WO2004092363A1 - Method of electrically stimulating cell - Google Patents

Method of electrically stimulating cell Download PDF

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Publication number
WO2004092363A1
WO2004092363A1 PCT/JP2004/005159 JP2004005159W WO2004092363A1 WO 2004092363 A1 WO2004092363 A1 WO 2004092363A1 JP 2004005159 W JP2004005159 W JP 2004005159W WO 2004092363 A1 WO2004092363 A1 WO 2004092363A1
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WIPO (PCT)
Prior art keywords
needle
cell
diameter
cells
present
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PCT/JP2004/005159
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French (fr)
Japanese (ja)
Inventor
Atsushi Miyawaki
Original Assignee
Riken
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Priority to JP2005505382A priority Critical patent/JPWO2004092363A1/en
Publication of WO2004092363A1 publication Critical patent/WO2004092363A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Definitions

  • the present invention relates to a method for electrically stimulating a local area of a cell and an apparatus for electrically stimulating the cell for use in the method.
  • a cell stimulating electrode is used to stimulate cells while culturing living cells.
  • conventional cell stimulating electrodes use gold, platinum, or the like, and have been used to stimulate cells in groups. Stimulation of a single cell is possible, but it is not possible to stimulate only a specific part of the cell. Disclosure of the invention
  • An object of the present invention is to solve the above-mentioned problems of the conventional technology. That is, the present invention makes it possible to apply a desired electrical stimulus to each cell at the level of a single cell, and to apply a desired electrical stimulus to an arbitrary local site with a single cell. An object of the present invention is to provide a method for electrically stimulating cells to be stimulated.
  • the present inventors have studied to solve the above-mentioned problems, and as a result, by placing the tip of a needle having a diameter of 3 IX m or less, connected to the potential control means, on the surface of a cell site, The inventors have found that the above problems can be solved, and have completed the present invention.
  • a method for local electrical stimulation of a cell comprising placing a needle having a diameter of 3 ⁇ or less connected to a potential control means on a cell surface.
  • a needle having a diameter of 50 to 500 nm can be used, and a needle having a length of 5 ⁇ m or less can be used.
  • a needle made of carbon nanotubes can be used.
  • the device has a diameter of less than 3 ⁇ coupled to the potential control means.
  • an electrical stimulation device for cells for use in the method of the present invention described above, comprising a needle for controlling the movement of the needle, and a driving means for controlling the movement of the needle.
  • a cell holding means for holding cells in a predetermined place
  • a needle having a diameter of 3 ⁇ m or less connected to an electric potential control means, and the needle connected to the needle.
  • Driving means for controlling the movement of the needle; and
  • electricity for the cells for use in the above-described method of the present invention which comprises a microscope for observing the cells held in the cell holding means.
  • a stimulator is provided.
  • a needle having a diameter of preferably 50 to 500 nm can be used.
  • FIG. 1 shows the outline of the method of the present invention.
  • 1 is a cantilever
  • 2 is a needle
  • 3 is a cell
  • 4 is a cell nucleus
  • 5 is a petri dish
  • 6 is a cell holding means
  • 9 is a driving means
  • 10 is a potential control means.
  • an arbitrary local site of the cell is electrically stimulated by placing a needle having a diameter of 3 or less connected to the potential control means on the surface of the specific site of the cell.
  • the present invention is characterized by using a very thin needle (several needles exceeding optical resolution) for electrical stimulation of cells, and specifically, has a diameter of 3 ⁇ or less. Needles can be used.
  • the needle used in the present invention is preferably a needle whose electrical properties such as chargeability are controlled.
  • the cell can be electrically stimulated as desired by controlling the charge on the needle after placing the needle on the surface of the cell.
  • any electrical stimulation can be applied to any desired cells in the cell population. For example, 100 cells are prepared, a needle is pierced into the cell from directly above each cell, and the potential of the needle is controlled by a potential control means connected to the needle, whereby the desired electric power is supplied to the cell. Can provide a stimulus.
  • the material of the needle used in the present invention is not particularly limited as long as it has the above-mentioned properties, and examples thereof include a carbon nanotube.
