JP2010011824A - Cell fusion vessel, cell fusion apparatus, and method for cell fusion using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new cell fusion vessel capable of more efficiently and surely carrying out cell fusion of two cells in a pair, to provide a cell fusion apparatus, and to provide a method for cell fusion using the same in electric cell fusion. <P>SOLUTION: The cell fusion vessel is provided with a pair of electrodes composed of electroconductive members oppositely disposed in a cell fusion region; and a platy insulator disposed through a platy spacer between the pair of electrodes, and having a plurality of micropores passing through the direction of the oppositely disposed electrodes. In the cell fusion vessel, the insulator is disposed on an electrode surface on the side of either one of cell fusion regions of the electrodes, and each cross section of the micropores is in a shape having the area of an opening surface reducing from the upper part of the opening to the opening bottom contacting the electrode. The cell fusion vessel, the cell fusion apparatus, and the method for cell fusion using the same are used. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell fusion container, a cell fusion device, and a cell fusion method using them for efficiently performing cell fusion.

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

  In contrast, the electric cell fusion method does not require advanced technology, can be easily and efficiently fused, has little toxicity to cells, and fuses cells with high activity. There is an advantage that you can. 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. Also, the force that draws cells in the direction of high current density, that is, the direction in which the lines of electric force concentrate, is generally called dielectrophoresis. In this state, when a DC pulse voltage of several μsec to several tens μsec is applied between the electrodes, the electric conductivity of the cell membrane is instantaneously reduced, and the reversible disturbance of the cell membrane constituted by the lipid bilayer and its reconstruction As a result, cell fusion occurs.

  For the electrical 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 looking at a fusion of a pair of two cells while looking through a microscope, and is extremely reliable. Examples of electrodes used in the microelectrode method are also included. Although it is reported (for example, refer to Patent Document 1), it is a time-consuming method, which requires skill and is not practical for handling a large amount 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 fused by applying a DC pulse voltage. As in the chemical fusion method, the contact of the two cells is accidental, and there is a problem that it is difficult to reliably control the cell fusion in a pair of two cells.

  In order to solve the problem of the parallel electrode method, a pair of electrodes made of a conductive member disposed so as to face a cell fusion region of a cell fusion container, a pair of electrodes disposed between the pair of electrodes, and the pair of electrodes An example of a cell fusion container made of an insulator having fine holes penetrating in the direction has been reported (for example, see Patent Document 2).

  FIG. 1 is a conceptual diagram showing a cross-sectional view of the cell fusion container of the above example. In FIG. 1, for example, electrodes (2) made of conductive members are arranged on both sides of a cell fusion region (1) of a cell fusion container made of a resin material, and these electrodes are provided outside via conductive wires (3). Connected to the power source (4). The power supply provided outside is an AC power supply (5) that outputs a high-frequency AC voltage with an electric field strength of about 400 V / cm to 700 V / cm and a frequency of about 1 MHz, and a DC pulse of about 7 kV / cm and a pulse width of about 50 μsec. A DC pulse power supply (6) for outputting a voltage and a changeover switch (7) having a switching mechanism for switching the electrical connection between the electrode and the AC power supply or the DC pulse power supply.

  Here, as a waveform of the AC voltage output from the AC power supply, a sine wave waveform is generally used unless otherwise specified. The cell fusion container is divided into two spaces by a partition wall (35) made of an electrically insulating material such as silicone resin. Here, the insulator is provided with a fine hole (9) having a minimum diameter of 1 μm to several tens of μm. The cells A (10) and B (11) are each placed in a cell suspension in the cell fusion region of the cell fusion container.

  The operation of the above example will be described with reference to FIGS. First, the changeover switch (7) of the power supply (4) is connected to an AC power supply (5) that outputs a high-frequency voltage having an electric field strength of about 400 V / cm to 700 V / cm and a frequency of 1 MHz. In this state, the electric lines of force (12) are concentrated in the fine holes (9) as shown in FIG. The cells A (10) and B (11) receive a dielectrophoretic force due to the electric lines of force (12) concentrated here, and are fixed near the center of the micropore (9) as shown in FIG. Here, cell A (10) and cell B (11) meet and come into contact. Next, the selector switch (7) of the power source (4) is switched to the DC pulse power source (6). The cell A (10) and the cell B (11) placed in the state shown in FIG. 22 undergo reversible destruction of the cell membrane at the contact point of the cell A (10) and the cell B (11) by the DC pulse voltage. Fusion occurs as shown in FIG. By doing in this way, the cell A and the cell B can be cell-fused by a pair of cells in the micropore.

  However, in the method of performing cell fusion using the cell fusion container described in Patent Document 2, when the diameter of the micropore is larger than the diameter of the cell A and larger than the diameter of the cell B, each cell becomes a micropore. When trapped and contacted, as shown in FIG. 3, there is a problem that the probability of cell fusion decreases because the probability that the cell A and the cell B contact in the same direction as the direction of the electric lines of force decreases. On the contrary, when the diameter of the micropore is smaller than the diameter of the cell A and smaller than the diameter of the cell B, both cells are trapped and contacted and fused in the micropore, but after the fusion, as shown in FIG. There was a problem that the cells could not be removed from the pores, and the cells could not be taken out from the micropores, and the fused cells were broken when trying to forcibly remove them.

  Furthermore, the method of performing cell fusion using the cell fusion container described in Patent Document 2 has a problem that it is difficult to fix two cell pairs simultaneously in the micropores. For example, when a cell suspension containing cells B is introduced into a cell fusion container in order to further introduce cells B into the micropores after the cells A are put into the micropores, the cells are preliminarily refined by liquid feeding at the time of introduction. The cells A fixed in the pores are detached from the micropores. In addition, it is very difficult to fix the cells A and B to the micropores at the same time because it requires skill in feeding the cell suspension. Furthermore, when a plurality of cells are fused simultaneously by the method described in Patent Document 2, it is necessary to fix two cells one by one in a plurality of micropores formed in an array on an insulator.

  Here, the array shape means that the vertical and horizontal intervals of the plurality of micro holes are arranged at almost equal intervals. However, when an AC power supply is connected and cells are fixed in the micropores, there are micropores in which a plurality of cells are concentrated and fixed, or micropores in which cells are not fixed at all, and are formed in a plurality of arrays. There was a problem that it was very difficult to perform the desired cell fusion of a pair of two cells with micropores.

  On the other hand, an example of a method of fixing cells one by one in a plurality of micropores formed in an array has been reported (for example, see Patent Document 3). In the method described in Patent Document 3, the inner diameter and depth of a micropore (described as a microwell in Patent Document 3) are the diameters of cells (described as a subject lymphocyte in Patent Document 3), respectively. A process of adding a liquid containing a plurality of cells to a plurality of micropores 1 to 2 times larger than the size of the micropore so as to cover the micropores and waiting for the cells to sink into the micropores, and washing away the cells outside the micropores One cell is fixed to one micropore by repeating the washing process.

