WO2018105748A1

WO2018105748A1 - Method for introducing foreign substance into cell using laser

Info

Publication number: WO2018105748A1
Application number: PCT/JP2017/044259
Authority: WO
Inventors: 恵彦祐村
Original assignee: 国立大学法人山口大学
Priority date: 2016-12-09
Filing date: 2017-12-08
Publication date: 2018-06-14
Also published as: JPWO2018105748A1; JP6981666B2

Abstract

The present invention addresses the problem of providing a method which enables the introduction of a foreign substance into a single cell with high efficiency and easily and is less likely to be influenced by the damage of the cell. A cell is left standing on a thin film which coats the upper surface of a coverslip and which is made from any one metal selected from gold, platinum, silver and aluminum, then a liquid containing a foreign substance to be introduced is brought into contact with the cell, and then laser light is emitted onto the thin film adjacent to the cell to cause the absorption of the laser light by the thin film. As a result, a fine pore is formed in the cell membrane, and the foreign substance is introduced into the cell through the fine pore.

Description

Method for introducing foreign substances into cells using laser

The present invention relates to a method for introducing a foreign substance into a cell by forming pores in a cell membrane by laser light irradiation.

Introducing a foreign gene or the like into a cell to produce a cell or organism having a new trait or bioimaging analysis in which a fluorescent dye is introduced into the cell to measure the function in the cell by fluorescence. In order to differentiate iPS (artificial pluripotent stem) cells, which have been researched in recent years, into cells having arbitrary differentiation traits, a substance introduction technique into the cells is essential.

Conventional methods for introducing genes and proteins into cells include methods using transfection reagents, methods using viruses, electroporation methods, particle gun methods, sonoporation methods, liposome fusion methods, methods using micromanipulators, etc. Has been used.

The method using a transfection reagent is a method in which a hydrophilic gene or protein is covered with a fat-soluble reagent so that the gene or protein can pass through a cell membrane made of lipid. Although the introduction efficiency is high, it is not applicable to all cells, and there is a problem that it cannot be applied to primary cultured cells, nerve cells, blood cells, slime mold cells, and the like. In addition, since transfection reagents remain in cells, there are concerns about cytotoxicity and carcinogenicity. In addition, commercially available transfection reagents do not disclose the components of the introduction agent. There is a problem that it cannot be used by humans for therapeutic purposes, and it is necessary for humans to check the safety of foods using plants introduced and modified using such transfection reagents.

The method using a virus is a method of introducing a gene into a virus and introducing it into a cell by infecting the cell. The problem is that it cannot be applied to all cell types and is limited to cells that can be infected by viruses, and because some of the virus remains in the cells, cells modified by this method cannot be used in medicine or food. There was a problem.

Electroporation is a method in which pores are formed in a cell membrane by passing a current transiently through the cell, and a gene or protein in a liquid brought into contact with the cell membrane is introduced into the cell. Although the operation is relatively simple, a dedicated device is required, the cell viability is as low as 10-50%, a large amount of cells and a large amount of foreign substances are required, and the introduction efficiency is 1 There was a problem that it was as low as × 10 ⁻⁴ % or less.

The particle gun method is mainly used for plant cells, and is a method in which genes and the like are attached to metal particles and introduced while passing the cells at a high speed with a dedicated gun. The introduction efficiency was as low as about 1 × 10 ⁻⁷ % or less, and the survival rate was as low as 20% or less.

The sonoporation method is a method in which a hole is made in a cell membrane with an ultrasonic wave and a gene or the like in an external solution is introduced until the hole is closed. The introduction efficiency was as low as about 1 × 10 ⁻⁷ % or less, and the survival rate was as low as 20% or less.

The liposome fusion method is a method of introducing a gene or protein inside a liposome into a cell by placing a gene or protein inside a lipid liposome (vesicle) and fusing the cell and the liposome into a membrane. Although versatility and simplicity are high, there is a problem that preparation of liposomes is difficult and preparation takes time.

The method using a micromanipulator is a method of introducing a foreign substance by direct microinjection into cells using a micromanipulator. It is a classic physical introduction method to one target cell, and is used for a relatively large cell such as an egg cell. However, the problem is that advanced operation techniques are required and introduction takes time, the introduction efficiency and survival rate depend on the technology of the person performing the introduction work, and many cells (for example, 10 cells). There was a problem that the introduction was extremely time consuming and labor intensive.

In addition, as a method for introducing a gene or protein into a cell, a method of forming a hole in a cell membrane by laser light irradiation is also used.

For example, a cell or living tissue is irradiated with laser light through an optical fiber, and the cell wall and / or cell membrane or whole cell of the irradiated cell is cut, removed or opened, and the cell and / or living tissue is passed through the irradiation site. A method of introducing a foreign substance into a cell (see Patent Document 1) or placing a small particle carrying a foreign substance on a part of the cell surface of a living cell, and irradiating a part of the cell surface with laser light for processing. Has disclosed a method for introducing a foreign substance into the living cell at the same time as perforating the cell wall and / or cell membrane (see Patent Document 2). However, a laser that is strong enough to cause perforation in the cell wall and / or cell membrane is disclosed. Since the cells are directly irradiated, there is a possibility that damage in the cells irradiated with the laser light is increased. In addition, cells have a three-dimensional and complicated three-dimensional structure, and are constantly changing in shape, and it is difficult to irradiate a laser beam to a specific location on the cell membrane. There was a problem that it was difficult to introduce the substance.

