first cell fixed to the micropore. is there. A concept showing a case where the diameter of the maximum circle inscribed in the planar shape of the micropore is about 1 to 2 times larger than the first cell and the depth of the micropore is equal to or less than the diameter of the first cell fixed to the micropore. FIG. It is the figure which showed the electric field strength of a micropore vicinity, a horizontal axis (X-axis) shows the distance (unit is arbitrary) from an electrode surface, and a vertical axis | shaft (Y-axis) shows electric field strength (unit is arbitrary). It is the schematic of general photolithography and the etching method. FIG. 4 is a diagram showing a typical waveform of a sine wave as an example of an AC voltage waveform used in the present invention, where the horizontal axis (X axis) indicates time and the vertical axis (Y axis) indicates voltage. As an example of the waveform of the AC voltage used in the present invention, it is a diagram showing a typical waveform of a triangular wave, the horizontal axis (X axis) shows time, and the vertical axis (Y axis) shows voltage. It is the figure which showed the typical waveform of the trapezoid wave as an example of the waveform of the alternating voltage used for this invention, a horizontal axis (X-axis) shows time and a vertical axis | shaft (Y-axis) shows a voltage. FIG. 2 is a diagram showing a typical waveform of a rectangular wave as an example of an AC voltage waveform used in the present invention, where the horizontal axis (X axis) indicates time and the vertical axis (Y axis) indicates voltage.

  Examples of the present invention will be described in detail below. 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. 4 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. 4, 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. As will be described later, the fine holes were formed in the insulating film disposed on the lower electrode (15) by general photolithography and etching.

  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. In addition, as shown in FIG. 4, in order to introduce and discharge cells into the cell fusion container, the spacer has an introduction flow path (29) for introducing cells and an introduction port (19) communicating with the cells, and discharge for discharging cells. A flow path (30) and a discharge port (20) communicating therewith were provided. Further, as shown in FIG. 4, the introduction port has a 1 mL capacity syringe A (34), a 1 mL capacity syringe B (35), and a 10 mL capacity syringe C (36) via the cell fusion solution introduction channel (2). ) Were connected via valves (21) as cell fusion solution introduction switching means, respectively.

  Further, the insulator (8) having a plurality of fine holes was produced by integrally forming 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 (33). 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), spacer (16), and fine hole-integrated insulator-integrated lower electrode (28) thus produced were laminated and pressure bonded as shown in FIG. FIG. 5 is a cross-sectional view taken along the line AA ′ of the cell fusion container shown in FIG. 4. The surface of the silicon sheet, which is a spacer, was sticky, and the parts were brought into close contact with each other by pressure bonding, and the cell fusion solution containing 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.

  As a power source for applying a voltage between the electrodes, a signal generator (NF circuit design block, WF1966) is used as an AC power source (5), and a cell fusion power source (manufactured by Nepagene, LF101) is used as a conductive wire (NF1). 3), the connection between the AC power supply and the DC pulse power supply can be switched by the power supply switching mechanism (7).

  Using the above-described cell fusion apparatus, cell fusion was performed using mouse spleen cells (diameter about 6 μm) and mouse myeloma cells (diameter about 10 μm). Myeloma cells were treated with sialic acid degrading enzyme before cell fusion, and mouse spleen cells were introduced into the cell fusion region and fixed in micropores, then treated with sialic acid degrading enzyme in the cell fusion region, Cell fusion was performed and the fusion regeneration probability was confirmed.

  First, spleen cells were removed from the mouse. Kim towel was placed in a sealed bottle, and the mouse was euthanized using sevoflurane (manufactured by Maruishi Pharmaceutical). After 70% disinfectant ethanol was sufficiently sprayed on the mouse, it was fixed to a dissection table in a clean bench with an injection needle. Next, the outer skin was picked up with tweezers and cut with scissors for dissection, and the outer skin was first cut out. The endothelium was then cut open with another new scissors to expose the spleen, and the spleen was cut from the mouse body with scissors while the spleen was pulled out of the body using tweezers. A medium for animal cell culture (hereinafter referred to as medium A) containing 10 mL of 10% FBS (bovine serum) was placed in a 50 mL centrifuge tube, and the spleen was moved and shaken therein to wash the surface. Next, the spleen was transferred onto a lid of a petri dish made of φ9 cm Sumilon, and fat adhered around the extracted spleen was removed using two tweezers. Place 10 mL of medium A in a Petri dish made of φ9 cm Sumilon, cut the spleen with new scissors into 4-5 pieces in a 40 mL mesh cell strainer (Falcon), and cut the flat part of the tail of the 5 mL Terumo syringe. The spleen was ground thoroughly. Spleen cells adhering to the cell strainer and the tail of the Terumo syringe were washed away with medium A. The suspension containing the spleen cells in the petri dish was transferred to a 50 mL centrifuge tube, the petri dish was washed twice with medium A, and the washing solution was also mixed in the centrifuge tube. The suspension containing the spleen cells was centrifuged at 1500 rpm for 5 minutes at room temperature, and then the supernatant was aspirated with an aspirator to loosen the spleen cell pellets. Next, after suspending the undissolved spleen cells in 1 mL of FBS, 9 mL of erythrocyte disruption solution (manufactured by SIGMA) was added and mixed well, and the mixture was allowed to stand at room temperature for 3 minutes to disrupt the erythrocytes. The suspension containing the spleen cells was again centrifuged at 1500 rpm for 5 minutes at room temperature, and the supernatant was aspirated with an aspirator to unravel the cell pellet and then suspended in 20 mL of medium A (the suspension in this state). Is hereinafter referred to as a spleen cell extract). Using a cell strainer, put the spleen cell extract into a 50 mL centrifuge tube, filter, and centrifuge 10 mL of this spleen cell-containing suspension at 1500 rpm at room temperature for 5 minutes, and then aspirate the supernatant with an aspirator. Then, the spleen cell pellet was loosened. Immediately after suspending the undissolved spleen cells in 10 mL of serum-free animal cell culture medium (hereinafter referred to as medium B), the mixture was centrifuged at 1500 rpm for 5 minutes at room temperature, and the supernatant was aspirated with an aspirator. The pellet of spleen cells was unwound and the spleen cells were washed with medium B.

