JPH0336508B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method and apparatus for assembling a plurality of polarizable microparticles, particularly cells, in contact with each other in a non-uniform electric field.

Technical Background Generally, when living cells are placed in an electric field, an electric double layer is formed in the cell membrane, that is, the cell membrane becomes polarized. Conventionally, such polarizable microparticles are placed in a nonuniform electric field and moved toward a pole point where the electric flux density or electric displacement is maximum, that is, dielectrophoresed, and the polarizable microparticles are brought into contact with each other at the pole point. It is known that it aggregates in

As a specific example of using the above assembly method, an electrical cell fusion method using dielectrophoresis is known. That is, when cells are suspended in a non-electrolyte solution such as a mannitol solution and this suspension is placed in a non-uniform electric field formed, for example, between a flat electrode and a rod electrode or between rod electrodes, the Each cell in the solution undergoes dielectrophoresis and is connected in a beaded manner at the tip and surface of the rod-shaped electrode where the electric displacement in the non-uniform electric field is maximum, and then the cells in contact are stimulated with an appropriate electric pulse to separate the cells. Fusion is accomplished.

However, in the conventional method described above, the polarizable fine particles to be brought into contact are brought into direct contact with the electrode surface, which results in the electrode surface being contaminated and maintaining the electric field strength constant. Therefore, it was difficult to perform the collective operation continuously and stably. In particular, when performing cell aggregation operations such as cell fusion, there is a great risk of not only contaminating the electrode surface but also damaging cells that come into direct contact with the electrode surface. Furthermore, the combinations of cell pairs brought into contact with each other are irregular; for example, when performing cell fusion of heterogeneous cells A and B, the combination of cells arranged near the electrode surface is A-A. , B-B, A-B, etc. appear irregularly, making it difficult to obtain only the required cell pair A-B. Therefore, for example, a microscope or the like is used to select the required cell pair A-B. The necessity of such an extra sorting step made the overall fusion operation less efficient.

Problems to be Solved This invention was made to solve the various problems mentioned above, and in principle, electric displacement or electric flux density is maximized at the intermediate portion between a pair of electrodes for generating an electric field. While forming a maximum point where It is an object of the present invention to provide a method for assembling polarizable fine particles by which a desired combination of polarizable fine particles can be obtained at distant pole points.

Furthermore, the present invention provides a conductive path with an extremely small cross-sectional area at the intermediate portion between the electrodes by interposing an electrical insulator between a pair of flat electrodes for generating an electric field installed in an electrically insulating container. This forms a local maximum point at which the electric displacement or electric flux density is maximum in the narrow part of the conductive path, and at the same time, a polarizable fine particle is provided at opposite side positions of the narrow part of the conductive path. It is an object of the present invention to provide a collecting device for polarizable fine particles having an inlet portion formed therein.

Although this invention is very useful especially for the fusion of different types of cells, it is not limited to this, but it is possible to encapsulate a necessary substance such as DNA (deoxyribonucleic acid) in a microcapsule, and combine the microcapsule with a cell. It is useful for the introduction of a substance by the so-called capsule method, in which the above substances are brought into contact with each other in a solution, and for the introduction of a DNA-vector into cells in a solution.

In this specification, cells include not only complete cells but also cells that have been subjected to a predetermined treatment for the purpose of cell fusion, such as protoplasts, unless there is a contradiction from the surrounding text.

In addition, the term "cell fusion" is used for convenience to refer to the fusion of cells and, for example, microcapsules encapsulating DNA.
Furthermore, it also refers to the introduction of a DNA-vector into cells. The microcapsules may be either artificial or natural.

Embodiments The present invention will be described with reference to the accompanying drawings showing embodiments. Note that this example is applied to the assembly and fusion of plant protoplasts.

1 and 2, reference numeral 1 denotes a frame integrally molded from acrylic resin into a substantially rectangular plate shape. Symmetrical four-sided truncated pyramid-shaped protrusions 2-1 on two opposing long side walls of the frame 1,
2-2 are formed to face each other.
The tapered surfaces 3, 3 and the tip surfaces 4 of both the protrusions 2-1, 2-2 form a tapered conduction path 5 whose cross-sectional area is minimum at the center of the frame 1. In addition,
The protrusions 2-1 and 2-2 may be separate from the frame 1. In addition, these protrusions 2-1, 2-
2 is not limited to acrylic resin, but is made of other synthetic plastic materials such as highly electrically insulating Teflon (trade name of DuPont, USA), Mylar (trade name of DuPont, USA), ceramics, mica, etc. It's okay.

