JPH0937762A - Flow chamber - Google Patents

Abstract

PURPOSE: To prevent attachment of a sample cell to an electrode plate and obtain an already treated cell in large amounts, compared with a conventional method by satisfactorily carrying out electric treatment to the sample cell. CONSTITUTION: In this flow chamber equipped with a flat plate spacer from which a part corresponding to a spacer for carrying out electric cell fusing method or a method for introducing nucleic acid to a cell is cut out, two sheets of electric plates which hold the flat spacer and a pressing means enabling pressing or its release to these electrode plates which hold the flat plate spacer, the electrode plates are formed as stripe-like electric plates 51A and 51B in which a non-electrode part 54B is formed in equal width or below, compared with an electrode part 54A, and at least the electrode part 54A of the electrode plates 51A and 51B are polished to mirror face.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a flow chamber, particularly to a flow chamber suitable for use in an electric cell fusion method and a method for introducing nucleic acid into cells.

[0002]

2. Description of the Related Art As a cell fusion method, a chemical fusion method mainly using polyethylene glycol (hereinafter referred to as PEG) is used. But this way

(1) PEG is highly toxic to cells, (2) it takes time to find optimal conditions for fusion, and (3) specific technology is required for fusion. It can only be used by people who are skilled in technology,
(4) It has drawbacks such as low fusion efficiency.

On the other hand, the electric fusion method does not require advanced technology, can be easily and efficiently fused, and is capable of fusing cells while maintaining high activity without toxicity to cells. The advantage is that The electric fusion method was established by Zimmermann of West Germany in 1981, and the principle thereof is as follows.

That is, an AC voltage is applied between the parallel electrodes,
When cells are introduced there, the cells are attracted to the one with the higher current density and line up on the beads. By applying a direct current pulse current of several μsec to several tens of μsec unit between the electrodes in this state, the electrical conductivity of the cell membrane is momentarily lowered, and reversible disorder and reconstitution of the lipid bilayer constituting the cell membrane The result is cell fusion, which results in cell fusion.

Conventionally, as the electric fusion method, (a) microelectrode method, (b) parallel electrode method, (c) Zimmermann method, etc. are known. However, the microelectrode method has a drawback that the fusion efficiency is low and it takes time, and the parallel electrode method has a low fusion rate (1 to 3%), but it can handle a large amount of fused cells (protoplasts) at a time, Has the advantage that it does not adhere to the electrodes.

The Zimmermann method is used as the most practical method because it has a higher fusion rate than the microelectrode method and the parallel electrode method, but since the fused cells adhere to the electrode surface, the fused cells It was an important subject how to develop an electric material for recovering cells without damaging them, and how to prepare fused cells in a large amount and rapidly.

On the other hand, the method of introducing electric nucleic acid was introduced in 1982.
This is a method developed by Neumann et al. In which cells and nucleic acids are mixed and suspended in an electrode, and then a DC pulse potential of several μsec to several tens of μsec is applied to introduce the nucleic acid into cells. Even in this case, improving the introduction efficiency was an important issue.

[0009]

DISCLOSURE OF THE INVENTION The inventors of the present invention have proposed various drawbacks of conventional parallel electrodes and operation chambers of an electrical cell manipulation device capable of cell fusion and nucleic acid introduction, that is, (A)
Low fusion rate or introduction rate, (B) damage the fused cells,
A new device was proposed in advance to solve the problems such as (C) cells adhering to electrodes and (D) inability to rapidly and rapidly prepare a large number of fused cells.

However, the chamber used in the proposed apparatus must be assembled by disassembling and assembling the spacers and parallel electrodes that form the chamber each time cell fusion or nucleic acid introduction is performed, and thus many operations can be performed quickly. Was inconvenient.
The present invention has been made in view of the above problems of the prior art, and an object thereof is to provide a flow chamber that enables rapid manipulation of a large amount of cells.