Next, spleen cells were suspended in 20 mL of a cell fusion solution (hereinafter referred to as cell fusion solution A) containing 300 mM mannitol, 0.1 mM CaCl ₂ , 0.1 mM MgCl ₂ , and 0.1 mg / mL BSA. After turbidity, the mixture was centrifuged at 1500 rpm for 5 minutes at room temperature, and the supernatant was aspirated. Then, the spleen cell pellet was unwound and the spleen cells were washed with the cell fusion solution A. Again, spleen cells are suspended in a small amount of cell fusion solution A, filtered into a 50 mL Falcon tube using a cell strainer, diluted with cell fusion solution A, and the final concentration of spleen cells is 0.9 × 10 ⁶ cells. A cell fusion solution containing spleen cells adjusted to / mL was prepared. A cell fusion solution containing 300 mM mannitol as a main component is almost isotonic with the osmotic pressure of cells.

In addition, the myeloma cells were suspended in the medium A in a φ15 cm suspension culture dish so that the concentration was always 1 × 10 ⁶ cells / mL or less, and the cells were subcultured in a CO ₂ incubator at 37 ° C. and 5%. It was. The day before cell fusion, suspend myeloma cells in medium A so that the concentration of myeloma cells is 2.0 × 10 ⁵ cells / mL, and use 40 mL of medium A as a myeloma culture solution in a φ15 cm suspension culture dish. Three cultures were performed. The myeloma culture was transferred from the petri dish to a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was aspirated with an aspirator to loosen the myeloma cell pellet. Immediately add 40 mL of medium B to each tube to suspend the myeloma cells, combine them into one tube, centrifuge again at 1000 rpm at room temperature for 5 minutes, and aspirate the supernatant with an aspirator. Then, the pellet of the myeloma cells was loosened. Myeloma cells were resuspended in 10 mL of medium B containing 0.2 U / mL sialic acid-degrading enzyme (manufactured by Sigma) and treated at 37 ° C. for 1 hour. Next, after centrifugation at 1000 pm for 5 minutes, the supernatant was aspirated and the myeloma cell pellets were unwound. Immediately add 50 mL of medium B to each tube to suspend the myeloma cells, combine them into one tube, centrifuge again at 1000 rpm and room temperature for 5 minutes, and aspirate the supernatant with an aspirator. Then, the pellet of the myeloma cells was loosened. The myeloma cells were resuspended in 20 mL of medium B, centrifuged at 1000 rpm at room temperature for 5 minutes, the supernatant was aspirated with an aspirator, and the myeloma cell pellets were loosened.

Next, the myeloma cells were resuspended in 40 mL of the cell fusion solution A, centrifuged at 1000 rpm at room temperature for 5 minutes, the supernatant was aspirated with an aspirator, and the myeloma cell pellet was unwound. The myeloma cells are suspended again in a small amount of the cell fusion solution A, filtered in a 50 mL falcon tube using a cell strainer, diluted with the cell fusion solution A, and the final concentration is 3.7 × 10 ⁶ cells / mL. A cell fusion solution containing myeloma cells treated with the prepared sialic acid-degrading enzyme was prepared.

  The cell fusion solution containing spleen cells is placed in syringe A of the cell fusion device, the cell fusion solution containing myeloma cells treated with sialic acid-degrading enzyme is placed in syringe B, and 0.2 U / mL sialic acid degradation is performed. Animal cell culture medium containing the enzyme was placed in syringe C, and cell fusion solution A was placed in syringe D.

  First, 600 μL of the cell fusion solution containing the spleen cells (the number of spleen cells is approximately 400,000 considering the loss) is introduced into the cell fusion region using syringe A, and the spleen cells are introduced into the cell fusion region. It was allowed to settle sufficiently. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are fixed in an array of approximately 400,000 micropores and one in each micropore. did. Subsequently, 3 μL of animal cell culture medium containing 0.2 U / mL sialic acid-degrading enzyme was added using syringe C, while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes by an AC power source. The cells were introduced into the cell fusion region in batches, and the AC power supply was cut off and allowed to stand at room temperature for 45 minutes without applying a voltage. As a result, spleen cells could be treated with sialic acid degrading enzyme in the cell fusion region.

  Next, the cell fusion solution A was introduced into the cell fusion region 2 to 3 times using the syringe D, and the animal cell culture medium containing the sialic acid degrading enzyme contained in the cell fusion region was replaced with the cell fusion solution A.

  Next, 600 μL of the cell fusion solution containing the myeloma cells treated with the above-mentioned sialic acid-degrading enzyme (the number of myeloma cells is about 2 million considering the loss of cells) is introduced into the cell fusion region using syringe B. did. In this case, since about 4 to 5 times as many myeloma cells as the number of spleen cells were introduced into the cell fusion region, at least one myeloma cell is in contact with the spleen cells fixed in the micropores. Estimated.