Further, the cells are arranged in a liquid, a gel, or a gel surface in which a target substance to be introduced is present, and the liquid, the gel, or the cells are irradiated with a focused laser beam, and the cells are generated by a shock wave generated thereby. Discloses a method of temporarily changing the structure of the cell membrane and introducing the substance into cells (see Patent Document 3). However, irradiating a laser beam aiming at fine particles or near the fine particles requires not only a high level of technology, but also has a problem that introduction efficiency is low, and it is necessary to use a large and expensive femtosecond laser.

Furthermore, in a living tissue, the drug is localized in the vicinity of a cell into which the drug is introduced, and a laser beam having a wavelength that is absorbed by a light absorber existing in the vicinity of the cell is irradiated to the cells of living organisms other than humans. A drug introduction method (see Patent Document 4) characterized by introducing a drug is disclosed. However, this is a method of directly irradiating a living tissue with laser light, and there is a problem that it is necessary to inject a drug to be introduced by a syringe into the living tissue before laser light irradiation.

In addition, the cells are allowed to stand on the thin film of the cover slip whose upper surface is coated with a thin film made of a light condensing agent, and a liquid containing a foreign substance to be introduced is brought into contact with the cells to form a thin film adjacent to the cells. On the other hand, a method is disclosed in which a laser beam is irradiated to form pores in a cell membrane, and the foreign substance is introduced into the cells from the pores (see Patent Document 5 and Non-Patent Document 1). However, for example, when carbon is used as a light condensing agent, carbon absorbs about 10% of transmitted light, and there is a problem in that fluorescence loss occurs during fluorescence observation.

A) maintaining one or more cells in a substantially stationary position within an effective distance from the solid surface; and b) the one or more specific three-dimensional positions on the solid surface in advance. Directing electromagnetic radiation sufficient to induce permeation of the membrane of the one or more cells without recognition, wherein the one or more cells are aligned with the path of the electromagnetic radiation A method for making the above cells temporarily permeable (see Patent Document 6) is disclosed. However, there is a problem that not only the cell membrane but also the cell itself is damaged, and that a foreign substance cannot be introduced targeting only one cell.

By the way, in recent years, bio-based analysis techniques using the resonance plasmon effect have been advanced. For example, in an immunochromatographic analysis method in which a test substance in a sample is labeled with a labeling substance and immobilized with a capture substance and detected, a method of sensitizing the coloration of the two labeling reagents by the resonance plasmon effect (see Patent Document 6) Or a substrate, an enhanced electromagnetic field forming layer in which a large number of metal fine particles are dispersed and arranged independently of each other on the surface of the substrate, and a protective layer formed on the substrate and the enhanced electromagnetic field forming layer. A sensor (see Patent Document 7) including a sensor chip constituting an enhancement element is disclosed.

JP 2002-281970 A JP 2002-325572 A JP 2005-168495 A JP 2005-330194 A JP2015-167551A JP 2009-162558 A JP 2014-228322 A

In bioimaging analysis, observation at the single cell level is necessary, but in the conventional method of introducing a foreign gene into a cell, a pore is formed in the cell membrane aiming at a specific single cell and Therefore, it has been difficult to introduce a substance into a cell easily and efficiently without being affected by damage. Accordingly, an object of the present invention is to provide a method that can introduce foreign substances into single cells with high efficiency and easily, and that is less affected by damage to cells.

The inventors of the present invention have been diligently studied to solve the above-mentioned problems. The surface of the coverslip is coated with a thin film of gold or platinum, the cells are allowed to stand on the thin film, and the thin film adjacent to the cells is coated. It was found that by irradiating the thin film with laser light and absorbing the laser light into the thin film, it is possible to form pores in the cell membrane with little influence by damage to the cells. Furthermore, the present invention has been completed by discovering that a foreign substance to be introduced into the cell is introduced into the cell by bringing a liquid containing the foreign substance to be introduced into the cell into contact with the cell.

That is, the present invention is as follows.
(1) A method for introducing a foreign substance into a cell, wherein the cell is placed on the thin film of a coverslip having a top surface coated with a thin film of any metal selected from gold, platinum, silver and aluminum. Then, a liquid containing a foreign substance to be introduced is brought into contact with the cells, and a laser beam is irradiated to the thin film adjacent to the cells to absorb the laser light, thereby forming pores in the cell membrane. A method for introducing a foreign substance into a cell, wherein the foreign substance is introduced into the cell from the pore.
(2) The method for introducing a foreign substance into a cell as described in (1) above, wherein the thin film adjacent to the cell is irradiated with laser light from the lower surface of the cover slip.
(3) The method for introducing a foreign substance into cells according to (1) or (2) above, wherein the thin film is irradiated with laser light through an objective lens.
(4) The method for introducing a foreign substance into cells according to any one of (1) to (3) above, wherein the spot diameter of the laser beam is 0.2 μm to 2 μm.
(5) The method for introducing a foreign substance into cells according to any one of the above (1) to (4), wherein a cover slip having an upper surface coated with a thin film of gold or platinum is used.
(6) The method for introducing a foreign substance into cells according to any one of the above (1) to (5), wherein the thin film is formed by sputtering.
(7) Any of (1) to (6) above, wherein the foreign substance is any one or more of DNA, RNA, protein, polypeptide, amino acid, saccharide, lipid, drug, and fluorescent dye A method for introducing a foreign substance into the cell.
(8) A transformed cell obtained by the method for introducing a foreign substance into a cell according to any one of (1) to (7) above.
(9) A thin film made of any metal selected from gold, platinum, silver and aluminum on the upper surface for use in the method for introducing a foreign substance into cells according to any one of (1) to (7) above Is coated coverslip.