  However, the method of fixing one cell in one micropore by the method described in Patent Document 3 has a long waiting time of about 5 minutes for the cell to sink by gravity, and the cell sinks in the micropore. It is necessary to repeat the process of waiting for and washing the cells outside the micropores, which is cumbersome and requires more time, and cells may be lost in the process of washing cells that did not enter the micropores There is a problem that it is difficult to use all the cells effectively. In general, when performing cell fusion, it is preferable that the treatment time is as short as possible in order to maintain the activity of the cell, and in order to find the specificity of each cell, the loss of the cell is as much as possible. Preferably not.

  In order to solve the problems and problems in the above-described conventional technology, the present inventors, as shown in the comparative example described later, from a conductive member arranged to face the cell fusion region of the cell fusion chamber. A pair of electrodes, and an insulator having a plurality of fine holes penetrating in the direction of the pair of electrodes, and the insulator is on the cell fusion region side electrode surface of one of the electrodes The first cell is introduced into the cell fusion region using the arranged cell fusion device, and the first cell is fixed in the micropore by applying an alternating voltage, and then the second cell is Then, a method of cell fusion was studied by bringing a second cell into contact with the first cell at the position of the micropore and applying a DC pulse voltage. In this study, the size of the micropores is optimized for the size of the cells to be fused, and the waveform of the AC voltage applied when fixing the cells to the micropores is optimized to form an array. In the plurality of micropores, it was found that one cell could be fixed very easily per one micropore, and a pair of two cells were fused to obtain a fusion probability of 1/10000. This is a fusion probability that is 5 times the fusion probability of 0.2 / 10000 in normal electric cell fusion, and an efficient fusion in a pair of two cells could be confirmed (for example, see Patent Document 4). .

  However, in general, the range of cell diameters is wide. For example, mouse spleen cells, which are examples of the first cell, have the largest number of cells having a diameter of about 6.2 μm as shown in FIG. The range of cell diameters ranges from about 5.5 μm to about 10.0 μm in diameter, with an average diameter of about 6.7 μm. In addition, mouse myeloma cells, which are examples of the second cell, have the largest number of cells having a diameter of about 11.3 μm, as shown in FIG. 2 (b), but the cells have a diameter of about 9.0 μm to about 15.0 μm. The range of diameters is broad and the average diameter is about 11.8 μm. In addition, the diameter distribution of each cell was measured using the Beckman Coulter make and Coulter Z2 type. Here, in Patent Document 4, the diameter of the micropore is determined in accordance with the average diameter of the spleen cell as the first cell. In this case, spleen cells having a diameter larger than the micropore can be put into the micropore. As a result, there is a problem that the fusion probability is lowered.

  In general, in the electric cell fusion method, the electric conductivity of the cell membrane is instantaneously reduced by applying a DC pulse voltage of several μsec to several tens of μsec between the electrodes, and the reversibility of the cell membrane composed of lipid bilayers is reduced. Cell disruption is generated at the contact surface of the two cells, and the cell membrane is reconstituted to perform cell fusion. In this case, the magnitude of the DC pulse voltage is generally determined in accordance with the average diameter of the spleen cells. Here, as shown in the following (formula 1), the DC pulse voltage Vp causing reversible disturbance of the cell membrane requires a higher voltage as the cell diameter R becomes smaller. Here, Vm is a cell membrane breakdown voltage (generally about 1 V), and d is a distance between electrodes.

Vp = (4 × Vm × d) / (3 × R) (Formula 1)
Therefore, spleen cells smaller than the average diameter do not undergo cell fusion because reversible disturbance of the cell membrane does not occur at the set DC pulse voltage. In addition, spleen cells larger than the average diameter will not be fused because they will be completely destroyed until the cell membrane cannot be repaired with the set DC pulse voltage, and the cells will die. As a result, there is a problem that the fusion probability is lowered.

Japanese Patent Publication No. 7-40914 Japanese Patent Publication No. 7-4218 Japanese Patent No. 3723882 JP 2007-295912 A

  The object of the present invention has been proposed in view of the conventional situation, and in the electric cell fusion, a novel cell fusion container and cell fusion capable of more efficiently and surely carrying out cell fusion of a pair of two cells. An object is to provide a device and a cell fusion method using them.

  As a means for solving the above-mentioned problems, the present invention provides a pair of electrodes made of conductive members arranged opposite to each other in the cell fusion region, and a plate-like spacer disposed between the pair of electrodes and facing each other. A cell fusion container comprising a flat insulator having a plurality of micropores penetrating in the direction of the arranged electrode, the insulator being arranged on the electrode surface on the cell fusion region side of one of the electrodes And using the cell fusion container in which the cross section of each micropore has a shape in which the area of the opening surface decreases from the top of the opening toward the bottom of the opening contacting the electrode, the pair of the cell fusion container and the cell fusion container A cell fusion device having a power source for applying an AC voltage and a DC pulse voltage to the electrode of the first electrode and a switching mechanism for switching between the AC power source and the DC pulse power source, and the first cell and the first cell using the cell fusion device. 1 A method of cell fusion with a second cell having an average diameter larger than the average diameter of the cell, wherein the first cell is introduced into the cell fusion region, and an alternating voltage is applied to the second cell in the micropore. After fixing one cell, a second cell is introduced into the cell fusion region, and an AC voltage is applied to bring the second cell into contact with the first cell at the position of the micropore. It has been found that the above-described problems of the prior art can be solved by using a cell fusion method in which the above is applied, and the present invention has finally been completed. Hereinafter, the present invention will be described in detail.

  The present invention relates to a pair of electrodes made of conductive members arranged opposite to each other in a cell fusion region, and a plate-like spacer arranged between the pair of electrodes, and the electrodes arranged opposite to each other A cell fusion container comprising a flat insulator having a plurality of fine holes penetrating in the direction, wherein the insulator is disposed on the electrode surface on the cell fusion region side of one of the electrodes In the cell fusion container, the cross-section of each of the micropores has a shape in which the area of the opening surface decreases from the top of the opening toward the bottom of the opening in contact with the electrode.

  Further, the present invention is the cell fusion container according to the above-mentioned cell fusion container, wherein each cross section of the micropore has a shape having a diameter and a depth of two or more steps from the top of the opening toward the bottom of the opening in contact with the electrode.

  Further, the present invention has a shape in which each cross-section of the micropore has a shape having a two-stage diameter and depth on the upper opening side and the lower opening side, and cell fusion that fuses two types of cells having different average diameters. Among the two cells in which the diameter of the micropore on the bottom side of the opening is a cell fusion, the depth of the micropore is equal to or greater than the average diameter of the cells having a small average diameter and the average diameter of the cells having a large average diameter. In the above cell fusion container, the diameter of the micropores having a small average diameter is less than the average diameter of the two cells, and the diameter of the micropore on the upper side of the opening is larger than the average diameter of the cells having the large average diameter among the two cells to be fused is there.