  Next, the power source is switched to a direct current pulse power source (manufactured by Nepagene Co., Ltd., LF101) by the power source switching mechanism, a direct current pulse voltage with a voltage of 80 V and a pulse width of 30 μs is applied between the electrodes, cell fusion is performed, and the state is left for 10 minutes. After that, the cell suspension in the cell fusion container is placed in a HAT medium (H: medium containing hypoxanthine, A: aminopterine, T: thymidine). Went. The HAT medium is a medium for selectively growing only fused cells (the above is referred to as fusion method A).

  Next, another cell fusion container having the same specification was used, and cell fusion was performed in a different fusion process. 600 μL of the cell fusion solution containing the spleen cells (the number of spleen cells is approximately 400,000 considering the loss) is introduced into the cell fusion region using syringe A, and the spleen cells are sufficiently precipitated in the cell fusion region. I let you. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are fixed in an array of approximately 400,000 micropores and one in each micropore. did. Subsequently, 600 μL of cell fusion solution containing myeloma cells treated with the above-mentioned sialic acid-degrading enzyme was applied while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes with an AC power source (considering cell loss). Then, about 2 million myeloma cells) were introduced into the cell fusion region using syringe B. Next, the power source is switched to a direct current pulse power source (manufactured by Nepagene Co., Ltd., LF101) by the power source switching mechanism, a direct current pulse voltage with a voltage of 80 V and a pulse width of 30 μs is applied between the electrodes, cell fusion is performed, and the state is left for 10 minutes Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured (the above is referred to as fusion method B).

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, 96 fused cells were obtained in fusion method A, and 54 fused cells were obtained in fusion method B. The fusion regeneration probability of 2.4 / 10000 for fusion method A and 1.8 / 10000 for fusion method B was obtained for approximately 400,000 spleen cells of mice introduced into the cell fusion container. It was. The fusion regeneration probability of fusion method A is 12 times the fusion regeneration probability of 0.2 / 10000 in the normal electrical cell fusion method shown in Comparative Example 1, and the cells are sialized in the normal electrical cell fusion method of Comparative Example 2. The fusion regeneration probability when treated with an acid-degrading enzyme was about 10 times the 0.25 / 10000 fusion regeneration rate, and a very high fusion regeneration probability could be obtained. In addition, the fusion regeneration probability of fusion method A is 1.3 times that of fusion method B, and sialic acid-degrading enzyme treatment is performed with the spleen cells fixed in the micropores in the fusion region. Better fusion regeneration probability could be obtained by solution replacement and fusion.

(Comparative Example 1)
As Comparative Example 1, a normal electric cell fusion method was performed. As an electrode for performing the electric cell fusion method, a gold wire electrode (Neppagene Co., Ltd., MS Gold Wire Electrode) having a 1 mm gap between the electrodes was used, and a cell fusion power source (Neppagene, LF101) was connected to this electrode. .

As the cells, mouse spleen cells and mouse myeloma cells used in Example 1 were used. Spleen cells and myeloma cells were mixed at a ratio of 4: 1, suspended in the cell fusion solution A used in Example 1, and the cell fusion solution was adjusted to a density of 1.7 × 10 ⁷ cells / mL.

40 μL of the cell fusion solution (approximately 600,000 spleen cells, approximately 150,000 myeloma cells) is injected between the electrodes, and a sine wave AC voltage with a peak voltage of 10 V and a frequency of 3 MHz is applied to the electrodes using a cell fusion power source. A DC pulse voltage having a voltage value of 200 V and a pulse width of 30 μs was applied in order to perform cell fusion. After standing for 10 minutes, the cell suspension in the cell fusion container was placed in HAT medium. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured. The number of fused cells was counted after 6 days. As a result, 12 fused cells could be confirmed, and 600,000 total mouse antibody-producing cells were obtained. As a result, a fusion reproduction probability of 0.2 / 10000 was obtained.

(Comparative Example 2)
As Comparative Example 2, cell fusion was performed by treating cells with a sialic acid-degrading enzyme in a normal electric cell fusion method. As an electrode for performing the electric cell fusion method, a gold wire electrode (Neppagene Co., Ltd., MS Gold Wire Electrode) having a 1 mm gap between the electrodes was used, and a cell fusion power source (Neppagene, LF101) was connected to this electrode. .

As the cells, mouse spleen cells and mouse myeloma cells used in Example 1 were used. Spleen cells and myeloma cells were mixed at a ratio of 4: 1, suspended in the cell fusion solution A used in Example 1, and further cultured with animal cells containing 0.2 U / mL sialic acid-degrading enzyme used in Example 1. An appropriate amount of the medium was added and the cell fusion solution was adjusted to a density of 1.7 × 10 ⁷ cells / mL, and allowed to stand at room temperature for 45 minutes. When the state of the cells in the cell fusion solution was confirmed after standing for 45 minutes, many cell aggregates were observed.

40 μL of the cell fusion solution (approximately 600,000 spleen cells, approximately 150,000 myeloma cells) is injected between the electrodes, and a sine wave AC voltage with a peak voltage of 10 V and a frequency of 3 MHz is applied to the electrodes using a cell fusion power source. A DC pulse voltage having a voltage value of 200 V and a pulse width of 30 μs was applied in order to perform cell fusion. After standing for 10 minutes, the cell suspension in the cell fusion container was placed in HAT medium. 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, 15 fused cells could be confirmed, and 600,000 total mouse antibody-producing cells were produced. As a result, a fusion reproduction probability of 0.25 / 10000 was obtained.