According to the method for introducing a foreign substance into a cell of the present invention, it is possible to introduce the foreign substance into a single cell with high efficiency and little influence due to damage to the cell.

(A) It is explanatory drawing of the method of irradiating a laser beam with respect to the thin film which adjoined the cell membrane from the lower surface of the cover slip. (B) It is a figure which shows a mode that the pore was formed in the cell membrane. (C) It is a figure which shows a mode that the foreign substance was introduce | transduced in the cell from the pore. It is the schematic which irradiates a laser to the cell left still in the glass bottom dish. It is the schematic of pulse laser beam irradiation by a pulse laser beam irradiation apparatus. (A) It is a figure which shows the observation result (when using the cover slip which coated the thin film of gold | metal | money) by the computer image of the cell which introduce | transduced the fluorescent dye in Example 1. FIG. (B) It is a figure which shows the relative fluorescence amount of the cell which introduce | transduced the fluorescent pigment | dye in Example 1. FIG. (C) It is a figure which shows the observation result (when using the cover slip which coated the thin film of platinum) by the computer image of the cell which introduce | transduced the fluorescent pigment | dye in Example 1. FIG. It is a figure which shows the observation result (when using the cover slip which coated the thin film of gold | metal | money) by the computer image of the cell which introduce | transduced the fluorescent pigment | dye in Example 2. FIG. It is a figure which shows the fluorescence observation result by the microscope of the cell which introduce | transduced the plasmid in Example 3. FIG. (A) shows a phase contrast micrograph, and (b) shows a fluorescence micrograph of the same field of view. It is a figure which shows the observation result of the cover slip in a reference example.

The method for introducing a foreign substance into a cell according to the present invention is a method for introducing a foreign substance into a cell, wherein a thin film of any metal selected from gold, platinum, silver and aluminum is coated on the upper surface. A cell is allowed to stand on the thin film of the cover slip, a liquid containing a foreign substance to be introduced is brought into contact with the cell, a laser beam is irradiated to the thin film adjacent to the cell, and the thin film is irradiated with the laser beam. There is no particular limitation as long as it is a method for introducing a foreign substance into a cell, characterized in that it forms a pore in the cell membrane by absorption and introduces the foreign substance into the cell from the pore. According to the method, it is possible to introduce a foreign substance into a single cell with high efficiency and with little influence by damage to cells.

In the present invention, the cell may be a single cell, a tissue cell, or a plurality of cultured cells, and the types of cells are protists, animal cells, plant cells, bacteria, yeasts. Can be illustrated.

Examples of protists include cellular slime molds, amoebae, algae, and ciliates, and suitable examples of cellular slime molds include cells of Dictyostelium discoideum. When using animal cells, examples of the origin include humans, monkeys, mice, rats, hamsters, rabbits, goats, sheep, horses, pigs, dogs and other mammals, and the types of cells are primary cells, Mammalian cells such as cell lines, fertilized eggs, and nerve cells can be exemplified. When animal cells are used, adherent cells or suspension cells may be used, but adherent cells can be preferably exemplified. Examples of plant cells include cells derived from Nicotiana tabacum and Arabidopsis thaliana. Examples of bacteria include Escherichia coli, archaea, and mycoplasma.

In the present invention, the foreign substance is not particularly limited, but is preferably one or more of DNA, RNA, peptide, amino acid, saccharide, lipid, drug, and fluorescent dye, and any of DNA, RNA, and fluorescent dye More preferably, it is one or more substances. Further, these substances may be used alone or in combination as foreign substances, and these substances may be used by being bonded to the surface of fine particles such as metals, inorganic substances, and organic polymers.

The thin film in the present invention may be formed of any metal selected from gold, platinum, silver and aluminum, and is preferably formed of gold or platinum, and is formed of gold from the viewpoint of permeability. More preferably. The thin film formed of the metal may be formed of the metal particles. By irradiating the irradiated laser light with a metal selected from gold, platinum, silver and aluminum, the cell membrane is close to the position where it was absorbed with low-power laser light without affecting the cells. It becomes possible to form pores. The pores are quickly closed by the cell membrane repair mechanism inherent to the cell. Examples of the coating method of any metal selected from gold, platinum, silver, and aluminum include a method of coating by sputtering or vacuum deposition, and a method of coating by applying the above metal particles. The sputtering method is preferable from the viewpoint of the ease of the manufacturing process.

