Electrode for plasma type molecular introduction device and plasma type molecular introduction device

Abstract

To provide an electrode for a plasma-type molecule introduction device that can improve control over an introduction target area.SOLUTION: An electrode for a plasma-type molecule introduction device that introduces molecules into an object to be introduced using plasma generated between spaced apart electrodes includes an electrode body made of a conductor with one end as a tip, and a covering portion that covers at least the tip of the electrode body, and is made of a dielectric material. The covering portion preferably covers an electric field concentration portion of the electrode body, and the thickness of the covering portion is preferably 0.1 to 2 mm.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an electrode for a plasma-type molecule introduction device and a plasma-type molecule introduction device. [Background technology]

[0002] In genetic engineering and pharmaceutical development, molecules are introduced into target cells or target tissues (hereinafter sometimes referred to as "target cells, etc.") and the functions and physiological activities of the molecules in the target cells, etc. are tested. Molecules include polynucleotides, signal transduction proteins, transcriptional regulators, small molecule bioactive substances, drug candidates, and the like.

[0003] Methods for introducing molecules include electroporation, gene gun, ribosome, cell fusion, viral vector, etc. However, the efficiency of introducing molecules into target cells etc. by these methods for introducing molecules is still not satisfactory.

[0004] In response to these problems, Patent Documents 1 and 2 propose a molecular introduction device having a capillary electrode, a counter electrode provided on the opposite side of the capillary electrode, and a power source connected to the capillary electrode and the counter electrode. According to the inventions of Patent Documents 1 and 2, molecules can be efficiently introduced into target cells, etc., by using plasma generated between the electrodes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6189019 [Patent Document 2] Patent No. 5737828 Summary of the Invention [Problem to be solved by the invention]

[0006] However, in conventional technology, the target cells directly under the electrode are significantly affected by heat from the plasma and shock waves generated when the discharge starts, and the target cells into which molecules have been introduced (hereinafter, sometimes referred to as "introduced cells") are damaged, their survival rate decreases, or they are blown away from the area directly under the electrode. For this reason, there was a problem that it was difficult to increase the efficiency of introducing molecules into the target cells in the targeted area (introduction target area) and to increase the survival rate of the introduced cells (improving control over the introduction target area). The present invention aims to provide an electrode for a plasma-type molecule introduction device that can improve control over an introduction target area. [Means for solving the problem]

[0007] The present invention has the following aspects. <1> An electrode for a plasma-type molecular introduction device that introduces molecules into an introduction target object using plasma generated between separated electrodes, The electrode body has one end as a tip and is made of a conductor; and a covering portion that covers at least the tip of the electrode body. The electrode for a plasma-type molecule introduction device, wherein the covering portion is made of a dielectric material. <2> The covering portion covers the electric field concentration portion of the electrode body. <1> An electrode for use in the plasma-type molecular introduction device described in . <3> The thickness of the coating portion is 0.01 to 2 mm. <1> or <2> An electrode for use in the plasma-type molecular introduction device described in .

[0008] <4> A first electrode; a second electrode spaced apart from the first electrode and positioned in a direction in which a tip of the first electrode faces; an illumination field located between the first electrode and the second electrode and spaced apart from a tip of the first electrode; A power supply unit that applies a voltage between the first electrode and the second electrode; having The first electrode has an electrode body made of a conductor and a covering portion covering at least a tip of the electrode body, The plasma type molecule introduction device, wherein the covering portion is made of a dielectric material. <5> The covering portion covers the electric field concentration portion of the electrode body. <4> The plasma-type molecular introduction device according to claim 1. <6> The thickness of the coating portion is 0.01 to 2 mm. <4> or <5> The plasma-type molecular introduction device according to claim 1. Effect of the Invention

[0009] According to the electrode for a plasma-type molecule introduction device of the present invention, it is possible to improve the controllability of the introduction target region. [Brief description of the drawings]

[0010] [Figure 1] 1 is a schematic cross-sectional view of a plasma-type molecular introduction device according to a first embodiment. [Diagram 2] FIG. 4 is a schematic diagram illustrating a second electrode according to another embodiment. [Diagram 3] 1 is a photograph showing the results of Example 1. [Figure 4] 3 is a photograph showing the results of Comparative Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In this specification and claims, the use of "to" indicating a range of numerical values ​​means that the numerical values ​​before and after it are included as the lower limit and upper limit.

