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WO2004092363A1 - Stimulation electrique d'une cellule - Google Patents

Stimulation electrique d'une cellule 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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WO
WIPO (PCT)
Prior art keywords
needle
cell
diameter
cells
present
Prior art date
Application number
PCT/JP2004/005159
Other languages
English (en)
Japanese (ja)
Inventor
Atsushi Miyawaki
Original Assignee
Riken
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Riken filed Critical Riken
Priority to JP2005505382A priority Critical patent/JPWO2004092363A1/ja
Publication of WO2004092363A1 publication Critical patent/WO2004092363A1/fr

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Classifications

    • 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.

Landscapes

  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Nanotechnology (AREA)
  • Materials Engineering (AREA)
  • General Physics & Mathematics (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Composite Materials (AREA)
  • Electromagnetism (AREA)
  • Cell Biology (AREA)
  • Sustainable Development (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention concerne un procédé permettant de stimuler électriquement une cellule, la stimulation pouvant être appliquée en un point arbitraire pour chaque cellule à son niveau propre. Plus spécifiquement, le procédé consiste à stimuler topiquement et électriquement une cellule en amenant sur la surface de cette dernière une aiguille de 3νm de diamètre ou moins connectée à une unité de commande de tension.
PCT/JP2004/005159 2003-04-11 2004-04-09 Stimulation electrique d'une cellule WO2004092363A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005505382A JPWO2004092363A1 (ja) 2003-04-11 2004-04-09 細胞の電気刺激方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2003-107268 2003-04-11
JP2003107268 2003-04-11

Publications (1)

Publication Number Publication Date
WO2004092363A1 true WO2004092363A1 (fr) 2004-10-28

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Family Applications (1)

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PCT/JP2004/005159 WO2004092363A1 (fr) 2003-04-11 2004-04-09 Stimulation electrique d'une cellule

Country Status (2)

Country Link
JP (1) JPWO2004092363A1 (fr)
WO (1) WO2004092363A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2005116184A1 (ja) * 2004-05-26 2008-04-03 株式会社ユニソク 生体試料操作方法
JPWO2015107761A1 (ja) * 2014-01-16 2017-03-23 浜松ホトニクス株式会社 細胞観察装置、電気刺激装置、及び細胞観察方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0099253A2 (fr) * 1982-07-08 1984-01-25 Shionogi & Co., Ltd. Micro-électrodes et procédé de fabrication
JPH04141087A (ja) * 1990-10-03 1992-05-14 Toshiba Corp 細胞の分化方法
WO1997016545A1 (fr) * 1995-11-03 1997-05-09 Massachusetts Institute Of Technology Stimulation neuronale faisant appel a des polymeres electriquement conducteurs
WO1998005920A1 (fr) * 1996-08-08 1998-02-12 William Marsh Rice University Dispositifs a nano-echelle, maniables de façon macroscopique et realises a partir d'ensembles nanotubes
JP2002214112A (ja) * 2001-01-15 2002-07-31 Fuji Xerox Co Ltd 走査型プローブ顕微鏡
US20030157708A1 (en) * 2001-11-13 2003-08-21 Riken Cell stimulation apparatus and a cell stimulation method
WO2004019012A1 (fr) * 2002-08-26 2004-03-04 Osaka University Unite de sonde et methode de commande

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0099253A2 (fr) * 1982-07-08 1984-01-25 Shionogi & Co., Ltd. Micro-électrodes et procédé de fabrication
JPH04141087A (ja) * 1990-10-03 1992-05-14 Toshiba Corp 細胞の分化方法
WO1997016545A1 (fr) * 1995-11-03 1997-05-09 Massachusetts Institute Of Technology Stimulation neuronale faisant appel a des polymeres electriquement conducteurs
WO1998005920A1 (fr) * 1996-08-08 1998-02-12 William Marsh Rice University Dispositifs a nano-echelle, maniables de façon macroscopique et realises a partir d'ensembles nanotubes
JP2002214112A (ja) * 2001-01-15 2002-07-31 Fuji Xerox Co Ltd 走査型プローブ顕微鏡
US20030157708A1 (en) * 2001-11-13 2003-08-21 Riken Cell stimulation apparatus and a cell stimulation method
WO2004019012A1 (fr) * 2002-08-26 2004-03-04 Osaka University Unite de sonde et methode de commande

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2005116184A1 (ja) * 2004-05-26 2008-04-03 株式会社ユニソク 生体試料操作方法
JP4645912B2 (ja) * 2004-05-26 2011-03-09 株式会社ユニソク 生体試料操作方法
JPWO2015107761A1 (ja) * 2014-01-16 2017-03-23 浜松ホトニクス株式会社 細胞観察装置、電気刺激装置、及び細胞観察方法
US10415010B2 (en) 2014-01-16 2019-09-17 Hamamatsu Photonics K.K. Cell observation device, electrostimulation device, and cell observation method
US11248202B2 (en) 2014-01-16 2022-02-15 Hamamatsu Photonics K.K. Cell observation device, electrostimulation device, and cell observation method

Also Published As

Publication number Publication date
JPWO2004092363A1 (ja) 2006-07-06

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