Further, the present invention provides the cell fusion container according to the above, wherein the plurality of micropores formed in the insulator are formed at the same position on the surface of the insulator and adjacent to any micropore. Yes,
Moreover, this invention is said cell fusion container in which the several micropore formed in an insulator is formed in the array form in the surface of the insulator.

  Moreover, this invention is said cell fusion container whose space | interval which adjoins a micropore is 0.5 to 6 times the average diameter of the small diameter cell put into a micropore.

  Moreover, this invention is said cell fusion container whose spacer has a through-hole which forms the said cell fusion area | region.

  Moreover, this invention is said cell fusion container in which a spacer has the introduction flow path which introduce | transduces a cell, and the discharge flow path which discharges | emits a cell.

  The present invention also provides a cell fusion device comprising the above cell fusion container and a power source for applying a voltage to the pair of electrodes of the cell fusion container, wherein the power source exchanges with the pair of electrodes of the cell fusion container. A cell fusion device comprising an AC power source for applying a voltage and a DC pulse power source for applying a DC pulse voltage, and having a switching mechanism for switching between the AC power source and the DC pulse power source.

  The present invention also provides a method for cell fusion of a first cell and a second cell having an average diameter larger than the average diameter of the first cell using the cell fusion device described above. The first cell is introduced into the cell, and the first cell is fixed in the micropore by applying an alternating voltage, and then the second cell is introduced into the cell fusion region and the alternating voltage is applied. In the cell fusion method, the second cell is brought into contact with the first cell at the position of the micropore and a DC pulse voltage is applied.

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

  The cell fusion container of the present invention and the cell fusion device provided with this cell fusion container are as described above.

  Here, the configuration of the cell fusion container and the cell fusion device of the present invention will be described in detail with reference to FIG.

  FIG. 6 is a diagram showing a conceptual diagram of the cell fusion device of the present invention. FIG. 6 shows the components of the cell fusion device of the present invention in a separated state for easy understanding. In FIG. 7 to be described later, the cell fusion device of the present invention is shown as an AA ′ cross-sectional view after the components are assembled as an original configuration. In the figure, the cell fusion device of the present invention comprises a cell fusion container (13) and a power source (4).

  As shown in FIG. 6, 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). A glass substrate, a resin substrate, a ceramic substrate or the like on which a transparent conductive material such as tin) is formed may be used. However, in order to observe the introduction of the fine particle suspension, a glass substrate on which a transparent conductive material such as ITO is formed. Is preferably used as the 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 (13) via a conductive wire (3).

  The spacer is provided so that the upper electrode and the lower electrode are not in direct contact with each other as in the case of the fine particle suspension introduction container, and a cell fusion region for securing a space for storing the cell suspension in the cell fusion container. The material may be any insulating material, such as glass, ceramic, resin, and the like. In addition, 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.

  In addition, fine holes (9) are formed in the insulator (8). The material of the insulator is not particularly limited as long as it is an insulating material such as glass, ceramic, or resin, but a material that is relatively easy to process, such as a resin, is preferable because it is necessary to form through holes. . 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.

  Here, FIG. 7 is a schematic view showing an AA ′ cross-sectional view of the cell fusion container of FIG. 6. As a means for bonding the upper electrode (14), the spacer (16), the insulator (8), and the lower electrode (15) as shown in FIG. 7, each of them is bonded with an adhesive or heated in a pressurized state. For example, a known method may be used, such as a method in which the spacer is fused and a method in which the spacer is bonded using a surface adhesive PDMS (poly-dimethylsiloxane) or a resin such as a silicon sheet. . In this way, the cell fusion region (1) shown in FIG. 7 can be formed.

  The cross-sectional shape of each of the micropores formed in the cell fusion container of the present invention has a diameter and depth of two or more steps from the top of the opening toward the bottom of the opening in contact with the electrode. It is preferable to have a shape with a diameter and depth of two or more steps, and it is particularly preferable to have a shape with a two-step diameter and depth on the opening upper side and the opening bottom side. The upper part of the opening or the lower part of the opening is, as an example, described with reference to FIG. 7. In the fine hole (9) serving as the opening provided in the insulator (8), the upper electrode (14) side is the upper part of the opening or the opening. The side that is in contact with the lower electrode (15) is the opening bottom or the opening bottom.

  Further, as shown in FIG. 7, the diameter of the largest circle inscribed in the planar shape of the fine hole is reduced toward two or more steps toward the lower electrode (15) that is the electrode surface on which the insulator is disposed. The fine holes having the diameter and the depth, and the fine holes having the diameter and the depth in two or more stages are fine holes having the diameter and the depth in two stages, and are fine on the bottom side of the opening. The diameter of the hole, that is, the micropore on the lower electrode (15) side which is the electrode surface on which the insulator is disposed (hereinafter referred to as the first micropore (17)) fuses the cells. Of the two cells, the average diameter is not less than the average diameter of cells having a small average diameter and not more than the average diameter of cells having a large average diameter, The depth of micropores (first micropores) fuses cells 2 A micropore on the upper side of the opening, that is, a micropore on the stage located on the side of the cell fusion region (1) (hereinafter referred to as the second micropore (21)). The diameter is larger than the average diameter of the cells having a larger average diameter among the two cells to be fused. The cell fusion target of the cell fusion container of the present invention is two types of cells having different average diameters, and each of the two types of cells has a predetermined cell diameter distribution as described later. I have it.

  Here, the number of stages of the fine holes is not particularly limited. However, if the number of stages is too large, it is difficult to manufacture the microholes, and a remarkable effect corresponding to the number of stages cannot be obtained. Moreover, the planar shape of the fine holes is not particularly limited, and may be a circular shape, a polygonal shape such as a triangular shape or a quadrangular shape, or a complex polygonal shape such as a star shape. In the case of a polygonal shape, when the apex corner is an acute angle, there is an effect of increasing the dielectrophoretic force for attracting cells by concentrating the lines of electric force at that portion. In addition, when a plurality of micropores are formed, it is necessary that cells be fixed uniformly in each micropore, and it is preferable that the dielectrophoretic force that attracts the cells to the micropores is uniform in the plane direction. The hole shape is preferably point-symmetric. Therefore, considering the ease of manufacturing the microholes, the planar shape of the micropores is preferably square or circular.

  Regarding the diameter of the first stage micropores, it is preferable that cells having a small average diameter enter the first stage micropores among the two cells to be fused, and cells having a large average diameter do not enter the first stage micropores. Therefore, it is preferable that the cell diameter is greater than or equal to the average diameter of the cells having a small average diameter and less than or equal to the average diameter of the cells having a large average diameter.

  In addition, if the depth of the first stage micropore (17) is too deep, cells having a small average diameter fixed to the first stage micropore and the second stage micropore (21) are fixed. Since it becomes impossible to contact cells with a large average diameter, there is no particular limitation as long as it is less than the average diameter of cells with a small average diameter, but if it is too shallow, the effect of concentrating the electric lines of force in the micropores will be weakened, Since it becomes difficult to reliably fix the cells to the micropores, the minimum depth of the first micropores is preferably about 1 μm.