An electrically insulating transparent glass plate 8 is adhered to the bottom surface 1-1 of the frame 1, while a lid 9 made of an electrically insulating material is attached to the top surface 1-2. In this way, the side walls of the frame 1, the transparent glass plate 8 and the lid 9 create two symmetrical sealed chambers 6-
1, 6-2 are formed. Double sealed chamber 6
-1 and 6-2 are connected via a conductive path 5 that converges at the center of the frame 1. A portion of the conductive path 5 where the cross-sectional area is minimal is referred to as a narrow portion 7. As shown in FIGS. 1 and 2, the length 1, width d, and height h of this narrow portion 7 are set appropriately depending on the cells to be aggregated.

Flat plate electrodes 11-1, 11-2 are installed in the sealed chambers 6-1, 6-2 in parallel and facing each other. Both plate electrodes 11-1 and 11-2 are both made of platinum (Pt), and have the same planar shape and area. Note that as the electrode material, for example, stainless steel, silver-silver chloride, or a liquid junction electrode such as Azabritsu may be used. And both flat plate electrodes 11-1, 1
1-2 is connected to an alternating current (AC) power supply circuit 13 for electric field generation, as will be described in detail later, and when an AC voltage is applied from the AC power supply circuit 13, the plate electrode 11
Chamber 6-1, 6- between -1, 11-2
2 and the internal region of the conductive path 5.
This electric field is applied to the frame 1, the transparent glass plate 8 and the lid 9.
The chambers 6-1, 6-2 and the conductive path 5
As shown in FIG. 3, electric flux density or electricity The displacement becomes maximum. In this way, the sealed chamber 6-
A non-uniform electric field is formed in the internal region of the conductive path 5 and the conductive path 5.

Cell loading ports 21-1 and 21-2 are provided in the side walls of the sealed chambers 6-1 and 6-2, respectively. Conduits 22-1 and 22-2 are connected to each charging port 21-1 and 21-2, respectively, and a valve 23-2 is connected by a pump (not shown).
The cell suspension is injected through 1 and 23-2. Further, a fused cell outlet 25 is provided on the distal end surface of one of the protrusions forming the narrow portion 7 of the conductive path 5, for example 2-1. A conduit 26 is connected to this outlet 25, and the fused cells are taken out via a valve 27 by a pump (not shown). The cells to be fused are preferably treated with mannitol, sorbitol, glucose, sucrose, etc. for adjusting osmotic pressure and specific gravity, and calcium chloride (CaCl ₂ ), etc. as a cell stability and conductivity adjusting agent.
It is suspended in an electrolyte solution containing a pH buffer salt and the like, and introduced into the above-mentioned non-uniform electric field in a suspension state.

On the other hand, the above-mentioned opposing flat plate electrodes 11-1, 11-2
is connected to an alternating current (AC) power supply circuit 13 via a lead wire 12 and a switch means 14, and to a pulse generator 15 via a lead wire 12 and a switch means 16. Both switch means 14 and 16 are electronic switches made of, for example, transistors, and are controlled to be turned on and off in accordance with a predetermined time chart set in the control circuit 17 by a known method. With this configuration, a frequency of approximately 10K is supplied from the AC power supply circuit 13 to the flat plate electrodes 11-1 and 11-2 via the switch means 14.
Hz~500KHz, peak voltage about 5V~2000V (volts)
A sine wave alternating voltage of is applied for about 1 second to 600 seconds,
As described above, a non-uniform electric field is formed in the internal regions of the chambers 6-1, 6-2 and the conductive path 5 so that the electric displacement is maximized at the narrow portion 7 of the conductive path 5. Note that the output waveform of the AC power supply circuit 13 is not limited to a sine wave, but may be a triangular wave, a square wave, a sawtooth wave, or the like. In addition, the narrow part 7
After the cells have assembled in contact with each other, the pulse generator 1
5 to the flat plate electrode 11- via the switch means 16.
1, 11-2 pulse width approximately 1.0μsec (microseconds)
~10msec (milliseconds), peak voltage approximately 10V (volts)
~5KV (kilovolt) rectangular pulse approximately 1m
1 to 1 at intervals of sec (milliseconds) to several msec (several milliseconds)
Added several times. In this way, the narrow part 7
Stimulation by electrical pulses is applied to the assembled cell pairs, and cell fusion is achieved through a known growth process. Note that the output pulses of the pulse generator 15 may be superimposed on the output voltage of the AC power supply circuit 13 and applied to the plate electrodes 11-1 and 11-2. Thereby, the time required for cell fusion processing can be shortened.

Next, using the apparatus with the above configuration, the heterologous cells A,
The operation for merging B will be explained.