[0012] (Plasma-type molecular introduction device) The plasma-type molecular introduction device of the present invention (hereinafter sometimes referred to as the "molecule introduction device") has spaced electrodes and a container for the plasma-type molecular introduction device positioned between the electrodes. The molecule introduction device introduces molecules into the object to be introduced, such as a target cell, a target tissue, etc. (collectively referred to as "target cell, etc."). The molecule introduction device generates plasma between separated electrodes, irradiates an irradiation object in which the target cell, etc. and the molecule coexist, and introduces the molecule into the target cell, etc. In the molecular introduction device of the present invention,5 Since the plasma is generated under atmospheric pressure of 10 Pa, there is no need to create a vacuum environment. In addition, since the first electrode is covered with a dielectric, the current to the target cells is appropriately controlled, suppressing thermal damage to molecules and target cells caused by the plasma and shock waves at the start of discharge. Hereinafter, the molecule introduction device of the present invention will be described with reference to one embodiment.

[0013] The molecule introduction device shown in FIG. 1 has a first electrode 10, a second electrode 20, a power supply section 30, and a container 40.

[0014] The second electrode 20 is spaced apart from the first electrode 10 and is located below the lower end (tip) 10a of the first electrode 10. That is, the second electrode 20 is located ahead of the tip 10a of the first electrode 10, and the first electrode 10 and the second electrode 20 face each other. The area directly below the lower end (tip) 10a of the first electrode 10 and its vicinity may be referred to as "directly below the plasma irradiation portion."

[0015] The power supply unit 30 is connected to the first electrode 10 and the second electrode 20 by a wiring 16 .

[0016] The container 40 is located between the first electrode 10 and the second electrode 20. The container 40 is placed on the second electrode 20 and is spaced apart from the first electrode 10.

[0017] The first electrode 10 has an electrode body 11 made of a conductor and a covering portion 12 made of a dielectric material. In this embodiment, the first electrode 10 is a high voltage electrode.

[0018] The electrode body 11 is a rod-shaped body extending in one direction. The electrode body 11 may have a hollow structure such as a cylindrical or polygonal tube, or a solid structure such as a cylindrical or polygonal column. In particular, when the electrode body 11 has a solid structure, durability is increased and manufacturing is easy. When the electrode body 11 has a hollow structure, the electrode body 11 may have a structure in which the end surface facing the second electrode 20 is closed. Examples of conductors that are the material of the electrode body 11 include metals such as stainless steel, copper, and tungsten, and non-metals such as carbon. As the conductor that is the material of the electrode body 11 in this embodiment, stainless steel is preferable because it has high corrosion resistance, is relatively inexpensive, and is easy to process.

[0019] The thickness (outer diameter) R11 of the electrode body 11 is within a range of, for example, 1 to 50 mm. When the electrode body 11 is cylindrical, the outer diameter is the diameter of a circle in the cross section, and when the electrode body 11 is polygonal, the outer diameter is the diameter of a circumscribing circle in the cross section.

[0020] The first electrode 10 has a covering portion 12 at one end of an electrode body 11 . The covering portion 12 covers the surface (opposing surface) 11a facing the second electrode 20. That is, the covering portion 12 covers the tip of the electrode body 11. In this embodiment, the covering portion 12 covers a part of the side surface 11c adjacent to the opposing surface 11a. As a result, the covering portion 12 covers the edge 11b of the electrode body 11. The edge 11b is the boundary between the opposing surface 11a and the side surface 11c, and is a portion (electric field concentration portion) where an electric field concentration occurs when a voltage is applied between the electrodes in the "Method of Using a Molecule Introduction Device" described later. The covering portion 12 only needs to cover the opposing surface 11a of the electrode body 11, and preferably covers the edge 11b.

[0021] The dielectric material of the covering portion 12 is a solid dielectric. Examples of materials for the covering portion 12 include metal oxides, ceramics (excluding metal oxides), mica, thermoplastic resins, thermosetting resins, rubber, and thermoplastic elastomers. As materials for the covering portion 12 of this embodiment, metal oxides such as alumina (aluminum oxide) and ceramics are preferable because they are highly durable, can be easily formed into a film, and are unlikely to cause dielectric breakdown due to a voltage applied between the electrodes. Two or more types of materials may be used in combination for the covering portion 12. Furthermore, the covering portion 12 may have a single-layer structure or a multi-layer structure. In the case of a multi-layer structure, the materials of the layers may be the same or different from each other.

[0022] The thickness t12 of the covering portion 12 is, for example, preferably 0.01 to 2 mm, more preferably 0.1 to 0.7 mm. When the thickness t12 is equal to or greater than the lower limit, the survival rate of the introduced cells can be further increased. When the thickness t12 is equal to or less than the upper limit, the controllability of the area directly below the plasma irradiation portion can be further increased.