[0011]

In order to achieve the above object, a flow chamber according to the present invention is a flat plate spacer in which a portion corresponding to a space for performing an electric cell fusion method or a nucleic acid introduction method into cells is cut out. In the flow chamber, the two electrode plates sandwiching the flat plate spacer, and the crimping means capable of crimping and releasing the flat plate spacer are sandwiched between the electrode plates. A flow chamber characterized in that a striped electrode plate is formed in which the electrode portion has a width equal to or less than that of the electrode portion, and at least the electrode portion of the electrode plate is mirror-polished.

Further, in the flow chamber according to the present invention, in the flat plate spacer, a space for performing the electric cell fusion method or the nucleic acid introduction method into cells is provided with a plurality of wide passages and narrow passages in the moving direction of the sample cell. The electrode plate is formed such that its electrode portion is located in the wide path portion of the flat plate spacer and its non-electrode portion is located in the narrow path portion of the flat plate spacer. It is characterized by using a striped electrode plate.

[0013]

According to the flow chamber of the present invention, as described above, the electrode plate is a striped electrode plate in which the non-electrode part is formed with a width equal to or less than that of the electrode part, and at least the electrode plate is formed. Since the electrode part is mirror-polished, it is possible to perform good electrical treatment on the sample cells and prevent the sample cells from adhering to the electrode plate, and it is possible to obtain a large number of treated cells. Become.

According to the flow chamber of the present invention, as described above, in the flat plate spacer,
The space for performing the electric cell fusion method or the method of introducing a nucleic acid into a cell is a substantially gourd-shaped space in which a plurality of wide and narrow paths are connected in the moving direction of the sample cell, and the electrode plate is The position of the electrode part of the electrode plate is formed by forming a striped electrode plate in which the electrode part is located in the wide path part of the flat plate spacer and the non-electrode part is located in the narrow path part of the flat plate spacer. It becomes possible to perform better electrical treatment of the sample cells in the wide part of the flat plate spacer, and by moving the sample cells to the next broad part through the narrow part, the sample cells are removed. Since it is possible to move well, it is possible to obtain a larger amount of treated cells.

[0015]

[First Embodiment] First, the construction of the entire electrical cell manipulating apparatus is described below.
It is shown schematically in the figure. The flow chamber used in the present invention is basically composed of a flat plate spacer 3 and electrodes 1 and 2 sandwiching the flat plate spacer 3 as has been proposed by us. AC oscillator 4 and pulse oscillator 5 on electrodes 1 and 2
The AC cycle, voltage and pulse voltage, and pulse width are monitored by the oscilloscope 6. The process of electrical cell fusion using this device will be described with reference to FIG.

In FIG. 2, the upper column shows the signal sequence applied to the electrodes, and the lower column schematically shows the corresponding cell fusion process.

In step (1), no voltage is applied to the electrodes. In this state, cells are introduced into the chamber space of the spacer 3 sandwiched between the plate electrodes 1 and 2 in FIG. In the process (2), cells are arranged in a row on the beads by applying the potential of the AC component from the transmitter 4 (Fig. 1).

In step (3), the direct current pulse causes a temporary disruption of the cell membrane at the cell contact surface. In the process (4), cell fusion occurs with the reconstitution of the cell membrane.

Next, the flow chamber will be described. Using parallel plate electrodes in which the surface of a substance that is less likely to undergo electrolysis is mirror-like, the electric field itself becomes homogeneous, and cells do not adhere to the electrode surface and Similar to the previous proposal by the applicant, the uniformly exposed cells are configured to exhibit a high fusion rate. 3 and 4 show respective embodiments of the flow chamber, (a) is an exploded perspective view, and (b) is a perspective view of a use state after assembly.

In these examples, the spacers 13 and 23 are sandwiched by two electrodes 11, 12;
It is crimped at 4, 24. The electrode wire 15 for the two electrodes,
Turn on 25, and the potential is applied. Further, the spacer has a thickness of 200 μm and uses a polyvinyl chloride plate having a central portion cut out, and the cut portion and the portion sandwiched by both electrodes serve as spaces 16 and 26 for cell manipulation.