  • Carbon nanotubes are small crystals composed of 100% carbon atoms, having a cylindrical shape with a rounded graphite layer (graphin).
  • graphin graphite layer
  • Examples of research using carbon nanotubes include the development of screens that use nanotubes for electron guns instead of liquid crystal and plasma displays, application to fuel cells and solar cells, and hydrogen storage materials. No. These are unique properties that are different from the conventional ones due to the combination of various features such as the small size of the carbon nanotube itself, the quantum physical properties obtained from its three-dimensional structure, and pure carbon.
  • Carbon nanotubes also consist of purely carbon and contain few impurities unlike carbon black. It also has the characteristic that it does not change when exposed to high temperatures during molding or during use.
  • multi-wall carbon nanotubes having a diameter of about 50 to 100 nm and a length of 3 zm or more are available, and such a carbon nanotube can be used in the present invention. preferable.
  • 3 / zm or less for example, Use a needle with a diameter of 500 or less.
  • the needle having a diameter of 3 m or less as described above can be attached to the tip of a cantilever of an atomic force microscope (AFM) to make an electrical connection.
  • the electrical connection referred to here is an electrical connection for controlling the electric charge of the needle to be positive or negative.
  • the electrical connection can be made by connecting the potential control means and the needle with an electric wire or the like.
  • the potential control means used in the present invention is not particularly limited as long as it can control the electric charge of the needle.
  • the cantilever can apply a desired electrical stimulus to only the target cells by moving between the target cells or between the sites in conjunction with the image processing of the microscope.
  • the needle is always oriented in the vertical direction, and the position of the needle tip can be controlled with high accuracy.
  • the movement of the needle described above can be performed by driving means for controlling the movement of the needle. That is, according to the present invention, there is provided a cell electric stimulator having a needle connected to a potential control means and having a diameter of 3 ⁇ or less, and a driving means for controlling the movement of the needle.
  • the cell electrical stimulator of the present invention comprises: (a) cell holding means for holding cells in a predetermined place; (b) a diameter of 3 ⁇ or less connected to potential control means. And a driving means for controlling the movement of the needle connected to the needle; and (c) a microscope for observing the cells held in the cell holding means.
  • FIG. 1 shows an outline of the method of the present invention.
  • FIG. 1 shows how the needle 2 attached to the cantilever 1 connected to the driving means 9 comes into contact with the cell surface from a position right above the cell 3 using vertical arrows.
  • the cells 3 are cultured inside a petri dish 5, and the dishes 5 are set on cell holding means 6.
  • the substrate on which the cells inside the petri dish 5 are formed is made of a transparent electrode so that a current flows.
  • the electric charge on the surface of needle 2 is controlled by potential control means 10 electrically connected to needle 2.
  • the needle 2 descends from the position right above the target cell 3 to be subjected to the electrical stimulation by the driving means 9 and comes into contact with the cell 3.
  • Needle 2 on cell 3 The charge on the surface is controlled by the means 10, whereby the desired electrical stimulus is applied to the cells 3. After applying electrical stimulation to the cells, the needle is withdrawn from the cell surface. Thereafter, desired electrical stimulation can be applied to desired cells by repeating the above operation for different cells. All the movements of the needle 2 described above are controlled by the driving means 9.
  • PC12 cells (clonal cells of the nervous system isolated from the rat adrenal medulla chromaffin cell type) were used as the nerve cells.
  • the medium used was DMEM (Dlbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS).
  • the culture was performed under the conditions of 37 ° C. and 5% CO 2 .
  • the needle used in the apparatus described in FIG. 1 is a needle made of carbon nanotubes having a diameter of 50 nm and a length of 3 m.
  • the needle was brought into contact with the surface of the nerve cell, and electrical stimulation (10 Hz pulse) was continuously applied for 10 seconds. Tension depolarization extending from the stimulation site during electrical stimulation was observed.

Abstract

It is intended to provide a method of electrically stimulating a cell by which cells can be electrically stimulated at an arbitrary site for each cell at the individual cell level. Namely, a method of topically and electrically stimulating a cell wherein a needle having a diameter of 3 μm or less which is connected to a potential-controlling unit is provided on cell surface.