  Further, the diameter of the second stage micropore (21) is not particularly limited as long as cells having a large average diameter can enter the second stage micropores, so long as it is larger than the average diameter of the cells having a large average diameter. However, if it is too large, a large number of cells will enter the micropores, making it difficult to fuse the two types of cells on a one-to-one basis. The average diameter is preferably not more than twice the average diameter. In addition, the depth of the micropores in the second stage is not particularly limited, but if it is too shallow, the concentration effect of the electric lines of force in the micropores is weakened, making it difficult to reliably fix the cells in the micropores. If it is too deep, cells with a small average diameter that are first fixed in the second micropores and cells with a large average diameter that are fixed in the vicinity of the micropore surface (31) cannot come into contact with each other, and cell fusion becomes impossible. Accordingly, the minimum value of the depth of the second stage micropores is preferably about 1 μm, more preferably about the average diameter of cells having a small average diameter, and the maximum value of the depth of the second stage micropores. Is preferably about the average diameter of cells having a small average diameter. Specifically, for example, when a mouse spleen cell (average diameter: about 6 μm) is used as a cell having a small average diameter and a mouse cancer cell (average diameter: about 10 μm) is used as a cell having a large average diameter as cells to be fused, The diameter of the fine hole in the step is about 8.5 μm, the depth of the fine hole in the first step is about 4 μm, the diameter of the fine hole in the second step is about 12 μm, and the depth of the fine hole in the second step is about 6 μm. It becomes. By adopting such an embodiment, even if there is a wide range of diameters of cells with a small average diameter fixed to the micropores, the range of cell diameters that can be fused with the set DC pulse voltage is expanded, and the fusion probability is increased. It becomes possible to make it higher.

  Before describing the effect of the cross-sectional shape of the micropores in the present invention, the characteristics of the cell fusion method of the present invention will be described.

  The cell fusion method of the present invention uses the above cell fusion device to introduce the first cell into the cell fusion region shown in FIG. 6 and immobilize the first cell in the micropore by applying an alternating voltage. Then, a second cell having an average diameter larger than the average diameter of the first cell is introduced into the cell fusion region, and an alternating voltage is applied to finely adjust the second cell to the first cell. This is a cell fusion method in which cells are fused by applying a direct-current pulse voltage at the position of the hole.

  That is, it is preferable that the average diameter of the second cell is larger than the average diameter of the first cell. When introducing the cell, a cell having a small average diameter is first introduced as the first cell, and a cell having a large average diameter is introduced. Is then preferably introduced as a second cell. In this way, it is possible for the first time to apply a voltage suitable for each size so that the cell membranes of two types of cells with different average diameters can be reversibly destroyed so that they can be repaired. You can get a probability. The feature of the cell fusion method of the present invention is that “the average diameter of the second cells is preferably larger than the average diameter of the first cells. It is preferable to introduce first as a cell, and then introduce a cell having a large average diameter as a second cell next, and the characteristic of the fine fusion container of the present invention, “having the two-stage diameter and depth. In the micropore, the diameter of the micropore on the electrode surface side on which the insulator is disposed (the micropore on the first stage) is equal to or greater than the average diameter of the cells having a small average diameter among the two cells to be fused. The average diameter of cells having a large average diameter is not more than the average diameter, and the depth of the micropores in the first stage is not more than the average diameter of cells having a small average diameter among the two cells to be fused, and is located on the cell fusion region side. Micro hole of step (second stage micro hole) Diameter larger average diameter greater than "it is preferable for reasons of cell average diameter of 2 cells cell fusion is described in further detail below.

  First, effects and problems when the cross-sectional shape of the micropore described in Patent Document 4 is one stage will be described.

  In the case of a microhole having a one-step cross-sectional shape, the lines of electric force are concentrated in the microhole, so that the electric field strength in the vicinity of the microhole is the highest in the electric field strength of the electrode surface in the microhole as shown in FIG. The electric field strength gradually decreases from the insulating film surface toward the other electrode. FIG. 12 shows a finite element method for calculating the electric field strength when an arbitrary voltage is applied between electrodes by arranging one minute hole having an arbitrary diameter and depth in an insulating film of an arbitrary 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. 12 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, as shown in (Formula 1), generally, the smaller the average cell diameter, the higher the DC pulse voltage that causes reversible disturbance of the cell membrane. Therefore, if a cell having a small average diameter is put in the micropore as a first cell and a cell having a large average diameter is fixed as a second cell from above the first cell fixed in the micropore, a DC pulse to be applied is applied. Although the voltage is the same, as shown in FIG. 12, since the electric field strength in the micropores is higher than the electric field strength on the surface of the micropores, the average fixed in the micropores (9) as shown in FIG. A higher voltage is applied to the first cell (18) having a small diameter, and a second cell (22) having a large average diameter fixed to the micropore surface (31) is applied to the first cell. A voltage that is about 20% lower than the applied voltage is applied. In this way, it is possible for the first time to apply a voltage appropriate for each cell to each cell so that the cell membranes of two types of cells with different average diameters can be reversibly destroyed. Thus, a high fusion probability can be obtained.

  However, as shown in FIG. 9, when the range of the diameter of the first cell (18) is wide, in the micropore having a one-step cross-sectional shape, a cell having a relatively large diameter among the first cells is a micropore. Since it does not enter (9), the second cell (22) is arranged further above or next to the first cell, and the effect described in Patent Document 4 cannot be obtained sufficiently, and cell fusion Because there are cells that do not, there is a problem that the fusion probability decreases.

  Therefore, as a result of intensive studies by the present inventors, the cross-sectional shape of the micropore is such that the diameter of the maximum circle inscribed in the planar shape of the micropore toward the electrode surface on which the insulator is disposed is reduced. The cross-sectional shape of the micropore having two or more stages, preferably two stages of the diameter and depth, has led to solving this problem.

  Here, in the micropores having the two-stage diameter and depth, the diameters of the micropores (first micropores) located on the electrode surface side where the insulator is disposed are fused 2. The average of the cells having a smaller average diameter among the two cells that are not less than the average diameter of the cells having the smaller average diameter and not larger than the average diameter of the cells having the larger average diameter, and the depth of the first micropore is cell fusion. More preferably, the diameter is equal to or smaller than the diameter, and the diameter of the micropore in the stage located on the cell fusion region side (second stage micropore) is larger than the average diameter of the cells having a larger average diameter among the two cells to be fused.

  Next, the feature of the present application is that the shape of the cross-section of each microhole is a shape having a diameter and depth of two or more steps from the top of the opening toward the bottom of the opening in contact with the electrode at the opening of the microhole in the insulator. It is preferable that the shape has a diameter and depth of two or more steps, and it is particularly preferable to have a shape having a two-step diameter and depth on the opening upper side and the opening bottom side and the effect thereof. Will be described with reference to FIGS.