The flow chamber of FIG. 3 has an elongated rectangular shape as a whole, and accordingly, the chamber space 16 is also elongated and has a capacity of 5 μl, for example.
Overhanging portions 17 for facilitating attachment of 5 are projected in opposite directions.

Further, there are provided introducing / extracting means for continuously guiding cells into the space 16 and discharging the cells therefrom, and these introducing / extracting means are similar to the spacer 13 in both electrodes. Connecting conduits 18, 1 sandwiched between
8'and the slanted groove 19 starting from the end face on the inner surface of the non-electrode and terminating at a point enabling communication with the space 16
a, 19b; 19a ', 19b' (not shown), respectively.

Reference numerals 20, 20 'denote connection conduits 18, 1
The cutouts are formed at both ends of the spacer 13 for receiving 8 '. By assembling the respective constituent members thus configured as shown in FIG. 3 (b) and connecting the connecting conduits 18 and 18 ′ to a pump, a cell reservoir, a cell collection tube and the like not shown respectively, In the case of cell fusion, cells are introduced into the flow chamber, once stopped, electrical treatment is performed, and then they are discharged, and at the same time, new cells are introduced.

Further, in introducing the nucleic acid, a procedure such as cell fusion is not required, and the nucleic acid-introduced cells can be collected by continuously performing electrical treatment while continuously flowing the cells and the nucleic acid solution in the flow chamber. . On the other hand, the flow chamber shown in FIG. 4 has a substantially square shape as a whole, and the volume of the oval chamber space 26 corresponding thereto is large.

The cell introducing / extracting means comprises an introducing tube 27 and an extracting tube 28, and the inner ends of the introducing / extracting tubes 27, 28 are open at both ends of the space 26. Therefore, by assembling the respective components as shown in FIG. 4 (b) and connecting the introducing / extracting pipes 27 and 28 to a pump, a cell reservoir, a cell collecting pipe, etc., which are not shown, respectively, Cell fusion and nucleic acid transfer can be performed in the same manner as in.

Since the flow chamber of FIG. 3 has a relatively small capacity, it can be operated with a low power source.
If the power supply can be powered up, the flow chamber of FIG. 4 having a large capacity is simpler and easier to handle, and the amount of operation per operation can be increased.

The space between the electrodes and the volume of the chamber space can be adjusted by changing the thickness of the spacer, and the size of the electrodes can be freely increased. further,
The number of spaces provided in one chamber is not limited to one, and a plurality of flat plate spacers having different space sizes and thicknesses and a plurality of flat plate electrodes corresponding thereto are prepared, and the flat plate spacers are sandwiched between them. Assemble two flat plate electrodes, provide independent cell introduction / extraction means for each space,
It is also possible to have a configuration having a plurality of spaces in one chamber.

FIG. 5 shows a chamber in which a large number of counter electrodes are arranged at certain intervals. FIG. A shows an electrode 54A and a non-electrode 5
4B shows a striped chamber. Two electrodes, non-electrodes, striped plates 51A and 52A of equal width sandwich a spacer 53, the inside of which is cut out in a rectangular shape, and the two electrodes are displaced by an amount corresponding to the portion connecting the electrodes to the outside of the chamber. Is configured.

The sample is introduced into such a chamber, an AC potential is applied to all the electrodes, and a DC pulse is applied to carry out electrical treatment, and then the sample is moved by the width of the electrode, and the electrical treatment is performed again. The cells are manipulated by replacing the sample in the chamber with a new sample after performing the electrical treatment on the electrode.

FIG. 3B shows a non-electrode narrow electrode striped chamber. Compared to the electrode width 54B, the non-electrode width 55B is made narrower, and two electrode plates in which a large number of electrodes are arranged are provided with a plurality of portions 57-1 to 57-5 into which a sample having a width equal to or less than the electrode width is placed. A flow path portion 58 passing through the sample space is arranged,
A chamber is formed by sandwiching a spacer by shifting a portion corresponding to a portion connecting the two electrode plates to an external line.

