JP2010022360A - Vessel for introducing fine particle suspension, cell fusion vessel using the same, and cell fusion device - Google Patents

Abstract

Provided are a fine particle suspension introduction container, a cell fusion container, and a cell fusion device that uniformly and evenly introduce into a fine particle suspension introduction container and efficiently discharges without leaving any fine particle suspension during discharge. .
SOLUTION: A pair of flat plates 2 and 3 disposed opposite to a fine particle suspension introduction region 1 and a flat spacer 4 formed so as to penetrate the fine particle suspension introduction region 1 between the pair of flat plates. The provided fine particle suspension introduction container 13 includes an introduction port 5 and an introduction flow path 7 through which the spacer 4 introduces the fine particle suspension, a discharge port 6 and a discharge flow path 8 through which the fine particle suspension is discharged. The fine particle suspension in which the introduction flow path 7 is arranged in communication with one end of the turbid liquid introduction area 1 and the discharge flow path 8 is arranged in communication with the other end of the fine particle suspension introduction area 1. An introduction container 13 having a shape in which the flow velocity at the center in the introduction direction of the fine particle suspension from the introduction flow path 7 to the discharge flow path 8 and the flow speeds of the wall surfaces on both sides are substantially equal. 13 is used.
[Selection] Figure 1

Description

  The present invention relates to a fine particle suspension introduction container for efficiently introducing and discharging a fine particle suspension, a cell fusion container using the same, and a cell fusion device.

  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 conditions for fusion at the time of cell fusion, such as electrical conditions, are easy to set, so that the fusion regeneration probability is higher than that of the PEG method. 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, 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 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 cells with a microscope, and is an extremely reliable example of an electrode used for the microelectrode method. (For example, refer to Patent Document 1) is a time-consuming method, which requires skill and is not practical in 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 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. The Zimmermann method is used as the most practical method because of its high fusion rate compared to the microelectrode method and the parallel electrode method. However, since the fused cells adhere to the electrode surface, The important issues were how to develop electrical materials that can be recovered without damaging them, and how to quickly and rapidly produce fused cells.

  In order to solve the above-mentioned problems of the electric fusion method, a plate-shaped spacer obtained by cutting out a region for cell fusion or introduction of nucleic acid into a cell, and a conductive member disposed so as to face the plate-shaped spacer An example of a flow chamber comprising a pair of electrodes and a crimping means that enables crimping and releasing of the electrode sandwiched between the flat plate spacers, and a cell fluid delivery means for continuously moving sample cells Has been reported. (For example, refer to Patent Document 2).

  However, in the chamber described in Patent Document 2, since cells are introduced and discharged by a pump, the damage to the cells is great, and the activity of the cells decreases or the rate at which the cells die increases. In addition, due to the form of the conduit connection to the rectangular or oblong chamber, the cell introduction efficiency and the discharge efficiency are poor, such as the air remaining in the chamber during cell introduction and the presence of cells left behind during cell discharge. As a result, there is a problem that the fusion probability is lowered.

  Therefore, in order to solve the problems and problems in the above-described conventional technology, the present inventors have used a conductive member arranged so as to face the cell fusion region of the cell fusion chamber as shown in a comparative example described later. A pair of electrodes and an insulator having a plurality of fine holes disposed between the pair of electrodes and penetrating in the direction of the pair of electrodes, and the insulator is a cell fusion of one of the electrodes. Using the cell fusion device arranged on the electrode surface on the region side, the first cells were introduced into the cell fusion region, and the first cells were fixed in the micropores by applying an alternating voltage. Thereafter, a method for cell fusion by introducing a second cell, bringing the second cell into contact with the first cell at the position of the micropore, and applying a pulse voltage was studied. 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 five times the fusion probability of 0.2 / 10000 in the normal electric cell fusion method, and an efficient fusion in a pair of two cells could be confirmed (for example, see Patent Document 3). ).

  However, in the method described in Patent Document 3, it is difficult to introduce cells uniformly throughout the cell fusion region when introducing the cell suspension solution into the cell fusion container. There was a problem that a pair of two cells could not be fused in a micropore, resulting in a low fusion probability.

Japanese Patent Publication No. 7-40914 Japanese Patent No. 2691230 JP 2007-295912 A

  The object of the present invention has been proposed in view of the conventional situation, and in the introduction of the fine particle suspension, the fine particle suspension is uniformly and uniformly introduced into the fine particle suspension introduction container, and the fine particles are suspended in the discharge. It is an object of the present invention to provide a fine particle suspension introduction container, a cell fusion container using the same, and a cell fusion device for efficiently discharging without turbid liquid remaining.

  In order to solve the above-mentioned problems, the present invention provides a pair of flat plates arranged opposite to the fine particle suspension introduction region, and a flat plate formed by penetrating the fine particle suspension introduction region between the pair of flat plates. In the fine particle suspension introduction container provided with the spacer, the spacer includes an introduction port for introducing the fine particle suspension and an introduction flow path communicating therewith, and a discharge port for discharging the fine particle suspension and a discharge communicating therewith A flow path, wherein the introduction flow path is arranged in communication with one end of the fine particle suspension introduction area, and the discharge flow path is communicated with the other end of the fine particle suspension introduction area. A fine particle suspension introduction container disposed, wherein the flow velocity at the center in the introduction direction of the fine particle suspension from the introduction flow channel to the discharge flow channel is substantially equal to the flow velocity of the wall surfaces on both sides. A fine particle suspension introduction container having By have found that it is possible to solve the above problems of the prior art, thereby completing the last present invention. Hereinafter, the present invention will be described in detail.

  That is, the fine particle suspension introduction container of the present invention includes a pair of flat plates arranged to face the fine particle suspension introduction region, and a flat plate formed by penetrating the fine particle suspension introduction region between the pair of flat plates. In the fine particle suspension introduction container having a spacer, the spacer includes an introduction port for introducing the fine particle suspension and an introduction flow path communicating with the introduction port, and a discharge port for discharging the fine particle suspension and communicates with the discharge port. A discharge flow path, the introduction flow path is disposed in communication with one end of the fine particle suspension introduction region, and the discharge flow path is communicated with the other end of the fine particle suspension introduction region. In which the flow velocity at the center in the direction of introduction of the fine particle suspension from the introduction flow path to the discharge flow path is substantially equal to the flow speed of the wall surfaces on both sides. Fine particle suspension introduction container having A.