Description

明細書  Specification
細胞の電気刺激方法 技術分野  Electrical stimulation of cells
本発明は、 細胞の局所を電気的に刺激する方法及びそれに用レ、るための細胞の 電気刺激装置に関するものである。 背景技術  TECHNICAL FIELD The present invention relates to a method for electrically stimulating a local area of a cell and an apparatus for electrically stimulating the cell for use in the method. Background art
生体の細胞を培養しながら細胞に刺激を与えるために細胞刺激電極が用いられ る。 しかし、従来の細胞刺激電極には、 金や白金などが用いられており、 細胞を 集団で刺激することがなされていた。 細胞 1個の刺激も可能ではあるが、 細胞の 特定部位のみに刺激を加えることは不可能である。 発明の開示  A cell stimulating electrode is used to stimulate cells while culturing living cells. However, conventional cell stimulating electrodes use gold, platinum, or the like, and have been used to stimulate cells in groups. Stimulation of a single cell is possible, but it is not possible to stimulate only a specific part of the cell. Disclosure of the invention
本発明は上記した従来技術の問題点を解消することを解決すべき課題とした。 即ち、 本発明は、 1個ずつの細胞レベルで細胞毎に所望の電気的刺激を付与する こと、 更に 1個の細胞で任意の局所部位に所望の電気的刺激を付与することを可 能にする細胞の電気刺激方法を提供することを解決すべき課題とした。  An object of the present invention is to solve the above-mentioned problems of the conventional technology. That is, the present invention makes it possible to apply a desired electrical stimulus to each cell at the level of a single cell, and to apply a desired electrical stimulus to an arbitrary local site with a single cell. An object of the present invention is to provide a method for electrically stimulating cells to be stimulated.
本発明者らは上記課題を解決するために検討した結果、 電位制御手段に違結さ れた 3 IX m以下の直径を有する針の先端を細胞のある部位の表面に置くことによ つて、 上記課題を解決できることを見出し、 本発明を完成するに至った。  The present inventors have studied to solve the above-mentioned problems, and as a result, by placing the tip of a needle having a diameter of 3 IX m or less, connected to the potential control means, on the surface of a cell site, The inventors have found that the above problems can be solved, and have completed the present invention.
即ち、 本発明によれば、 電位制御手段に連結された 3 μ πι以下の直径を有する 針を細胞表面に設置することを含む、 細胞の局所的電気刺激方法が提供される。 好ましくは、 5 0〜 5 0 0 n mの直径を有する針を使用することができ、 5 μ m以下の長さを有する針を使用することができる。 好ましくは、 カーボンナノチ ユーブから成る針を使用することができる。  That is, according to the present invention, there is provided a method for local electrical stimulation of a cell, comprising placing a needle having a diameter of 3 μπι or less connected to a potential control means on a cell surface. Preferably, a needle having a diameter of 50 to 500 nm can be used, and a needle having a length of 5 μm or less can be used. Preferably, a needle made of carbon nanotubes can be used.
本発明の別の側面によれば、 電位制御手段に連結された 3 μ πι以下の直径を有 する針、 及ぴ該針の移動を制御するための駆動手段を有する、 上記した本発明の 方法で使用するための細胞の電気刺激装置が提供される。 According to another aspect of the invention, the device has a diameter of less than 3 μπι coupled to the potential control means. There is provided an electrical stimulation device for cells for use in the method of the present invention described above, comprising a needle for controlling the movement of the needle, and a driving means for controlling the movement of the needle.
好ましくは、 （a ) 細胞を所定の場所に保持するための細胞保持手段； （b ) 電 位制御手段に連結された 3 μ m以下の直径を有する針、 及ぴ該針に連結された該 針の移動を制御するための駆動手段；及ぴ （c ) 細胞保持手段内に保持された細 胞を観察するための顕微鏡を有する、 上記した本発明の方法で使用するための細 胞の電気刺激装置が提供される。  Preferably, (a) a cell holding means for holding cells in a predetermined place; (b) a needle having a diameter of 3 μm or less connected to an electric potential control means, and the needle connected to the needle. Driving means for controlling the movement of the needle; and (c) electricity for the cells for use in the above-described method of the present invention, which comprises a microscope for observing the cells held in the cell holding means. A stimulator is provided.