First, open the valve 23-1 and open the charging port 21-1.
Inject a suspension of cells A into the chamber 6-1 from above, open the valve 23-2, and suspend cells B similar to the above suspension into the chamber 6-2 from the charging port 21-2. Inject the suspension.

Next, the control circuit 17 closes the switch means 14 in a known manner, and the flat electrodes 11-1, 11 are connected from the AC power supply circuit 13 through the switch means 14.
For example, an alternating voltage such as a sine wave, a triangular wave, or a square wave with a frequency of about 100 KHz (kilohertz) and a peak voltage of about 40 V to 80 V is applied between -2. In this case, the interelectrode distance was 2 millimeters (mm). A region partitioned by both flat plate electrodes 11-1, 11-2, frame 1 and projections 2-1, 2-2,
That is, a non-uniform electric field is formed in the internal regions of the sealed chambers 6-1, 6-2 and the conductive path 5, which maximizes the electrical displacement in the narrow portion 7 of the conductive path 5.
Hereinafter, the portion where the electrical displacement is maximum in this narrow portion 7 will be referred to as a local maximum point. The distance between the flat electrodes, the dimensions of the flat electrodes, and the dimensions of the conductive path of the above device were as follows. : Distance between flat electrodes: 2.0 mm, flat electrode dimensions (width x height): 0.8 mm x 0.5 mm, conduction path dimensions (d x h x l): (0.1 to 0.25 mm) x (0.5
mm)×(0.01mm) When the above-mentioned non-uniform electric field acts on the cells A and B in the chambers 6-1 and 6-2 and the conduction path 5, an electric double layer is induced in the cell membrane portion of each cell A and B, That is, it is polarized. These polarized cells A, B
is the narrow part 7 based on the energy of the non-uniform electric field.
It moves toward the maximum point of , so-called dielectrophoresis. FIG. 3 shows a simulated state of dielectrophoresis at a certain instant. That is, cell A in chamber 6-1
moves toward the maximum point from the flat electrode 11-1 side, while cell B in the chamber 6-2 moves toward the maximum point from the flat electrode 11-2 side in the opposite direction to the moving direction of the cell A. do. Cells A and B that are close to each other are attracted to each other based on the Coulomb force that acts between the polarized charges of different polarities in each cell.
It becomes a contact state. This state can be observed through a microscope (not shown) installed on the transparent glass plate 8 side of the apparatus.

In addition, in the narrow part 7, three or more cells A and B are arranged in a rosary shape, for example, a cell pair A-
When A-B-B, A-A-A-B, A-B-B, etc. occur, one cell A and one cell It is possible to obtain a cell pair A-B consisting of cells B and B.

Next, the switch means 16 is closed, and a peak voltage of approximately 50 V to 50 V is applied from the pulse generator 15 to the flat electrodes 11-1 and 11-2 via the switch means 16.
A rectangular pulse of 200 V and a pulse width of about 20 msec to 200 μsec is applied once to several times at intervals of 1 second to several seconds. This allows the conduction path 5 to pass through the cell suspension solution.
Electrical pulse stimulation is applied to the cell pair A-B gathered in the narrow part 7 of the cell, and these cell pairs A-B fuse, respectively.

Next, the valve 27 is opened, and the conduits 22-1 and 22-2 are pumped by a pump (not shown), respectively.
Transfer the suspension of cells A and B through chamber 6-
1,6-2. This completes one cycle of the cell fusion operation.

Thereafter, as described above, the operations of generating a nonuniform electric field, gathering cells A and B, applying electrical stimulation to the cell pair A-B, taking out the fused cells, and introducing new cells A and B are repeated.

Note that three or more cells can also be aggregated and fused in the same manner as described above.

Next, some modifications of the above device are shown in FIGS.
This will be explained with reference to FIG. In these drawings, the same reference numerals are given to the same parts as those in the apparatus shown in FIGS. 1 and 2, and the explanation thereof will be omitted.

The device shown in FIG. 4 uses an electrically insulating plate 30 having a plurality of through holes 31 instead of the protrusions 2-1 and 2-2.
is installed between flat plate electrodes 11-1 and 11-2 for generating an equal electric field. Each through hole 31
As with the conductive path 5 formed by the projections 2-1 and 2-2, a tapered surface is formed in which the cross-sectional area becomes minimum or extremely small at the center. In this way, when a voltage is applied between both flat plate electrodes 11-1 and 11-2, the electrical displacement becomes maximum at the narrow portion 32 of each through hole 31. Moreover, both flat plate electrodes 11-1, 1
The area between the electrically insulating plate 30 and the electrically insulating plate 30 is used as chambers 6-1 and 6-2. With this configuration, cell fusion of different types of cells can be performed efficiently.