In the present invention, examples of the thickness of the thin film include 5 nm to 200 nm, preferably 8 nm to 50 nm, and more preferably 10 nm to 20 nm. If the thickness of the thin film exceeds 200 nm, the transparency of the cover slip is reduced, which hinders microscopic observation of cells into which foreign substances have been introduced, and if it is less than 5 nm, the ability to absorb laser light is insufficient. Sufficient pores will not be formed in the cell membrane for introducing foreign substances. The thickness of the thin film and the output of the laser beam that forms pores in the cells are inversely proportional, and it is necessary to increase the output if the thickness of the thin film is reduced. Can be adjusted according to.

In the present invention, the thin film has an OD value of 0.005 to 0.1, preferably 0.01 to 0.08 when measured with a spectrophotometer (532 nm).

Further, the average particle diameter of the metal particles selected from gold, platinum, silver and aluminum is not particularly limited, but may be 2 nm to 200 nm, preferably 5 nm to 100 nm.

The thin film may be coated with collagen, gelatin, fibronectin, elastin, or other extracellular matrix as necessary for cell growth. Moreover, when using a floating cell as a cell, it is preferable to coat the upper surface of the thin film with polylysine, polyethyleneimine, spermidine, spermine, or other polycations.

In the present invention, the material of the cover slip is not particularly limited, but glass, polystyrene, and polymethacrylamide may be exemplified from the viewpoint of permeability when observing under a microscope. Further, the thickness of the cover slip is not particularly limited, but may be 0.05 mm to 0.3 mm.

In the present invention, leaving the cells on the thin film means that the cells are placed on the stationary thin film. A cell has a three-dimensional and complicated three-dimensional structure, and is constantly changing its shape. However, when the cell is left on the thin film as described above, a part of the cell membrane comes into contact with the thin film. As a result, the cell membrane on the surface in contact with the thin film becomes flat and has almost no morphological change, and it becomes easy to irradiate laser light aiming at a certain place of the cell membrane.

In the present invention, the method of allowing the cells to stand still and bringing the liquid containing the foreign substance to be introduced into contact with the cells includes culturing the cells on a thin film, and then removing the supernatant medium and culturing containing the foreign substance A method of adding the liquid can be exemplified. In many animal cultured cells, it is common to create suspension cells by removing them from the substrate where the cells are cultured by adding a divalent cation chelating agent such as enzyme treatment or EDTA. Cost. Therefore, in order to eliminate the need for the above work, for example, cells are cultured in advance in a glass bottom dish, which will be described later, and a small amount of a culture solution containing a foreign substance except for the supernatant culture solution may be added.

In addition, the cells are suspended in a liquid, and the suspension is added onto the thin film. After the cells adhere or contact the thin film by gravity, a liquid containing a foreign substance is dropped or injected into the suspension to A method of making the cell covered with a liquid containing a foreign substance, or suspending cells in a liquid containing a foreign substance in advance, adding the suspension to the thin film, and allowing the cell to adhere to or contact the thin film, so that the cell is foreign The method of making it the state covered with the liquid containing a substance can be illustrated. Such a method is preferable in the case of cells that can be easily peeled off from the substrate and are easier to be suspended, such as slime mold cells or blood cells. Further, in this method, when the number of cells is small, the liquid containing the foreign substance may be a small amount of about 10 μl to 30 μl. Therefore, it is preferable when an expensive or rare foreign substance is used.

The concentration of the foreign substance in the liquid containing the foreign substance to be introduced can be appropriately adjusted depending on the amount of the foreign substance to be introduced and the number of cells, but is 1 ng / ml to 10 mg / ml, preferably 0.1 μg / ml to 100 μg. / Ml.

The light source of the laser beam used in the present invention is not particularly limited, and examples thereof include a pulse laser and a continuous wave (CW) laser, but a pulse laser is preferable.

The wavelength of the laser beam used for irradiation of the thin film used in the present invention is not particularly limited, but examples thereof include 380 nm to 1100 nm, preferably 450 nm to 700 nm. By irradiating laser light, pores are formed in the cell membrane due to heat and plasmon effect, but by setting it to 450 nm to 700 nm, it becomes possible to easily cause plasmon effect, and laser light is irradiated at a lower output. Even so, pores are formed in the cell membrane. Further, the frequency of the laser beam can be 0.1 KHz to 100 KHz, preferably 1 KHz to 10 KHz, and the output of the laser beam is within a range where pores can be formed without being affected by damage. Preferably, 0.1 mW to 20 mW, more preferably 0.5 mW to 5 mW, and still more preferably 0.8 mW to 1.8 mW can be exemplified. Note that, as described above, the thickness of the thin film and the output of the laser beam that forms pores in the cells are inversely proportional, and the output of the laser beam can be adjusted according to the thickness of the thin film.

The number of times of laser light irradiation is not particularly limited, and may be one or more times depending on the amount of foreign substance to be introduced and the type of foreign substance. In addition, when introducing a plurality of foreign substances into cells, a liquid containing a plurality of foreign substances to be introduced may be brought into contact with the cell membrane and introduced into the cells by a single laser beam irradiation. The liquid containing the foreign substance to be introduced every time may be replaced, and each liquid may be brought into contact with the cell membrane and sequentially introduced into the cell by multiple times of laser light irradiation.