  First, the diameter of the first cell having a small average diameter first introduced into the cell fusion container is positioned on the electrode surface side where the insulator is disposed among the fine holes having the two-stage diameter and depth. FIG. 10 shows an example of the case where the diameter of the microhole (the first micropore (17) of the first stage) is equal to or smaller than the diameter. In addition, the electric field strength in the vicinity of the fine holes at this time is shown in FIG. As shown in FIG. 13, the electric field strength of the first-stage microhole (17) is the highest, and then the electric field strength of the second-stage microhole (21) is higher than the electric field strength of the first-stage microhole. The electric field strength of the micropore surface (31) is about 15% lower and about 20% lower than the electric field strength of the second micropore. In this case, the second cell having a large average diameter introduced into the cell fusion container is a micropore (second step) located on the cell fusion region side among the micropores having the two-stage diameter and depth. And contact with the first cell. However, if the depth of the first stage micropores is larger than the average diameter of the first cells, it will not be possible to contact the second cells that have entered the second stage micropores. The depth of the micropores is preferably smaller than the average diameter of the first cells. When a DC pulse voltage is applied in this state, the electric field strength in the first microhole is higher than the electric field strength in the second microhole, as in Patent Document 4, so that the first stage A higher voltage is applied to the first cells having a small average diameter fixed in the micropores, and the first cells are applied to the second cells having a large average diameter fixed in the second micropores. A voltage about 15% lower than the applied voltage is applied. In this way, it is possible for the first time to apply a voltage appropriate for each cell to each cell so that the cell membranes of two types of cells with different average diameters can be reversibly destroyed. Thus, a high fusion probability can be obtained.

  Next, FIG. 11 shows an example in which the diameter of the first cell having a small average diameter first introduced into the cell fusion container is larger than the diameter of the first micropore. At this time, the electric field strength in the vicinity of the fine holes is the same as that in FIG. In this case, the first cell (18) is fixed to the upper side of the first-stage micropore (17), that is, the second-stage micropore (21). Subsequently, when the second cell (22) having a large average diameter is introduced into the cell fusion container, the second cell is arranged on the micropore surface (31) as shown in FIG. 11, and comes into contact with the first cell in the previous period. . When a DC pulse voltage is applied in this state, the electric field strength in the second micropore is higher than the electric field strength on the surface of the micropore, so that the first average diameter fixed in the second micropore is small. Although a higher voltage is applied to one cell, a voltage that is about 15% lower than that in the case where the cell is fixed in the first micropore is applied to the first cell. The first cells having a relatively large diameter that does not enter the first stage micropores and are fixed in the second stage micropores than the first cells having a relatively small diameter to enter. A voltage that destroys the cell membrane to such an extent that it cannot be repaired is not applied, but a voltage that causes reversible disturbance that can repair the cell membrane is applied. Further, a voltage about 20% lower than the voltage applied to the first cell is applied to the second cell having a large average diameter fixed to the surface of the micropore. By doing so, even if there is a spread in the range of the diameter of the first cell, so that the cell membrane of two types of cells with different average diameters can be reversibly destroyed to the extent that it can be repaired, It becomes possible for the first time to apply a voltage suitable for each size to each cell, and a high fusion probability can be obtained.

  In addition, since the cell fusion container of the present invention can obtain a higher fusion regeneration probability when one cell is fixed in one micropore, a plurality of micropores formed in the insulator described above. However, on the surface of the insulator, the positions of the adjacent microholes from any one of the microholes are formed at the same position, that is, as shown in FIG. 6, a plurality of microholes are arrayed on the surface of the insulator. It is preferable to be formed. 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 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. 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 by fixing one cell in one micropore. Here, the waveform of the alternating voltage for fixing one cell per one micropore is preferably a rectangular wave. The reason for this is that, as shown in FIGS. 14 to 17, the waveform of the AC voltage is instantaneous compared to the sine wave (FIG. 14), the triangular wave (FIG. 15), and the trapezoidal wave (FIG. 16). Reaches the peak voltage Vp (38) set to, so that the cells move rapidly into the micropores, so the probability that the cells overlap and enter the micropores is low, and thus one cell is fixed per micropore. Probability increases. 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.

  Moreover, it is preferable that the waveform of the alternating voltage used for the cell fusion device of the present invention does not have a direct current component. This is because the cells move by receiving a biased force in a specific direction due to the electrostatic force generated by the DC component, and it becomes difficult to fix the cells in the micropores due to the dielectrophoretic force. 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.

  By the way, in the cell fusion method of the present invention, after fixing the first cell in one micropore, the second cell is fixed further from above the fixed first cell. Dielectric migration force, gravity, and electrostatic force of the first cell act on the second cell and come into contact with the first cell. However, for the reasons described above, the first cells block the micropores, making it difficult for the current to flow, thereby suppressing the generation of electric lines of force and weakening the dielectrophoretic force acting on the second cells. Therefore, the probability of bringing the second cells into contact with the first cells fixed in the micropores one by one is reduced. However, it is possible to increase the contact probability between the first cell and the second cell by making the concentration of the second cell higher than that of the first cell and introducing it excessively into the cell fusion region. . In this case, as shown in FIG. 13, the electric field strength is highest near the fine hole and becomes weaker as the distance from the fine hole becomes smaller. Therefore, the first pulse contacted in the vicinity of the fine hole can be achieved by appropriately adjusting the DC pulse voltage. The cell membrane of only the second cell and the second cell cause reversible disturbance and cell fusion. Accordingly, 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, and a high fusion probability can be obtained. In addition, if the number of first cells is larger than the number of micropores, there are cells that are not fixed in the micropores, and as a result, the proportion of cells involved in cell fusion decreases, so the number of first cells is the number of micropores. The number is preferably equal to or less than the number. As described above, when the number of the second cells is smaller than the number of the first cells, there is a first cell that cannot contact the second cell, and as a result, there are few combinations of one set of two cells that fuse cells. Therefore, it is preferable that the number of the second cells is larger than the number of the first cells. However, if the number of the second cells is too large, it may be impossible to actually introduce the cells. The number of cells is preferably the same number as the first cell number to about 4 times.

According to the present invention, the following effects can be obtained.
(1) According to the cell fusion container and the cell fusion device of the present invention, the diameter of the cell that can be fused with the set DC pulse voltage even if the range of the diameter of the cell having a small average diameter fixed to the micropore is wide. And the fusion probability can be increased.
(2) According to the cell fusion container and the cell fusion device of the present invention, the voltage suitable for each size is applied so that the cell membranes of two types of cells having different average diameters can be reversibly destroyed to the extent that they can be repaired. It becomes possible for the first time to apply to each cell, and a high fusion probability can be obtained.
(3) According to the cell fusion container and the cell fusion device of the present invention, it is possible to simultaneously fuse a pair of two cells fixed in a plurality of micropores, and efficiently perform cell fusion with a pair of two cells. Can be done.
(4) According to the cell fusion container and the cell fusion device of the present invention, the probability of fixing one cell in one micropore can be increased, and the probability of making a pair of two cells contact in each micropore can be increased. It becomes possible.
(5) According to the cell fusion method of the present invention, two cell pairs in the vicinity of the micropores can be selectively fused, and cell fusion with two cell pairs can be performed efficiently.
(6) According to the cell fusion method of the present invention, a voltage suitable for each size is applied to each cell so that the cell membranes of two types of cells having different average diameters can be reversibly destroyed to the extent that they can be repaired. It becomes possible for the first time to apply, and a high fusion probability can be obtained.