FIG. 6 shows an example in which a plurality of chambers are arranged for the sample flow. FIG. 7 shows an example in which a plurality of chambers are arranged in series with respect to the sample flow. In the drawings, the same numbers indicate the same things. In FIG. 6 and FIG. 7, an AC oscillator 62 and a pulse oscillator 63
Are connected to the electrode wires 69 to 73 of the respective chambers 64 to 68.

The sample reservoir 61 has an agitator 60, and the agitator 60 agitates the sample in the sample reservoir 61. The manipulated cells derived from each chamber are pump 8
The manipulated cells harvested in the manipulated cell harvester 81 by the suction force of 0 and the manipulated cells collected in the manipulated cell harvester 81 are kept at a constant temperature by a thermostat.

The output from the AC oscillator 62 is applied to the chambers 64 to 68, and the output from the pulse oscillator 63 is
Electrodes 69 of each chamber 64-68 while switching sequentially
To 73 for electrical treatment. 6 and 7
The pump 80 shown in the figure is of a suction type, but it is also possible to place the pump on the sample inlet side of the chamber to send the sample.

Further, instead of introducing the sample into each chamber at the same time, the cells are caused to flow in one chamber,
After that, an electrical treatment for cell fusion or diffusion introduction is performed with the flow stopped, and then the treated cells are allowed to flow and flow, and the operation of introducing new cells into the flow chamber is sequentially switched for each chamber. A large amount of manipulated cells can also be obtained by carrying out. To do this, a switching valve can be installed in the output tube of the sample reservoir 61 and this switching valve can be switched.

Up to now, the flow path and the electric field application for cell fusion have been mainly shown for each flow chamber, but this is for explaining that the cell fusion is performed by stopping the flow. Next, the flow chamber of FIG.
An example of cell fusion and RNA introduction (nucleic acid infection) using 0 μl) will be described. FIG. 8 shows a flowchart in the case of cell fusion.

In the figure, first, the pump is operated to
After feeding 0 μl of the cell sample, AC6V is applied for 1 minute, and the cells are arranged in a beaded pattern. Next, while applying AC2V, a DC pulse of 40 V and 50 μs is applied twice to cause temporary collapse of the cell membrane, and then AC2V is applied for 1 minute to cause cell fusion. After collecting the fused cells obtained next, the above process was repeated 20 times to obtain a total of 1 m.
1, 2 × 10 ⁵ protoplasts were fused. The time required is 2 minutes per cycle x 20 times = 40 minutes, and the fusion rate is 4
It was 0%.

FIG. 9 shows a flow chart in the case of RNA introduction (nucleic acid infection). In the figure, 2 × 10 ⁵
TMV-RNA in 10 μg / ml of protoplasts / ml.
ml was added, and while continuously feeding this at a flow rate of 50 μl / secmp, a DC pulse of 40 V and 50 μs was applied 10 times /
It was continuously applied at a rate of 2 seconds. This operation was continued for 80 seconds to collect a total of 4 ml of 8 × 10 ⁵ protoplasts. As a result, the infection rate was 96% and the survival rate was 85%.

[0038]

【The invention's effect】 [Brief description of drawings]

As described above, according to the flow chamber of the present invention, continuous flow operation is possible, and fusion of a large number of cells and nucleic acid introduction required in the field of biotechnology can be efficiently and promptly operated.

FIG. 1 is a block diagram showing the overall configuration of an electrical cell manipulation device.

FIG. 2 is a diagram for explaining the process of cell fusion.

3 (a) and 3 (b) are an exploded perspective view of an embodiment of a flow chamber according to the present invention and a perspective view showing a use state after assembly.

4 (a) and (b) are an exploded perspective view of another embodiment of the flow chamber according to the present invention and a perspective view showing a use state after assembly.