In the present invention, the laser beam may be irradiated to the thin film adjacent to the cell from the upper surface of the cover slip or to the thin film adjacent to the cell from the lower surface of the cover slip. From the viewpoint of reducing cell damage, it is preferable to irradiate the thin film adjacent to the cell from the lower surface of the cell. In the present invention, irradiating the thin film adjacent to the cell with the irradiation laser light from the lower surface of the cover slip means that the laser light from the lower surface of the cover slip is applied to a part of the thin film in the region where the cell is adjacent. Refers to irradiation. Moreover, when irradiating a laser beam, it is preferable to previously focus the laser beam irradiation on the thin film. FIG. 1A shows a method of irradiating a part of the thin film 2 in the region 4 where the cells 7 are close to the laser beam from the lower surface of the cover slip 1.

As a result of the irradiation, the thin film 2 and / or the cell membrane 3 of the cell 7 absorbs the laser light, and the energy of the laser light is concentrated on the thin film 2 and / or the cell membrane 3, as shown by the arrow in FIG. The pore 5 is formed in the cell membrane 3, and the foreign substance 6 can be introduced into the cell from the pore 5 by passive diffusion as shown in FIG. 1 (c). Since the formed pores are repaired and closed in a short time, the formation of the pores hardly affects the growth of the cells. In the present invention, the formation of pores in the cell membrane is not included in cell damage.

In the present invention, the laser beam irradiation is preferably performed through the objective lens on the thin film. The pupil diameter of the laser light when passing through the objective lens can be 3 to 18 m, preferably 5 to 15 mm, more preferably 11 to 13 mm.

In the present invention, the spot (focal point) diameter of the laser beam when irradiating the thin film close to the cell with the laser beam is not particularly limited, but may be 0.2 μm to 2 μm, more preferably 0. .5 μm to 1.5 μm can be exemplified.

As the cover slip in which the upper surface of the present invention is coated with a thin film of any metal selected from gold, platinum, silver and aluminum, the cells are allowed to stand on the thin film of the cover slip, and foreign substances to be introduced are introduced. Forming pores in the cell membrane by contacting the contained liquid with the cells, irradiating the thin film adjacent to the cells with laser light and absorbing the laser light into the thin film; The cover slip for use in the method for introducing a substance into the cell is not particularly limited as long as a thin film of any metal selected from gold, platinum, silver and aluminum is coated on the upper surface. By using a cover slip coated with a thin film of any metal selected from gold, platinum, silver and aluminum on the top surface of the present invention, the method for introducing a foreign substance into cells of the present invention is highly efficient, and Can be done easily.

As the use of the cover slip in which the upper surface of the present invention is coated with a thin film of any metal selected from gold, platinum, silver and aluminum, any metal selected from gold, platinum, silver and aluminum is used on the upper surface. The cells are allowed to stand on the thin film of the coverslip coated with the thin film, the liquid containing the foreign substance to be introduced is brought into contact with the cells, and the thin film adjacent to the cells is irradiated with laser light to The upper surface is selected from gold, platinum, silver and aluminum for use in a method of forming a pore in the cell membrane by absorbing a laser beam into a thin film and introducing the foreign substance into the cell from the pore. There are no particular restrictions on the use of a cover slip coated with any metal thin film, but a cover coated with gold or platinum It is preferable that the lip.

The transformed cell obtained by the method for introducing a foreign substance into a cell of the present invention includes the cover slip of which the upper surface is coated with a thin film made of any metal selected from gold, platinum, silver and aluminum. The cells are allowed to stand on the thin film, a liquid containing a foreign substance to be introduced is brought into contact with the cells, and the thin film adjacent to the cells is irradiated with laser light to absorb the laser light. The cell is not particularly limited as long as it is a transformed cell obtained by forming pores in the cell membrane and introducing the foreign substance into the cells from the pores. In addition to being useful as a cell having a characteristic, it is useful for analyzing the function of a cell by bioimaging.

[Introduction of fluorescent dye into cells (cellular slime molds)]
(Preparation of a cover slip coated with a thin gold film)
Using an ion sputtering apparatus (manufactured by Sanyu Electronics Co., Ltd.), with a gold (Au) target, a cover slip No. of 0.17 mm in thickness under the conditions of 14 mA, 20, 30, 35, 40, 50, or 60 seconds. A cover slip in which a gold thin film was coated on the upper surface of No. 1 (manufactured by Matsunami Glass Industry Co., Ltd.) was produced. The thicknesses of the prepared gold thin films were 6.6, 10, 11.6, 13.3, 16.6, and 20 nm in the order of 20, 30, 35, 40, 50, and 60 seconds, respectively. Further, when the absorbance of the transmitted light was examined with a spectrophotometer, all of them were below the measurement limit at 532 nm. On the other hand, in the case of a cover slip coated with a thin film made of carbon produced by the method of Patent Document 5, the absorbance of transmitted light based on OD532 nm was 10%. Therefore, it was clarified that the cover slip coated with the gold thin film has less loss of fluorescence, and the fluorescence signal is not lost in fluorescence observation, and the SN (signal / noise) ratio can be increased.