本発明の電気刺激装置では、 好ましくは 5 0〜 5 0 0 n mの直径を有する針を 使用することができる。 図面の簡単な説明  In the electrical stimulator of the present invention, a needle having a diameter of preferably 50 to 500 nm can be used. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 本発明の方法の概要を示す。 図 1において、 1はカンチレバー、 2は 針、 3は細胞、 4は細胞核、 5はシャーレ、 6は細胞保持手段、 9は駆動手段、 1 0は電位制御手段を示す。 発明を実施するための最良の形態  FIG. 1 shows the outline of the method of the present invention. In FIG. 1, 1 is a cantilever, 2 is a needle, 3 is a cell, 4 is a cell nucleus, 5 is a petri dish, 6 is a cell holding means, 9 is a driving means, and 10 is a potential control means. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の実施の形態について詳細に説明する。  Hereinafter, embodiments of the present invention will be described in detail.
本発明の方法においては、 電位制御手段に連結された 3 以下の直径を有す る針を細胞の特定部位の表面に置くことによって、 細胞の任意の局所部位を電気 的に刺激する。  In the method of the present invention, an arbitrary local site of the cell is electrically stimulated by placing a needle having a diameter of 3 or less connected to the potential control means on the surface of the specific site of the cell.
本発明は、 細胞の電気刺激のために非常に細い針 （光学的分解能を超えるくら いの針） を使用することを特徴とするが、 具体的には、 3 μ πι以下の直径を有す る針を使用することができる。 本発明で使用する針は、 その帯電性などの電気的 性質をコントロールしゃすい針であることが好ましい。 本発明では、 針を細胞の 表面に置いた後に針の電荷を制御することによって、 細胞を所望の通りに電気的 に刺激することができる。 本発明では、 細胞集団の中でさらに任意の狙った細胞 に任意の電気的刺激を与えることができる。 例えば、 1 0 0個の細胞を用意し、針を個々の細胞の真上から細胞に突き刺し、 針の電位を針に連結された電位制御手段により制御することにより、 該細胞に所 望の電気的刺激を与えることができる。 このように針を 1個の細胞に接触させて 電気的に刺激する操作を繰り返すことによって、 異なる細胞にそれぞれ所望の電 気的刺激を与えることができる。 従って、 本発明の方法によれば、 薬剤スクリー ニングゃ生体分子間相互作用の網羅的解析を、 従来のように 9 6穴プレートや 3 8 4穴プレート等を用いてゥエル毎に行なうのではなく、 一細胞レベルで行なう ことが可能になる。 また、 1個の培養神経細胞のある特異的な部位 （例えば、 樹 状突起） に注目して、 その中の特定部位に電気刺激を与えることができる。 神経 細胞がシナプスを介して受ける入力刺激に近い電気刺激を空間的分解能を高めな がら与えることができる。 The present invention is characterized by using a very thin needle (several needles exceeding optical resolution) for electrical stimulation of cells, and specifically, has a diameter of 3 μπι or less. Needles can be used. The needle used in the present invention is preferably a needle whose electrical properties such as chargeability are controlled. In the present invention, the cell can be electrically stimulated as desired by controlling the charge on the needle after placing the needle on the surface of the cell. In the present invention, any electrical stimulation can be applied to any desired cells in the cell population. For example, 100 cells are prepared, a needle is pierced into the cell from directly above each cell, and the potential of the needle is controlled by a potential control means connected to the needle, whereby the desired electric power is supplied to the cell. Can provide a stimulus. By repeating the operation of bringing the needle into contact with one cell and electrically stimulating in this manner, it is possible to apply desired electric stimulus to different cells. Therefore, according to the method of the present invention, drug screening--exhaustive analysis of interactions between biomolecules is not performed for each well using a 96-well plate, a 384-well plate, or the like as in the prior art. This can be done at the single cell level. Also, by focusing on a specific site (eg, dendrites) of a single cultured neuron, electrical stimulation can be applied to a specific site. An electrical stimulus similar to an input stimulus that a nerve cell receives via a synapse can be given while increasing the spatial resolution.