(Example 2)
Using the cell fusion device used in Example 1, cell fusion was performed using mouse spleen cells and mouse myeloma cells. Cell fusion solution A based on 300 mM mannitol used in Example 1, 200 mM mannitol hypotonic than osmotic pressure, 0.1 mM CaCl ₂ , 0.1 mM MgCl ₂ , 0.1 mg / mL Cell fusion was performed using spleen cells and myeloma cells suspended in cell fusion solution B containing BSA.

Spleen cells were suspended in cell fusion solution A or cell fusion solution B to adjust the final concentration to 0.8 × 10 ⁶ cells / mL. The myeloma cells were suspended in the cell fusion solution A and the final concentration was adjusted to 6.7 × 10 ⁶ cells / mL.

  The cell fusion solution A containing the spleen cells is the syringe A of the cell fusion device, the cell fusion solution B containing the spleen cells is the syringe B of the cell fusion device, and the cell fusion solution A containing the myeloma cells is the cell fusion device. Syringe C and cell fusion solution B containing myeloma cells were placed in syringe D of the cell fusion device. Moreover, the cell fusion solution B was put into the syringe E.

  First, 600 μL of the cell fusion solution A containing the spleen cells (the number of spleen cells is approximately 400,000 considering the loss) is introduced into the cell fusion region using the syringe A, and the spleen cells are introduced into the cell fusion region. Was allowed to settle sufficiently. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are fixed in an array of approximately 400,000 micropores and one in each micropore. did. Subsequently, 600 μL of the cell fusion solution A containing the myeloma cells was applied while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes with an AC power supply (considering the loss of cells, the number of myeloma cells was About 3.5 million) were introduced into the cell fusion region using syringe C. In this case, since about 4 to 5 times as many myeloma cells as the number of spleen cells were introduced into the cell fusion region, at least one myeloma cell was in contact with the spleen cells fixed in the micropores. Estimated. Next, 600 μL of the cell fusion solution B was introduced twice into the cell fusion region using the syringe E while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes by an AC power source, The cell fusion solution in the fusion region was replaced (the above is referred to as fusion method C).

  Next, the power source is switched to a direct current pulse power source (manufactured by Nepagene Co., Ltd., LF101) by the power source switching mechanism, a direct current pulse voltage with a voltage of 80 V and a pulse width of 30 μs is applied between the electrodes, cell fusion is performed, and the state is left for 10 minutes. Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium to culture the fused cells.

  Next, another cell fusion container having the same specification was used, and cell fusion was performed in a different fusion process. Using the syringe B, introduce 600 μL of the cell fusion solution B containing the spleen cells (the number of spleen cells is approximately 400,000 considering the loss) into the cell fusion region. Allowed to settle. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are fixed in an array of approximately 400,000 micropores and one in each micropore. did. Subsequently, while applying a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz between the electrodes by an AC power source, 600 μL of the cell fusion solution B containing the myeloma cells (in consideration of cell loss, the number of myeloma cells is About 4 million) were introduced into the cell fusion region using syringe D. In this case, since about 4 to 5 times as many myeloma cells as the number of spleen cells were introduced into the cell fusion region, at least one myeloma cell is in contact with the spleen cells fixed in the micropores. Estimated.

  Next, the power source is switched to a direct current pulse power source (manufactured by Nepagene Co., Ltd., LF101) by the power source switching mechanism, a direct current pulse voltage with a voltage of 80 V and a pulse width of 30 μs is applied between the electrodes, cell fusion is performed, and the state is left for 10 minutes. Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured (the above is referred to as fusion method D).

After culturing cells in a CO ₂ incubator with HAT medium containing the cell suspension and counting the number of fused cells after 6 days, 304 fused cells in fusion method C and 220 fused cells in fusion method D should be confirmed. The fusion regeneration probability of 7.6 / 10000 for fusion method C and 5.5 / 10000 for fusion method D was obtained for approximately 400,000 spleen cells of mice introduced into the fusion container. The fusion regeneration probability of the fusion method C is the same as that in the normal electric cell fusion method of Comparative Example 3, using the cell fusion solution B mainly composed of 200 mM mannitol, which is hypotonic than the osmotic pressure of the cells from the beginning. In this case, the fusion reproduction probability was about 33 times the 0.23 / 10000, and a very high fusion reproduction probability could be obtained. In addition, the fusion regeneration probability of fusion method C is 1.4 times that of fusion method D, and the spleen cells are fixed in the micropores in the fusion region, and the hypotonicity is lower than the osmotic pressure immediately before the fusion. A better fusion regeneration probability could be obtained by substituting with a sugar solution for fusion.

(Comparative Example 3)
As Comparative Example 3, cell fusion was performed using a cell fusion solution B composed mainly of 200 mM mannitol, which is hypotonic from the beginning of the cell osmotic pressure in a normal electric cell fusion method. As an electrode for performing the electric cell fusion method, a gold wire electrode (Neppagene Co., Ltd., MS Gold Wire Electrode) having a 1 mm gap between the electrodes was used, and a cell fusion power source (Neppagene, LF101) was connected to this electrode. .