FIG. 5 shows a case where a plurality of flow chambers are used in parallel at the same time, and shows a chamber in which a large number of counter electrodes are arranged at certain intervals.
FIG. B shows a non-electrode narrow electrode.

FIG. 6 is a schematic explanatory view showing an example of multiple chambers connected in parallel to the flow of a sample.

FIG. 7 is a schematic explanatory view showing an example of multiple chambers connected in series to the flow of a sample.

FIG. 8 is a flowchart showing an example of cell fusion.

FIG. 9 is a flowchart showing an example of RNA introduction (nucleic acid infection).

[Explanation of symbols]

 1, 2; 11, 12, 12; 21, 22 electrodes 3, 13, 23 spacer 4 AC oscillator 5 pulse oscillator 6 oscilloscope 14, 24 crimping means (clip) 15, 25 electrode wire 16, 26 chamber space 18, 19a, 19b; 27 cell introduction means 18 ', 19'a, 19'b; 28 cell derivation means

[Procedure amendment]

[Submission date] July 25, 1995

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction contents]

That is, an AC voltage is applied between the parallel electrodes,
When cells are introduced there, the cells are attracted to the one with the higher current density and line up on the beads. In this state, a few μsec
By applying a DC pulse voltage of several tens of μsec units between the electrodes, the electrical conductivity of the cell membrane is instantaneously lowered, reversible disturbance and its reconstitution of the lipid bilayer that constitutes the cell membrane is carried out, as a result , Cell fusion occurs.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction contents]

On the other hand, electrically nucleic acid transfer method was developed by the like Neumann in 1982, was suspended between a mixture of cells and nucleic electrodes, several μsec~ tens μsec
This is a method of introducing a nucleic acid into cells by applying the DC pulse potential of 1., but also in this case, it was an important issue to improve the introduction efficiency.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction contents]

[0011]

[Means for Solving the Problems] A flat plate spacer in which a portion corresponding to a space for performing an electric cell fusion method or a nucleic acid introduction method into cells is cut off, and two electrode plates sandwiching the flat plate spacer. In a flow chamber provided with a crimping means capable of crimping and releasing the electrode plate sandwiching the flat plate spacer, the electrode plate is formed so that the non-electrode part has a width equal to or less than that of the electrode part. Striped electrode plate, at least the electrode part of the electrode plate is mirror-polished, and when performing cell fusion, sample cells are
After introduction into the cell, the cell flow was stopped and the sample was electrically treated.
The sample is processed and electrically processed, and then the sample is moved by the width of the electrode section.
Repeatedly, when introducing nucleic acid into cells
Is a means for delivering cell fluid that continuously moves sample cells
It is characterized by having.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigeyuki Kimura 5-16-10 Nishihirayama, Hino-shi, Tokyo (72) Inventor Takeshi Kazami 1616 Fujisawa, Shinji-mura, Shinji-gun, Ibaraki

Claims (2)

[Claims] 1. A flat plate spacer in which a portion corresponding to a space for carrying out an electric cell fusion method or a nucleic acid introduction method into a cell is cut off, two electrode plates sandwiching the flat plate spacer, and the flat plate spacer. In a flow chamber provided with a crimping means capable of crimping and releasing the sandwiched electrode plate, the electrode plate is formed into a striped pattern in which the non-electrode part is formed to have a width equal to or less than that of the electrode part. A flow chamber comprising an electrode plate, wherein at least an electrode portion of the electrode plate is mirror-polished. 2. In the flat plate spacer, a space for performing an electric cell fusion method or a nucleic acid introduction method into cells is formed into a substantially gourd-like shape in which a plurality of broad and narrow passages are connected in a moving direction of a sample cell. The electrode plate is a striped electrode plate formed such that its electrode portion is located in the wide path portion of the flat plate spacer and its non-electrode portion is located in the narrow path portion of the flat plate spacer. The flow chamber according to claim 1, wherein: