JPS6349065A - Electrode for electrical cell operation apparatus and operation chamber - Google Patents

Abstract

PURPOSE:To attain high rate of fusion and to prevent the adhesion of cells to electrode, by constituting the surface of a pair of electrodes with a chemically stable substance hardly causing electrolysis and having uniform and flat surface with negligible unevenness compared with the size of cell. CONSTITUTION:Two electrodes 1, 2 of a chamber of electrical cell fusion or introduction of uncleic acid are applied with AC from an oscillator 4 generating AC having controllable voltage and frequency and with DC pulse from a pulse oscillator 5 generating pulses having controllable pulse voltage and pulse width to effect the cell fusion or introduction of uncleic acid. The electrode has uniform and flat surface having negligible unevenness compared with the size of cell and is made of a chemically stable substance (e.g. mirror-polished flat glass plate metallized with platinum) hardly causing electrolysis. High cell fusion ratio and high uncleic acid introduction rate can be attained by the above apparatus preventing the adhesion of cell to the electrode without damaging the fused cell.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an electrical cell manipulation device for effectively performing cell fusion and nucleic acid introduction by electrical methods.

The E Toguchi group cell fusion method mainly uses polyethylene glycol (PE).
G) A chemical fusion method using PEG is used, but this method requires (i) PEG is highly toxic to cells, and (11) it takes time and effort to find the optimal conditions for fusion. (iii) Advanced technology is required for fusion, and it can only be used by people who are proficient in a specific technology.
(iv) It has drawbacks such as low fusion efficiency.

On the other hand, the electrical cell fusion method does not require advanced technology, can be easily and efficiently fused, has no toxicity to the cells, and can fuse cells while maintaining high activity. It has the advantage of being able to

The electrical cell fusion method was developed in 1981 by Zimme in West Germany.
-r+wann has been established, and its principle is as follows. That is, when an alternating current voltage is applied between parallel electrodes and cells are introduced therein, the cells are attracted to the direction of higher current density and line up in a beaded pattern. In this state, several μsec ”-
By applying a DC pulse current of several tens of microseconds between the electrodes, the electrical conductivity of the cell membrane drops instantaneously, causing reversible disturbance and reorganization of the lipid bilayer that makes up the membrane, resulting in cell fusion. is what happens.

Conventionally, this electrical fusion method includes [al microelectrode method, (b
The l parallel electrode method, fc + the method of Zimmermann et al. mentioned above, etc. are known, but (al has the disadvantage of low fusion efficiency and is time-consuming, and t'bl has a low fusion rate (
1 to 3%) has the advantage of being able to handle a large amount of fused cells (protoplasts) at once, and that the fused cells do not adhere to the electrode. In addition, (c+ is used as the most practical method because it has a higher fusion rate than (δ1.(bl), but since the fused cells adhere to the electrode surface, it is difficult to recover the fused cells without damaging them. Important issues were how to develop electrode materials and how to rapidly produce fused cells in large quantities.

On the other hand, the electrical nucleic acid transfer method was developed by Neuman in 1982.
This is a method in which nucleic acids are introduced into cells by mixing cells and nucleic acids, suspending them on an electrode, and then applying a DC pulse potential of several μsec to several tens of μsec to this mixture using the method developed by N. However, even in this case, increasing implementation efficiency was an important issue.

The present invention solves various drawbacks of conventional parallel electrodes and operation chambers of electric cell manipulation devices capable of cell fusion and nucleic acid transfer, namely: 1) low fusion rate and occupation rate; ii) low fusion rate and occupancy rate; The purpose of the present invention is to provide a novel electrode and operation chamber that can solve all of the problems such as: (mountain) cells adhering to the electrode; and (iv) inability to rapidly produce large quantities of fused cells.

As a result of repeated research on electrode materials and the structure of the operation chamber, the inventors of the present invention found that they used a chemically stable substance that does not easily cause electrolysis, and created a mirror-like surface with surface irregularities that are almost negligible compared to the cell size. A flat plate electrode was created. The operation chamber is an electrode and operation chamber for an electric cell manipulation device that overcomes the drawbacks of conventional devices by sandwiching a flat plate spacer with a space used for cell fusion and nucleic acid introduction between two flat plate electrodes. It has been successful in providing.

That is, the present invention provides voltage and cycle control (11
In an electric cell production device that performs cell fusion or nucleus 61 introduction by applying an AC potential and a pulse voltage from a possible oscillator, and a DC pulse potential from a pulse oscillator whose pulse width and number of pulses can be controlled, the electrode These electrodes are characterized by having a uniformly flat surface that is almost negligible compared to the cell size and being composed of a chemically stable substance that does not easily cause electrolysis. Gold on flat substrates such as non-metallic materials such as polished glass, quartz, sapphire, and plastic, and metallic materials such as aluminum, stainless steel, and silver.

