JPH07194366A - Operation chamber for electric cell manipulation apparatus - Google Patents

Abstract

PURPOSE:To obtain a manipulation chamber useful for an electric cell manipulation apparatus capable of effectively performing the cell fusion and the introduction of nucleic acid. CONSTITUTION:This manipulation chamber is produced by preparing a flat plate spacer having a cut space corresponding to the space to be used in the cell fusion or the introduction of nucleic acid and a pair of flat plate electrodes for sandwiching the flat plate spacer and sandwiching the flat plate spacer with the electrodes by the aid of a pressing means to facilitate the assembling and disassembling of the plates. The distance between the electrodes and the capacity of the chamber are arbitrarily selected by preparing plural flat plate spacers having different size, thickness and cut space volume and plural flat plate electrodes having different size and combining a flat plate spacer and a pair of flat plate electrodes selected from the above spacers and electrodes.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electric cell manipulator for effectively performing cell fusion and nucleic acid introduction by an electric method.

[0002]

2. Description of the Related Art As a cell fusion method, a chemical fusion method using polyethylene glycol (PEG) is mainly used. In this method, (i) PEG has a strong toxicity to cells ( ii) It takes time and effort to find the optimal conditions for fusion, (iii) advanced technology is required for fusion, and it can be used only by those who are familiar with specific technology, (iv) low fusion efficiency, etc. It has drawbacks. On the other hand, the electrical cell fusion method does not require advanced technology, can be easily and efficiently fused, does not cause toxicity to cells, and fuses cells while maintaining high activity. The advantage is that The electrical cell fusion method was introduced in 1981 by Zimmerman in West Germany.
n is established, and its principle is as follows.
That is, when an AC voltage is applied between the parallel electrodes to introduce cells into the cells, the cells are attracted to the side having a higher current density and are arranged in a bead shape. In this state, by applying a direct current pulse current of several μsec to several tens of μsec between the electrodes, the electrical conductivity of the cell membrane is instantaneously decreased, and the reversible disorder of the lipid bilayer constituting the membrane and its reconstitution are performed. That is, cell fusion occurs as a result. Conventionally, in this electrical fusion method,
(A) Microelectrode method, (b) Parallel electrode method, (c) Zi
Although methods such as mmmermann are known, (a)
Has the drawback of low fusion efficiency and time-consuming
(B) has a low fusion rate (1 to 3%), but has an advantage that a large amount of cell fusion (protoplast) can be handled at one time and the fused cells do not adhere to the electrode. Also, (c) is (a),
Since it has a higher fusion rate than (b), it is used as the most practical method, but since fused cells adhere to the electrode surface, how to develop an electrode material for recovering fused cells without damaging them It has been an important subject how to produce fused cells in large quantities and rapidly. on the other hand,
The electrical nucleic acid introduction method is a method developed by Neumann et al. In 1982, in which cells and nucleic acids are mixed and suspended in an electrode, and then a direct current pulse potential of several μsec to several tens μsec is applied to the mixture. Although this is a method of introducing a nucleic acid into cells, in this case as well, increasing the introduction efficiency was an important issue.

[0003]

DISCLOSURE OF THE INVENTION The present invention has various drawbacks of conventional parallel electrodes and operation chambers of an electric cell manipulation device capable of cell fusion and nucleic acid introduction, namely, i) low fusion rate and introduction rate, ii) The purpose of the present invention is to provide a new operation chamber for solving problems such as damage to fused cells, iii) cells attached to electrodes, and iv) inability to produce fused cells in large quantities and rapidly. is there.