[0023] The length L12 of the covering portion 12 (the length from the upper end to the lower end) is not particularly limited and is, for example, 1 to 50 mm.

[0024] The second electrode 20 may be any electrode that functions as a ground electrode, and may be, for example, a flat electrode. Examples of materials for the second electrode 20 include metals such as stainless steel, copper, and tungsten, and non-metals such as carbon.

[0025] In a plan view, the area of ​​the covering portion 12 is preferably equal to or larger than the area of ​​the opposing surface 11a of the electrode body 11, and more preferably larger than the area of ​​the opposing surface 11a. When the area of ​​the second electrode 20 is larger than the opposing surface 11a, the controllability of the introduction target range can be further improved.

[0026] The power supply unit 30 only needs to apply a voltage between the first electrode 10 and the second electrode 20 and generate plasma between the two electrodes. The power supply unit 30 switches between starting and stopping the supply of electricity to the electrodes. The power supply unit 30 also adjusts the voltage and frequency applied to the electrodes. An example of the power supply unit 30 is a circuit that is connected to an external power source and includes an inverter. An example of the power supply unit 30 is a secondary battery.

[0027] In this embodiment, the container 40 functions as an irradiation field. The irradiation field may be any place where an irradiation target is positioned in a molecule introduction method described later. Therefore, when the container 40 is not a part of the configuration of the molecule introduction device 1, the second electrode 20 on which the container 40 is placed may be the irradiation field. The container 40 may be any container that can accommodate an irradiation target, such as each well of a microplate. Moreover, the upper end of the container 40 may be open, or the upper end may be partially or entirely closed.

[0028] The material of the container 40 is preferably a dielectric material such as a thermoplastic resin, a thermosetting resin, rubber, a thermoplastic elastomer, ceramics, or glass. Examples of the thermoplastic resin include polystyrene, polyolefin, and polyester.

[0029] (How to use the molecular introduction device (molecular introduction method)) An example of a method for using the above-mentioned molecule introduction device of the present invention (that is, a molecule introduction method) will be described. An example of the molecule introduction method is a molecule introduction method including a pre-incubation step, a contact step, and an irradiation step.

[0030] The pre-culture step is a step of culturing target cells etc. before introducing a molecule. When the number of target cells etc. is sufficiently large, the pre-culture step may be omitted. The method for culturing the target cells in the pre-culturing step can be a conventionally known culturing method. An example of the culturing method is a method for culturing the target cells in a container 40. For example, when a multiple well plate is used as the container 40, the inside of the wells of the multiple well plate are coated with a scaffold material, and the target cells are cultured in the medium in the coated wells. After the pre-culture, the medium is removed from each well of the container 40 to obtain the pre-cultured target cells and the like.

[0031] Examples of target cells include human-derived cells, non-human animal-derived cells, plant cells, bacterial cells, yeast cells, algae cells (excluding plant cells and bacterial cells), and fungal cells (excluding yeast cells). Human-derived cells include cells that are not intended to be returned to a human individual, and cells that are intended to be returned to a human individual for use in regenerative medicine, etc. The above-mentioned human-derived cells also include cells cultured from cells taken from a human individual.

[0032] The medium used for pre-culture is not particularly limited and may be appropriately selected depending on the purpose. The culture conditions for the pre-culture step are, for example, 30 to 40° C. and 24 to 72 hours.

[0033] The number of target cells 44 after pre-culture is appropriately determined taking into consideration the capacity of the container 40. For example, if the container 40 is a 96-well culture plate, the number of target cells 44 after pre-culture is 1×10 3 ~1×10 6 For example, if the container 40 is a 6-well culture plate, the number of target cells 44 after pre-culture is 1×10 per well. 4 ~1×10 7 It is considered to be one.

[0034] The contact step is a step in which target cells or the like and the molecule are allowed to coexist in an aqueous solution. The molecule 42 is added to the container 40 from which the medium has been removed. At this time, the molecule 42 may be made into an aqueous solution or dispersion (these may be collectively referred to as "aqueous liquid"), and this aqueous liquid may be added to the container 40. As a result, the target cells, etc. 44 and the molecule 42 coexist in the aqueous liquid, and the molecule 42 is located in the vicinity of the target cells, etc. 44.