As the cells, mouse spleen cells and mouse myeloma cells used in Example 2 were used. Spleen cells and myeloma cells were mixed at a ratio of 4: 1, suspended in the cell fusion solution B used in Example 2, and the cell fusion solution was adjusted to a density of 1.7 × 10 ⁷ cells / mL. In addition, after the adjustment, when the state of the cells in the cell fusion solution was confirmed, many dead cells were seen.

40 μL of the cell fusion solution (approximately 600,000 spleen cells, approximately 150,000 myeloma cells) is injected between the electrodes, and a sine wave AC voltage with a peak voltage of 10 V and a frequency of 3 MHz is applied to the electrodes using a cell fusion power source. A DC pulse voltage having a voltage value of 200 V and a pulse width of 30 μs was applied in order to perform cell fusion. After standing for 10 minutes, the cell suspension in the cell fusion container was placed in HAT medium. 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, 15 fused cells could be confirmed, and 600,000 total mouse antibody-producing cells were produced. As a result, a fusion reproduction probability of 0.23 / 10000 was obtained.

(Example 3)
In Example 3, using the cell fusion apparatus used in Example 1, cell fusion was performed using mouse spleen cells and mouse myeloma cells. However, as the cell fusion container, one having a micropore interval of 20 μm and a number of micropores in the portion where the spacer was hollowed out was 1 million. The spleen cells were frozen in a cell banker at −80 ° C. and thawed at 37 ° C., and the cell fusion solution A mainly composed of 300 mM mannitol used in Example 1 and 500 mM of hypertonicity higher than osmotic pressure were used. Suspensions in cell fusion solution C containing mannitol, 0.1 mM CaCl ₂ , 0.1 mM MgCl ₂ , and 0.1 mg / mL BSA were prepared. In addition, cell fusion was performed using myeloma cells suspended in cell fusion solution A.

Spleen cells were suspended in cell fusion solution A or cell fusion solution B to adjust the final concentration to 1.7 × 10 ⁶ cells / mL. The myeloma cells were suspended in the cell fusion solution A and the final concentration was adjusted to 6.7 × 10 ⁶ cells / mL.

  The cell fusion solution A containing the spleen cells is the syringe A of the cell fusion device, the cell fusion solution B containing the spleen cells is the syringe B of the cell fusion device, and the cell fusion solution A containing the myeloma cells is the cell fusion device. Placed in syringe C.

  First, 600 μL of the cell fusion solution containing the spleen cells (about 1 million spleen cells considering the loss) is introduced into the cell fusion region using syringe A, and the spleen cells are introduced into the cell fusion region. It was allowed to settle sufficiently. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are fixed in an array of about 1 million micropores, one in each micropore. did. Subsequently, 600 μL of the cell fusion solution containing the myeloma cells was applied while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes with an AC power supply (considering the loss of cells, the number of myeloma cells was about 4 million) were introduced into the cell fusion region using syringe C. In this case, since about 4 to 5 times as many myeloma cells as the number of spleen cells were introduced into the cell fusion region, at least one myeloma cell is in contact with the spleen cells fixed in the micropores. Estimated.

  Next, the power source switching mechanism is used to switch the power source to a DC pulse power source (LF101, manufactured by Nepagene Co., Ltd.), and a cell is fused by applying a DC pulse voltage with a voltage of 100 V and a pulse width of 30 μs between the electrodes, and left for 10 minutes. Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured (the above is referred to as fusion method E).

  Next, another cell fusion container having the same specification was used, and cell fusion was performed in a different fusion process. 600 μL of the cell fusion solution C containing the spleen cells (the number of spleen cells is about 1 million considering the loss) is introduced into the cell fusion region using the syringe B, and the spleen cells are sufficiently contained in the cell fusion region. Allowed to settle. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are fixed in an array of about 1 million micropores, one in each micropore. did. Subsequently, 600 μL of the cell fusion solution A containing the myeloma cells was applied while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes with an AC power supply (considering the loss of cells, the number of myeloma cells was About 4 million) were introduced into the cell fusion region using syringe C. In this case, since about 4 to 5 times as many myeloma cells as the number of spleen cells were introduced into the cell fusion region, at least one myeloma cell is in contact with the spleen cells fixed in the micropores. Estimated.

  Next, the power source switching mechanism is used to switch the power source to a DC pulse power source (LF101, manufactured by Nepagene Co., Ltd.), and a cell is fused by applying a DC pulse voltage with a voltage of 100 V and a pulse width of 30 μs between the electrodes, and left for 10 minutes. Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured (the above is referred to as fusion method F).

After culturing the cells in the HAT medium containing the cell suspension in a CO ₂ incubator and counting the number of fused cells after 6 days, 1794 fusion cells in Fusion Method E and 2293 fused cells in Fusion Method F should be confirmed. The fusion regeneration probability of 17.9 / 10000 for fusion method E and 22.9 / 10000 for fusion method F was obtained for about 1 million spleen cells of mice introduced into the cell fusion container. The fusion regeneration probability of the fusion method F uses the cell fusion solution A mainly composed of 300 mM mannitol that is isotonic and isotonic with the spleen cells that were freeze-thawed from the beginning in the normal electric cell fusion method of Comparative Example 4. The fusion regeneration probability when the cells were fused was 143.1 times the 0.16 / 10000 fusion regeneration probability, and a very high fusion regeneration probability could be obtained. Further, the fusion regeneration probability of fusion method F is 1.3 times the fusion regeneration probability of fusion method E, and the freeze-thawed spleen cells are suspended in cell fusion solution B that is hypertonic than osmotic pressure before entering the cell fusion region. By introducing the myeloma suspended in the cell fusion solution A and finally, the composition of the cell fusion solution in the cell fusion region is fused as a sugar solution that is isotonic with the cells. Probability could be obtained.