  In the fine particle suspension introduction container of the present invention, in the fine particle suspension introduction container described above, the introduction angle of the introduction channel extending from the introduction port is 0 degree or more and 60 degrees or less, and the discharge channel extends from the discharge port. In the fine particle suspension introduction container, the discharge angle is 0 degree or more and 60 degrees or less, and the introduction flow path and the discharge flow path communicate with the fine particle suspension introduction region.

  The fine particle suspension introduction container of the present invention has a fine particle suspension introduction region in which the introduction flow channel communicating with one end of the fine particle suspension introduction region and the discharge flow channel communicating with the other end are connected. And the fine particle suspension introduction container is a fine particle suspension introduction container having a fine particle suspension introduction direction length longer than a vertical length as a shape of the fine particle suspension introduction container.

  The fine particle suspension introduction container of the present invention is a fine particle suspension introduction container in which the shape of the fine particle suspension introduction region is a curved shape in the fine particle suspension introduction container described above.

  The fine particle suspension introduction container of the present invention is a fine particle suspension introduction container in which the periphery of the introduction port is hydrophobic in the fine particle suspension introduction container described above.

  The fine particle suspension introduction container of the present invention includes a horizontal confirmation mechanism for confirming that the fine particle suspension introduction container is horizontal in the fine particle suspension introduction container, and information detected by the horizontal confirmation mechanism. The particulate suspension introducing container is provided with a level adjusting means for maintaining the level of the particulate suspension introducing container.

  The cell fusion container of the present invention comprises the above-described fine particle suspension introduction container, wherein the fine particle is a cell, and the flat plate disposed facing the fine particle suspension introduction region is formed from the conductive member. A flat plate-like insulator having a plurality of fine holes penetrating in the direction of the electrodes arranged opposite to each other, and the insulator is a fine particle suspension of one of the electrodes It is a cell fusion container arranged on the electrode surface on the liquid introduction region side.

  Moreover, the cell fusion device of the present invention is a cell fusion device comprising the above-described cell fusion container and a power source for applying a voltage to a pair of electrodes of the cell fusion container.

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

  The fine particle suspension introduction container of the present invention has a pair of flat plates arranged to face the fine particle suspension introduction region, and a flat plate shape formed by penetrating the fine particle suspension introduction region between the pair of flat plates. In the fine particle suspension introduction container provided with the spacer, the spacer includes an introduction port for introducing the fine particle suspension and an introduction flow path communicating therewith, and a discharge port for discharging the fine particle suspension and a discharge communicating therewith A flow path, wherein the introduction flow path is arranged in communication with one end of the fine particle suspension introduction area, and the discharge flow path is communicated with the other end of the fine particle suspension introduction area. A fine particle suspension introduction container disposed, wherein the flow velocity at the center in the introduction direction of the fine particle suspension from the introduction flow channel to the discharge flow channel is substantially equal to the flow velocity of the wall surfaces on both sides. A fine particle suspension introduction container having Further, in the fine particle suspension introduction container, the introduction angle at which the introduction channel extends from the introduction port is 0 ° or more and 60 ° or less, and the discharge angle at which the discharge channel spreads from the discharge port is 0 ° or more and 60 ° or less. The fine particle suspension introduction container in which the introduction channel and the discharge channel communicate with the fine particle suspension introduction region. The material of the upper plate and the lower plate is not particularly limited as long as it is a chemically stable member, and may be a glass substrate, metal, resin, or the like. The spacer may be a stable member that maintains its shape, and includes, for example, glass, ceramic, resin, and the like.

  Here, FIGS. 1 and 2 are conceptual diagrams of the fine particle suspension introduction container of the present invention.

  As shown in FIG. 1, the fine particle suspension introduction container (13) according to the present invention introduces a fine particle suspension by arranging a spacer (4) between an upper plate (2) and a lower plate (3). The region (1) is secured, and the spacer is provided so that the upper plate and the lower plate are not in direct contact, and the fine particle suspension is introduced to and discharged from the fine particle suspension introduction container. An introduction channel (7) and an introduction port (5) communicating therewith, a discharge channel (8) for discharging the fine particle suspension, and a discharge port (6) communicating therewith are provided. Further, as shown in FIG. 2, the introduction angle (16) where the introduction channel extends from the introduction port is 0 degree or more and 60 degrees or less, and the discharge angle (17) where the discharge channel spreads from the discharge port is 0 degree or more and 60 degrees. The following is preferable. Further, it is preferable that the introduction channel and the discharge channel communicate with the fine particle suspension introduction region in a substantially curved shape.

  Here, the “introduction angle” is an angle at which the fine particle introduction flow channel extends to the left and right with respect to the introduction direction when connecting to the fine particle suspension introduction region, and the “discharge angle” is the same as the fine particle discharge flow. When the path is connected to the fine particle suspension introduction region, it is an angle that spreads to the left and right with respect to the introduction direction.

  As a result, as shown in FIG. 2, the flow velocity (11) at the center in the introduction direction (10) of the fine particle suspension from the introduction flow path to the discharge flow path is substantially equal to the flow velocity (12) on both wall surfaces.

  Here, the flow velocity at the center in the direction of introduction of the fine particle suspension from the introduction flow path to the discharge flow path is substantially equal to the flow velocity at the wall surfaces on both sides. It means less than about 13%. This is because, with such an embodiment, the introduction angle is 60 degrees or less, and there is no remaining bubble, so that uniform filling can be achieved.

  The central flow rate and the wall surface flow rate can be evaluated by measuring the linear velocity in the introduction direction of the fine particle suspension.

