WO2020110735A1 - Electrode retention member - Google Patents

Electrode retention member Download PDF

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Publication number
WO2020110735A1
WO2020110735A1 PCT/JP2019/044577 JP2019044577W WO2020110735A1 WO 2020110735 A1 WO2020110735 A1 WO 2020110735A1 JP 2019044577 W JP2019044577 W JP 2019044577W WO 2020110735 A1 WO2020110735 A1 WO 2020110735A1
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Prior art keywords
electrode
holding member
substrate
cell culture
working
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PCT/JP2019/044577
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French (fr)
Japanese (ja)
Inventor
徳幸 中谷
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株式会社Ｓｃｒｅｅｎホールディングス
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Publication of WO2020110735A1 publication Critical patent/WO2020110735A1/en

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    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance

Definitions

  • the present invention relates to an electrode holding member for measuring the electric resistance of cultured cells.
  • the cell culture container described in Patent Document 1 has an electrode extending from the lid portion to the inside of the insert container in which cells are cultured, and an electrode arranged on the bottom surface of the cell culture container below the insert container. In such an electrode structure, it is necessary to use a dedicated cell culture container equipped with an electrode.
  • the present invention has been made in view of such circumstances, and an object thereof is to provide a technique capable of easily measuring the electric resistance of cultured cells using a general-purpose cell culture container. ..
  • a first invention of the present application is an electrode holding member for arranging an electrode in the recess of a well plate having a plurality of recesses, and a plurality of observation openings arranged in a grid pattern. And a flat plate-like and insulative substrate extending in the horizontal direction, and a plurality of electrode portions arranged in the vicinity of a part or all of the observation opening, each of the electrode portions. , One end of which is fixed to the substrate and extends downward from the substrate.
  • 2nd invention of this application is an electrode holding member of 1st invention, Comprising:
  • the said 1st electrode part is an insulating 1st columnar member,
  • the 1st working electrode provided in the side surface of the 1st columnar member,
  • a second reference electrode provided on a side surface of the first columnar member, the second electrode portion having an insulating second columnar member, and a second working electrode provided on a side surface of the second columnar member.
  • a second reference electrode provided on the side surface of the second columnar member.
  • 3rd invention of this application is an electrode holding member of 2nd invention, Comprising:
  • substrate has several conductor wire at least one part was printed on the upper surface of the said board
  • the said 1st electrode part and the said 2nd The upper end portion of each of the electrode portions is disposed above the upper surface of the substrate, and the upper surface of the lead wire printed on the upper surface of the substrate, the first working electrode, the first reference electrode, and the second working electrode.
  • the electrode and any one of the second reference electrodes are electrically connected via a substantially rectangular parallelepiped conductive connecting member.
  • the fourth invention of the present application is the electrode holding member according to any one of the first invention to the third invention, wherein the number of the electrode portions is the same as the number of the observation openings.
  • the fifth invention of the present application is the electrode holding member according to any one of the first to third inventions, wherein the number of the electrode portions is smaller than the number of the observation openings.
  • the direction parallel to the substrate of the electrode holding member is called “horizontal direction”
  • the direction orthogonal to the horizontal direction is called “vertical direction”.
  • the postures of the cell culture container and the electrode holding member during use may be such that the substrate of the electrode holding member is not horizontal.
  • FIG. 1 is a top view of a cell culture container 2 used with an electrode holding member 1 according to an embodiment of the present invention.
  • FIG. 2 is a top view of the electrode holding member 1 according to the embodiment of the present invention.
  • FIG. 2 the positions of the respective parts of the electrode holding member 1 when the electrode holding member 1 is used are shown by broken lines. Note that, in FIG. 2, the conductive connecting member 15 is omitted except for the enlarged portion.
  • FIG. 3 is a cross-sectional view taken along the line AA of the electrode holding member 1 and the cell culture container 2.
  • FIG. 4 is a partial cross-sectional view of the electrode holding member 1 taken along the line BB. In FIG. 4, each component is exaggerated for easy understanding of the positional relationship, and the dimensions are different from the actual ones.
  • the cell culture container 2 is a container for culturing cells whose electric resistance value is to be measured using the electrode portion 10 of the electrode holding member 1.
  • the cell culture container 2 is a so-called well plate having a plurality of wells 21. As shown in FIGS. 1 and 3, the well 21 is a recess recessed from the upper surface of the cell culture container 2.
  • a total of 6 wells 21 arranged in 2 rows and 3 columns are arranged in a lattice.
  • the number of wells of the cell culture container used with the electrode holding member of the present invention is not limited to 6, and may be 12, 24, 96, 384, etc., for example.
  • the insert cup 20 is arranged inside each well 21 of the cell culture container 2.
  • the insert cup 20 has a tubular portion 31, a support portion 32, and a cell culture portion 33.
  • the tubular portion 31 is an insulating portion formed in a truncated cone shape and a tubular shape.
  • the support portion 32 extends outward from the upper end of the tubular portion 31.
  • the support part 32 and the cell culture part 33 should be arranged inside the well 21 at a position where the cell culture part 33 does not contact the bottom surface of the well 21.
  • the support portions 32 are radially arranged at three locations in the circumferential direction of the tubular portion 31.
  • the support portion 32 may have another shape as long as it can support the tubular portion 31 and the cell culture portion 33 at a predetermined position.
  • the support portions 32 may be arranged at only two positions in the circumferential direction of the insert cup 20, or may be arranged at four or more positions.
  • the support portion 32 may have a flange shape that extends outward from a part of the insert cup 20 in the circumferential direction.
  • the cell culture section 33 is a film that covers the opening below the tubular section 31.
  • a film having cell adhesiveness is used for the cell culture unit 33.