[0004]

As a result of repeated studies on the structure of the electrode material and the operation chamber, the present inventors have used a chemically stable substance that is unlikely to cause electrolysis, and A flat plate electrode having a mirror surface state in which unevenness is almost negligible was prepared. The operation chamber has a structure in which a flat plate spacer having a space portion used for cell fusion and nucleic acid introduction is sandwiched between the two flat plate electrodes to provide an electrode for an electric cell operation device that overcomes the conventional drawbacks. It was a successful one. That is, the present invention relates to two electrodes of a chamber used for electrical cell fusion or nucleic acid introduction, in which an AC potential from an oscillator capable of controlling voltage and cycle, a pulse pressure, a pulse width, and a pulse oscillator capable of controlling the number of pulses are controlled. In an electric cell manipulation device that applies cell fusion or nucleic acid introduction by applying DC pulse potentials respectively, the electrode surface is chemically flat and almost even compared to the cell size to a level that is almost negligible, and chemically stable against electrolysis. These electrodes are made of a non-metallic material such as glass, quartz, sapphire, or plastic whose surface is mirror-polished, or a flat substrate made of a metallic material such as aluminum, stainless, or silver. ,
It is characterized by using a vapor-deposited metal such as platinum. As these electrodes, on a flat metal substrate,
The metal flat plate having a mirror-finished surface plated with gold, platinum, titanium or the like may be used as the electrode, or the metal flat plate of platinum, titanium, stainless steel or the like may be electrolytically polished and the metal flat plate may be used as the electrode. good. Ion-plating a flat plate that has been machined or electropolished,
The processed substrate may be used as an electrode.

The structure of the operation chamber of the present invention is such that a plate spacer having a portion corresponding to a space used for cell fusion or nucleic acid introduction and a plate electrode sandwiching the plate spacer are easily assembled and disassembled. It is configured to be crimped by crimping means so that it can be performed. By preparing a plurality of flat plate spacers having different sizes, thicknesses and cut spaces and a plurality of flat plate electrodes having different sizes, and assembling two flat plate electrodes sandwiching the flat plate spacer, The distance and chamber volume can be made variable.

[0006]

EXAMPLE A concrete 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. AC oscillator 4 on electrodes 1 and 2
And a pulse oscillator 5 are connected, and 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. 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, the cells are introduced into the chamber of the spacer 3 sandwiched between the plate electrodes 1 and 2 in FIG. In the process (2), cells are arranged in a beaded pattern by applying the potential of the AC component from the oscillator 4 (FIG. 1). In the process (3), the direct current pulse causes a temporary collapse of the cell membrane at the cell contact surface. In the process (4), cell fusion occurs with the reconstitution of the cell membrane. As shown in FIG. 3, there are various types of electrodes and chambers conventionally used in electric cell manipulation devices. a) is a parallel platinum wire. In b), the electrode at the center of the circle and the electrodes surrounding it concentrically are arranged. c) is an array of electrodes so as to obtain a large amount of fused cells, and d) is a hybrid fused cell of a different type. The electrodes used for these were platinum wire, platinum plate, stainless steel plate, etc., and all had the disadvantage that the electric field current was non-uniform because the surface was not a smooth mirror surface, and cells adhered to the surface. .

In the present invention, by devising the structure of the operation chamber by using a parallel plate electrode in which the surface of a substance which is less likely to undergo electrolysis is mirror-finished, the electric field itself becomes homogeneous and cells are brought into contact with the electrode surface. It was observed that cells that were not attached and were uniformly exposed to the electric field showed a high fusion rate. In the example, a spacer is sandwiched between two electrodes vapor-deposited with gold on a glass substrate, and they are pressure-bonded with clips. Electrodes are attached to the two electrodes and a potential is applied.

As the spacer, a polyvinyl chloride plate having a thickness of 200 μm and a central portion cut off is used. The part cut out to 10 mm × 10 mm and the part sandwiched between both electrodes become a space for cell manipulation.
The electrode interval and the capacity of the operating chamber can be adjusted by changing the thickness of the spacer, and the size of the electrode can be freely increased. Experiments of cell fusion and nucleic acid introduction were carried out using the above-mentioned operation 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 was tested. (2) Cell fusion method: about 2 × 10 ⁵ / m of protoplasts
in 100 μM CaCl ₂ -0.5M mannitol at a voltage of 400 V / cm to 500 V / cm at a frequency of 500 KHz in the chamber having an electrode interval of 200 μm.
After alternating current is applied between the electrodes to orient the cells,
Pulse width 50 at a potential of 6 kV / cm to 1.0 kV / cm
A direct current pulse of μsec was applied once. (3) Nucleic acid introduction method: As in the case of the cell fusion method, protoplasts were added at about 2 × 10 ⁵ / ml to 100 μM CaC.
was suspended in l _2-0.5 M mannitol, 10 [mu] g / ml
After adding TMV-RNA, a DC pulse having a pulse width of 50 μsec was applied several times at a potential of 0.6 kV / cm to 1.0 kV / cm without applying an AC component to the electrodes.