The electrical insulating plate 30 and the flat electrode 11-
1 and 11-2 may be arranged horizontally as shown in FIG. As a result, the weight of the fused cells can be effectively utilized to lower the chamber 6-1.
The chamber 6-1 can also be used as a conduit for removing fused cells.

Further, as shown in FIGS. 6 and 7, the lower end 34 of the electrically insulating plate 33 is formed into a convex shape, and the lower end 36 and the transparent glass plate 8 on the bottom of the device form a central part. The conductive path 35 may be formed to have a minimum or extremely small cross-sectional area. This electrically insulating plate 33 can be adjusted to move up or down as indicated by the white arrow, depending on the size of the cells to be fused. This makes it possible to generate a plurality of linear cell groups that are aligned horizontally and in a single layer in the narrow portion 37 in the upward and downward directions along the surface of FIG. 7, and are in contact with each other.

Furthermore, in the apparatus shown in FIG. 8, an electrically insulating plate 38 having a through hole 39 is installed in place of the transparent glass plate 8, and an electrode plate is installed so as to close the lower opening of the through hole 39. 41 will be installed. The upper opening 40 of the elongated through hole 39 has a shape similar to the convex portion of the lower end portion 34 of the electrically insulating plate 33, and the convex portion of the lower end portion 34 is inserted into the opening 40 to form the elongated through hole. 39 is sealed, and this sealed part is used as a conduit for cell removal. A non-uniform electric field for cell aggregation is formed by applying a voltage between the opposing flat electrodes 11-1 and 11-2 in the same manner as in the apparatus shown in FIGS. A voltage is applied between the flat electrodes 11-1 and 11-2 and the electrode plate 41.

In addition, in the embodiment described above, the chamber 6-
1 and 6-2 are preferably sealed to facilitate solution loading, but may be open without a lid.

In the embodiment described above, a DC voltage is applied instead of an AC voltage between opposing electrodes to form a non-uniform electric field, and a vector having a negative charge is charged into a chamber on the negative flat plate electrode side, while a vector with a negative charge is charged in the chamber on the negative flat plate electrode side. Cells are loaded into the chamber on the electrode side, and the cells are placed in the narrow part 7 of the conduction path 5.
On the other hand, a negatively charged vector is electrophoresed toward the narrow part 7 based on a DC electric field. In this way, the cell and the vector can be brought into contact with each other in the narrow region 7, and the vector can be introduced into the cell.

Further, microcapsules containing cells and a predetermined substance such as DNA are charged into chambers 6-1 and 6-2, respectively, and the cells and microcapsules are fused together in the same manner as in the cell fusion of different types described above. A predetermined substance can also be introduced into the cell by bringing them into contact and then fusing the two.

Effects of the Invention According to the present invention, a maximum point where the electric displacement or electric flux density is maximum or maximum is formed at the intermediate position of the region between opposing electrodes, and the portion charged from the side position opposite to the maximum point is formed. Since the polar fine particles are dielectrophoresed based on the non-uniform electric field and brought into contact with each other at the maximum point, the polarized fine particles being dielectrophoresed do not come into direct contact with the electrode as in the conventional method. Therefore, a stable non-uniform electric field can be formed without contaminating the electrodes, and a more stable collective operation can be performed. In particular, when the polarizable fine particles are cells, it is possible not only to prevent the electrode from being contaminated but also to effectively prevent damage to the cells themselves.

In addition, since different types of polarizable particles are gathered from opposite side positions of their maximum points toward the maximum point, it is possible to obtain a predetermined combination of polarizable particles that are in contact with each other. be able to.
In particular, when used to fuse two different types of cells, if cells of each type are introduced into the nonuniform electric field from opposite lateral positions of the maximum point, only pairs of cells of different types can be combined. Therefore, since no sorting work is required, fusion of heterologous cells can be achieved with high efficiency.

Note that the present invention is not limited to cell fusion, but also applies to other aggregation operations of polarizable fine particles, such as introduction of a predetermined substance into cells by microcapsule method, DNA-
If used for vector introduction, etc., the introduction operation can be achieved with extremely high efficiency.