  Hereinafter, embodiments of the present invention will be described in detail. 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)
The conceptual diagram of the cell fusion apparatus used for the Example at FIG. 6 is shown. The cell fusion device is roughly divided into a cell fusion container (13) and a power source (4). In the cell fusion container, as shown in FIG. 6, a spacer (16) is arranged between the upper electrode (14) and the lower electrode (15), and a plurality of circular micropores (9) are formed in an array. It has a structure in which an insulator (8) is sandwiched between a spacer and a lower electrode. FIG. 7 shows the AA ′ cross-sectional view of the cell fusion container shown in FIG. 6, that is, the cross-sectional shape of the micropores. As shown in FIG. 7, the cross-sectional shape of the micropore is such that the diameter of the micropore on the lower electrode (15) side where the insulator is disposed is small (diameter: about 8.5 μm, depth: about 4 μm), and the cell fusion region (1) The micropores having two-stage diameters (hereinafter referred to as stepped micropores) were formed so that the diameter of the micropores on the side was large (diameter: about 12 μm, depth: about 6 μm). In this embodiment, as will be described later, stepped microholes are produced by general photolithography as shown in FIG. 18, and stepped microholes are formed in an array in the lower electrode as shown in FIG. It was manufactured as an insulator-integrated lower electrode (36) with stepped micropores in which the formed insulator was integrally formed.

  The upper electrode and the lower electrode were formed by depositing ITO (film thickness 150 nm) on a Pyrex (registered trademark) substrate 70 mm long × 40 mm wide × 1 mm thick. The spacer was used by hollowing out the center of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm into a length of 20 mm and a width of 20 mm. Further, as shown in FIG. 6, an introduction port (19) and a discharge port (20) for introducing and discharging the cell suspension were provided. Here, the insulator (8) having a plurality of stepped micropores was fabricated as follows by being integrally formed on the lower electrode by a photolithography and etching method shown in FIG.

  First, a resist (25) was applied to the ITO film-formed 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 4 μm. After drying, prebaking (65 ° C., 1 minute → 95 ° C., 3 minutes) was performed using a hot plate. The resist used was SU-8 (manufactured by Kayaku Microchem), which is an epoxy negative resist. Next, in the area of 30 mm length × 30 mm width, a micro hole pattern with a diameter of 8.5 μm arranged in an array of 1500 vertical x 1500 horizontal, with the vertical and horizontal spacing of the fine holes being 20 μm is drawn. The resist was exposed (27) with a UV exposure machine using the exposure photomask (26), and developed with a developer (33). Prior to development, pre-development baking (65 ° C., 1 minute → 95 ° C., 2 minutes) was performed using a hot plate. The exposure time and development time were adjusted so that the depth of the micropores was 4 μm, which was equal to the resist film thickness, so that the ITO was exposed on the bottom surface of the micropores. After development, the substrate was rinsed with isopropyl alcohol (IPA) and hard-baked (155 ° C., 30 minutes) using a hot plate to harden the resist and produce an insulator-integrated lower electrode (28) with fine holes. Subsequently, a resist (25) is further applied on the produced microporous insulator-integrated lower electrode (28) using a spin coater so as to have a film thickness of 6 μm. After naturally drying for 1 minute, a hot plate is used. And prebaking (65 ° C., 1 minute → 95 ° C., 3 minutes). Similarly, SU-8 (manufactured by Kayaku Microchem), which is an epoxy negative resist, was used as the resist. Next, with respect to the fine holes having a diameter of 8.5 μm arranged in an array of 1500 vertical × 1500 horizontal, the vertical and horizontal distance between the fine holes is 20 μm, and the vertical 1500 × horizontal 1500 An exposure photomask (26) on which fine hole patterns with a diameter of 12 μm arranged in an array are drawn so that the centers of the fine holes coincide with each other, the resist is exposed (27) with a UV exposure machine, and a developer (33 ) And developed. Prior to development, pre-development baking (65 ° C., 1 minute → 95 ° C., 2 minutes) was performed using a hot plate. Also, the exposure time and development time are adjusted so that the depth of the micropores is 10 μm, which is equal to the sum of the film thicknesses of the first-stage resist and the second-stage resist, and the ITO is exposed on the bottom surface of the microholes. I did it. After development, the substrate rinsed with IPA was used as the insulator-integrated lower electrode (36) with stepped micropores. The stepped microporous insulator-integrated lower electrode (36) thus produced was laminated together with the upper electrode (14) and the spacer (16) as shown in FIG. 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 suspension containing the cells could be put into the cell fusion container without leakage. Here, since the area formed by hollowing out the spacer is 20 mm long × 20 mm wide, the number of micropores existing in this space is about 1 million. In addition, the power source for applying a voltage between the electrodes is a signal generator (NF circuit design block, WF1966) as an AC power source, and a cell fusion power source (Neppagene, LF101) as a pulse power source via a conductive wire (3). The AC power supply and the pulse power supply can be switched to the electrode by a changeover switch.

  Subsequently, 600 μL of 0.1 mg / mL bovine serum albumin (BSA) aqueous solution was injected into the cell fusion device from the introduction port of the spacer using a 1 mL pipette, and a voltage of 900 V between the electrodes was pulsed by a DC pulse power supply. A pulse voltage having a width of 30 μs was applied 99 times to hydrophilize the surface of the insulator of the stepped microporous insulator-integrated lower electrode. Here, the hydrophilization treatment plays a role of making the surface of the insulating insulator hydrophobic by making it hydrophilic with BSA and preventing adhesion between the cells and the insulating film.

The experiment described below was performed using the above-described cell fusion device composed of the insulator-integrated lower electrode with stepped micropores subjected to the hydrophilic treatment. As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Both cells were suspended in 300 mM mannitol aqueous solution containing BSA (1 mg / mL), and mouse antibody-producing cells and mouse myeloma cells were suspended at a density of 1.7 × 10 ⁶ cells / mL. A suspension was prepared. Here, BSA plays a role of reducing damage to cells due to voltage application. Both cell suspensions were added with 0.1 mM calcium chloride and 0.1 mM magnesium chloride to promote cell membrane regeneration during cell fusion.

  600 μL of the cell suspension of mouse antibody-producing cells (number of mouse antibody-producing cells: about 1 million) was injected from the introduction port of the spacer using a 1 mL pipette, and a rectangular wave with a voltage of 10 Vpp and a frequency of 3 MHz from an AC power source. When an alternating voltage is applied between the electrodes, about 1 to 2 mouse antibody-producing cells can be fixed in each of a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. A plurality of cells could be arranged in an array. At this time, the probability that about 1 to 2 mouse antibody-producing cells enter one micropore was about 90%.