It is characterized by using a metal such as platinum deposited by vapor deposition. These electrodes are made of metal flat plate substrates,
The 1i pole may be a flat metal plate plated with gold, platinum, titanium, etc. to give a mirror surface, or platinum, titanium, etc.
A flat metal plate of titanium, stainless steel, or the like may be electrolytically polished and used as an electrode.

Alternatively, a flat plate substrate that has been machined or electrolytically polished may be subjected to ion plating processing, and the processed substrate may be used as an electrode.

The structure of the operation chamber of the present invention includes a flat plate spacer with a portion corresponding to the space used for cell fusion or nucleic acid introduction cut out, and two flat plate electrodes sandwiching the flat plate spacer, and a pressure bonding means for easy assembly and disassembly. It has a crimped structure.

By preparing a plurality of flat plate spacers with different sizes, thicknesses, and cut spaces and a plurality of flat plate electrodes with different sizes, and assembling the two flat plate electrodes sandwiching the flat plate spacer, the electrode The distance between the chambers and the chamber capacity can be varied.

Salmon A specific example of the present invention is schematically shown in FIG.

The operation chamber is composed of a flat plate spacer 3 and electrodes 1.2 sandwiching it. An alternating current oscillator 4 and a pulse oscillator 5 are connected to the electrode 1゜2, and the alternating current period,
The voltage, pulse voltage, and pulse width are monitored with an oscilloscope 6. The process of electrical cell fusion using this device will be explained using FIG. 2.

The upper column schematically shows the signal sequence applied to the electrode, and the lower column schematically shows the corresponding cell fusion process.

In step (1), cells are introduced into the chamber of the spacer 3 sandwiched between the plate electrodes 1.2 in FIG. 1 in this state with no voltage applied to the electrodes.

In step (2), the cells are arranged in a beaded pattern by applying an alternating current component potential from the oscillator 4 (FIG. 1).

In step (3), the direct current pulse causes temporary collapse of the cell membrane at the cell contact surface.

In step (4), cell fusion occurs with the reorganization of the cell membrane.

There are various types of TiH and chambers conventionally used in electric cell manipulation devices, as shown in FIG.

a) has parallel platinum wires; b) has an electrode at the center of a circle and electrodes surrounding it concentrically; C) has electrodes arranged to obtain a large number of fused cells; d) is for creating different types of hybrid fused cells. The electrodes used in these are ■Platinum wire ■Platinum temporary ■Stainless steel plate, etc. None of them have a smooth mirror surface, so the electric field current is It has the disadvantage that it becomes non-uniform and cells adhere to the surface.

In the present invention, by devising the structure of the operation chamber using a parallel plate electrode made of a material that is difficult to cause electrolysis and having a mirror-like surface, the electric field itself becomes homogeneous and cells do not adhere to the electrode surface. It was observed that cells uniformly exposed to the electric field showed a high fusion rate. In the embodiment, a spacer is sandwiched between two electrodes made of gold vapor-deposited on a glass substrate, and the spacers are crimped with clips. Electrode wires are attached to the two electrodes and a potential U is applied to them.

The spacer is a polyvinyl chloride plate with a thickness of 200 μm and a cutout in the center. 10■1
The part cut out at xlQmm and the part sandwiched between both electrodes become a space for cell manipulation. The electrode spacing and the capacity of the operation chamber can be adjusted by changing the thickness of the spacer, and the size of ti can be freely increased.

Cell fusion and nucleic acid introduction experiments were carried out using the above operation chamber and the apparatus shown in FIG.

1. Experimental method +11 Test materials: Tobacco mesophyll protoplasts (variety: Xanthi NN) and tobacco mosaic virus (TM
V)-RNA was used.

(2) Cell fusion method: protoplasts at approximately 2XIO'
/ml of 100 μM Ca Cl z -suspended in 0.5 M mannitol at a frequency of 500 KHz and a voltage of 400 V/ml in the above chamber with an electrode spacing of 200 μm.
After applying an alternating current of ~500 V/cm between the electrodes to orient the cells, a single DC pulse of 50 μsec in pulse width was applied at a potential of 0.6 k V/c+m to 1.0 k V/cm. .

(3) Chain acid introduction method: As in the case of the cell fusion method, protoplasts are
100 II M Ca Cl at X 10'/ml
x suspended in 0.5M mannitol, 10μg/
After adding ml"r'M V-RNA, 0.6 to 1.O
A DC pulse with a pulse width of 50 μsec was applied several times at a potential of k/cI11.

2. Experimental results (1) As a preliminary test, we investigated the effect of pulse voltage on fusion. When the pulse voltage was increased in the presence of 100 μM Ca, the fusion rate increased from 400 V/am to 850 V/am.
In the mouth, about 90% or more of the protoplasts that were internally arranged in all protoplasts fused.

At higher voltages, the number of fragmented protoplasts increased and the overall fusion rate decreased (Figure 5).