  • a plate-shaped member provided with a large number of minute through holes may be used for the cell culture section 33.
  • the electrode holding member 1 is a member for disposing electrodes in the well 21 of the cell culture container 2. Specifically, the electrode holding member 1 is placed on the cell culture container 2 and each electrode 42, 43, for measuring the electric resistance value of the cells cultured on the insert cup 20 in each well 21. Hold 52 and 53. The electrodes 42, 43, 52, 53 will be described later.
  • the electrode holding member 1 has a substrate 11, a plurality of conducting wires 12 formed on the substrate, and a plurality of electrode portions 10.
  • the substrate 11 is a flat plate-like insulating substrate that extends in the horizontal direction.
  • the substrate 11 is formed of, for example, glass epoxy resin.
  • the substrate 11 is provided with a plurality of observation openings 13 arranged in a grid pattern. Each observation opening 13 is formed in accordance with the position of each well 21 of the cell culture container 2.
  • each conductive wire 12 is an electrode connection terminal 121 electrically connected to any of the electrodes 42, 43, 52, 53 of the electrode unit 10 described later.
  • the other end of each conductor 12 is an external connection terminal 122 that can be electrically connected to the outside.
  • At least a part of the conductive wire 12 including the electrode connection terminal 121 is formed by being printed on the upper surface of the substrate 11. Other portions of the conductive wire 12 may be printed on a layer inside the substrate 11 formed in multiple layers, or may be embedded inside when the substrate 11 is formed.
  • Each of the electrode parts 10 is arranged near the observation opening 13.
  • the electrode portion 10 is arranged for every observation opening 13.
  • the electrode portion 10 may be arranged only for some of the observation openings 13.
  • the electrode parts 10 each include a first electrode part 40 and a second electrode part 50.
  • the first electrode portion 40 has a first columnar member 41, a first working electrode 42, and a first reference electrode 43. One end of the first electrode portion 40 is fixed to the substrate 11. Further, the first electrode portion 40 extends downward from the substrate 11.
  • the first columnar member 41 is a prismatic insulating member extending in the vertical direction.
  • the first columnar member 41 is made of glass, for example.
  • the first working electrode 42 is provided on the entire one of the four side surfaces of the first columnar member 41.
  • the first reference electrode 43 is provided on the entire other side surface of the first columnar member 41.
  • the first working electrode 42 and the first reference electrode 43 are not adjacent to each other and are arranged with a space.
  • each of the first working electrode 42 and the first reference electrode 43 is a metal thin film formed on the side surface of the first columnar member 41.
  • each of the first working electrode 42 and the first reference electrode 43 may have a structure in which a metal plate is bonded to the first columnar member 41, or may have another configuration.
  • the second electrode section 50 has a second columnar member 51, a second working electrode 52, and a second reference electrode 53.
  • the second electrode portion 50 has one end fixed to the substrate 11. In addition, the second electrode portion 50 extends downward from the substrate 11.
  • the second columnar member 51 is a prismatic insulating member extending in the vertical direction.
  • the second columnar member 51 is made of, for example, glass.
  • the second working electrode 52 is provided on the entire one of the four side surfaces of the second columnar member 51.
  • the second reference electrode 53 is provided on the entire other side surface of the second columnar member 51.
  • the second working electrode 52 and the second reference electrode 53 are not adjacent to each other and are arranged with a space.
  • each of the second working electrode 52 and the second reference electrode 53 is a metal thin film formed on the side surface of the second columnar member 51.
  • each of the second working electrode 52 and the second reference electrode 53 may have a structure in which a metal plate is adhered to the second columnar member 51, or may have another configuration.
  • the substrate 11 is provided with a through hole 14 around the observation opening 13 into which the first electrode portion 40 and the second electrode portion 50 are inserted.
  • the upper ends of the first electrode portion 40 and the second electrode portion 50 are arranged above the upper surface of the substrate 11, respectively.
  • the electrode connection terminals 121 of the two conductive wires 12 are arranged at positions adjacent to each through hole 14. As shown in FIG. 4, each of the first working electrode 42 and the first reference electrode 43 is electrically connected to the electrode connection terminal 121 of the lead wire 12 via the conductive connection member 15.
  • Each conductive connecting member 15 has a substantially rectangular parallelepiped shape. As shown in the enlarged portion of FIG. 2 and FIG. 4, the conductive connection member 15 is arranged on the electrode connection terminal 121 of each conductor 12. Further, as shown in FIG. 4, the vicinity of the upper end portion of the first working electrode 42 is bonded to the side surface of one conductive connecting member 15 by the conductive adhesive 16. Further, the lower surface of the conductive connecting member 15 and the upper surface of the electrode connecting terminal 121 of the one conducting wire 12 are bonded by the conductive adhesive 16. As a result, the first working electrode 42 and the one conductive wire 12 are electrically connected to each other via the conductive connecting member 15 and the conductive adhesive 16.
  • the vicinity of the upper end of the first reference electrode 43 is bonded to the side surface of the other conductive connecting member 15 with the conductive adhesive 16. Further, the lower surface of the conductive connecting member 15 and the upper surface of the electrode connecting terminal 121 of the other one conducting wire 12 are adhered by the conductive adhesive 16. As a result, the first reference electrode 43 and the other one conducting wire 12 are electrically connected via the conductive connecting member 15 and the conductive adhesive 16.
  • first working electrode 42 and the first reference electrode 43 are thus adhered to the substrate 11 via the conductive connecting member 15 so that the upper end of the first electrode portion 40 is fixed to the substrate 11.
  • the second working electrode 52 and the second reference electrode 53 are also connected to the conducting wire 12 via the conductive connecting member 15.