[0035] The molecule 42 is a molecule selected for introduction into a target cell or the like 44. Examples of the molecule 42 include a polymer compound, a low-molecular-weight biologically active substance, and a drug candidate. Examples of polymer compounds include polynucleotides such as DNA and RNA, or derivatives thereof, proteins such as signal transduction proteins and transcriptional regulatory factors, or peptides, or derivatives thereof. The DNA or RNA is preferably one capable of expressing a gene in a target cell, such as a construct, a plasmid, or a phagemid. The molecule 42 may be of one type alone or of two or more types in combination.

[0036] The irradiation step is a step of irradiating plasma into the container 40 in a state in which the target cells, etc. 44 and the molecules 42 coexist in the container 40. The molecules 42 are introduced into the target cells, etc. 44 through the irradiation step.

[0037] In the irradiation step, power is supplied from the power supply unit 30 to the first electrode 10 and the second electrode 20 , and a voltage is applied between the first electrode 10 and the second electrode 20 . When a voltage is applied between the electrodes, an electric field is concentrated at the edge 11b of the electrode body 11, a discharge occurs between the first electrode 10 and the second electrode 20, and plasma is generated between the electrodes (the application of a voltage between the first electrode 10 and the second electrode 20 to generate plasma between the electrodes is also referred to as "plasma irradiation"). During plasma irradiation, a discharge occurs between the electrodes through the covering portion 12, thereby limiting the current during plasma irradiation, thereby reducing damage to target cells or introduced cells directly below the plasma irradiation portion, and increasing the survival rate of the introduced cells.

[0038] The distance L40 from the tip 10a of the first electrode 10 to the irradiation target (a liquid in which the molecules 42 and the target cells 44 coexist) is preferably 0.1 to 5 mm. When the distance L40 is equal to or greater than the lower limit and equal to or less than the upper limit, stable plasma can be generated.

[0039] The AC voltage applied between the first electrode 10 and the second electrode 20 is preferably 1 to 30 kVpp, more preferably 5 to 20 kVpp. When the AC voltage is equal to or higher than the lower limit, the introduction efficiency can be further improved. When the AC voltage is equal to or lower than the upper limit, damage to the target cells or the introduced cells during plasma irradiation can be suppressed, and the survival rate of the introduced cells can be further improved. The unit "Vpp (Volt peak to peak)" representing the AC voltage is the potential difference between the maximum value and the minimum value of the AC voltage waveform.

[0040] The frequency of the alternating current applied between the first electrode 10 and the second electrode 20 is preferably 1 to 500 kHz, more preferably 10 to 200 kHz. When the frequency is equal to or higher than the lower limit, the introduction efficiency can be further increased. When the frequency is equal to or lower than the upper limit, the survival rate of the introduced cells can be further increased.

[0041] The plasma irradiation time in the irradiation step is, for example, preferably 10 nanoseconds to 1 second, and more preferably 100 to 0.1 seconds. When the irradiation time is equal to or more than the above lower limit, the efficiency of introducing the molecules 42 into the target cells 44 can be further increased. When the irradiation time is equal to or less than the above upper limit, the survival rate of the introduced cells can be further increased.

[0042] In the irradiation step, the environment inside the container 40 is not particularly limited, but may be, for example, an environment in which CO2 is present. The CO2 concentration is, for example, 0.1 to 10% by volume.

[0043] The method for introducing a molecule may include a post-culture step after the irradiation step. The post-culture step is a step of culturing the target cells or the like into which the molecule has been introduced (hereinafter sometimes referred to as "introduced cells or the like"). The culture conditions for the post-culture step are the same as those for the pre-culture step, and can be appropriately determined depending on the type of the introduced cells, etc., the purpose of use of the introduced cells, etc. The culture conditions for the post-culture step may be the same as or different from the culture conditions for the pre-culture step.

[0044] The resulting transfected cells are used, for example, for substance production, gene therapy, and the like.

[0045] According to the molecule introduction device of the present embodiment, the first electrode has a covering portion, which reduces damage to the irradiation target directly below the first electrode and improves controllability.

[0046] (Other embodiments) The electrode body in the above-mentioned embodiment has a substantially uniform thickness from the upper end to the lower end, but the present invention is not limited thereto. The electrode body may have, for example, a conductive wire extending in the vertical direction and a flat conductor located at the tip (lower end) of the conductive wire. In this case, the covering part may cover only the lower end surface of the flat conductor, or may cover the entire flat conductor. From the viewpoint of further increasing the survival rate of the introduced cells, etc., it is preferable that the electric field concentration part of the flat conductor is covered. Alternatively, for example, the electrode body may be a conductive wire extending in the vertical direction and a conductive thin film connected to the tip of the conductive wire. The conductive thin film may be, for example, a pattern-printed conductive thin film.