(Comparative Example 4)
As Comparative Example 4, cell fusion was performed using a normal electric cell fusion method in which freeze-thawed spleen cells were suspended in a cell fusion solution. As an electrode for performing the electric cell fusion method, a gold wire electrode (Neppagene Co., Ltd., MS Gold Wire Electrode) having a 1 mm gap between the electrodes was used, and a cell fusion power source (Neppagene, LF101) was connected to this electrode. .

As the cells, mouse spleen cells A suspended in the cell fusion solution A used in Example 3 and mouse myeloma cells suspended in the cell fusion solution A were used. Spleen cells and myeloma cells were mixed at a ratio of 4: 1, suspended in the cell fusion solution A used in Example 1, and the cell fusion solution was adjusted to a density of 1.7 × 10 ⁷ cells / mL. In addition, after the adjustment, when the state of the cells in the cell fusion solution was confirmed, many dead cells were seen.

40 μL of the cell fusion solution (approximately 600,000 spleen cells, approximately 150,000 myeloma cells) is injected between the electrodes, and a sine wave AC voltage with a peak voltage of 10 V and a frequency of 3 MHz is applied to the electrodes using a cell fusion power source. A DC pulse voltage having a voltage value of 200 V and a pulse width of 30 μs was applied in order to perform cell fusion. After standing for 10 minutes, the cell suspension in the cell fusion container was placed in HAT medium. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured. The number of fused cells was counted after 6 days. As a result, 10 fused cells could be confirmed, and 600,000 total mouse antibody-producing cells were obtained. As a result, a fusion reproduction probability of 0.16 / 10000 was obtained.

Example 4
Using the cell fusion device used in Example 3, cell fusion was performed using mouse spleen cells and mouse myeloma cells. The spleen cells were extracted from the mouse, and the spleen cell extract was washed once with Medium B and once with Cell Fusion A and suspended in Cell Fusion A as shown in Example 1. (Hereinafter referred to as spleen cell A), the spleen cell extract was centrifuged at 1500 rpm for 5 minutes at room temperature, the supernatant was aspirated, the spleen cell pellet was unwound and immediately suspended in the cell fusion solution A Prepared (hereinafter referred to as spleen cells B). In addition, as shown in Example 1, myeloma cells were washed once with medium B and once with cell fusion solution A, and suspended in cell fusion solution A to perform cell fusion. It was.

Spleen cells A and spleen cells B were suspended in cell fusion solution A to adjust the final concentration to 0.8 × 10 ⁶ cells / mL. The myeloma cells were suspended in the cell fusion solution A and the final concentration was adjusted to 6.7 × 10 ⁶ cells / mL.

  The cell fusion solution A containing the spleen cell A is fused with the syringe A of the cell fusion device, the cell fusion solution A containing the spleen cell B is used with the syringe B of the cell fusion device, and the cell fusion solution A containing the myeloma cell is fused with the cell. Placed in syringe C of the device.

  First, 600 μL of the cell fusion solution A containing the spleen cells A (approximately 500,000 spleen cells considering the loss) is introduced into the cell fusion region using the syringe A, and the spleen is introduced into the cell fusion region. Cells were fully sedimented. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and about one minute is divided into about one half of 500,000 fine holes. Spleen cells were fixed in an array, one per hole. Subsequently, 600 μL of the cell fusion solution containing the myeloma cells was applied while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes with an AC power supply (considering the loss of cells, the number of myeloma cells was about 4 million) was introduced into the cell fusion region using syringe C. In this case, since about 8-10 times as many myeloma cells as the number of spleen cells are introduced into the cell fusion region, at least one myeloma cell is in contact with the spleen cells fixed in the micropores. Estimated.

  Next, the power source switching mechanism is used to switch the power source to a DC pulse power source (LF101, manufactured by Nepagene Co., Ltd.), and a cell is fused by applying a DC pulse voltage with a voltage of 100 V and a pulse width of 30 μs between the electrodes, and left for 10 minutes. Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured (the above is referred to as fusion method G).

  Next, another cell fusion container having the same specification was used, and cell fusion was performed in a different fusion process. 600 μL of the cell fusion solution A containing the spleen cells B (the number of spleen cells is about 500,000 considering the loss) is introduced into the cell fusion region using the syringe B, and the spleen cells are sufficiently contained in the cell fusion region. It was allowed to settle. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are fixed in an array of about 1 million micropores, one in each micropore. did. Subsequently, 600 μL of the cell fusion solution A containing the myeloma cells was applied while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes with an AC power supply (considering the loss of cells, the number of myeloma cells was About 4 million) were introduced into the cell fusion region using syringe C. In this case, since about 8-10 times as many myeloma cells as the number of spleen cells are introduced into the cell fusion region, at least one myeloma cell is in contact with the spleen cells fixed in the micropores. Estimated.

  Next, the power source switching mechanism is used to switch the power source to a DC pulse power source (LF101, manufactured by Nepagene Co., Ltd.), and a cell is fused by applying a DC pulse voltage with a voltage of 100 V and a pulse width of 30 μs between the electrodes, and left for 10 minutes. Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured (the above is referred to as fusion method H).