  • the insert cup 20 is placed on one side in a predetermined direction (on the right side in FIG. 1). ) And place it. Then, by placing the electrode holding member 1 on the cell culture container 2, the electrode portion 10 is inserted into the desired well 21.
  • each observation opening 13 is arranged at substantially the center of each well 21.
  • the first electrode portion 40 is arranged on one side (right side in FIG. 2) of the observation opening 13, and the second electrode portion 50 is disposed on the other side of the observation opening 13 ( It is arranged on the left side in FIG.
  • the insert cup 20 is biased to one side of the well 21, the first electrode portion 40 is inserted inside the insert cup 20, and the second electrode portion 50 is outside the insert cup 20. Inserted in.
  • the second electrode section 50 projects below the first electrode section 40.
  • the lower end portion of the first electrode portion 40 is arranged above the cell culture portion 33 of the insert cup 20 and the second electrode portion 50.
  • the lower end of the cell culture section is arranged below the cell culture section 33.
  • FIG. 5 is a schematic diagram showing the electrical connection of the measurement unit 9. Note that FIG. 5 shows the electrical connection of the measurement unit 9 when the number of the electrodes 42, 43, 52, 53 is only one set. It should be noted that the configuration of the measurement unit 9 is different when simultaneously measuring the plurality of electrode portions 10. For example, the connection destinations of the power supply device 91 and the voltmeter 92 of the measuring device 90 described later can be switched for each electrode unit 10.
  • the measurement unit 9 includes the electrode holding member 1, the cell culture container 2, the culture solution in the cell culture container 2, and the measuring device 90 connected to the electrode holding member 1.
  • the measuring device 90 has a power supply device 91 and a voltmeter 92.
  • the two output terminals of the power supply device 91 are connected to the first working electrode 42 and the second working electrode 52 via the lead wire 12. Further, the two input terminals of the voltmeter 92 are connected to the first reference electrode 43 and the second reference electrode 53 via the lead wire 12.
  • the resistance Rm is the electrical resistance of the cell culture section 33 and the cells cultured on the cell culture section 33 (hereinafter, referred to as “cell section”).
  • the resistance Rw1 is the electric resistance of the culture solution between the first working electrode 42 and the cell part.
  • the resistance Rw2 is the electric resistance of the culture solution between the second working electrode 52 and the cell part.
  • the resistance Rr1 is the electric resistance of the culture solution between the first reference electrode 43 and the cell part.
  • the resistance Rr2 is the electrical resistance of the culture solution between the second reference electrode 53 and the cell part.
  • the resistance values of the resistances Rw1, Rr1, Rw2, Rr2 between the electrodes 42, 43, 52, 53 and the cell part and the resistance Rm of the cell culture part 33 in the state where the cells are not cultured are respectively set in advance. Measure as a control.
  • the power supply device 91 is driven to apply a potential between the first working electrode 42 and the second working electrode 52, and at the same time, the voltage between the first reference electrode 43 and the second reference electrode 53 is measured by the voltmeter 92. Measure the value.
  • the electrical resistance value between the working electrodes 42 and 52 is calculated by estimating the accurate voltage value between the working electrodes 42 and 52 from the measured voltage value. Further, the resistance Rm of the cell portion is calculated from the electric resistance value between the working electrodes 42 and 52. Thereby, the electrical characteristics of the cells cultured on the cell culture section 33 can be obtained.
  • the oxidation reaction of the culture solution and the oxidation reaction of the culture solution occur on the electrode surfaces of the first working electrode 42 and the second working electrode 52.
  • a reduction reaction occurs.
  • an electric double layer is formed on the electrode surfaces of the first working electrode 42 and the second working electrode 52. Therefore, the output potential of the power supply device 91 does not become an accurate voltage value between the working electrodes 42 and 52. Therefore, the first reference electrode 43 and the second reference electrode 53 are arranged near the first working electrode 42 and the second working electrode 52, respectively, and the potential between the reference electrodes 43 and 53 is measured.
  • the resistance Rm of the cell part can be more accurately measured by performing calculation using the measured potential.
  • the electrode holding member 1 is provided with an observation opening 13 corresponding to each well 21. This makes it easy to observe the cells cultured on the cell culture unit 33. Furthermore, by arranging the first electrode portion 40 and the second electrode portion 50 of the electrode portion 10 at positions that do not vertically overlap the observation opening 13 of the observation opening 13, it is possible to measure the electric resistance of the cultured cells. , And observation of cells can be performed at the same time.
  • the number of electrode parts of the electrode holding member is the same as the number of observation openings. That is, the electrode holding member had the electrode portion corresponding to each of all the observation openings.
  • the number of electrode portions included in the electrode holding member of the present invention may be smaller than the number of observation openings. That is, the electrode holding member may have an electrode portion corresponding to only a part of the plurality of observation openings.
  • the present invention is not limited to this.
  • the conductor portion is not limited to the one printed on the substrate. Further, the arrangement of the conductor portion can be changed as appropriate.
  • the first electrode portion and the second electrode portion are arranged in a straight line in the plurality of electrode portions arranged in the same row, but the present invention is not limited to this.
  • the second electrode portion may be arranged at the corner of the well.
  • the shape of the well of the cell culture container on which the electrode holding member is placed is substantially quadrangular in a top view, but the present invention is not limited to this.
  • the shape of the well of the cell culture container may be round when viewed from above.
  • the detailed configuration of the electrode holding member may be different from the drawings of the present application. Further, the respective elements appearing in the above-described embodiments and modifications may be appropriately combined within a range where no contradiction occurs.