(Preparation of a cover slip coated with a platinum thin film)
Using a magnetron sputtering apparatus (JFC-1600: manufactured by JEOL Ltd.), platinum (Pt) as a target, 20 mA-10 seconds, 20 mA-20 seconds, 30 mA-20 seconds, 30 mA-30 seconds, 40 mA-20 seconds, Or a cover slip No. of 0.17 mm in thickness under the condition of 40 mA-30 seconds. Sputtering platinum particles on the upper surface of 1 (manufactured by Matsunami Glass Industrial Co., Ltd.), six types of cover slips coated with a platinum thin film were produced. When the produced cover slip was measured with a spectrophotometer (532 nm), the OD values were 0.002, 0.018, 0.026, 0.039, 0.058, and 0.062, respectively.

(Production of glass bottom dish)
As shown in FIG. 2, a glass bottom dish 9 is produced by making a hole with a diameter of 12 mm in the bottom of the plastic dish 8 and attaching the cover slip 1 with the adhesive so that the side coated with the thin film 2 is inside (upper side). did.

(Leave cells)
A cell culture medium containing cells of Dictyostelium discoideum, which is a kind of cellular slime mold, was added to a sterilized glass bottom dish 9, so that the cells of the yellow mold were left on a gold thin film. In addition, since there is a culture solution around the cells of the static mold that has been allowed to stand, the cells of the mold are in a state where they can grow.

(Pulse laser light irradiation to the thin film from the bottom surface of the cover slip)
FIG. 3 shows a method of irradiating the thin film from the lower surface of the cover slip with the FDSS532-Q pulse laser light irradiation device (manufactured by Crylas). The pulse laser beam 11 introduced from the laser 10 passes through the ND filter 12 having a variable transmittance, the lens 13, the shutter 14, the dichroic mirror 15, and the objective lens 16 to form a thin film from the lower surface of the cover slip 1 attached to the glass bottom dish 9. 2 is irradiated. When the pore 5 is formed in the cell 7, the laser beam 11 is irradiated from the lower surface of the cover slip 1 to the thin film 2 in the area adjacent to the cell 7 and the pore is not formed in the cell 7. Irradiates the thin film 2 in a region where the cells are not in proximity from the lower surface of the cover slip 1 with the laser beam 11. The glass bottom dish 9 can be slid in the horizontal direction by moving the stage of an inverted microscope. The position of the cell 7 and the absorption position of the pulse laser beam 11 can be adjusted, or the pulse laser beam 11 can be observed while observing the cell 7. Can be irradiated. The irradiation time was controlled by the shutter 14, and the output of the pulse laser beam 11 was controlled by the ND filter 12 with variable transmittance. When the pores 5 were formed in the cells 7, an ND filter with variable transmittance was used, and when adjusting the focus, irradiation was performed without the ND filter with variable transmittance. One laser irradiation time was 1/125 seconds. In the case where an upright microscope is used instead of the inverted microscope, the pulse laser beam may be irradiated from the upper surface of the cover slip in the same flow as described above.

(Adjusting the focus of pulsed laser light irradiation)
On the stage of an inverted microscope (IX70I manufactured by Olympus), the above-mentioned yellow-spotted mold was placed, the cultured glass bottom dish 9 was placed, and the focal point of the objective lens 16 (ApoN 60 ×: manufactured by Olympus) was spotted ( The focal point was adjusted to the surface of the cover slip 1 so that the diameter was 0.5 μm. Next, the laser beam 11 (532 nm, 15 mW, 1 nsec pulse, 4.8 KHz) is irradiated from the lower surface of the cover slip 1 to the thin film 2 in the region where the cell membrane is not close, and the position where the thin film 2 is peeled is used as an index. The focal point of irradiation with the pulse laser beam 11 was adjusted to the thin film 2 so that the spot (focal point) diameter was 1 μm. The pupil diameter of the laser beam that passed through the objective lens was 12 mm. In the following examples, the cover slip was coated under the condition of 14 mA-35 seconds as the cover slip coated with the gold thin film, and the cover slip coated with the platinum thin film was performed under the condition of 30 mA-20 seconds. The coated one was used.

(Pulse laser light irradiation)
Fluorescent dye 17 (PI (propidium iodide: manufactured by Dojin Chemical Co., Ltd.)) as a foreign substance is added to the culture solution in the glass bottom dish 9 so as to be 0.1 mg / ml, and a liquid containing the fluorescent dye 17 is added to the cell membrane 3. A part of the thin film 2 in a region close to the cell 7 is irradiated with a pulse laser beam 11 (532 nm, 15 mW, 1 nsec pulse, 4.8 KHz) by the method shown in FIG. The number of cells irradiated with 100 was 100. At the time of irradiation, the output was reduced to 1.5 mW using an ND filter with variable transmittance, and the time of one irradiation was 1/125 seconds. Using a plastic dish 9 to which a cover slip 1 that is not coated with a thin film is pasted, as in the above, from standing on the cover slip 1 of the yellow mold fungus, a region close to the cell 7 Up to the irradiation of the pulse laser beam 11. The observation of the cell 7 and the irradiation of the pulse laser beam 11 can be performed with the naked eye through the eyepiece 18 or a camera (ORCA-ER Hamamatsu Photonics) ) Was connected and images were acquired on a computer.