本発明で用いる針の材料は、 上記した性質を有するものであれば特に限定され ないが、 例えば、 カーボンナノチューブなどが挙げられる。 カーボンナノチュー ブは、グラフアイトの一層（グラフイン）を丸めた円筒形の形状を有し、 1 0 0 % 炭素原子から構成される微小な結晶である。 近年、 ナノテクノロジーが脚光を浴 ぴ、 このカーボンナノチューブも多方面から注目されている。 カーボンナノチュ ーブを使用する研究例には、 液晶、 プラズマディスプレイに代わり、 ナノチュー ブを電子銃に使用する画面の開発、 燃料電池おょぴ太陽電池への応用、 または水 素貯蔵材料などが挙げられる。 これらは、 カーボンナノチューブ自体が有する微 小さ、 その立体構造から得られる量子物性、 ならびに純粋に炭素からのみなると いう各種の特徴の組み合わせから、 従来とは異なるユニークな性質を有するため である。 カーボンナノチューブはまた、 純粋に炭素のみからなり、 カーボンブラ ックなどと異なり不純物をほとんど含有しない。 また、 成形時おょぴノまたは使 用時に高温下に曝されても、 変化しないという特徴をも有する。  The material of the needle used in the present invention is not particularly limited as long as it has the above-mentioned properties, and examples thereof include a carbon nanotube. Carbon nanotubes are small crystals composed of 100% carbon atoms, having a cylindrical shape with a rounded graphite layer (graphin). In recent years, nanotechnology has been in the limelight, and this carbon nanotube has also attracted attention from many angles. Examples of research using carbon nanotubes include the development of screens that use nanotubes for electron guns instead of liquid crystal and plasma displays, application to fuel cells and solar cells, and hydrogen storage materials. No. These are unique properties that are different from the conventional ones due to the combination of various features such as the small size of the carbon nanotube itself, the quantum physical properties obtained from its three-dimensional structure, and pure carbon. Carbon nanotubes also consist of purely carbon and contain few impurities unlike carbon black. It also has the characteristic that it does not change when exposed to high temperatures during molding or during use.
現在、 Multi wallカーボンナノチューブとしては、 直径が 5 0〜1 0 0 n m程 度で、 長さが 3 z m以上のものが入手可能であり、 本発明ではこのようなカーボ ンナノチューブを使用することが好ましい。 本発明では、 3 /z m以下、 例えば、 5 0 0 以下の直径を有する針を使用する。 At present, multi-wall carbon nanotubes having a diameter of about 50 to 100 nm and a length of 3 zm or more are available, and such a carbon nanotube can be used in the present invention. preferable. In the present invention, 3 / zm or less, for example, Use a needle with a diameter of 500 or less.
本発明では、 上記したような 3 m以下の直径を有する針を、 原子間力顕微鏡 (A F M)のカンチレバーの先に装着し、 電気的接続を行なうことができる。 こ こで言う電気的接続とは、 針の電荷を正又は負に制御するための電気的接続のこ とを言う。 電気的接続は、 電位制御手段と針を電線などで接続することにより行 なうことができる。 本発明で用いる電位制御手段は、 針の電荷を制御できるもの であれば特に限定されずない。 また、 このカンチレパーは、 顕微鏡の画像処理と 連動させて、 目的の細胞の間や部位の間を移動させることにより、 所望の電気的 刺激を目的の細胞のみに与えることができる。 本発明の好ましい態様では、 針は 常に垂直方向を向き、 高い精度で針先位置をコントロールすることができる。 上 記した針の移動は、 針の移動を制御するための駆動手段により行なうことが 、き る。 即ち、 本発明によれば、 電位制御手段に連結された 3 μ πι以下の直径を有す る針、 及び該針の移動を制御するための駆動手段を有する細胞の電気刺激装置が 提供される。 さらに具体的には、本発明の細胞の電気刺激装置は、 （a ) 細胞を所 定の場所に保持するための細胞保持手段；（b )電位制御手段に連結された 3 μ πι 以下の直径を有する針、 及ぴ該針に連結された該針の移動を制御するための駆動 手段；及び （c ) 細胞保持手段内に保持された細胞を観察するための顕微鏡から 構成することができる。  In the present invention, the needle having a diameter of 3 m or less as described above can be attached to the tip of a cantilever of an atomic force microscope (AFM) to make an electrical connection. The electrical connection referred to here is an electrical connection for controlling the electric charge of the needle to be positive or negative. The electrical connection can be made by connecting the potential control means and the needle with an electric wire or the like. The potential control means used in the present invention is not particularly limited as long as it can control the