  • Electrode holding member 2 Cell culture container 9 Measurement unit 10 Electrode part 11 Substrate 12 Conductive wire 13 Observation opening 14 Through hole 15 Conductive connection member 16 Conductive adhesive 20 Insert cup 21 Well 31 Tube part 32 Support part 33 Cell culture part 40 1st electrode part 41 1st columnar member 42 1st working electrode 43 1st reference electrode 50 2nd electrode part 51 2nd columnar member 52 2nd working electrode 53 2nd reference electrode

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Abstract

Provided is a technology with which it is possible to easily measure the electrical resistance of cultured cells, by using a generic cell culture vessel. This electrode retention member (1) is used to arrange electrodes in multiple recesses formed in a well plate. The electrode retention member (1) has a tabular and insulating substrate (11) and a plurality of electrode parts (10). The substrate (11) has a plurality of observation openings (13) arranged in a grid configuration, and extends horizontally. The respective electrode parts (10) are arranged in the vicinity of some or all of the observation openings (13). Each of the electrode parts (10) has a first electrode section (40) and a second electrode section (50). The first electrode section (40) and the second electrode section respectively have one end thereof fixed to the substrate (11), and extend downward from the substrate (11).

Description

電極保持部材Electrode holding member
　本発明は、培養した細胞の電気抵抗を測定するための電極保持部材に関する。 The present invention relates to an electrode holding member for measuring the electric resistance of cultured cells.
　従来、培養した細胞の性質や、培養状態を調べるために細胞の電気抵抗値を計測するための技術が知られている。例えば、上皮細胞抵抗（ＴＥＥＲ）計測では、培養液中において細胞培養用の膜の一方側と他方側とに電極を配置して電気抵抗値を計測することにより、膜状に培養された細胞自体の電気抵抗値を計測する。このように細胞の電気抵抗値を計測する技術については、例えば、特許文献１に記載されている。 Conventionally, a technique for measuring the electrical resistance value of cells in order to investigate the properties of cultured cells and the state of culture is known. For example, in the measurement of epithelial cell resistance (TEER), cells cultured in a film form are obtained by arranging electrodes on one side and the other side of a cell culture membrane in a culture medium to measure an electric resistance value. Measure the electrical resistance value of. A technique for measuring the electric resistance value of a cell in this way is described in, for example, Patent Document 1.
国際公開第２０１２／１４７４６３号International Publication No. 2012/147463
　特許文献１に記載の細胞培養容器は、蓋部から細胞が培養されるインサート容器の内部まで延びる電極と、インサート容器の下方において細胞培養容器の底面に配置される電極とを有する。このような電極構造では、電極を備えた専用の細胞培養容器を用いる必要がある。 The cell culture container described in Patent Document 1 has an electrode extending from the lid portion to the inside of the insert container in which cells are cultured, and an electrode arranged on the bottom surface of the cell culture container below the insert container. In such an electrode structure, it is necessary to use a dedicated cell culture container equipped with an electrode.
　本発明は、このような事情に鑑みなされたものであり、汎用的な細胞培養容器を用いて、培養された細胞の電気抵抗を簡便に計測することができる技術を提供することを目的とする。 The present invention has been made in view of such circumstances, and an object thereof is to provide a technique capable of easily measuring the electric resistance of cultured cells using a general-purpose cell culture container. ..
　上記課題を解決するため、本願の第１発明は、複数の凹部を有するウェルプレートの前記凹部内に電極を配置するための電極保持部材であって、格子状に配置された複数の観察用開口を有し、水平方向に延びる平板状かつ絶縁性の基板と、前記観察用開口の一部または全てのそれぞれの近傍に配置される、複数の電極部と、を有し、前記電極部はそれぞれ、一端を前記基板に固定され、前記基板から下方へ延びる第１電極部および第２電極部を有する。 In order to solve the above-mentioned problems, a first invention of the present application is an electrode holding member for arranging an electrode in the recess of a well plate having a plurality of recesses, and a plurality of observation openings arranged in a grid pattern. And a flat plate-like and insulative substrate extending in the horizontal direction, and a plurality of electrode portions arranged in the vicinity of a part or all of the observation opening, each of the electrode portions. , One end of which is fixed to the substrate and extends downward from the substrate.
　本願の第２発明は、第１発明の電極保持部材であって、前記第１電極部は、絶縁性の第１柱状部材と、前記第１柱状部材の側面に設けられる第１作用電極と、前記第１柱状部材の側面に設けられる第１参照電極と、を有し、前記第２電極部は、絶縁性の第２柱状部材と、前記第２柱状部材の側面に設けられる第２作用電極と、前記第２柱状部材の側面に設けられる第２参照電極と、を有する。 2nd invention of this application is an electrode holding member of 1st invention, Comprising: The said 1st electrode part is an insulating 1st columnar member, The 1st working electrode provided in the side surface of the 1st columnar member, A second reference electrode provided on a side surface of the first columnar member, the second electrode portion having an insulating second columnar member, and a second working electrode provided on a side surface of the second columnar member. And a second reference electrode provided on the side surface of the second columnar member.
　本願の第３発明は、第２発明の電極保持部材であって、前記基板は、少なくとも一部が前記基板の上面にプリントされた複数の導線を有し、前記第１電極部および前記第２電極部はそれぞれ、その上端部が前記基板の上面よりも上方に配置され、前記基板の上面にプリントされた前記導線の上面と、前記第１作用電極、前記第１参照電極、前記第２作用電極、および前記第２参照電極のいずれかとは、略直方体の導電性接続部材を介して電気的に接続される。 3rd invention of this application is an electrode holding member of 2nd invention, Comprising: The said board|substrate has several conductor wire at least one part was printed on the upper surface of the said board|substrate, The said 1st electrode part and the said 2nd The upper end portion of each of the electrode portions is disposed above the upper surface of the substrate, and the upper surface of the lead wire printed on the upper surface of the substrate, the first working electrode, the first reference electrode, and the second working electrode. The electrode and any one of the second reference electrodes are electrically connected via a substantially rectangular parallelepiped conductive connecting member.