  Here, as a result of intensive studies by the present inventors, in order to efficiently introduce the particles into the fine particle suspension introduction container uniformly and evenly in every corner, and to efficiently discharge the particles without leaving any fine particle suspension. Requires that the flow velocity at the center in the direction of introduction of the fine particle suspension from the introduction flow path to the discharge flow path is substantially equal to the flow velocity of the wall surfaces on both sides, and if the introduction angle is 60 degrees or more, the fine particle suspension During the introduction of the turbid liquid, when the fine particle suspension in the center in the direction of introduction of the fine particle suspension reaches the fine particle suspension introduction region, the fine particle suspension on both sides of the wall is not , About 87% of the length from the inlet to the connection portion with the fine particle suspension introduction region is not reached, so that the fine particle suspension of the fine particle suspension on the center and both wall surfaces The difference in the time to enter the liquid introduction area is large, and the particle suspension It occurs the incoming container bubble remains. Similarly, when the discharge angle is 60 degrees or more, when discharging the fine particle suspension, the fine particle suspension in the center in the introduction direction of the fine particle suspension comes out of the fine particle suspension introduction region and is discharged from the discharge port. The particle suspension on both wall surfaces is approximately 87% of the length from the connection between the discharge channel and the particle suspension introduction region to the discharge port at the side of the wall of the discharge channel. As a result, there is a large difference in the time for entering the fine particle suspension discharge port between the center and both wall surfaces, and bubbles may remain in the fine particle suspension introduction container. Accordingly, it is preferable that the introduction angle at which the introduction channel extends from the introduction port is 0 ° to 60 ° and the discharge angle at which the discharge channel extends from the discharge port is 0 ° to 60 °. That is, since the introduction angle is 0 ° to 60 ° and the discharge angle is 0 ° to 60 °, the difference between the flow velocity at the center in the introduction direction and the flow velocity at the wall surfaces on both sides is less than about 13% and becomes almost equal. The fine particle suspension can be uniformly introduced and discharged at the center in the introduction direction of the fine particle suspension introduction region and the wall surfaces on both sides, and air remains in the fine particle suspension introduction region. In addition, it is possible to substantially eliminate the remaining of the fine particles in discharging the fine particles.

  In Table 1 below, when the introduction angle is changed, the ratio (%) of the flow velocity near the wall surface to the central flow velocity of the fine particle suspension and the fine particle suspension introduction region when introducing the fine particle suspension Table 2 shows the relationship between the presence and absence of bubbles and the ratio of the flow rate near the wall surface to the central flow rate of the fine particle suspension (%) and the fine particle suspension discharge rate when the discharge angle is changed. 2 shows an example of the relationship between the presence or absence of a phenomenon of fine particles remaining in the fine particle suspension introduction region.

表１および表２において、壁面付近と中心流速の測定方法は微粒子懸濁溶液の導入方向における線速で評価でき、壁面付近とは微粒子懸濁液導入領域の壁側近傍を意味する。また、線速を測定する器具・計測装置は、例えば、微粒子懸濁溶液の端面を目視によりストップウォッチ等で計測する方法などで測定できる。さらに、中心流速の計算方法は、中心流路を液の端面が導入口から排出口へ向かって到達する時間とその流路長さから、線速を求めることが例示できる。壁面付近の流速の場合も同様に、液の端面が壁をつたって進む時間と壁側流路の長さ（角から角までの辺の長さ）から、線速を求めることができる。流速は、導入口からの微粒子懸濁溶液の導入速度によって違ってくることがあるため、導入方向の中心の流速と両側の壁面の流速の差（割合）を明記により求めることもできる。

In Tables 1 and 2, the method for measuring the vicinity of the wall surface and the central flow velocity can be evaluated by the linear velocity in the direction of introduction of the fine particle suspension. The vicinity of the wall surface means the vicinity of the wall side of the fine particle suspension introduction region. The instrument / measuring device for measuring the linear velocity can be measured by, for example, a method of visually measuring the end surface of the fine particle suspension with a stopwatch or the like. Further, the calculation method of the center flow velocity can be exemplified by obtaining the linear velocity from the time required for the liquid end surface to reach the center channel from the inlet to the outlet and the channel length. Similarly, in the case of the flow velocity near the wall surface, the linear velocity can be obtained from the time during which the liquid end face travels through the wall and the length of the wall-side flow path (the length of the side from the corner to the corner). Since the flow rate may vary depending on the introduction speed of the fine particle suspension solution from the introduction port, the difference (ratio) between the flow rate at the center in the introduction direction and the flow rates on the wall surfaces on both sides can also be determined explicitly.

  The shape of the fine particle suspension introduction container in the present invention may be a curved shape as shown in FIG.

  Here, the shape of the fine particle suspension introduction region is a curved shape when the connection portion between the introduction flow path and the fine particle suspension introduction region and the connection portion between the discharge flow passage and the fine particle suspension introduction region have corners. Means that the fine particle suspension introduction region has no corners and has a swelled shape like an ellipse.

  Further, here, the introduction channel and the discharge channel communicate with the particulate suspension introduction region in a substantially curved shape means that the connection between the introduction channel and the particulate suspension introduction region, and the discharge channel and the particulate suspension. It means that the connection part of the liquid introduction region is smoothly connected substantially in a curved line with no corners.

  In this way, in the introduction, the fine particle suspension near the wall surface on both sides in the introduction direction proceeds more smoothly, the flow velocity at the center and the flow velocity on the wall surfaces on both sides become more equal, and it is easier without bubbles remaining. The fine particle suspension introduction container can be uniformly and evenly introduced into every corner, and can be efficiently discharged without any remaining fine particle suspension in the discharge.

  In addition, as shown in FIG. 3, the shape of the fine particle suspension introduction container in the present invention is such that an introduction flow channel communicating with one end of the fine particle suspension introduction region and a discharge flow channel communicating with the other end are provided. Are arranged linearly across the fine particle suspension introduction region, and the fine particle suspension introduction container has a shape in which the length of the fine particle suspension introduction direction (10) is longer than the length in the vertical direction. It is preferable that

  Here, the center of the introduction flow path and the center of the discharge flow path are arranged substantially linearly with respect to the introduction direction of the fine particle suspension from the introduction flow path to the discharge flow path. Means that the straight line connecting the center of the discharge flow path and the wall surfaces on both sides of the fine particle suspension introduction region are parallel to each other.

  In this way, the fine particle suspension can flow more efficiently in a short time along the introduction direction of the fine particle suspension from the introduction flow path to the discharge flow path, and evenly introduce and discharge the fine particles. It becomes possible.

  In order to uniformly fill the fine particles, it is assumed that the fine particle suspension is completely filled in the fine particle suspension introduction container before the fine particles settle. This is because if the fine particles settle on the bottom of the fine particle suspension introduction container before the fine particle suspension is completely filled in the fine particle suspension introduction container, the accumulation of fine particles occurs and the fine particles are uniformly distributed in the container. It is because it becomes difficult to introduce. Accordingly, the height H of the fine particle suspension introduction container and the linear velocity u when the suspension is fed are parameters for completing the filling of the fine particle suspension into the fine particle suspension introduction container before the fine particles settle. Need to be adjusted accordingly. In this regard, the case where the fine particles are assumed to be cells will be described in detail below as an example.