4 (a) and 4 (c) show representative examples of observation results by computer images from the lower surface direction of cells irradiated with pulsed laser light. (A) is a case where a cover slip coated with a gold thin film is used, and (c) is a case where a cover slip coated with a platinum thin film is used. The numerical values in the photographs in FIGS. 4A and 4C indicate the elapsed time (seconds) when the time of irradiation with the pulse laser beam is 0.0 second. Moreover, the graph which calculated | required the relative fluorescence amount from the cell photograph of Fig.4 (a) is shown in FIG.4 (b). In FIG. 4B, the horizontal axis indicates the elapsed time (seconds) when the laser beam irradiation time is 0.0 second, and the vertical axis indicates the relative fluorescence amount. Relative fluorescence was quantified using ImageJ software (National Institutes of Health: NIH).

(result)
As shown in FIG. 4, the amount of fluorescence in the cell increased after irradiation with laser light. In addition, intracellular fluorescence was observed in 99 cells out of 100 cells irradiated with laser light. Furthermore, as a result of observing the movement of the cells before and after laser light irradiation with a microscope, the change in cell morphology after laser light irradiation was not different from the normal cell morphology change before laser light irradiation. Further, the introduced fluorescent dye was maintained as it was and did not leave the cells even after several hours. Although not shown in the figure, when the above control cover slip that was not coated with a thin film was used, no pores were formed in the cell membrane, and there was no change in the amount of fluorescence in the cell.

Therefore, it was clarified that pores were formed in the cell membrane and the fluorescent dye was introduced into the cell with very high efficiency by the method for introducing a foreign substance into the cell of the present invention. Furthermore, it has been clarified that the method for introducing a foreign substance into a cell of the present invention has little influence due to damage to the cell.

In addition, in the method for introducing a foreign substance into a cell of the present invention, the cell is placed on the thin film, and the cell membrane adjacent to the thin film is located on the thin film without a large change in shape. It was easy to irradiate with a pulsed laser beam aiming at the place.

[Introduction of fluorescent dye into cells (Cos-1)]
The same method as in Example 1, except that Cos-1 cells derived from African green monkeys were used instead of cellular slime molds, and FM4-64 was added as a fluorescent dye as a foreign substance instead of PI so as to be 10 μM. A fluorescent dye was introduced into the cells. As the cover slip, a cover slip coated with a gold thin film was used. The results are shown in FIG. The numerical value in the photograph of FIG. 5 indicates the elapsed time (second) when the time of irradiation with the pulsed laser beam is 0.0 second, and the arrow indicates the position where the laser is spotted.

As shown in FIG. 5, it was confirmed that the fluorescent dye FM4-64 was incorporated into the cells even when Cos-1 cells were used. Therefore, it has been clarified that the method for introducing a foreign substance into cells of the present invention can also be used in mammalian cells.

[Introduction of plasmid into cells]
(Pulse laser light irradiation)
In the same manner as described in Example 1, a cell bottom dish was placed on the stage of an inverted microscope (IX70I Olympus), and then a cultured glass bottom dish was placed, and an objective lens (ApoN 60 ×: The focus of Olympus was adjusted to the surface of the cover slip so that the spot (focal point) diameter was 0.5 μm, and the focus of the pulse laser beam irradiation was adjusted to the gold thin film. Next, the GFP-lifeact plasmid prepared by the present inventors using the pBIG expression vector (Ruppel et al., Journal of Biological Chemistry, 269: 18773-187780, 1994) as a foreign substance in the culture solution in the glass bottom dish. (Yumura, S. Scientific Reports, 6: 22055 (2016)) is added at 0.05 mg / ml to bring the cell membrane into contact with a liquid containing the GFP-lifeact plasmid, and then the region close to the cells A part of the gold thin film was irradiated with a pulse laser beam (532 nm, 15 mW, 1 nsec pulse, 4.8 KHz) by the method shown in FIG. The pupil diameter of the laser beam that passed through the objective lens was 12 mm. The number of cells irradiated with pulsed laser light was 12. At the time of irradiation, the output was reduced to 1.5 mW using an ND filter with variable transmittance, and the irradiation time for one time was 1/125 seconds. One week after the introduction of the GFP-lifeact plasmid into the cells by pulse laser light irradiation, the expression of GFP-lifeact was observed with a fluorescence microscope IX71 (Olympus). FIG. 6 shows the observation results after 1 week of 1 cell out of 12 cells into which the GFP-actin plasmid was introduced. In FIG. 6, (a) is a phase contrast micrograph, and (b) is a fluorescence micrograph of the same field.

(result)
As shown in FIG. 6, one cell was divided to 10 cells or more, and fluorescence was observed in all the cells in the fluorescence micrograph. Although not shown in the figure, the other 11 cells into which the GFP-lifeact plasmid was introduced into the cells had similar results. Therefore, a cell type (for example, a nerve cell) in which a large amount of cells cannot be used because a gene can be introduced into the cell with high efficiency by the method of introducing a foreign substance into the cell of the present invention and there is almost no fertilized egg or divisional proliferation This is particularly effective when a foreign substance is introduced into a very valuable cell. In addition, since one cell has been divided into 10 cells or more, and cell division was observed as shown by the arrows in FIG. 6, the method for introducing a foreign substance into the cells of the present invention It has been confirmed that the cell has the ability to divide like normal cells and is less affected by damage to the cells.

[Reference example]
In order to introduce a foreign substance at the level of one cell, it is necessary to absorb the pulse laser beam in a very narrow range as compared with the cell size. Therefore, the absorption level was examined when the thin film was irradiated with pulsed laser light from the bottom surface of the cover slip whose upper surface was coated with a gold thin film.