　本願の第４発明は、第１発明ないし第３発明のいずれかの電極保持部材であって、前記電極部の数は、前記観察用開口の数と同一である。 The fourth invention of the present application is the electrode holding member according to any one of the first invention to the third invention, wherein the number of the electrode portions is the same as the number of the observation openings.
　本願の第５発明は、第１発明ないし第３発明のいずれかの電極保持部材であって、前記電極部の数は、前記観察用開口の数よりも少ない。 The fifth invention of the present application is the electrode holding member according to any one of the first to third inventions, wherein the number of the electrode portions is smaller than the number of the observation openings.
　本願の第１発明～第５発明によれば、汎用的な細胞培養容器を用いて、培養された細胞の電気抵抗を簡便に計測することができる。 According to the first to fifth inventions of the present application, it is possible to easily measure the electric resistance of cultured cells using a general-purpose cell culture container.
細胞培養容器の上面図である。It is a top view of a cell culture container. 電極保持部材の上面図である。It is a top view of an electrode holding member. 細胞培養容器および電極保持部材の断面図である。It is sectional drawing of a cell culture container and an electrode holding member. 電極保持部材の部分断面図である。It is a fragmentary sectional view of an electrode holding member. 計測ユニットの電気的な接続を示した概略図である。It is the schematic which showed the electrical connection of a measurement unit.
　以下、本発明の実施形態について、図面を参照しつつ説明する。なお、以下の説明において電極保持部材の基板に平行な方向を「水平方向」、水平方向に直交する方向を「上下方向」と称する。しかしながら、細胞培養容器および電極保持部材の使用時の姿勢は、電極保持部材の基板が水平方向とならなくてもよい。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description, the direction parallel to the substrate of the electrode holding member is called "horizontal direction", and the direction orthogonal to the horizontal direction is called "vertical direction". However, the postures of the cell culture container and the electrode holding member during use may be such that the substrate of the electrode holding member is not horizontal.
　＜１．細胞培養容器および電極保持部材の構成＞
　図１は、本発明の一実施形態に係る電極保持部材１とともに用いられる細胞培養容器２の上面図である。図２は、本発明の一実施形態に係る電極保持部材１の上面図である。図２中に、電極保持部材１の使用時における電極保持部材１の各部の位置が破線で示されている。なお、図２中、拡大部を除いて、導電性接続部材１５が省略して示されている。図３は、電極保持部材１および細胞培養容器２のＡ－Ａ断面における断面図である。図４は、電極保持部材１のＢ－Ｂ断面における部分断面図である。なお、図４は、位置関係の把握を容易にするために、各構成要素を誇張して表示しており、その寸法は実際と異なる。 <1. Structure of cell culture container and electrode holding member>
FIG. 1 is a top view of a cell culture container 2 used with an electrode holding member 1 according to an embodiment of the present invention. FIG. 2 is a top view of the electrode holding member 1 according to the embodiment of the present invention. In FIG. 2, the positions of the respective parts of the electrode holding member 1 when the electrode holding member 1 is used are shown by broken lines. Note that, in FIG. 2, the conductive connecting member 15 is omitted except for the enlarged portion. FIG. 3 is a cross-sectional view taken along the line AA of the electrode holding member 1 and the cell culture container 2. FIG. 4 is a partial cross-sectional view of the electrode holding member 1 taken along the line BB. In FIG. 4, each component is exaggerated for easy understanding of the positional relationship, and the dimensions are different from the actual ones.
　細胞培養容器２は、電極保持部材１の有する電極部１０を用いて電気抵抗値を計測する対象である細胞を培養するための容器である。細胞培養容器２は、複数のウェル２１を有する、いわゆるウェルプレートである。図１および図３に示すように、ウェル２１は、細胞培養容器２の上面から凹む凹部である。本実施形態の細胞培養容器２では、縦２行、横３列の合計６個のウェル２１が格子状に配列されている。なお、本発明の電極保持部材とともに用いられる細胞培養容器のウェルの数は、６個に限られず、例えば、１２個、２４個、９６個、３８４個等であってもよい。 The cell culture container 2 is a container for culturing cells whose electric resistance value is to be measured using the electrode portion 10 of the electrode holding member 1. The cell culture container 2 is a so-called well plate having a plurality of wells 21. As shown in FIGS. 1 and 3, the well 21 is a recess recessed from the upper surface of the cell culture container 2. In the cell culture container 2 of the present embodiment, a total of 6 wells 21 arranged in 2 rows and 3 columns are arranged in a lattice. The number of wells of the cell culture container used with the electrode holding member of the present invention is not limited to 6, and may be 12, 24, 96, 384, etc., for example.
　細胞培養容器２の各ウェル２１の内部には、インサートカップ２０が配置される。インサートカップ２０は、筒部３１と、支持部３２と、細胞培養部３３とを有する。筒部３１は、円錐台状かつ筒状に形成された絶縁性の部位である。 An insert cup 20 is arranged inside each well 21 of the cell culture container 2. The insert cup 20 has a tubular portion 31, a support portion 32, and a cell culture portion 33. The tubular portion 31 is an insulating portion formed in a truncated cone shape and a tubular shape.