After culturing the cells in the CO ₂ incubator with HAT medium containing the cell suspension and counting the number of fused cells after 6 days, 1825 fusion cells in Fusion Method G and 1603 fused cells in Fusion Method H should be confirmed. The fusion regeneration probability of 36.5 / 10000 for fusion method G and 32.1 / 10000 for fusion method H was obtained for approximately 500,000 mouse spleen cells introduced into the cell fusion container. The fusion regeneration probability of fusion method B is a mixture of spleen cells that are not washed with medium B and cell fusion solution A and myeloma cells that have been washed in the normal electrical cell fusion method of Comparative Example 5. Thus, the fusion regeneration probability of cell fusion was 107.0 times the 0.30 / 10000, and a very high fusion regeneration probability could be obtained. Further, the fusion regeneration probability of the fusion method H is 0.88 times the fusion regeneration probability of the fusion method G. Even when spleen cells not subjected to the washing operation are used, the washed myeloma is used as the cell fusion solution A. By suspending and introducing and fusing, a complicated cell washing operation can be omitted and a good fusion regeneration probability can be obtained.

(Comparative Example 5)
As Comparative Example 5, cell fusion was performed using spleen cells in which the cell washing operation was omitted and myeloma cells that had been washed in the usual electric cell fusion method. As an electrode for performing the electric cell fusion method, a gold wire electrode (Neppagene Co., Ltd., MS Gold Wire Electrode) having a 1 mm gap between the electrodes was used, and a cell fusion power source (Neppagene, LF101) was connected to this electrode. .

The cells used were mouse spleen B and mouse myeloma cells used in Example 4. Spleen cells and myeloma cells were mixed at a ratio of 4: 1, suspended in the cell fusion solution A used in Example 1, and the cell fusion solution was adjusted to a density of 1.7 × 10 ⁷ cells / mL.

40 μL of the cell fusion solution (approximately 600,000 spleen cells, approximately 150,000 myeloma cells) is injected between the electrodes, and a sine wave AC voltage with a peak voltage of 10 V and a frequency of 3 MHz is applied to the electrodes using a cell fusion power source. A DC pulse voltage having a voltage value of 200 V and a pulse width of 30 μs was applied in order to perform cell fusion. After standing for 10 minutes, the cell suspension in the cell fusion container was placed in HAT medium. 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, 18 fused cells could be confirmed, and 600,000 total mouse antibody-producing cells were obtained. As a result, a fusion reproduction probability of 0.30 / 10000 was obtained.

(Example 5)
Using the cell fusion device used in Example 3, cell fusion was performed using mouse spleen cells and mouse myeloma cells. After extracting spleen cells from the mouse, as shown in Example 1, the spleen cell extract was centrifuged at 1500 rpm for 5 minutes at room temperature, the supernatant was aspirated with an aspirator, the spleen cell pellet was unwound, and immediately cell fusion What was prepared by suspending in the liquid A was prepared. The myeloma cells were prepared by washing the myeloma culture solution used in Example 1 once with the medium B and once with the cell fusion solution A and suspending in the cell fusion solution A (hereinafter, myeloma cells). A), the supernatant of the myeloma culture solution is aspirated with an aspirator, the myeloma cell pellet is unwound, and immediately suspended in the cell fusion solution A (hereinafter referred to as myeloma cell B). did.

Spleen cells were suspended in cell fusion solution A and the final concentration was adjusted to 1.6 × 10 ⁶ cells / mL. In addition, myeloma cell A and myeloma cell B were suspended in cell fusion solution A to adjust the final concentration to 6.5 × 10 ⁶ cells / mL.

  The cell fusion solution A containing the spleen cells is fused with the syringe A of the cell fusion device, the cell fusion solution A containing the myeloma cells A is used with the syringe B of the cell fusion device, and the cell fusion solution A containing the myeloma cells B is used with the cell fusion. Placed in syringe C of the device. In addition, cell fusion solution A containing neither spleen cells nor myeloma cells was placed in syringe D.

  First, 600 μL of the cell fusion solution A containing the spleen cells (the number of spleen cells is about 1 million considering the loss) is introduced into the cell fusion region using the syringe A, and the spleen cells are introduced into the cell fusion region. Was allowed to settle sufficiently. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are arranged in an array with about one million micropores, one in each micropore. Fixed.

  Subsequently, 600 μL of the cell fusion solution containing the myeloma cells A was applied while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes with an AC power source (considering the loss of cells, the number of myeloma cells was About 4 million) were introduced into the cell fusion region using syringe B. In this case, since about four times as many myeloma cells as the number of spleen cells were introduced into the cell fusion region, at least one myeloma cell was in contact with the spleen cells fixed in the micropores. Presumed.

  Next, the power source switching mechanism is used to switch the power source to a DC pulse power source (LF101, manufactured by Nepagene Co., Ltd.), and a cell is fused by applying a DC pulse voltage with a voltage of 100 V and a pulse width of 30 μs between the electrodes, and left for 10 minutes. Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured (the above is referred to as fusion method I).

