JPH06343478A - Micro-injection method and apparatus - Google Patents

Abstract

PURPOSE:To surely inject an extraneous substance into a cell while suppressing the damage on the cell using a micro-injection apparatus for injecting a substance into a biotissue or cell. CONSTITUTION:A plate 10 holding regularly arranged cells is placed on a micromanipulation stage and a needle 20 fixed to a fine-adjustment positioning apparatus 13 is approached to a cell 11 and slowly inserted into the cell while observing the cell with a stereo-microscope 14. A substance to be injected into the cell is preparatorily applied to the tip of the needle by electrodeposition. A needle 22 different from the needle coated with the injection substance is inserted into the cell at a side opposite to the coated needle and a voltage is applied between the needles with a power source 15. The injection substance fixed to the needle by electrodeposition is actively released into the cell by this process.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microinjection method and an apparatus for injecting a substance into living tissues and cells.

[0002]

2. Description of the Related Art Conventionally, a glass needle has been used as a technique for injecting a gene, protein, or physiologically active substance into a living tissue or cell by a physical mechanical operation or for extracting a substance from the living tissue or cell and detecting it. Now, a mechanical microinjection device for injecting substances (for example, Experimental Biology Course 8, Cell Biology, P277-297 "Micromanipulation" Maruzen Co., Ltd., Japanese Patent Laid-Open No. 3-119989 "Microinjection device and its injection control method". There is). Moreover, as a method of injecting a substance,
Electroporation in which a cell membrane is perforated by applying an electric pulse (for example, an experimental example regarding the introduction of a thymidine kinase gene into a mouse cell by Neumann et al. (Neumann, E., Schaefer-Ridder, M., Wang, Y., Ho
fschneider, PH: EMBO J., 1, 841-845 (1982)))
Or laser microinjection to make a hole in the cell membrane by laser (eg "Gene transfer by laser cell processor" Plant Cell Engineering, Vol.3 No.2 (1991)
135-138), and further, a particle gun in which a substance is applied to fine particles and bombarded in a tissue or a cell (for example, “Transfection and Expression of Gene into Plant Cell by Particle Gun”, Plant Cell Engineering, Vol.2 No. 5 (1991) 631-6
(See 37).

[0003]

In the conventional mechanical microinjection, it is possible to inject a substance into a specific cell, but since the hollow glass capillary is used as an injecting needle, its outer diameter is changed. There was a limit to making it smaller. Therefore, when the needle is injected into the cell, the cell may be ruptured or fatally damaged, and the operation is complicated. Further, in electroporation, laser microinjection, and particle gun, a substance can be injected into a large number of cells at one time, but it is difficult to inject the substance into only specific cells.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for injecting a substance into a specific cell or a plurality of cells with minimal damage.

In order to solve the above-mentioned problems, a substance is applied to the needle tip, the substance is injected into the cell while being held at the needle tip, and the substance is released from the needle tip at the target position in the cell. To be achieved.

[0006]

In the present invention, since the substance to be injected is retained on the surface of the needle, the needle used for injecting the substance into cells can be made thinner than that used conventionally. as a result,
When the needle is injected into the cell, the hole in the cell membrane becomes smaller, which reduces damage to the cell and reduces cell destruction.

[0007]

EXAMPLE A first example of the present invention will be described with reference to the drawings.

FIG. 1 shows an overall configuration diagram for carrying out this embodiment. FIG. 2 is an enlarged schematic diagram when the needle portion is inserted into the tissue. In the figure, 1 is an XY stage, 2 is a needle support base, 3
Is a Z-direction stage, 4 is an inverted microscope, 5 is a petri dish, 6
Is a petri dish containing biological tissue, 7 is a needle, and 8 is biological tissue. Next, the operation of this device will be described. The biological tissue is fixed in the petri dish 5. The fixing method can be performed by pressing on agar or gelatin. From the bottom of the petri dish 5 on which the living tissue is fixed, the living tissue can be observed with a microscope.

The petri dish on which the living tissue is fixed is fixed at a fixed position on the XY stage. Next, the tip of the needle fixed to the needle support is immersed in a solution in the dish 6, for example, a solution containing DNA, and the tip of the needle is coated with a substance to be injected into the tissue,
dry. As a method for fixing the substance at the tip portion of the needle 7, an electrodeposition method may be used in which the solution is fixed at the tip portion of the needle by freezing or applying an electric current. Subsequently, while observing the needle 7 with an inverted microscope, the needle 7 is placed on the desired living tissue, and the needle 7 is gently lowered by the Z-direction stage 3 and inserted into the living tissue as shown in FIG. The depth of penetration depends on the target tissue. After the substance to be injected diffuses into the cells, the needle is then pulled up to its original position, the solution is applied again to the tip of the needle by the above-described method, and the needle 7 is inserted into the tissue.