　支持部３２は、筒部３１の上端から外方へと延びる。支持部３２を細胞培養容器２の上面に載置すると、ウェル２１の内部であって、細胞培養部３３がウェル２１の底面に接触しない位置に、支持部３２および細胞培養部３３を配置することができる。本実施形態では、筒部３１の周方向の３箇所に支持部３２が放射状に配置される。しかしながら、支持部３２は、筒部３１および細胞培養部３３を所定の位置に支持できる構成であれば、他の形状であってもよい。例えば、支持部３２は、インサートカップ２０の周方向の２箇所のみに配置されてもよいし、４箇所以上に配置されてもよい。また、支持部３２は、インサートカップ２０の周方向の一部から外方へ延びるフランジ状であってもよい。 The support portion 32 extends outward from the upper end of the tubular portion 31. When the support part 32 is placed on the upper surface of the cell culture container 2, the support part 32 and the cell culture part 33 should be arranged inside the well 21 at a position where the cell culture part 33 does not contact the bottom surface of the well 21. You can In this embodiment, the support portions 32 are radially arranged at three locations in the circumferential direction of the tubular portion 31. However, the support portion 32 may have another shape as long as it can support the tubular portion 31 and the cell culture portion 33 at a predetermined position. For example, the support portions 32 may be arranged at only two positions in the circumferential direction of the insert cup 20, or may be arranged at four or more positions. Further, the support portion 32 may have a flange shape that extends outward from a part of the insert cup 20 in the circumferential direction.
　細胞培養部３３は、筒部３１の下方の開口を覆う膜である。細胞培養部３３には、細胞接着性を有する膜が用いられる。なお、細胞培養部３３には、微細な貫通孔が多数設けられた板状の部材が用いられてもよい。計測対象である細胞の培養時には、ウェル２１の内部に、少なくとも細胞培養部３３が浸る位置まで培養液を注入する。 The cell culture section 33 is a film that covers the opening below the tubular section 31. A film having cell adhesiveness is used for the cell culture unit 33. A plate-shaped member provided with a large number of minute through holes may be used for the cell culture section 33. At the time of culturing cells to be measured, the culture solution is injected into the well 21 at least to the position where the cell culture section 33 is immersed.
　電極保持部材１は、細胞培養容器２のウェル２１内に電極を配置するための部材である。具体的には、電極保持部材１は、細胞培養容器２に載置して、各ウェル２１内のインサートカップ２０上で培養された細胞の電気抵抗値を計測するための各電極４２，４３，５２，５３を保持する。電極４２，４３，５２，５３については、後述する。 The electrode holding member 1 is a member for disposing electrodes in the well 21 of the cell culture container 2. Specifically, the electrode holding member 1 is placed on the cell culture container 2 and each electrode 42, 43, for measuring the electric resistance value of the cells cultured on the insert cup 20 in each well 21. Hold 52 and 53. The electrodes 42, 43, 52, 53 will be described later.
　電極保持部材１は、基板１１と、基板上に形成された複数の導線１２と、複数の電極部１０とを有する。 The electrode holding member 1 has a substrate 11, a plurality of conducting wires 12 formed on the substrate, and a plurality of electrode portions 10.
　基板１１は、水平方向に延びる平板状かつ絶縁性の基板である。基板１１は、例えば、ガラスエポキシ樹脂により形成される。基板１１には、格子状に配置された複数の観察用開口１３が設けられる。各観察用開口１３は、細胞培養容器２の各ウェル２１の位置に合わせて形成されている。 The substrate 11 is a flat plate-like insulating substrate that extends in the horizontal direction. The substrate 11 is formed of, for example, glass epoxy resin. The substrate 11 is provided with a plurality of observation openings 13 arranged in a grid pattern. Each observation opening 13 is formed in accordance with the position of each well 21 of the cell culture container 2.
　各導線１２の一端は、電極部１０の後述する電極４２，４３，５２，５３のいずれかと電気的に接続される電極接続端子１２１である。各導線１２の他端は、外部と電気的に接続可能な外部接続端子１２２である。導線１２のうち、電極接続端子１２１を含む少なくとも一部は、基板１１の上面にプリントされることにより形成される。導線１２のその他の部分は、複数層に形成された基板１１の内部の層にプリントされたり、基板１１の形成時に内部に埋め込まれたりしていてもよい。 An end of each conductive wire 12 is an electrode connection terminal 121 electrically connected to any of the electrodes 42, 43, 52, 53 of the electrode unit 10 described later. The other end of each conductor 12 is an external connection terminal 122 that can be electrically connected to the outside. At least a part of the conductive wire 12 including the electrode connection terminal 121 is formed by being printed on the upper surface of the substrate 11. Other portions of the conductive wire 12 may be printed on a layer inside the substrate 11 formed in multiple layers, or may be embedded inside when the substrate 11 is formed.
　電極部１０はそれぞれ、観察用開口１３の近傍に配置される。本実施形態では、全ての観察用開口１３ごとに、電極部１０が配置されている。しかしながら、一部の観察用開口１３のみに対して、電極部１０が配置されていてもよい。電極部１０は、それぞれ、第１電極部４０と、第２電極部５０とを有する。 Each of the electrode parts 10 is arranged near the observation opening 13. In the present embodiment, the electrode portion 10 is arranged for every observation opening 13. However, the electrode portion 10 may be arranged only for some of the observation openings 13. The electrode parts 10 each include a first electrode part 40 and a second electrode part 50.
　第１電極部４０は、第１柱状部材４１と、第１作用電極４２と、第１参照電極４３とを有する。第１電極部４０は、その一端が基板１１に固定される。また、第１電極部４０は、基板１１から下方へ延びる。 The first electrode portion 40 has a first columnar member 41, a first working electrode 42, and a first reference electrode 43. One end of the first electrode portion 40 is fixed to the substrate 11. Further, the first electrode portion 40 extends downward from the substrate 11.
　第１柱状部材４１は、上下方向に延びる角柱状の絶縁性部材である。第１柱状部材４１は、例えば、ガラスにより形成される。 The first columnar member 41 is a prismatic insulating member extending in the vertical direction. The first columnar member 41 is made of glass, for example.