electric charge of the needle. In addition, the cantilever can apply a desired electrical stimulus to only the target cells by moving between the target cells or between the sites in conjunction with the image processing of the microscope. In a preferred aspect of the present invention, the needle is always oriented in the vertical direction, and the position of the needle tip can be controlled with high accuracy. The movement of the needle described above can be performed by driving means for controlling the movement of the needle. That is, according to the present invention, there is provided a cell electric stimulator having a needle connected to a potential control means and having a diameter of 3 μπι or less, and a driving means for controlling the movement of the needle. . More specifically, the cell electrical stimulator of the present invention comprises: (a) cell holding means for holding cells in a predetermined place; (b) a diameter of 3 μπι or less connected to potential control means. And a driving means for controlling the movement of the needle connected to the needle; and (c) a microscope for observing the cells held in the cell holding means.
以下、 本発明の実施態様の一例を図面を参照して説明する。  Hereinafter, an example of an embodiment of the present invention will be described with reference to the drawings.
図 1に本発明の方法の概要を示す。 図 1は、 駆動手段 9に接続されたカンチレ パー 1に装着された針 2が、 細胞 3の真上の位置から細胞表面に接触する様子を 上下方向の矢印を用いて示す。 細胞 3はシャーレ 5の内部で培養されており、 シ ヤーレ 5は細胞保持手段 6の上に設置されている。 また、 シャーレ 5の内部の細 胞の生えている基板は、 電流が流れるように透明電極から構成されている。 針 2 の表面の電荷は、 針 2に電気的に接続されている電位制御手段 1 0によって制御 されている。 電気刺激を与えるべき目的の細胞 3の真上の位置から針 2は、 駆動 手段 9によって真下に降下して細胞 3に接触する。 細胞 3上で針 2は、 電位制御 手段 10によって表面の電荷を制御され、 それにより細胞 3に所望の電気的刺激 が付与される。細胞に電気的刺激を与えた後、針は細胞表面から引き上げられる。 以後、 異なる細胞に上記の操作を繰り返すことにより、 所望の細胞に所望の電気 的刺激を与えることができる。 上記した針 2の移動は全て駆動手段 9により制御 されている。 FIG. 1 shows an outline of the method of the present invention. FIG. 1 shows how the needle 2 attached to the cantilever 1 connected to the driving means 9 comes into contact with the cell surface from a position right above the cell 3 using vertical arrows. The cells 3 are cultured inside a petri dish 5, and the dishes 5 are set on cell holding means 6. The substrate on which the cells inside the petri dish 5 are formed is made of a transparent electrode so that a current flows. The electric charge on the surface of needle 2 is controlled by potential control means 10 electrically connected to needle 2. The needle 2 descends from the position right above the target cell 3 to be subjected to the electrical stimulation by the driving means 9 and comes into contact with the cell 3. Needle 2 on cell 3 The charge on the surface is controlled by the means 10, whereby the desired electrical stimulus is applied to the cells 3. After applying electrical stimulation to the cells, the needle is withdrawn from the cell surface. Thereafter, desired electrical stimulation can be applied to desired cells by repeating the above operation for different cells. All the movements of the needle 2 described above are controlled by the driving means 9.
以下の実施例により本発明をさらに具体的に説明するが、 本発明は以下の実施 例によって限定されるものではない。 実施例  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the following examples. Example
培養シャーレ内で培養している神経細胞に対して、 図 1に記載の装置を用いて 電気的刺激を与えた。  Electrical stimulation was applied to the nerve cells cultured in the culture dish using the apparatus described in FIG.