2. Experimental results (1) As a preliminary test, when the effect of pulse voltage on fusion was examined, when the pulse voltage was increased in the presence of 100 μM Ca ^{++, the} fusion rate increased from 400 V / cm, and at 850 V / cm, within all protoplasts. About 90% or more of the protoplasts in the double sequence were fused. At higher voltage, the fragmentation protoplasts increased and the overall fusion rate decreased (Fig. 5). (2) When the fusion experiment was performed under the optimum conditions (pulse application of 850 V / cm) using the operation chamber of the present invention as this experiment, about 46% of all protoplasts were fused. (3) When tobacco mesophyll protoplasts and carrot root protoplasts were fused, hybrid fused protoplasts of both were formed. (4) In the nucleic acid introduction experiment, after pulse application, protoplasts were cultured in Aoki and Takebe (1969) culture solution at 28 ° C. for 40 hours, and the infection rate was measured by the fluorescent antibody method.
/ Cm, and at 800 V / cm, about 95% of all protoplasts were infected (Fig. 5). The protoplast survival rate at this time was about 95%, which was the same as before the pulse treatment.

[0012]

EFFECTS OF THE INVENTION By using the operation chamber according to the present invention in the electrical cell operation device, a high fusion rate is obtained, and excellent effects such as no damage to the fused cells and no adhesion of the cells to the electrodes are revealed. Became. As a result of further experiment of nucleic acid introduction using the electrode of the present invention,
It was revealed that the optimum electrical conditions for electric cell fusion and electric nucleic acid introduction are almost common. Furthermore, as a result of introducing viral RNA into plant protoplasts, about 95% of all protoplasts were infected, and a very high nucleic acid introduction rate was obtained as compared with the conventional method. It turned out to be very effective. This shows that it is extremely excellent as a method for efficiently introducing genetically modified nucleic acid such as DNA into animal and plant cells and microbial cells.

[0013]

[Brief description of drawings]

FIG. 1 is a schematic view of the whole 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 view of various types of operating chambers. FIG. 4 is a structural diagram of an operation chamber according to the present invention. FIG. 5 is a diagram showing the relationship between the electrofusion rate of protoplasts, the nucleic acid introduction rate of protoplasts by TMV-RNA, the protoplast crushing rate, and the pulse voltage as an example according to the present invention. 1, 2 ... Electrodes, 3 ... Spacers, 4 ... AC oscillator, 5 ... Pulse oscillator, 6 ... Oscilloscope.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Miyoji Sugiura 4-chome, 1050, Namiki, Sakuramura, Shinji-gun, Ibaraki 926 Room 104 (72) Inventor Takeshi Kazami 1616, Fujisawa, Shinji-mura, Ibaraki (72) Invention Shigeyuki Kimura 5-16-10 Nishihirayama, Hino City, Tokyo

Claims (2)

[Claims] 1. An AC potential from an oscillator capable of controlling a voltage and a period, a pulse voltage, a pulse width, and a pulse oscillator capable of controlling a pulse width and a pulse number are provided to two electrodes of a chamber used for electrical cell fusion or nucleic acid induction. A plate spacer for use in an electrical cell manipulation device for cell fusion or nucleic acid introduction by applying a DC pulse potential, respectively, and a flat plate spacer in which a space used for cell fusion or nucleic acid introduction is cut off, and two flat plate spacers sandwiching the flat plate spacer. An operation chamber characterized in that a flat plate electrode is press-contacted so that it can be assembled and disassembled. 2. The flat plate spacer is a plurality of flat plate spacers having different sizes, thicknesses, and cut spaces, and the flat plate electrode is a flat plate electrode having different sizes, and the two flat plate spacers sandwiching the flat plate spacer are sandwiched between the flat plate spacers. The operation chamber according to claim 1, wherein the distance between the electrodes and the chamber capacity can be easily changed by assembling the flat plate electrodes.