　第１作用電極４２は、第１柱状部材４１の４つの側面のうちの１つの全体に設けられる。第１参照電極４３は、第１柱状部材４１の他の側面の全体に設けられる。第１作用電極４２と第１参照電極４３とは、互いに隣り合わず、間隔を空けて配置される。本実施形態では、第１作用電極４２および第１参照電極４３はそれぞれ、第１柱状部材４１の側面に形成された金属薄膜である。しかしながら、第１作用電極４２および第１参照電極４３はそれぞれ、第１柱状部材４１に金属板を接着したものや、その他の構成であってもよい。 The first working electrode 42 is provided on the entire one of the four side surfaces of the first columnar member 41. The first reference electrode 43 is provided on the entire other side surface of the first columnar member 41. The first working electrode 42 and the first reference electrode 43 are not adjacent to each other and are arranged with a space. In the present embodiment, each of the first working electrode 42 and the first reference electrode 43 is a metal thin film formed on the side surface of the first columnar member 41. However, each of the first working electrode 42 and the first reference electrode 43 may have a structure in which a metal plate is bonded to the first columnar member 41, or may have another configuration.
　第２電極部５０は、第２柱状部材５１と、第２作用電極５２と、第２参照電極５３とを有する。第２電極部５０は、その一端が基板１１に固定される。また、第２電極部５０は、基板１１から下方へ延びる。 The second electrode section 50 has a second columnar member 51, a second working electrode 52, and a second reference electrode 53. The second electrode portion 50 has one end fixed to the substrate 11. In addition, the second electrode portion 50 extends downward from the substrate 11.
　第２柱状部材５１は、上下方向に延びる角柱状の絶縁性部材である。第２柱状部材５１は、例えば、ガラスにより形成される。 The second columnar member 51 is a prismatic insulating member extending in the vertical direction. The second columnar member 51 is made of, for example, glass.
　第２作用電極５２は、第２柱状部材５１の４つの側面のうちの１つの全体に設けられる。第２参照電極５３は、第２柱状部材５１の他の側面の全体に設けられる。第２作用電極５２と第２参照電極５３とは、互いに隣り合わず、間隔を空けて配置される。本実施形態では、第２作用電極５２および第２参照電極５３とはそれぞれ、第２柱状部材５１の側面に形成された金属薄膜である。しかしながら、第２作用電極５２および第２参照電極５３はそれぞれ、第２柱状部材５１に金属板を接着したものや、その他の構成であってもよい。 The second working electrode 52 is provided on the entire one of the four side surfaces of the second columnar member 51. The second reference electrode 53 is provided on the entire other side surface of the second columnar member 51. The second working electrode 52 and the second reference electrode 53 are not adjacent to each other and are arranged with a space. In the present embodiment, each of the second working electrode 52 and the second reference electrode 53 is a metal thin film formed on the side surface of the second columnar member 51. However, each of the second working electrode 52 and the second reference electrode 53 may have a structure in which a metal plate is adhered to the second columnar member 51, or may have another configuration.
　図２～図４に示すように、基板１１には、観察用開口１３の周囲に、第１電極部４０および第２電極部５０が挿入される貫通孔１４が設けられている。図３および図４に示すように、第１電極部４０および第２電極部５０はそれぞれ、その上端部が基板１１の上面よりも上方に配置される。 As shown in FIGS. 2 to 4, the substrate 11 is provided with a through hole 14 around the observation opening 13 into which the first electrode portion 40 and the second electrode portion 50 are inserted. As shown in FIGS. 3 and 4, the upper ends of the first electrode portion 40 and the second electrode portion 50 are arranged above the upper surface of the substrate 11, respectively.
　図２に示すように、各貫通孔１４に隣接する位置に、２つの導線１２の電極接続端子１２１が配置される。図４に示すように、第１作用電極４２および第１参照電極４３はそれぞれ、導電性接続部材１５を介して導線１２の電極接続端子１２１に電気的に接続される。 As shown in FIG. 2, the electrode connection terminals 121 of the two conductive wires 12 are arranged at positions adjacent to each through hole 14. As shown in FIG. 4, each of the first working electrode 42 and the first reference electrode 43 is electrically connected to the electrode connection terminal 121 of the lead wire 12 via the conductive connection member 15.
　各導電性接続部材１５は、略直方体形状である。図２中の拡大部および図４に示すように、導電性接続部材１５は、各導線１２の電極接続端子１２１の上に配置される。また、図４に示すように、第１作用電極４２の上端部付近が、１つの導電性接続部材１５の側面と導電性接着剤１６により接着される。また、当該導電性接続部材１５の下面と１つの導線１２の電極接続端子１２１の上面とが、導電性接着剤１６により接着される。これにより、第１作用電極４２と１つの導線１２とが、導電性接続部材１５および導電性接着剤１６を介して電気的に接続される。 Each conductive connecting member 15 has a substantially rectangular parallelepiped shape. As shown in the enlarged portion of FIG. 2 and FIG. 4, the conductive connection member 15 is arranged on the electrode connection terminal 121 of each conductor 12. Further, as shown in FIG. 4, the vicinity of the upper end portion of the first working electrode 42 is bonded to the side surface of one conductive connecting member 15 by the conductive adhesive 16. Further, the lower surface of the conductive connecting member 15 and the upper surface of the electrode connecting terminal 121 of the one conducting wire 12 are bonded by the conductive adhesive 16. As a result, the first working electrode 42 and the one conductive wire 12 are electrically connected to each other via the conductive connecting member 15 and the conductive adhesive 16.