[Brief explanation of the drawing]

FIG. 1 is a partial sectional view showing the configuration of an apparatus according to an embodiment of the present invention, FIG. 2 is a sectional view taken along the line -- in FIG. 1, and FIG. 3 is an explanatory diagram of the non-uniform electric field formed in the above device , FIG. 4 is a partial sectional view of a modified example of the present invention, FIG. 5 is a sectional view of a main part of another modified example of the present invention, and FIG. 6 is a partially cutaway partial sectional view of yet another modified example. 7 is a sectional view taken along the line -- in FIG. 6, and FIG. 8 is a sectional view of a main part of yet another modification. 1... Frame made of electrical insulating material, 2-1, 2-2...
Projection, 3... Tapered surface (slanted surface), 5... Conduction path, 6-1, 6-2... Chamber, 7... Narrow portion, 8... Transparent glass plate, 9... Lid, 11 −
1, 11-2... Flat plate electrode, 12... Lead wire,
13... AC power supply circuit, 14... switch means,
15...DC pulse generator, 16...Switch means, 21-1, 21-2...Charging port section, 22-
1,22-2,26... conduit, 23-1,
23-2, 27...Valve, 25...Fused cell extraction port, 30...Electrical insulating plate, 31...Through hole, 32...Narrow portion, 33...Electrical insulating plate, 3
4... Convex lower end portion, 35... Conduction path, 37...
Narrow part, 39...through elongated hole, 40...opening.

Claims (1)

[Scope of Claims] 1. In dielectrophoresing a plurality of polarizable fine particles in a non-uniform electric field and bringing them together in contact with each other at a position where the electric displacement in the non-uniform electric field is maximum, a counter electrode for generating an electric field is provided. A non-uniform electric field having a maximum point of electric displacement is generated at an intermediate position in the intervening region, and polarizable fine particles are charged into the non-uniform electric field from a position intermediate between the maximum point and the above-mentioned two electrodes. Collection method. 2. Claim 1, in which different types of polarizable fine particles are charged into the non-uniform electric field from an intermediate position facing each other between the local maximum point and the counter electrode for electric field generation.
Collection method described in Section. 3. The assembly method according to claim 1 or 2, wherein an electrolyte solution is charged between opposing electrodes, and different types of polarizable fine particles are suspended in the electrolyte solution. 4. The aggregation method according to claim 3, wherein the different types of polarizable fine particles are two different types of cells. 5. The aggregation method according to claim 4, wherein an electric pulse is applied to a plurality of cells brought into contact with each other by aggregation in a non-uniform electric field to cause cell fusion. 6 Claim 3, wherein the electrolyte solution contains a cell fusion inducing agent and the different types of polarizable fine particles are cells.
Collection method described in Section. 7. The aggregation method according to claim 3, wherein the different types of polarizable fine particles are microcapsules containing cells and a predetermined substance. 8. The assembly method according to claim 3, wherein the different types of polarizable microparticles are cells and DNA-vectors. 9. In an apparatus for dielectrophoresing a plurality of polarizable fine particles in a non-uniform electric field and bringing them together in contact with each other at a position where the electric displacement in the non-uniform electric field is maximum, an electrically insulating container; flat electrodes for electric field generation that are opposed in parallel with a predetermined interval; a power supply circuit connected to the flat electrodes; and an electric field forming space interposed between the flat electrodes; an electrical insulator forming a conductive path with a minimum cross-sectional area at an intermediate position between the conductive paths, and a plurality of polarizable fine particle charging ports each communicating with the conductive path at positions on opposite sides of the narrow portion of the conductive path. An apparatus comprising: a non-uniform electric field in which electric displacement is maximized in a narrow portion of the conductive path by applying a predetermined voltage between the flat electrodes from the power supply circuit. 10 Claim 9, wherein the electrical insulator is a plate-shaped body, and at least one through portion is provided with a tapered surface whose opening cross-sectional area gradually decreases inward from both sides of the plate-shaped body. The device described in. 11. Claims in which the conductive path is formed by a convex inclined or circular curved surface on one end surface of an electrically insulating plate and one surface of another electrically insulating plate facing the surface Apparatus according to paragraph 9. 12 The power supply circuit is connected between the opposing flat electrodes at approximately 5KHz~
12. The apparatus according to claim 9, which outputs an alternating voltage that generates an electric field of 10 MHz and a field strength of about 10 V/cm to 2 KV/cm. 13 A predetermined electrolyte solution is loaded into the intermediate region between the plate-shaped electrodes of the container and cells are suspended in the electrolyte solution, while another pulse power circuit is provided, and from the pulse power supply circuit, the opposite plate-shaped electrode is charged. 13. The device according to claim 9, wherein a pulse voltage is applied to a plurality of cells brought into contact with each other in a conductive path via a conductive path to perform cell fusion. 14. The device according to claim 13, wherein a means for extracting fused cells is provided in the narrow part of the conductive path.