(Confirmation of pulse laser light absorption level)
A glass bottom dish was prepared in the same manner as described in Example 1, and after standing the yellow mold on the cover slip gold thin film, the glass bottom dish was placed on the stage of an inverted microscope (IX70I Olympus). installed. Next, the focus of the objective lens (ApoN 60 ×: Olympus) is focused on the surface of the cover slip, the focus of the pulse laser light irradiation is focused on the gold thin film, and then the glass bottom dish placed on the inverted microscope is slid. The cell was brought close to the focal point of the pulse laser beam irradiation. Thereafter, pulse laser light (532 nm, 15 mW, 1 nsec pulse, 4.8 KHz) was irradiated for 1/125 seconds by the method shown in FIG. 3 without using an ND filter with variable transmittance. FIG. 7 shows the observation result of the cover slip by the inverted microscope after the laser light irradiation. In FIG. 7, the arrow represents the position where the pulse laser beam is absorbed.

(result)
In FIG. 7, the gold thin film was peeled only at the position where the pulsed laser beam indicated by the arrow was absorbed (spot (focal point diameter: 0.5 μm)), and no damage was observed in the adjacent cells. Therefore, it is clear that when the cover slip thin film with the gold thin film coated on the top surface is irradiated with pulsed laser light, the pulse laser light is absorbed in a very narrow range and the absorption level is high. became.

From the above results, when the present invention is used, different foreign substances are introduced into two adjacent cells, the reaction between these cells is examined, or two or more types of cells are mixed together as in primary culture. It is also possible to introduce a desired foreign substance only into a specific type of cell.

In addition, the amount of foreign substances introduced can be adjusted by changing the laser light irradiation time and number of times, or multiple foreign substances can be introduced into the same cell step by step. The effect can be observed.

Furthermore, in the conventional method of introducing a foreign substance into a cell, on the premise of the growth of the cell into which the foreign substance has been introduced, the cells that have been introduced with the foreign substance are observed after the growth to investigate the effects of the introduction of the foreign substance. However, according to the method for introducing a foreign substance into a cell of the present invention, it is possible to introduce a foreign substance into a single cell, so that a single cell that grows very slowly or does not grow like a nerve cell. It is possible to introduce foreign substances into the cells and observe the single cells as they are to examine the effects of the introduction of foreign substances.

In addition, if plant cells are used as cells, it is possible to form callus from single cells prepared by the method for introducing foreign substances into the cells of the present invention and to produce plant bodies modified with foreign substances. .

In addition, the production of a cover slip coated with a thin film of gold or platinum can be produced in a short time of less than 1 minute with a sputtering apparatus, and the sputtering apparatus can be easily cleaned. Therefore, the manufacturing process is simple and the cost is low as compared with the production of a cover slip coated with a carbon thin film.

According to the method for introducing a foreign substance into a cell of the present invention, it becomes possible to introduce the foreign substance into a single cell with high efficiency and without damaging the cell, so that bioimaging, It is useful in fields such as regenerative medicine and production of useful plants.

DESCRIPTION OF SYMBOLS 1 Cover slip 2 Thin film 3 Cell membrane 4 The area | region which the cell membrane adjoins 5 Pore 6 Foreign substance 7 Cell 8 Plastic dish 9 Glass bottom dish 10 Laser 11 Pulse laser beam 12 Variable transmittance ND filter 13 Lens 14 Shutter 15 Dichromic Mirror 16 Objective lens 17 Fluorescent dye 18 Eyepiece

Claims

A method for introducing a foreign substance into a cell, wherein the cell is allowed to stand on the thin film of a cover slip coated with a thin film of any metal selected from gold, platinum, silver and aluminum on the upper surface and introduced A liquid containing a foreign substance is brought into contact with the cells, and the thin film adjacent to the cells is irradiated with laser light to absorb the laser light, thereby forming pores in the cell membrane, and A method for introducing a foreign substance into a cell, wherein the foreign substance is introduced into the cell from a hole.
The method for introducing a foreign substance into a cell according to claim 1, wherein the thin film adjacent to the cell is irradiated with laser light from the lower surface of the cover slip.
3. The method for introducing a foreign substance into cells according to claim 1, wherein the thin film is irradiated with laser light through an objective lens.
The method for introducing a foreign substance into cells according to any one of claims 1 to 3, wherein the spot diameter of the laser beam is 0.2 μm to 2 μm.
The method for introducing a foreign substance into a cell according to any one of claims 1 to 4, wherein a cover slip having an upper surface coated with a thin film of gold or platinum is used.
6. The method for introducing an exogenous substance into cells according to claim 1, wherein the thin film is formed by sputtering.
The foreign substance according to any one of claims 1 to 6, wherein the foreign substance is any one or more of DNA, RNA, protein, polypeptide, amino acid, saccharide, lipid, drug, and fluorescent dye. How to introduce foreign substances.
A transformed cell obtained by the method for introducing a foreign substance into a cell according to any one of claims 1 to 7.
A cover slip having a top surface coated with a thin film made of any metal selected from gold, platinum, silver and aluminum for use in the method for introducing a foreign substance into cells according to any one of claims 1 to 7. .