  Subsequently, 600 μL of the mouse myeloma cell suspension (number of mouse myeloma cells: about 1 million cells) was introduced into the spacer inlet while a rectangular wave AC voltage having a voltage of 10 Vpp and a frequency of 3 MHz was applied between the electrodes by an AC power source. When a 1 mL pipette was used for injection, mouse antibody-producing cells fixed in the micropores and mouse myeloma cells could be contacted in the micropores. At this time, the probability of one mouse myeloma cell entering one micropore was about 70%. When the mouse myeloma cells were introduced, it was hardly observed that the mouse antibody-producing cells put in the micropores were detached from the micropores. Therefore, the mouse antibody-producing cells and the mouse myeloma cells were paired in pairs in the micropores. Estimated contact is estimated to be approximately 63% (= 90% × 70%).

Next, the power source is switched to a DC pulse power source (manufactured by Nepagene Co., Ltd., LF101), a cell fusion is performed by applying a pulse voltage of 80 V and a pulse width of 30 μs between the electrodes, and the cell fusion container is allowed to stand for 15 minutes. The cell suspension was placed in a HAT medium (H: hypoxanthine, A: medium containing aminopterine, T: thymidine), and the fused cells were cultured. The HAT medium is a medium for selectively growing only fused cells. HAT medium containing the cell suspension was placed in a 5% CO ₂ incubator and cultured, and after 6 days, the number of fused cells was counted. As a result, about 4900 fused cells could be confirmed, and all mouse antibody-producing cells A fusion probability of about 49/10000 was obtained for 1 million pieces. This is a fusion probability that is about 1.3 times the fusion probability of about 38/10000 in the comparative example to be described later, confirming efficient fusion of two cell pairs with reduced influence of the particle size distribution of mouse antibody-producing cells. We were able to.

(Comparative example)
FIG. 19 shows a conceptual diagram of the cell fusion device used in the comparative example. FIG. 19 shows the components of the cell fusion device of the present invention in a separated state for easy understanding. Further, in FIG. 20, the cell fusion device of the present invention is shown as a cross-sectional view taken along the line BB ′ after the components are assembled in an original configuration. In the figure, the cell fusion device is roughly divided into a cell fusion container (13) and a power source (4). As shown in FIG. 19, the cell fusion container includes an insulator (8) in which a spacer (16) is arranged between an upper electrode (14) and a lower electrode (15), and a plurality of micropores are formed in an array. It has a structure sandwiched between a spacer and a lower electrode. In the comparative example, the lower electrode (15) and the insulator-integrated lower electrode with a fine hole integrally formed with the insulator in which a plurality of fine holes are formed in an array shape by general photolithography and etching as in the embodiment. (28) was used.

  The upper electrode and the lower electrode were formed by depositing ITO (film thickness 150 nm) on a Pyrex (registered trademark) substrate 70 mm long × 40 mm wide × 1 mm thick. The spacer was used by hollowing out the center of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm into a length of 20 mm and a width of 20 mm. Moreover, as shown in FIG. 7, the introduction port (19) and the discharge port (20) for introducing and discharging the cell suspension containing the cells were provided. Here, the insulator (8) having a plurality of fine holes was fabricated as follows by being integrally formed on the lower electrode by a photolithography and etching method shown in FIG.

  First, a resist (25) was applied to the ITO film-forming surface of Pyrex (registered trademark) glass (24) on which ITO (23) was formed using a spin coater so as to have a film thickness of 4 μm. Then, pre-baking (65 degreeC, 1 minute-> 95 degreeC, 3 minutes) was performed using the hotplate. The resist used was SU-8 (manufactured by Kayaku Microchem), which is an epoxy negative resist. Next, a microhole pattern having a diameter of 8.5 μm is arranged in an area of 30 mm in length and 30 mm in width and arranged in an array of 1000 in length and 1000 in width with an interval between the holes of 30 μm. The resist was exposed (27) with a UV exposure machine using the exposure photomask (26), and developed with a developer (33). The exposure time and development time were adjusted so that the depth of the micropores was 4 μm, which was equal to the resist film thickness, so that the ITO was exposed on the bottom surface of the micropores. After development, the substrate was rinsed with isopropyl alcohol (IPA) and hard-baked (155 ° C., 30 minutes) using a hot plate to harden the resist and produce an insulator-integrated lower electrode (28) with fine holes. The thus-produced insulator-integrated lower electrode (28) with fine holes was laminated and pressure-bonded together with the upper electrode (14) and the spacer (16) as shown in FIG. 20 is a BB ′ cross-sectional view of the cell fusion container shown in FIG. 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 suspension containing the cells could be put into the cell fusion container without leakage. Here, 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. The power source for applying a voltage between the electrodes is a signal generator (NF circuit design block, WF1966) as an AC power source, and a cell fusion power source (Neppagene, LF101) as a DC pulse power source via a conductive wire (3). The AC power supply and DC pulse power supply can be switched to the electrode by a changeover switch.

  Subsequently, 600 μL of 0.1 mg / mL bovine serum albumin (BSA) aqueous solution was injected into the cell fusion device from the introduction port of the spacer using a 1 mL pipette, and a voltage of 900 V between the electrodes was pulsed by a DC pulse power supply. A pulse voltage having a width of 30 μs was applied 99 times to hydrophilize the surface of the insulator of the microporous integrated insulator lower electrode. Here, the hydrophilization treatment plays a role of making the surface of the insulating insulator hydrophobic by making it hydrophilic with BSA and preventing adhesion between the cells and the insulating film.

An experiment described later was performed using the above-described cell fusion device constituted by the insulator-integrated lower electrode with micropores subjected to the hydrophilic treatment. As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Both cells were suspended in an aqueous mannitol solution having a concentration of 300 mM, and the cell suspension was adjusted to a density of 0.7 × 10 ⁶ cells / mL. To both cell suspensions, 0.1 mM calcium chloride and 0.1 mM magnesium chloride were added in order to promote cell membrane regeneration by cell fusion.

  First, 600 μL of the mouse antibody-producing cell suspension (number of mouse antibody-producing cells: about 400,000) was injected from the introduction port of the spacer using a 1 mL pipette, and the voltage was 10 Vpp and the frequency was 3 MHz from an AC power source. When applying the rectangular wave AC voltage between the electrodes, one mouse antibody-producing cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds, Multiple cells could be arranged in an array. At this time, the probability that one mouse antibody-producing cell enters one micropore was about 85%.

  Subsequently, 600 μL of the cell suspension of mouse myeloma cells (number of mouse myeloma cells: about 400,000 cells) was applied to the spacer inlet while a rectangular wave AC voltage having a voltage of 10 Vpp and a frequency of 3 MHz was applied between the electrodes by an AC power source. When injected with a 1 mL pipette, one mouse myeloma cell can be fixed one by one in a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. Cells could be arranged in an array. At this time, the probability of one mouse myeloma cell entering one micropore was about 70%. When the mouse myeloma cells were introduced, it was hardly observed that the mouse antibody-producing cells put in the micropores were detached from the micropores. Therefore, the mouse antibody-producing cells and the mouse myeloma cells were paired in pairs in the micropores. Estimated contact is estimated to be about 60% (= 85% × 70%).