[0047] In the above-described embodiment, the container is placed on the second electrode (that is, the second electrode is located on the lower surface of the bottom wall of the container), but the present invention is not limited to this. 2, an electrode may be provided that wraps around the side wall 46 of the container 40 near the bottom wall 47. When the second electrode 20c is used, it is preferable that the upper end 21 of the second electrode 20c is located below the upper surface 41 of the irradiation target. EXAMPLES

[0048] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0049] (Raw materials used) ·Target cells: HEK293F (Thermo Fisher Scientific, R79007) cells. · Aqueous solution of molecules: DNA solution (pCMC-eGFP). Culture medium: HE100 (Gmep). · Constituents: Penicin · Streptomycin.

[0050] (Evaluation method) <Productivity> The introduced cells obtained in each case were photographed using a fluorescent microscope, and the extent to which the molecules had been introduced was evaluated from the appearance of the fluorescent images.

[0051] Example 1 A molecule introduction device similar to the molecule introduction device 1 in FIG. 1 was produced according to the following specifications. <Specifications> First electrode: An electrode having a cylindrical stainless steel electrode body (outer diameter 3 mm) and a ceramic coating (thickness t12 = 0.5 mm, length L12 = 10 mm). Second electrode: 3mm thick copper plate. · Vessel: 6-well culture plate.

[0052] 2.0 x 10 cells in each well of a 6-well plate 6 Cells were seeded at 100 cells / well and cultured for 24 hours in a CO2 incubator (37°C, 5% CO2 by volume). Before molecule introduction, the medium was removed and 57.5 μL of EGFP plasmid was added. Molecular introduction was performed by plasma irradiation treatment, and the cells were cultured for 48 hours in a CO2 incubator (37°C, 5% CO2 by volume). The resulting transfected cells were observed for green fluorescence using a fluorescence microscope (BZ-X800, Keyence Corporation). The results are shown in Figure 3. The green bright spots indicate target cells in which genes were introduced and fluorescent molecules were expressed, and molecule introduction occurred uniformly, including in the area directly under the plasma irradiation area.

[0053] <Irradiation conditions> AC voltage: 16kVpp. Frequency of alternating current: 50kHz. -Plasma exposure time: 20msec. -Distance from the bottom of the first electrode to the object to be irradiated: 1mm.

[0054] Comparative Example 1 The introduced cells and the like were obtained in the same manner as in Example 1, except that no coating portion was provided on the first electrode. Culture medium was added to the obtained introduced cells, which were then cultured at 37°C for 24 hours, and green fluorescence was observed using a fluorescence microscope (BZ-X800, Keyence Corporation). The results are shown in Figure 4. As shown in Figure 4, a region where no molecule introduction occurred was generated directly under the discharge, and a donut-shaped introduction region was generated outside of this.

[0055] From the above results, it was confirmed that the application of the present invention can improve the controllability of the target area. [Explanation of symbols]

[0056] 1 Molecule introduction device 10 First electrode 11 Electrode body 11a Opposite surface 11b Edge 12 Covering part 20, 20c second electrode 30 Power supply unit 40 containers 42 molecules 44 Target cells etc.

Claims

1. An electrode for a plasma-type molecular introduction device that introduces molecules into an introduction target object using plasma generated between separated electrodes, The electrode body has one end as a tip and is made of a conductor; and a covering portion that covers at least the tip of the electrode body. The electrode for a plasma-type molecule introduction device, wherein the covering portion is made of a dielectric material.

2. The electrode for a plasma-type molecule introduction device according to claim 1 , wherein the covering portion covers an electric field concentration portion of the electrode body.

3. 3. The electrode for a plasma-type molecular introduction device according to claim 1, wherein the thickness of the coating portion is 0.01 to 2 mm.

4. A first electrode; a second electrode spaced apart from the first electrode and positioned in a direction in which a tip of the first electrode faces; an illumination field located between the first electrode and the second electrode and spaced apart from a tip of the first electrode; A power supply unit that applies a voltage between the first electrode and the second electrode; having The first electrode has an electrode body made of a conductor and a covering portion covering at least a tip of the electrode body, The plasma type molecule introduction device, wherein the covering portion is made of a dielectric material.

5. The plasma type molecule introduction device according to claim 4 , wherein the covering portion covers an electric field concentration portion of the electrode body.

6. 6. The plasma type molecular introduction device according to claim 4, wherein the thickness of the covering portion is 0.01 to 2 mm.

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