  Next, another cell fusion container having the same specification was used, and cell fusion was performed in a different fusion process. 600 μL of the cell fusion solution A containing the spleen cells (the number of spleen cells is about 1 million considering the loss) is introduced into the cell fusion region using the syringe A, and the spleen cells are sufficiently contained in the cell fusion region. Allowed to settle. Next, a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz is applied between the electrodes by an AC power source, and spleen cells are arranged in an array with approximately one million micropores, one in each micropore. Fixed. Subsequently, 600 μL of the cell fusion solution A containing the myeloma cells B was applied while a rectangular wave AC voltage having a peak voltage of 5 V and a frequency of 3 MHz was applied between the electrodes with an AC power supply (considering the loss of cells, the number of myeloma cells About 4 million) were introduced into the cell fusion region using syringe C. In this case, since about four times as many myeloma cells as the number of spleen cells were introduced into the cell fusion region, at least one myeloma cell was in contact with the spleen cells fixed in the micropores. Presumed. Next, the cell fusion solution A was introduced into the cell fusion region 2-3 times using the syringe D, and the spleen cells and myeloma cells that had entered the cell fusion region were washed with the cell fusion solution A.

  Next, the power source switching mechanism is used to switch the power source to a DC pulse power source (LF101, manufactured by Nepagene Co., Ltd.), and a cell is fused by applying a DC pulse voltage with a voltage of 100 V and a pulse width of 30 μs between the electrodes, and left for 10 minutes. Thereafter, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured (the above is referred to as fusion method J).

After culturing the cells in the CO ₂ incubator with the cell suspension in the CO ₂ incubator and counting the number of fused cells after 6 days, 1312 fusion cells in Fusion Method I and 3417 fused cells in Fusion Method J should be confirmed. The fusion regeneration probability of 13.1 / 10000 for fusion method I and 34.2 / 10000 for fusion method J was obtained for approximately 1 million mouse spleen cells introduced into the cell fusion container. The fusion regeneration probability of fusion method J is 2.6 times the fusion regeneration probability of fusion method I. When spleen cells without washing operation and myeloma cells without washing operation are used, spleen cells and myeloma cells are treated as cells. Spleen cells and myeloma cells can be easily washed in a short time by introducing the cell fusion solution A into the cell fusion region after introduction into the fusion region and fixation in the micropores, and obtaining a good fusion regeneration probability. did it. This is probably because the cell activity was maintained by omitting a complicated cell washing operation in the adjustment stage of each cell to be inserted into the cell fusion region, and therefore a good fusion regeneration probability could be obtained.

1: Cell fusion region 2: Cell fusion solution introduction flow path 3: Conductive wire 4: Power supply 5: AC power supply 6: DC pulse power supply 7: Power supply switching mechanism 8: Insulator 9: Micropore 10: Dielectrophoretic force 11: Electricity Concentration site of force lines 12: Electric force lines 13: Cell fusion container 14: Upper electrode 15: Lower electrode 16: Spacer 17: Cell fusion solution introduction switching means 18: First cell 19: Inlet 20: Discharge 21: Valve 22: Second cell 23: ITO
24: Pyrex (registered trademark) glass 25: Resist 26: Photomask for exposure 27: Exposure 28: Developer 29: Introduction channel 30: Discharge channel 31: Peak voltage 32: Fusion cell 33: Developer 34: Syringe A
35: Syringe B
36: Syringe C
37: Syringe D
38: Syringe E

Claims (9)

A cell fusion solution inlet and a cell fusion solution discharge port, a pair of electrodes made of a conductive member disposed to face the cell fusion region, and a plate-like spacer disposed between the pair of electrodes; and It consists of a flat insulator having a plurality of fine holes penetrating in the direction of the electrodes arranged facing each other, and the insulator is on the electrode surface on the cell fusion region side of one of the electrodes A cell fusion device comprising a cell fusion container, a power source, and a cell fusion solution introduction switching means for switching and introducing three or more cell fusion solutions, wherein the power source is connected to the pair of electrodes. A cell fusion device comprising a power source switching mechanism for switching and connecting an AC power source for applying an AC voltage and a DC pulse power source for applying a DC pulse voltage. When the first cell and the second cell are fused in the cell fusion region using the cell fusion device, a cell fusion solution containing the first cell is introduced into the cell fusion region, and the alternating voltage is applied. After applying and fixing the first cell in the micropore, a cell fusion solution containing the second cell is introduced into the cell fusion region, and the alternating voltage is applied to the first cell. A cell fusion between the first cell and the second cell by contacting the second cell in the micropore and applying a DC pulse voltage to the micropore, The time when the cell fusion solution containing the cell fusion treatment solution is introduced using the solution introduction switching means is at least one time before the introduction of the second cell, before the cell fusion and after the cell fusion. A cell fusion method. The cell fusion solution containing two or more cell fusion solutions containing the cell fusion treatment solution, wherein the cell fusion solutions containing the two or more cell fusion treatment solutions are all the same, partly different, or all different. The cell fusion method according to claim 2, which is a cell fusion solution containing a solution. The cell fusion method according to claim 2 or 3, wherein a specific component in the cell fusion solution in the cell fusion region is removed with the cell fusion solution containing the cell fusion treatment solution. The cell fusion solution containing the cell fusion treatment solution is a solution containing a specific component, and the specific component is introduced into the cell fusion solution in the cell fusion region by introducing the cell fusion solution containing the cell fusion treatment solution. The cell fusion method according to any one of claims 2 to 4, wherein is introduced. The cell fusion method according to claim 4 or 5, wherein the specific component is an enzyme. The cell fusion method according to claim 6, wherein the enzyme is a sialic acid degrading enzyme or a protease. The cell fusion method according to claim 4, wherein the specific component is calcium ion. The cell fusion method according to any one of claims 4 to 8, wherein the specific component is an additive for increasing the fusion regeneration probability.

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