Next, the power supply is switched to a DC pulse power supply (LF101, manufactured by Nepagene Corporation), a cell fusion is performed by applying a DC pulse voltage with a voltage of 100 V and a pulse width of 30 μs between the electrodes, and the cells are allowed to stand for 10 minutes, and then the cells are fused. The cell suspension in the container was placed in a HAT medium (H: hypoxanthine, A: medium containing aminopterine, T: thymidine), and the fused cells were cultured. The HAT medium is a medium for selectively growing only fused cells. The HAT medium containing the cell suspension was placed in a CO ₂ incubator and cultured, and after 6 days, the number of fused cells was counted. As a result, about 1520 fused cells could be confirmed. The fusion probability was about 38/10000 per piece.

It is the conceptual diagram which showed sectional drawing of the cell fusion container of patent document 1. It is the figure which showed the example of the diameter distribution of a mouse | mouth spleen cell and a mouse | mouth myeloma cell. In the cell fusion container of patent document 1, it is a conceptual diagram which shows the example in case the diameter of a micropore is larger than the diameter from two cells. In the cell fusion container of patent document 1, it is a conceptual diagram which shows the example in case the diameter of a micropore is smaller than the diameter from two cells. It is the schematic of general photolithography and an etching method when producing the insulator integrated lower electrode with a fine hole. It is a conceptual diagram which shows an example of the cell fusion apparatus of this invention, and the cell fusion apparatus used for the Example. FIG. 7 is an AA ′ sectional view of the cell fusion container shown in FIG. 6. In patent document 4, it is a figure which shows an example when the cell with a comparatively small diameter enters into a micropore among the 1st cells. In patent document 4, it is a figure which shows an example when the cell with comparatively large diameter among 1st cells does not enter in a micropore. In this invention, it is a figure which shows an example at the time of a cell with comparatively small diameter entering into the 1st step | paragraph micropore among the 1st cells. In this invention, it is a figure which shows an example at the time of a cell with comparatively large diameter among 1st cells not entering into the 1st step | paragraph micropore, but entering into the 2nd step | paragraph micropore. It is the figure which showed distribution of the electric field strength of a micropore vicinity. It is the figure which showed distribution of the electric field strength of the stair-like fine hole vicinity. It is the figure which showed the sine wave as an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the triangular wave as an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the trapezoid wave as an example of the waveform of the alternating voltage used for this invention. It is the figure which showed the rectangular wave as an example of the waveform of the alternating voltage used for this invention. It is the schematic of the general photolithography and the etching method when producing the insulator integrated lower electrode with a step-like fine hole. It is a conceptual diagram which shows the cell fusion apparatus used for the comparative example. FIG. 20 is a BB ′ sectional view of the cell fusion container shown in FIG. 19. FIG. 10 is a first diagram illustrating the operation of the cell fusion container described in Patent Document 1. It is the 2nd figure explaining operation of a cell fusion container given in patent documents 1. FIG. 10 is a third diagram illustrating the operation of the cell fusion container described in Patent Document 1.

Explanation of symbols

1: Cell fusion region 2: Electrode 3: Conductive wire 4: Power supply 5: AC power supply 6: DC pulse power supply 7: Changeover switch 8: Insulator 9: Micropore 10: Cell A
11: Cell B
12: Electric field line 13: Cell fusion container 14: Upper electrode 15: Lower electrode 16: Spacer 17: First stage micropore 18: First cell 19: Inlet 20: Outlet 21: Second stage micro Hole 22: Second cell 23: ITO
24: Pyrex (registered trademark) glass 25: Resist 26: Photomask for exposure 27: Exposure 28: Insulator-integrated lower electrode with micropores 29: Introduction channel 30: Discharge channel 31: Surface of micropores 32: Fusion cell 33: Developer 34: Peak voltage Vp
35: Partition 36: Insulator-integrated lower electrode with stepped micropores

Claims (10)

A pair of electrodes made of conductive members arranged opposite to each other in the cell fusion region, and a flat spacer disposed between the pair of electrodes and penetrating in the direction of the oppositely arranged electrodes A cell fusion container comprising a flat insulator having a plurality of micropores, wherein the insulator is disposed on the electrode surface on the cell fusion region side of the electrode, and the micropores A cell fusion container in which each of the cross-sections has a shape in which the area of the opening surface decreases from the top of the opening toward the bottom of the opening contacting the electrode. The cell fusion container according to claim 1, wherein the cross section of each of the micropores has a shape having a diameter and a depth of two or more steps from the top of the opening toward the bottom of the opening in contact with the electrode. A cross-section of each of the micropores has a shape with a two-stage diameter and depth on the upper side of the opening and the bottom side of the opening, and is a cell fusion container for fusing two types of cells having different average diameters. The diameter of the micropores on the bottom side of the opening is greater than the average diameter of the cells having the smaller average diameter and less than the average diameter of the cells having the larger average diameter, and the depth of the micropores The average diameter of the cells having a small average diameter is not more than the average diameter of the cells, and the diameter of the micropore on the upper side of the opening is larger than the average diameter of the cells having the large average diameter among the two cells to be fused. Cell fusion container. The plurality of micropores formed in the insulator are formed in the same position on the surface of the insulator, in which the positions of the micropores adjacent to each other are the same. Cell fusion container. The cell fusion container according to claim 4, wherein the plurality of micropores formed in the insulator are formed in an array on the surface of the insulator. The cell fusion container according to claim 4 or 5, wherein an interval between adjacent micropores is 0.5 to 6 times the average diameter of cells having a small diameter to be inserted into the micropores. The cell fusion container according to any one of claims 1 to 6, wherein the spacer has a through-hole forming the cell fusion region. The cell fusion container according to any one of claims 1 to 7, wherein the spacer has an introduction channel for introducing cells and a discharge channel for discharging cells. A cell fusion device comprising: the cell fusion container according to any one of claims 1 to 8; and a power source for applying a voltage to the pair of electrodes of the cell fusion container, wherein the power source is the cell fusion. A cell fusion device comprising an AC power source for applying an AC voltage to the pair of electrodes of the container and a DC pulse power source for applying a DC pulse voltage, and having a switching mechanism for switching between the AC power source and the DC pulse power source . A method for cell fusion of a first cell and a second cell having an average diameter larger than the average diameter of the first cell using the cell fusion device according to claim 9, wherein the cell fusion region The first cells are introduced into the cells, and the first cells are fixed in the micropores by applying an alternating voltage, and then the second cells are introduced into the cell fusion region. A cell fusion method, wherein a voltage is applied to bring the second cell into contact with the first cell at the position of the micropore and a DC pulse voltage is applied.

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