JPH0567276B2 - - Google Patents

Description

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

Conventional technology The chemical fusion method using polyethylene glycol (PEG) is mainly used as a cell fusion method, but in this method, PEG is highly toxic to cells, so it is not suitable for fusion. It has disadvantages such as: it takes time to find the appropriate conditions; advanced technology is required for fusion, so it can only be used by people who are proficient in specific techniques; and fusion efficiency is low.

On the other hand, the electrical cell fusion method does not require advanced technology, can be easily and efficiently fused, has no toxicity to 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 West Germany in 1981.
It was established by Zimmermann, 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, by applying a DC pulse current of several microseconds to several tens of microseconds between the electrodes, the electrical conductivity of the cell membrane decreases momentarily, and the lipid bilayer that makes up the membrane undergoes excessive disturbance and its reorganization. As a result, cell fusion occurs.

Conventionally, this electrical fusion method includes (a) microelectrode method;
(b) Parallel electrode method, (c) method of Zimmermann et al. mentioned above, etc. are known, but (a) has the disadvantage of low fusion efficiency and is time-consuming, and (b) has a low fusion rate (1
~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) has a higher fusion rate than (a) and (b), so it is used as the most practical method, but since the fused cells adhere to the electrode surface, it does not damage the fused cells. Important issues were how to develop electrode materials for recovery and how to rapidly produce fused cells in large quantities.

On the other hand, the electrical nucleic acid transfer method was developed in 1982.
After mixing cells and nucleic acids and suspending them on the electrode using the method developed by Neumann et al.
This is a method of introducing nucleic acids into cells by applying a DC pulse potential of several tens of microseconds, but in this case as well, increasing the introduction efficiency was an important issue.

Problems to be Solved by the Invention The present invention solves various drawbacks of conventional parallel electrodes and manipulation chambers of electrical cell manipulation devices capable of cell fusion and nucleic acid transfer, namely: low fusion rate and low transfer rate;
The purpose of the present invention is to provide a novel electrode that can solve all of the following problems: damage to fused cells, adhesion of cells to the electrode, and inability to rapidly produce fused cells in large quantities.

Means for Solving the Problems As a result of repeated research into electrode materials and the structure of the operating chamber, the inventors of the present invention have developed a method using chemically stable substances that do not easily cause electrolysis, and which have surface irregularities compared to the cell size. We created a flat plate electrode with a mirror surface state that can be almost ignored. The operation chamber provides an electrode for an electrical cell manipulation device that overcomes the conventional drawbacks by having a structure in which a flat plate spacer, in which a space used for cell fusion and nucleic acid introduction is cut out, is sandwiched between two flat plate electrodes. It was extremely successful.

That is, the present invention provides two electrodes of a chamber used for electrical cell fusion or nucleic acid introduction with an AC potential from an oscillator whose voltage and period can be controlled, and a pulse oscillator whose pulse pressure, pulse width, and number of pulses can be controlled. In electrical cell manipulation devices that perform cell fusion or nucleic acid copper introduction by applying a direct current pulse potential, the electrode surface is made uniformly flat to the extent that it can be ignored compared to the cell size, and is chemically resistant to electrolysis. These electrodes are made of a stable material, and these electrodes are made of flat substrates made of mirror-polished non-metallic materials such as glass, quartz, sapphire, plastic, etc., or metallic materials such as aluminum, stainless steel, and silver, and gold, etc. It is characterized by using a metal such as platinum deposited by vapor deposition. These electrodes may be made by plating a metal flat plate with gold, platinum, titanium, etc. to make the surface mirror-finished, or by electrolytically polishing a metal flat plate made of platinum, titanium, stainless steel, etc. However, the metal flat plate may be 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.
Prepare a plurality of flat plate spacers with different sizes, thicknesses, and cut-out spaces, a plurality of flat plate spacers with different sizes, and a plurality of flat plate electrodes with different sizes, and prepare two plates sandwiching the flat plate spacers. By assembling the plate electrodes, the distance between the electrodes and the chamber capacitance can be made variable.

Example 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 and 2 sandwiching it. electrode 1,
An AC oscillator 4 and a pulse oscillator 5 are connected to the AC oscillator 2, and the AC period, 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), no voltage is applied to the electrodes. In this state, cells are introduced into the chamber of the spacer 3 sandwiched between the flat electrodes 1 and 2 shown in FIG.

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

In process (4), cell fusion occurs as the cell membrane reorganizes.

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

(a) shows parallel platinum wires.

(b) shows an electrode at the center of a circle and electrodes surrounding it in a concentric circle.

(c) shows electrodes arranged to obtain a large amount of fused cells.

(d) shows the creation of different types of hybrid fusion cells.

The electrodes used in these devices are made of platinum wire, platinum plate, stainless steel plate, etc., and their surfaces are not smooth and mirror-like, resulting in uneven electric field and current, and they also have the drawback of cells adhering to their surfaces. .

In the present invention, by devising the structure of the operation chamber using parallel plate electrodes made of substances that are 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 this embodiment, a spacer is sandwiched between two electrodes deposited with gold on a glass substrate, and then crimped with a clip. Electrode wires are attached to the two electrodes and a potential is applied.

The spacer is a polyvinyl chloride plate with a thickness of 200 μm and a cutout in the center. The 10mm x 10mm cutout and the area sandwiched between the electrodes will be the space for cell manipulation. The electrode spacing and the capacity of the operating chamber can be adjusted by changing the thickness of the spacer, and the size of the electrodes can be freely increased.

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

1. Experimental method (1) Test materials: Tobacco mesophyll protoplasts (variety: Xanthi NN) and tobacco mosaic virus (TMV)-RNA were tested.

(2) Cell fusion method: protoplasts at approximately 2×10 ⁵ /ml
Suspended in 100 μM CaCl ₂ −0.5 M mannitol,
In the above chamber with an electrode spacing of 200 μm, alternating current with a voltage of 400 V/cm to 500 V/cm is applied between the electrodes at a frequency of 500 KHz to orient the cells, and then a pulse width is applied with a potential of 0.6 kV/cm to 1.0 kV/cm. A DC pulse of 50 μsec was applied once.

(3) Nucleic acid introduction method: As in the case of the cell fusion method, protoplasts were injected with 100 μM CaCl ₂ − at approximately 2×10 ⁵ /ml.
Suspended in 0.5M mannitol, 10μg/ml
After adding TMV-RNA, 0.6kV/cm to 1.0kV/cm without applying AC component to the electrode.
A DC pulse with a pulse width of 50 μsec was applied several times at a potential of cm.

2 Experimental results (1) When we investigated the effect of pulse voltage on fusion using a preliminary reagent, we found that when the pulse voltage was increased in the presence of 100 μM Ca ⁺⁺ , the fusion rate increased from 400 V/cm;
At 850V/cm, approximately 90% or more of the protoplasts, which were arranged in a multilayered arrangement within all protoplasts, fused.

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

(2) In this experiment, a fusion experiment was conducted using the operating chamber of the present invention under the above-mentioned optimal conditions (pulse application of 850 V/cm), and approximately 46% of all protoplasts fused.

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

(4) In nucleic acid transfer experiments, after pulse application, protoplasts were
and Takebe (1669) at 28°C.
After culturing for 40 hours, the infection rate was measured by fluorescent antibody method.
It increased from 400V/cm, and at 800V/cm, about 95% of all protoplasts were infected (Figure 5).
The protoplast survival rate at this time was about 95%, the same as before the pulse treatment.

Effects of the Invention By using the electrode according to the present invention in an electrical cell manipulation device, excellent effects such as a high fusion rate, no damage to the fused cells at all, and no cells adhering to the electrode have been revealed. .

Further, as a result of experiments on nucleic acid transfer using the electrode 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, resulting in an extremely high rate of nucleic acid transfer compared to conventional methods, indicating that the electrode of the present invention is also highly effective for nucleic acid transfer. was found to be effective. This shows that this method is also extremely excellent as a method for efficiently introducing nucleic acids such as genetically modified DNA into animal, plant, and microbial cells.

The present invention is not limited to the above-mentioned embodiments, and efficient results can be obtained by using the present invention as an electrode for electrophoresis, which has been widely used in the past. Further, it can be used as it 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 drawings]

FIG. 1 is a schematic diagram of the entire apparatus using 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. Fourth
The figure is a structural diagram of the operating chamber according to the invention. FIG. 5 is a diagram showing the relationship between the electrical fusion rate of protoplasts, the rate of nucleic acid introduction into 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... Oscilloscope.

Claims (1)

[Claims] 1. An alternating current potential and a pulse oscillator whose voltage and period can be controlled and a pulse oscillator whose pulse width and number of pulses can be controlled, applied to two electrodes of a chamber used for electrical cell fusion or nucleic acid induction. The electrode surface roughness or unevenness is smooth and is approximately 1/30 or less of the diameter of the cell, and is used in an electric cell manipulation device that performs cell fusion or nucleic acid introduction by applying a DC pulse potential from An electrode comprising at least one substance selected from the group consisting of , platinum, and titanium. 2. A patent claim in which the electrode is made of a non-metallic material such as glass, quartz, sapphire, or plastic with a mirror-polished surface, or a flat plate substrate made of a metallic material such as aluminum, stainless steel, or silver, on which a metal such as gold or platinum is vapor-deposited. The electrode according to item 1. 3. The electrode according to claim 1, wherein the electrode is made of a metal flat plate plated with gold, platinum, titanium, etc. to give the surface a mirror finish. 4. Claim 1, in which the electrode is an electrolytically polished flat metal plate made of platinum, titanium, stainless steel, etc.
Electrode as described in Section. 5. The electrode according to claim 1, wherein the electrode is formed by ion plating a flat plate substrate that has been machined or electrolytically polished.