The foreign substance can be injected into a large number of cells by repeating the above operation and sequentially injecting needles into different parts of the tissue.

Regarding the needle used in this apparatus, for example, a tungsten wire having a wire diameter of 25 to 50 microns is made by the electrolytic polishing method. According to this method, a needle having a diameter of several hundred angstroms can be produced. Further, by providing the depression at the tip of the needle, it is possible to hold more of the substance to be injected in the needle.

A second embodiment will be described with reference to the drawings. In this example, a method for surely injecting a foreign substance into individual cells will be described. FIG. 3 (a) is an overall view of a plate for fixing cells. 3 (b) is a front view of a part of the plate, FIG.
(C) is a sectional view of a part of the plate. FIG. 4 is a schematic view of this embodiment, FIG. 5 is a schematic view of a holder for fixing a plate, and FIG. 6 is an enlarged view of a main part.

The plate 9 for fixing cells is obtained by anisotropically etching a single crystal silicon wafer. In this plate, the cell-retaining holes 10 for fixing the cells 11 are in the shape of a skirt, and a square hole with a side of about 5 μm is opened on the bottom surface and communicates with the back surface. First, a method of fixing cells on the plate will be described with reference to FIG. The plate 9 is fixed to the plate holder 19. The plate is immersed in the buffer solution 21, and is gently sucked by the suction device 17 from the back surface of the plate. Aspirated liquid is silicone tube 1
Return to the plate holder 19 again through 8. When the cell suspension is spread on the plate 9, the cells are fixed in the cell holding holes 10 one by one. After confirming that the cells have been fixed with the stereoscopic microscope 12, excess cells are washed away. Next, the plate holder 19 is fixed to the XY stage 14.

The operation will be described with reference to FIG. While observing the needle 20 fixed to the positioning device 13 capable of finely moving these cells with the stereoscopic microscope 12, the needles 20 are slowly inserted into the cells. Here, a needle having a diameter of 0.5 micron or less, which is produced by electrolytic polishing a tungsten wire having a diameter of 25 micron, is used. A substance to be injected into cells is previously applied to the tip of the needle by an electrodeposition method. Next, as shown in FIG. 5, a needle 22 other than the needle 20 coated with the substance to be injected is inserted from the opposite side of the first needle, and a voltage is applied by a lead wire 16 from a power source 15 between both needles. The substance to be injected, which has been electrodeposited and fixed on the needle 20, is actively released into the cell from the needle 20.

Next, the needle is slowly removed from the cell, the substance to be reintroduced is applied to one needle, and the same operation is performed on the next cell. If you want to surely inject foreign substances into many cells at the same time, fix the cells on the plate,
This can be achieved by injecting into each cell needles arranged regularly at a pitch corresponding to the fixed cells.

[0016]

As described above, according to the present invention, damage to cells can be reduced, so that success rate at the time of injecting a substance into specific cells can be improved.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an entire example 1.

FIG. 2 is a schematic diagram showing a main part of the first embodiment.

3 (a), (b) and (c) are a schematic view of a cell fixing plate used in Example 2, a front view of the cell fixing plate and a sectional view of the cell fixing plate.

FIG. 4 is a schematic diagram showing an entire example 2.

FIG. 5 is a schematic diagram showing a main part of the second embodiment.

FIG. 6 is an enlarged view of a main part of the second embodiment.

[Explanation of symbols]

1 ... XY stage, 2 ... needle support, 3 ... Z direction stage,
4 ... Inverted microscope, 5 ... Petri dish, 6 ... Petri dish containing biological tissue, 7 ... Needle, 8 ... Living tissue, 9 ... Plate, 1
0 ... Cell holding hole, 11 ... Cell, 12 ... Stereomicroscope, 13
... Positioning device capable of fine movement, 14 ... XY stage, 15 ... Power supply, 16 ... Wire, 17 ... Suction device, 1
8 ... Silicon tube, 19 ... Plate holder, 20
... needle, 21 ... buffer solution, 22 ... needle.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location 8412-4B C12N 5/00 D

Claims (6)

[Claims] 1. A microinjection method comprising applying a nucleic acid, a protein or other physiologically active substance to the tip of a needle and inserting the needle into the target portion of cells or fine tissues to introduce the substance into the target portion. . 2. A needle having a tip coated with a nucleic acid, a protein, or other physiologically active substance, a container arranged at a position facing the needle and containing the nucleic acid, protein, or another physiologically active substance, and the needle inside the container. A microinjection device comprising means for inserting into a target portion of cells or fine tissues. 3. The microinjection device according to claim 2, wherein the injected substance is frozen and fixed on the needle surface by cooling the needle. 4. The microinjection device according to claim 2, wherein the injection substance is fixed to the needle surface by electrodeposition on the needle. 5. The microinjection device according to claim 2, further comprising a plurality of the needles. 6. The microinjection device according to claim 2, wherein a minute recess is provided at the tip of the needle.