神経細胞としては P C 12細胞 （ラット副腎髄質クロム親和性細胞種より単離 された神経系クローン細胞） を用いた。 培地は、 10 %胎児ゥシ血清 （ F B S ) を含む DMEM (D l b e c c o' s Mo d i f i e d E a g l e Me d i um) を用いた。 培養は 37°C、 5%C02の条件下で行なった。 PC12 cells (clonal cells of the nervous system isolated from the rat adrenal medulla chromaffin cell type) were used as the nerve cells. The medium used was DMEM (Dlbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS). The culture was performed under the conditions of 37 ° C. and 5% CO 2 .
図 1に記載の装置で使用した針は、 直径 50 nm及び長さ 3 mを有するカー ボンナノチューブから成る針である。  The needle used in the apparatus described in FIG. 1 is a needle made of carbon nanotubes having a diameter of 50 nm and a length of 3 m.
先ず、 針を神経細胞の表面に接触させて、 電気刺激 （10Hzパルス） を、 1 0秒間与え続けた。電気刺激中に刺激部位から拡がる緊張性脱分極が認められた。 産業上の利用可能性 '  First, the needle was brought into contact with the surface of the nerve cell, and electrical stimulation (10 Hz pulse) was continuously applied for 10 seconds. Tension depolarization extending from the stimulation site during electrical stimulation was observed. Industrial applicability ''
本発明により、 顕微鏡視野内の任意の細胞の任意の微小部位に電気的刺激を与 える方法を提供することが可能になった。  According to the present invention, it has become possible to provide a method for applying an electrical stimulus to an arbitrary microsite of an arbitrary cell in a microscope visual field.

Claims

請求の範囲 The scope of the claims
1. 電位制御手段に連結された 3 μ m以下の直径を有する針を細胞表面に設 置することを含む、 細胞の局所的電気刺激方法。 1. A method for local electrical stimulation of a cell, comprising placing a needle with a diameter of 3 μm or less on the cell surface connected to a potential control means.
2. 50〜500 nmの直径を有する針を使用する、 請求項 1又は 2に記載 の方法。  2. The method according to claim 1 or 2, wherein a needle having a diameter of 50 to 500 nm is used.
3. 5 m以下の長さを有する針を使用する、 請求項 1から 3の何れかに記 載の方法。  3. The method according to claim 1, wherein a needle having a length of 5 m or less is used.
4. カーボンナノチューブから成る針を使用する、 請求項 1から 3の何れか に記載の方法。  4. The method according to any one of claims 1 to 3, wherein a needle made of carbon nanotubes is used.
5. 電位制御手段に連結された 3 μ m以下の直径を有する針、 及ぴ該針の移 動を制御するための駆動手段を有する、 請求填 1から 5の何れかに記載の方法で 使用するための細胞の電気刺激装置。  5. Use according to any of claims 1 to 5, comprising a needle connected to the potential control means and having a diameter of 3 μm or less and a drive means for controlling the movement of the needle. Cell electrical stimulator for doing.
6. 50〜500 nmの直径を有する針を使用する、 請求項 5に記載の電気 刺激装置。  6. The electrical stimulator according to claim 5, wherein a needle having a diameter of 50 to 500 nm is used.
7. (a) 細胞を所定の場所に保持するための細胞保持手段；（b) 電位制御 手段に連結された 3 m以下の直径を有する針、 及び該針に連結された該針の移 動を制御するための駆動手段；及び （c) 細胞保持手段内に保持された細胞を観 察するための顕微鏡を有する、 請求項 1から 5の何れかに記載の方法で使用する ための細胞の電気刺激装置。  7. (a) cell holding means for holding cells in place; (b) a needle having a diameter of 3 m or less connected to potential control means, and movement of the needle connected to the needle. 6. A driving means for controlling the electric field; and (c) a microscope for observing cells held in the cell holding means, comprising: a microscope for observing cells held in the cell holding means. Stimulator.
8. 50〜500 nmの直径を有する針を使用する、 請求項 7に記載の電気 刺激装置。  8. The electrical stimulator according to claim 7, wherein a needle having a diameter of 50 to 500 nm is used.
PCT/JP2004/005159 2003-04-11 2004-04-09 Method of electrically stimulating cell WO2004092363A1 (en)

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