(2) The operation chamber of the present invention was used as the main experiment under the above optimal conditions (pulse application of 850 V/elm).
When we conducted a fusion experiment, we found that approximately 4 of all protoplasts
6% fused.

(3) When tobacco mesophyll protoplasts and carrot root protoplasts were fused, a hybrid fused protoplast of both was formed.

(4) In nucleic acid introduction experiments, after pulse application, protoplasts were
d in the culture medium of Tak ebe (1969), 2
After culturing at 8°C for 40 hours, the infection rate was measured by fluorescent antibody method, and the infection rate was 400 V/c.
At 800 V/mouth, approximately 95% of all protoplasts were infected (Fig. 5), and the survival rate of protoplasts at this time was approximately 95%, the same as before the pulse treatment.

- By using the electrode and operation chamber of the present invention in an electrical cell manipulation device, a high fusion rate can be obtained, the fused cells will not be damaged at all, and the cells will not adhere to the electrodes. The effect of this study was revealed.

Furthermore, as a result of experiments on nucleic acid transfer using the apparatus of the present invention, it was revealed that the optimal electrical conditions for electrical cell fusion and electrical nucleic acid transfer are almost the same. Furthermore, as a result of introducing viral RNA into plant protoplasts,
Approximately 95% of all protoplasts were infected, and a significantly higher nucleic acid transfer rate was obtained compared to conventional methods, indicating that the electrode and operation chamber of the present invention are extremely effective for nucleic acid transfer. did. This means that genetically modified DNA
This shows that this method is also extremely excellent as a method for efficiently introducing nucleic acids such as A into animal, plant, and microbial cells.

The present invention is not limited to the above embodiments (for example, efficient results can be obtained by using it as an electrode for electrophoresis, which has been widely used in the past.
It can be used as is as an electrode for cell electrophoresis, etc., and can also be applied as an electrode for a device for promoting cell proliferation by electrical stimulation. There are other things that can be easily modified and applied by those skilled in the art, and it goes without saying that these various applications are included within the scope of the present invention.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of the entire apparatus used in the present invention. FIG. 2 is a schematic diagram showing the relationship between the cell fusion process and the interelectrode potential waveform. FIG. 3 is a schematic diagram of various types of operating chambers. FIG. 4 is a structural diagram of the operating chaso bar according to the present invention. 51st? 1 is a diagram showing the relationship between the electrical fusion rate of protoplasts, the nucleic acid introduction rate of protoplasts by TMV-RNA, the protoplast disruption rate, and the pulse voltage as an example according to the present invention. 1.2... Electrode, 3... Spacer, 4...
- AC oscillator, 5... pulse oscillator, 6... onoroscope.

Claims (7)

[Claims] (1) The two electrodes of the chamber used for electrical cell fusion or nucleic acid introduction are supplied with an AC potential and a pulse voltage from an oscillator whose voltage and period can be controlled, and a DC pulse from a pulse oscillator whose pulse width and number of pulses can be controlled. In electrical cell manipulation devices that perform cell fusion or nucleic acid transfer by applying electric potential, the electrode surface is uniformly flat and almost negligible compared to the cell size, and is chemically stable and does not easily cause electrolysis. An electrode characterized in that it is made of a substance. (2) AC potential and pulse voltage from an oscillator whose voltage and cycle can be controlled, and DC pulses from a pulse oscillator whose pulse width and number of pulses can be controlled, are applied to the two electrodes of the chamber used for electrical cell fusion or nucleic acid introduction. In an electric cell manipulation device that performs cell fusion or nucleic acid transfer by applying electric potential, a flat plate spacer with a space used for cell fusion or nucleic acid transfer cut out and two flat plate electrodes sandwiching the flat plate spacer are assembled and disassembled. An operation chamber characterized by a structure that is crimped with a crimping means so that it can be easily performed. (3) Mirror-polished glass, quartz, sapphire,
2. The electrode according to claim 1, wherein the electrode is made of a flat substrate made of a non-metallic material such as plastic or a metallic material such as aluminum, stainless steel, silver, etc., on which a metal such as gold or platinum is vapor-deposited. (4) The electrode according to claim 1, characterized in that a metal flat plate plated with gold, platinum, titanium, etc. is used to make the surface mirror-finished. (5) The electrode according to claim 1, characterized in that a flat metal plate of platinum, titanium, stainless steel, etc. is electrolytically polished and used. (6) The electrode according to claim 1, characterized in that a flat plate substrate that has been machined or electrolytically polished is processed by ion plating, and the processed substrate is used. (7) By preparing multiple flat plate spacers with different sizes, thicknesses, and cut spaces and multiple flat plate electrodes with different sizes, and assembling the two flat plate electrodes sandwiching the flat spacer. Claim 2, characterized in that the distance between the electrodes and the chamber capacity can be made variable.
Operation chamber as described in section.