  For example, the cell suspension is temporarily introduced at a linear velocity of 1.8 mm / s into a fine particle suspension introduction container (corresponding to a cell fusion container in Example 2) used in Example 2 described later. At this time, the Reynolds number Re of the fluid flowing in the fine particle suspension introduction container is expressed by the following (formula 1). The Reynolds number is a dimensionless number (ratio of inertial force to viscous force) used to investigate the flow properties (turbulent flow, laminar flow). When the Reynolds number is sufficiently smaller than 1, the fluid is It can be regarded as a laminar flow.

Re = uLρ _m / η (Formula 1)
Further, the Stokes number S for the particles in the fluid in the fine particle suspension introduction container (corresponding to cells in Example 2) is expressed by the following (Equation 2). The Stokes number is a dimensionless number used for examining the followability to the flow of particles. When the Stokes number is sufficiently smaller than 1, the particles in the fluid follow the flow.

^{_{S = ρ p r 2 u /}} ηL ··· ( Equation 2)
Here, u is the linear velocity (about 1.8 mm / s), L is the representative length (here, cell diameter, about 10 μm), r is the cell diameter (about 10 μm), ρ _m is the medium density (water at room temperature) as density of about 1000 kg ^{/ m} 3), as the density of the [rho _p is cell density (mouse cancer cells, about 1160kg ^{/ m} 3), η is a viscosity coefficient (normal pressure, as viscosity of water at 25 ° C., about 0.9 mPa · s).

  The fluid in the fine particle suspension introduction container of Example 2 from (Equation 1) and (Equation 2) has a Reynolds number of about 0.022, which is sufficiently smaller than 1, and is a laminar flow, and the Stokes number is also about 0. Since the particles are sufficiently smaller than .026 and 1, the particles follow the flow. In particular, when the Reynolds number is sufficiently smaller than 1 and the Stokes number is sufficiently smaller than 1, the movement of the fine particles in the fluid follows the Stokes equation. That is, the fine particles in the fluid in the fine particle suspension introduction container of Example 2 can be regarded as following the Stokes equation.

Here, the Stokes equation is expressed by the following (Equation 3). Incidentally, the formula of Stokes, the sum of the buoyancy acting on the particles _{^{((4/3) πr 3 ρ m}} g) and the viscous drag force (6πηrV _t) is, the gravitational force acting on the particles ((4/3) πr ³ It expresses the [rho _{p g)} is equal state equation of balance.

_{^{(4/3) πr 3 ρ m g}} + 6πηrV t = (4/3) πr 3 ρ p g ··· ( Equation 3)
Here, r is the cell diameter (about 10 μm), ρ _m is the medium density (about 1000 kg / m ³ as the density of water at room temperature), and ρ _p is the cell density (about 1160 kg / m as the density of mouse cancer cells). ³ ), g is the acceleration of gravity (9.8 m / s ² ), η is the viscosity coefficient (approximately 0.9 mPa · s as the viscosity coefficient of water at normal pressure and 25 ° C.), and Vt is the cell sedimentation velocity (m / s).

  The cell sedimentation velocity Vt is calculated from the above Stokes equation, the height of the microparticle suspension introduction container is set to H, and the average cell sedimentation time T until the cells settle on the bottom surface (from the center of the height of the microparticle suspension introduction container) The equation for calculating the time until the cells settle to the bottom surface is expressed by the following (Equation 4).

T = (H / 2) / V _t = {18η (H / 2)} / {(2r) ² (ρ _p −ρ _m ) g} (Expression 4)
Therefore, it is necessary to complete the filling of the fine particle suspension within less than the average cell sedimentation time T shown in (Equation 4). Therefore, the height H of the fine particle suspension introduction container is used so that the fine particle suspension is completely filled in the fine particle suspension introduction container before the fine particles settle using the above (Formula 1) to (Formula 4). By appropriately adjusting the linear velocity u when the suspension is fed, fine particles can be more uniformly filled.

  In the fine particle suspension introduction container in the present invention, when the introduced fine particle suspension is hydrophilic, it is preferable that the periphery of the introduction port is hydrophobic.

  Here, the vicinity of the inlet of the fine particle suspension introduction container is hydrophobic means that the contact angle between the upper plate and the solution is 90 degrees or more when the solution is suspended from the introduction port. . Further, in order to make the introduction port hydrophobic, there is no particular limitation as long as it is a hydrophobic member and a chemically stable member. As shown in FIG. 5, a hydrophobic thin sheet such as a silicon rubber sheet (18) is preferably closely attached to the periphery of the inlet from above. Alternatively, there is no problem even if the periphery of the inlet of the upper plate is subjected to a hydrophobic treatment by silane coupling or the like.

  By doing so, the introduced hydrophilic fine particle suspension is quickly drawn into the hydrophilic fine particle suspension introduction container from the inlet of the hydrophobic fine particle suspension introduction container. The fine particle suspension introduction container can be efficiently introduced into the fine particle suspension introduction region without leaking and spreading around the inlet of the upper plate.

  When the fine particle suspension to be introduced is hydrophobic, the periphery of the introduction port is preferably hydrophilic, and the periphery of the introduction port of the fine particle suspension introduction container is preferably hydrophilized.

  In addition, the fine particle suspension introduction container according to the present invention has a horizontal confirmation mechanism for confirming that the fine particle suspension introduction container is horizontal and the level of the fine particle suspension introduction container based on the information detected by the horizontal confirmation mechanism. It is preferable to provide a horizontal adjustment means for maintaining.

  Here, the horizontal confirmation mechanism for confirming that the fine particle suspension introduction container is horizontal means that the fine particle suspension has no inclination in the fine particle suspension introduction container and has a corner in the fine particle suspension introduction container. This is a mechanism for measuring the level of the fine particle suspension introduction region in order to uniformly and uniformly introduce it, and specifically, the mechanism indicated by reference numeral 34 in FIG. Horizontal is, for example, relative to the normal plane of gravity.

  Further, the level adjusting means for keeping the level of the fine particle suspension introduction container is means for keeping the level of the fine particle suspension introduction container based on the information detected by the level confirmation mechanism. There are 35 means. This is for maintaining the level of the fine particle suspension introduction region by adjusting the level confirmed by the level confirmation mechanism.

  In general, if the horizontal suspension of the fine particle suspension introduction container is tilted without being maintained, the fine particles are biased in the inclined direction due to the influence of gravity, so that the fine particles are uniformly introduced into and discharged from the fine particle suspension introduction region. Is difficult. However, by maintaining the horizontality of the fine particle suspension introduction container, the fine particles spread uniformly in the horizontal direction, and the fine particles can be easily and uniformly introduced and discharged.

  As a specific mechanism for checking and adjusting the level, a commercially available level may be installed in the fine particle suspension introduction container, or the same function as a commercially available level as shown in FIG. You may incorporate the structure which has these in the fine particle suspension introduction container. For example, as shown in FIG. 5, a structure having the same function as a spirit level is provided with a space for enclosing a liquid containing bubbles in a specific part of a fine particle suspension introduction container, and screws or the like at four corners. By adjusting the length of the screw coming out from the fine particle suspension container so that the bubble comes to the center in the liquid of the level confirmation mechanism, it becomes possible to keep the level.

  The fine particle suspension introduction container in the present invention may be used as a cell fusion container for cell fusion. The cell fusion container in this case is a cell fusion container composed of a fine particle suspension introduction container, and a flat plate arranged facing the fine particle suspension introduction region is a pair of electrodes made of a conductive member, And a flat plate-like insulator having a plurality of fine holes penetrating in the direction of the electrodes arranged opposite to each other, the insulator being the fine particle suspension introduction region of any one of the electrodes It is the cell fusion container arrange | positioned on the electrode surface of the side.

  Moreover, since the fine particle suspension introduction container in the present invention may be used as a cell fusion container for performing cell fusion as described above, a cell fusion apparatus using the cell fusion container can be configured. The cell fusion device according to the present invention in this case is a cell fusion device comprising a power source for applying a voltage to the pair of electrodes of the cell fusion container.

  Here, the configuration of 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. The cell fusion device of the present invention comprises a cell fusion container (19) and a power source (20).

  As shown in FIG. 6, the cell fusion container secures the cell fusion region (21) by arranging the spacer (4) between the upper electrode (14) and the lower electrode (15), and the micropore (9). The insulator (22) formed with the 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 or the like on which a transparent conductive material such as tin) is formed may be used, but 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 used as an electrode. preferable.

  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 (20) is connected to the upper electrode and the lower electrode of the cell fusion container (19) via a conductive wire (30).

  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 (7) for introducing cells and an introduction port (5) communicating therewith, a discharge channel (8) for discharging cells, and a cell for introducing and discharging cells to and from the cell fusion container. A communicating outlet (6) is provided.

  In addition, fine holes (9) are formed in the insulator (22). The material of the insulator (22) 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.

  Here, the shape and size of the micropores are not particularly limited, but when the cell fusion device of the present invention is used, a higher fusion regeneration probability can be obtained 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 in the micropore (depending on the cell, about 1 μm to several tens of μm), or It is preferable that the diameter is in the range of about 1 to 2 times the diameter and the depth of the micropore is equal to or less 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. 8, when the diameter of the maximum circle inscribed in the planar shape of the micropore is larger than the diameter of the two cells fixed to the micropore, the first cell (31) and the second cell are inserted into the micropore. A plurality of cells (32) are contained, and the cell fusion in the one-to-one relationship between the first cell and the second cell becomes impossible, and the fusion regeneration probability is lowered. However, as shown in FIG. 9, 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 to the micropore, a pair of the first cell and the second cell 1 cell fusion is possible and the fusion regeneration probability is high. In addition, as shown in FIG. 10, 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. 11, 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. 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 addition, since the cell sorting apparatus of the present invention can perform more efficient cell sorting by fixing one cell 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 is inappropriate that the interval between micropores formed in an array is too narrow or too wide. When the interval between the micropores is too narrow, the probability that a plurality of cells are fixed in one micropore is increased, and as a result, the probability that a micropore that does not contain cells is increased. In addition, when the interval between the micropores is too wide, cells are left between the micropores, and the probability that micropores that do not contain cells are increased. Therefore, more specifically, 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.

  In addition, the shape of the fine holes 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 polygonal shape such as a triangular shape or a square shape, 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. 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 (4), the insulator (22), and the lower electrode (15) as shown in FIG. 7, each of them is bonded with an adhesive or heated in a pressurized state. 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 (21) shown in FIG. 7 can be formed.

According to the present invention, the following effects can be obtained.
(1) According to the fine particle suspension introduction container of the present invention, the flow velocity at the center in the introduction direction of the fine particle suspension from the introduction flow path to the previous discharge flow path is substantially equal to the flow velocity of the wall surfaces on both sides. The introduction angle is not less than 0 degrees and not more than 60 degrees, and the discharge angle is not less than 0 degrees and not more than 60 degrees, so that the particles can be uniformly and evenly introduced into the fine particle suspension introduction container. It is possible to discharge efficiently without any leftovers.
(2) According to the fine particle suspension introduction container of the present invention, the introduction channel and the discharge channel communicate with the fine particle suspension introduction region in a substantially curved shape, so that the wall surfaces on both sides in the introduction direction in the introduction. The fine particle suspension in the vicinity advances more smoothly, the flow velocity at the center and the flow velocity of the wall surfaces on both sides become more equal, and there is no air bubbles remaining, making it easier to evenly and uniformly introduce into the fine particle suspension introduction container. In addition, it is possible to efficiently discharge the particulate suspension without leaving any particulate suspension.
(3) According to the fine particle suspension introduction container of the present invention, the center of the introduction channel and the center of the discharge channel are arranged substantially linearly, and the fine particle suspension introduction region in the introduction direction Is longer than the length of the fine particle suspension introduction region in the direction perpendicular to the introduction direction, and further uniformly and uniformly introduced into the fine particle suspension introduction container and discharged. In this case, it is possible to efficiently discharge the particulate suspension without any remaining of the particulate suspension.
(4) The fine particle suspension introduction container of the present invention is a fine particle suspension introduction container, wherein the fine particle suspension introduction container has a curved shape in the shape of the fine particle suspension introduction region. In this way, it can be more easily and evenly introduced into the fine particle suspension introduction container evenly and evenly, and can be efficiently discharged without any remaining of the fine particle suspension in the discharge. Become.
(5) According to the fine particle suspension introduction container of the present invention, when the introduced fine particle suspension is hydrophilic, the fine particle suspension introduction container is characterized in that the periphery of the introduction port is hydrophobic; By doing so, it is possible to prevent the fine particle suspension solution introduced from the introduction port into the fine particle suspension introduction container from leaking out of the introduction port and efficiently introduce it.
(6) According to the fine particle suspension introduction container of the present invention, the fine particle suspension introduction container includes a fine particle having a level confirmation mechanism and a horizontal adjustment means for keeping the fine particle suspension introduction container horizontal. By using the suspension introduction container, there is no inclination in the fine particle suspension introduction container, and it becomes possible to uniformly and uniformly introduce every corner of the fine particle suspension introduction container.
(7) In the cell fusion container and the cell fusion device, which are composed of the fine particle suspension introduction container of the present invention, and the fine particles are cells, the flat plate disposed facing the fine particle suspension introduction region is composed of a conductive member. A pair of electrodes, and a flat insulator having a plurality of fine holes penetrating in the direction of the electrodes arranged to face each other, the insulator being one of the electrodes A cell fusion device comprising: a cell fusion container disposed on the electrode surface on the fine particle suspension introduction region side; and a power source for applying a voltage to the pair of electrodes of the cell fusion container. In this way, the cell suspension is uniformly and evenly introduced into the cell fusion container, and the cell suspension is efficiently discharged without any leftover of the cell suspension. Can be done It made.

It is a 1st conceptual diagram which shows the basic particulate suspension solution introduction container in this invention. It is a conceptual diagram of the basic fine particle suspension solution introduction method in the present invention. It is a 1st conceptual diagram which shows the basic fine particle suspension solution introduction | transduction area | region in this invention. It is a 2nd conceptual diagram which shows the basic particulate suspension solution introduction area | region in this invention. It is a 2nd conceptual diagram which shows the basic particulate suspension solution introduction container in this invention. It is the conceptual diagram of the cell fusion apparatus in this invention, and the conceptual diagram of the cell fusion apparatus used for Example 2. FIG. FIG. 7 is a cross-sectional view along AA ′ shown in FIG. 6. As an example of the micropore used in the present invention, it is a diagram showing a case where the diameter of the maximum circle inscribed in the planar shape of the micropore is larger than twice the diameter of the cell fixed in the micropore. As an example of the micropore used in the present invention, it is a diagram showing a case where the diameter of the maximum circle inscribed in the planar shape of the micropore is smaller than the diameter of cells fixed in the micropore. As an example of the micropore used in the present invention, the diameter of the maximum circle inscribed in the planar shape of the micropore is about 1 to 2 times larger than the cell fixed to the micropore and the depth of the micropore is fixed to the micropore. It is the figure which showed the case where it is larger than the diameter of 1 cell. As an example of the micropore used in the present invention, the diameter of the maximum circle inscribed in the planar shape of the micropore is about 1 to 2 times larger than the cell fixed to the micropore and the depth of the micropore is fixed to the micropore. It is the figure which showed the case where it is below the diameter of 1 cell. It is the schematic which shows the general photolithography and the etching method for manufacturing the insulator integrated lower electrode with a fine hole used in the present Example. It is a figure which shows the 1st example of the fine particle suspension solution introduction container of this invention. 5 is a view showing a fine particle suspension solution introduction container in Comparative Example 1. FIG. It is a figure which shows the cell fusion apparatus in the comparative example 2.

  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 1
FIG. 5 shows a conceptual diagram of the fine particle suspension introduction container used in Example 1. For the upper and lower plates, Pyrex (registered trademark) substrates of 125 mm long × 125 mm wide × 1 mm thick were used. As a spacer, a hexagonal fine particle suspension introduction region having a center of 50 mm in length and 100 mm in width as shown in FIG. 5 was used in a silicon sheet having a length of 120 mm × width of 120 mm × thickness of 1.5 mm. Here, in order to introduce and discharge the fine particle suspension, the fine particle suspension introduction region introduces and discharges the fine particle suspension, an introduction port communicating therewith, a discharge flow path for discharging the fine particle suspension, and A discharge port communicating therewith, an introduction angle of the introduction channel extending from the introduction port is 60 degrees, and an angle of the discharge channel extending from the discharge port is 60 degrees, and the introduction channel and the discharge channel Produced a fine particle suspension introduction container which communicated with the fine particle suspension introduction region in a substantially curved line. In addition, in order to make the introduction port hydrophobic, a silicon rubber sheet (length 125 mm × width 30 mm × thickness 0.5 mm) having a hole of almost the same size as the introduction port at the same position as the introduction port, It is in close contact with the periphery of the inlet from above. In addition, a space as a horizontal confirmation mechanism (34) that can enclose a liquid containing bubbles and check the inclination of the fine particle suspension introduction container according to the position of the bubbles in the liquid, at a predetermined position of the fine particle suspension introduction container, Further, horizontal adjustment means (35) capable of adjusting the inclination of the fine particle suspension introducing container so that bubbles are formed in the center of the liquid are provided at the four corners of the fine particle suspension introducing container, so that the fine particle suspension introducing container is not inclined. Similarly, the height of the fine particle suspension introduction container was adjusted.

  Here, when the fine particle suspension was introduced from the inlet of the fine particle suspension introduction container using a 10 mL capacity dispenser, it could be uniformly and uniformly introduced into the fine particle suspension introduction container and discharged. In this case, it was possible to efficiently discharge the particles without leaving any fine particle suspension.

(Example 2)
A conceptual diagram of the cell fusion device used in Example 2 is shown in FIG. The cell fusion device is roughly divided into a cell fusion container (19) and a power source (20). As shown in FIG. 6, the cell fusion container includes an insulator (22) in which a spacer (4) 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. As a spacer, a hexagonal cell suspension introduction region having a center of 17 mm in length and 33 mm in width was hollowed out on a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm as shown in FIG. Here, since the cell suspension introduction region introduces and discharges the cell suspension, an introduction channel for introducing the cell suspension and an introduction port communicating therewith, a discharge channel for discharging the cell suspension, and A discharge port communicating therewith, an introduction angle of the introduction channel extending from the introduction port is 60 degrees, and an angle of the discharge channel extending from the discharge port is 60 degrees, and the introduction channel and the discharge channel Produced a cell suspension introduction container which communicated with the cell suspension introduction region in a substantially curved shape. As in Example 1, in order to make the introduction port hydrophobic, a silicon rubber sheet (length 125 mm × width 30 mm × thickness 0. 0 mm) having a hole of the same size as the introduction port at the same position as the introduction port. 5 mm) is in close contact with the periphery of the inlet from the top of the upper plate.

  Further, the insulator (22) having a plurality of fine holes was produced by integrally forming the lower electrode by a method using photolithography and etching 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, in a 20 mm long × 20 mm wide area, a micro hole pattern having a diameter of 8.5 μm is arranged in an array of 1000 vertical × 1000 horizontal, with the vertical and horizontal spacing of the fine holes being 20 μm. The resist was exposed (27) with a UV exposure machine using the exposure photomask (26), and developed with a developer (29). The exposure time and development time were adjusted so that the depth of the micropores was 2.5 μm, which is equal to the film thickness of the resist, so that the ITO was exposed on the bottom surfaces of the micropores. After development, the resist was hardened by post-baking (115 ° C., 30 minutes) using a hot plate.

  The upper electrode (14), the spacer (4), and the insulator-integrated lower electrode (28) with fine holes were laminated and pressure-bonded as shown in FIG. FIG. 7 is an AA ′ cross-sectional view of the cell fusion container shown in FIG. 6. 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. In addition, the number of micropores existing in the space of the cell fusion region where the spacer is hollowed out is about 700,000.

  In addition, the cell fusion device includes a horizontal confirmation mechanism and a horizontal adjustment mechanism for keeping the cell fusion container horizontal in the cell fusion container. In the same manner as in Example 1, the liquid as the horizontal confirmation mechanism (34) in which the liquid containing the bubbles is sealed and the inclination of the fine particle suspension introduction container can be confirmed by the position of the bubbles in the liquid is introduced into the fine particle suspension. Level adjustment means (35) capable of adjusting the inclination of the fine particle suspension introduction container at predetermined locations of the container and in the center of the liquid so that bubbles are formed at the four corners of the fine particle suspension introduction container are provided. The height of the fine particle suspension introduction container was adjusted so that the inclination of the liquid introduction container disappeared.

Experiments described below were performed using the cell fusion device configured with the above-described microporous integrated insulating film lower electrode. As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Both cells were suspended in a 300 mM mannitol aqueous solution containing BSA (1 mg / mL). Mouse antibody-producing cells were 1.7 × 10 ⁶ cells / mL, and mouse myeloma cells were 3.4 × 10 ⁶ cells / mL. The cell suspension was adjusted to a density of mL. 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.

  When 900 μL of the cell suspension of mouse antibody-producing cells (number of mouse antibody-producing cells: about 600,000) was injected using a 1 mL volume dispenser from the introduction port of the spacer, it was thoroughly poured into the cell fusion container. When the AC voltage of 10 Vpp and frequency of 3 MHz is applied between the electrodes by an AC power supply (NF1966, manufactured by NF circuit design block), it can be introduced evenly and uniformly, and a plurality of arrays formed in an extremely short time of about 2 to 3 seconds About 1 to 2 mouse antibody-producing cells could be fixed to each of the micropores, and 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 85%. Here, the cell fixation rate means that 225 fine pores of 15 vertical x 15 horizontal are visible in the field of view of the microscope, and the fine cells containing 1-2 cells when cells are introduced and fixed. It was defined as a value obtained by dividing the number of holes by the number of fine holes of 225. In addition, the cell fixation rate in the following comparative example 2 is also the same definition.

  Subsequently, 1 μL of 900 μL of the mouse myeloma cell suspension (number of mouse myeloma cells: about 600,000 cells) was applied from the inlet of the spacer while an AC voltage of 10 Vpp and a frequency of 3 MHz was applied between the electrodes. When injected using a volumetric dispenser, mouse myeloma cells can be uniformly and evenly introduced to every corner of the cell fusion container, and one of a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. About 1 to 2 mouse myeloma cells could be fixed each, and a plurality of cells could be arranged in an array. At this time, the probability of about 1 to 2 mouse myeloma cells entering one micropore is about 80%, and when the mouse myeloma cell is introduced, the previously introduced mouse antibody-producing cells are detached from the micropore. Since almost no separation was observed, it is estimated that the probability that the mouse antibody-producing cells and the mouse myeloma cells are in contact with each other in a micropore is about 68% (= 85% × 80%). Is done.

Next, a DC pulse power source (manufactured by Nepagene Co., Ltd., LF101) applies a DC pulse voltage of 80 V and a pulse width of 30 μs between the electrodes to perform cell fusion, and left for 15 minutes, and then disassembles the cell fusion container. The fused cells are taken out, and the cell suspension in the cell fusion container is placed in a HAT medium (H: hypoxanthine, A: aminopterine, T: thymidine). 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 3 million fused cells could be confirmed, and total mouse antibody production A fusion probability of about 5.00 / 10000 was obtained for 600,000 cells.

(Comparative Example 1)
FIG. 13 shows a conceptual diagram of the fine particle suspension introduction container used in Comparative Example 1. As the upper and lower plates, Pyrex (registered trademark) substrates having a length of 70 mm, a width of 40 mm, and a thickness of 1 mm were used. The spacer is a silicon sheet 40 mm long x 40 mm wide x 1.5 mm thick with a shape in which the fine particle suspension introduction region is hollowed, and the fine particle suspension is introduced and discharged there. An introduction channel for introducing the suspension and an introduction port communicating therewith, a discharge channel for discharging the fine particle suspension and a discharge port communicating therewith are provided, and the introduction angle at which the introduction channel spreads from the introduction port is 120 degrees And the angle at which the discharge channel extends from the discharge port is 120 degrees, and the fine particle suspension introduction container in which the introduction flow channel and the discharge flow channel communicate with the fine particle suspension introduction region in a substantially curved shape. Was made.

  Here, when the fine particle suspension (33) was introduced from the inlet of the fine particle suspension introduction container using a 1 mL capacity dispenser, the introduction of the fine particle suspension introduction container as shown in FIG. Bubbles remained at the corners and could not be uniformly and evenly introduced into every corner of the fine particle suspension introduction container.

(Comparative Example 2)
FIG. 15 shows a conceptual diagram of the cell fusion device used in Comparative Example 2. The cell fusion device is roughly divided into a cell fusion container (19) and a power source (20). As shown in FIG. 15, the cell fusion container has an insulator (22) in which a spacer (4) 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 a spacer, a hexagonal cell suspension introduction region having a center of 17 mm in length and 33 mm in width was hollowed out on a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm as shown in FIG. Here, since the cell suspension introduction region introduces and discharges the cell suspension, an introduction channel for introducing the cell suspension and an introduction port communicating therewith, a discharge channel for discharging the cell suspension, and A discharge port communicating therewith, an introduction angle of the introduction channel extending from the introduction port is 120 degrees, and an angle of the discharge channel extending from the discharge port is 120 degrees, and the introduction channel and the discharge channel Produced a cell suspension introduction container which communicated with the cell suspension introduction region in a substantially curved shape. In addition, the number of micropores existing in the space of the cell fusion region where the spacer is hollowed out is about 700,000.

  The micropores, the upper electrode, and the lower electrode were produced in the same manner as in Example 2 to produce a cell fusion device.

Experiments described below were performed using the cell fusion device configured with the above-described microporous integrated insulating film lower electrode. As the cells, mouse antibody-producing cells (φ5 μm) and mouse myeloma cells (φ10 μm) were used. Both cells were suspended in a 300 mM mannitol aqueous solution containing BSA (1 mg / mL). Mouse antibody-producing cells were 1.7 × 10 ⁶ cells / mL, and mouse myeloma cells were 3.4 × 10 ⁶ cells / mL. The cell suspension was adjusted to a density of mL. 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.

  When 900 μL of the cell suspension of mouse antibody-producing cells (number of mouse antibody-producing cells: about 600,000) was injected from the introduction port of the spacer using a 1 mL volume dispenser, cell fusion was performed as shown in FIG. Bubbles remained at the corners of the container, and could not be uniformly and evenly introduced into every corner of the cell fusion container. When an AC voltage of 10 Vpp and a frequency of 3 MHz was applied between the electrodes by an AC power supply (manufactured by NF Circuit Design Block, WF1966), a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. One to two mouse antibody-producing cells could be fixed one by one, and 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 70%.

  Subsequently, 1 μL of 900 μL of the mouse myeloma cell suspension (number of mouse myeloma cells: about 600,000 cells) was applied from the inlet of the spacer while an AC voltage of 10 Vpp and a frequency of 3 MHz was applied between the electrodes. When injected using a volumetric dispenser, mouse myeloma cells could not be uniformly and evenly introduced to every corner of the cell fusion container. Here, about 1 to 2 mouse myeloma cells can be fixed to each of a plurality of micropores formed in an array in an extremely short time of about 2 to 3 seconds. I was able to arrange it. At this time, the probability of about 1 to 2 mouse myeloma cells entering one micropore is about 60%, and when the mouse myeloma cells are introduced, the previously put mouse antibody-producing cells escape from the micropores. Since almost no separation was observed, it was estimated that the probability that the mouse antibody-producing cells and the mouse myeloma cells are in contact with each other in a micropore is about 42% (= 70% × 60%). Is done.

Next, as in Example 2, cell fusion was performed by applying a DC pulse voltage of 80 V and a pulse width of 30 μs between the electrodes with a DC pulse power supply (LF101, manufactured by Nepagene Corporation), and then allowed to stand for 15 minutes. The cell fusion container was disassembled, the fused cells were taken out, the cell suspension in the cell fusion container was placed in a HAT medium, and the fused cells were cultured. 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 3 million fused cells could be confirmed, and total mouse antibody production A fusion probability of about 3.1 / 10,000 was obtained for 600,000 cells.

1: Fine particle suspension introduction area 2: Upper plate 3: Lower plate 4: Spacer 5: Introduction port 6: Discharge port 7: Introduction channel 8: Discharge channel 9: Fine hole 10: Introduction direction 11: Introduction direction Central flow velocity 12: Flow velocity near the wall 13: Fine particle suspension solution introduction container 14: Upper electrode 15: Lower electrode 16: Introduction angle 17: Discharge angle 18: Hydrophobic sheet 19: Cell fusion container 20: Power supply 21: Cell fusion Region 22: Insulator 23: ITO
24: Pyrex (registered trademark) glass 25: Resist 26: Photomask for exposure 27: Exposure 28: Insulator-integrated lower electrode with fine holes 29: Developer 30: Conductive wire 31: First cell 32: Second Cell 33: Fine particle suspension 34: Horizontal confirmation mechanism 35: Horizontal adjustment means

Claims (8)

A fine particle suspension introduction container comprising a pair of flat plates arranged opposite to the fine particle suspension introduction region, and a flat spacer formed so as to penetrate the fine particle suspension introduction region between the pair of flat plates. The spacer includes an inlet for introducing the fine particle suspension and an introduction flow path communicating therewith, and a discharge opening for discharging the fine particle suspension and a discharge flow path communicating therewith, the fine particle suspension A fine particle suspension introduction container in which the introduction flow path is disposed in communication with one end of the introduction region and the discharge flow path is disposed in communication with the other end of the fine particle suspension introduction region; A fine particle suspension introduction having a shape in which the flow velocity at the center in the introduction direction of the fine particle suspension from the introduction flow channel toward the discharge flow channel and the flow velocity of the wall surfaces on both sides are substantially equal. container. In the fine particle suspension introduction container, the introduction angle at which the introduction channel extends from the introduction port is 0 ° to 60 °, and the discharge angle at which the discharge channel extends from the discharge port is 0 ° to 60 °. 2. The fine particle suspension introduction container according to claim 1, wherein the introduction flow channel and the discharge flow channel communicate with the fine particle suspension introduction region. The introduction flow channel communicating with one end of the fine particle suspension introduction region and the discharge flow channel communicating with the other end are arranged linearly across the fine particle suspension introduction region, 3. The fine particle suspension introduction container according to claim 1, wherein the fine particle suspension introduction container has a shape in which the introduction direction length of the fine particle suspension is longer than the vertical length. The fine particle suspension introduction container according to any one of claims 1 to 3, wherein the fine particle suspension introduction region has a curved shape. The fine particle suspension introduction container according to any one of claims 1 to 4, wherein a periphery of the introduction port is hydrophobic in the fine particle suspension introduction container. In the fine particle suspension introduction container, a horizontal confirmation mechanism for confirming that the fine particle suspension introduction container is horizontal, and a horizontal adjustment for keeping the fine particle suspension introduction container horizontal based on information detected by the horizontal confirmation mechanism. The fine particle suspension introduction container according to any one of claims 1 to 5, further comprising means. A cell fusion container comprising the fine particle suspension introduction container according to any one of claims 1 to 6, wherein the fine particles are cells, and a flat plate disposed opposite to the fine particle suspension introduction region is electrically conductive. A pair of electrodes made of a member, and including a flat insulator having a plurality of fine holes penetrating in the direction of the electrodes arranged to face each other, and the insulator is one of the electrodes A cell fusion container disposed on the electrode surface of the electrode in the microparticle suspension introduction region side. A cell fusion device comprising the cell fusion container according to claim 7, further comprising a power source for applying a voltage to the pair of electrodes of the cell fusion container.

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