　同様に、第１参照電極４３の上端部付近が、他の１つの導電性接続部材１５の側面と導電性接着剤１６により接着される。また、当該導電性接続部材１５の下面と他の１つの導線１２の電極接続端子１２１の上面とが、導電性接着剤１６により接着される。これにより、第１参照電極４３と他の１つの導線１２とが、導電性接続部材１５および導電性接着剤１６を介して電気的に接続される。 Similarly, the vicinity of the upper end of the first reference electrode 43 is bonded to the side surface of the other conductive connecting member 15 with the conductive adhesive 16. Further, the lower surface of the conductive connecting member 15 and the upper surface of the electrode connecting terminal 121 of the other one conducting wire 12 are adhered by the conductive adhesive 16. As a result, the first reference electrode 43 and the other one conducting wire 12 are electrically connected via the conductive connecting member 15 and the conductive adhesive 16.
　また、このように第１作用電極４２および第１参照電極４３が導電性接続部材１５を介して基板１１に接着されることにより、第１電極部４０の上端部が基板１１に固定される。 In addition, the first working electrode 42 and the first reference electrode 43 are thus adhered to the substrate 11 via the conductive connecting member 15 so that the upper end of the first electrode portion 40 is fixed to the substrate 11.
　第２作用電極５２および第２参照電極５３についても、第１作用電極４２および第１参照電極４３と同様に、導電性接続部材１５を介して導線１２と接続される。 Similarly to the first working electrode 42 and the first reference electrode 43, the second working electrode 52 and the second reference electrode 53 are also connected to the conducting wire 12 via the conductive connecting member 15.
　電極保持部材１を細胞培養容器２に載置する際には、図１に示すように、電極部１０を挿入するウェル２１において、インサートカップ２０を所定の方向の一方側（図１中では右側）に寄せて配置する。そして、電極保持部材１を細胞培養容器２に載置することによって、電極部１０を所望のウェル２１内に挿入する。 When the electrode holding member 1 is placed on the cell culture vessel 2, as shown in FIG. 1, in the well 21 into which the electrode portion 10 is inserted, the insert cup 20 is placed on one side in a predetermined direction (on the right side in FIG. 1). ) And place it. Then, by placing the electrode holding member 1 on the cell culture container 2, the electrode portion 10 is inserted into the desired well 21.
　本実施形態では、電極保持部材１を細胞培養容器２に載置した際に、各観察用開口１３が各ウェル２１の略中央に配置される。ここで、図２に示すように、第１電極部４０は、観察用開口１３の一方側（図２中では右側）に配置され、第２電極部５０は、観察用開口１３の他方側（図２中では左側）に配置される。上記の通り、インサートカップ２０は、ウェル２１の一方側に偏って配置されるため、第１電極部４０は、インサートカップ２０の内部に挿入され、第２電極部５０は、インサートカップ２０の外部に挿入される。 In the present embodiment, when the electrode holding member 1 is placed on the cell culture container 2, each observation opening 13 is arranged at substantially the center of each well 21. Here, as shown in FIG. 2, the first electrode portion 40 is arranged on one side (right side in FIG. 2) of the observation opening 13, and the second electrode portion 50 is disposed on the other side of the observation opening 13 ( It is arranged on the left side in FIG. As described above, since the insert cup 20 is biased to one side of the well 21, the first electrode portion 40 is inserted inside the insert cup 20, and the second electrode portion 50 is outside the insert cup 20. Inserted in.
　図３に示すように、第２電極部５０は、第１電極部４０よりも下方に突出している。これにより、電極保持部材１を細胞培養容器２に載置した際に、第１電極部４０の下端部がインサートカップ２０の細胞培養部３３よりも上方に配置されるとともに、第２電極部５０の下端部が細胞培養部３３よりも下方に配置される。 As shown in FIG. 3, the second electrode section 50 projects below the first electrode section 40. As a result, when the electrode holding member 1 is placed on the cell culture container 2, the lower end portion of the first electrode portion 40 is arranged above the cell culture portion 33 of the insert cup 20 and the second electrode portion 50. The lower end of the cell culture section is arranged below the cell culture section 33.
　＜２．培養細胞の電気抵抗計測＞
　続いて、電極保持部材１に保持される電極４２，４３，５２，５３を用いた培養細胞の電気抵抗計測の方法について、図３および図５を参照しつつ説明する。図５は、計測ユニット９の電気的な接続を示した概略図である。なお、図５では、電極４２，４３，５２，５３が１組のみの場合における計測ユニット９の電気的な接続を示している。なお、複数の電極部１０に対して同時に計測を行う場合には、計測ユニット９の構成が異なる。例えば、後述する計測装置９０の電源装置９１や電圧計９２の接続先を、電極部１０ごとに切替可能とする。 <2. Measurement of electrical resistance of cultured cells>
Next, a method for measuring the electric resistance of cultured cells using the electrodes 42, 43, 52, 53 held by the electrode holding member 1 will be described with reference to FIGS. 3 and 5. FIG. 5 is a schematic diagram showing the electrical connection of the measurement unit 9. Note that FIG. 5 shows the electrical connection of the measurement unit 9 when the number of the electrodes 42, 43, 52, 53 is only one set. It should be noted that the configuration of the measurement unit 9 is different when simultaneously measuring the plurality of electrode portions 10. For example, the connection destinations of the power supply device 91 and the voltmeter 92 of the measuring device 90 described later can be switched for each electrode unit 10.
　図５に示すように、計測ユニット９は、上記の電極保持部材１、細胞培養容器２および細胞培養容器２内の培養液と、電極保持部材１と接続する計測装置９０とにより構成される。 As shown in FIG. 5, the measurement unit 9 includes the electrode holding member 1, the cell culture container 2, the culture solution in the cell culture container 2, and the measuring device 90 connected to the electrode holding member 1.
　計測装置９０は、電源装置９１と、電圧計９２とを有する。電源装置９１の２つの出力端子は、導線１２を介して第１作用電極４２および第２作用電極５２に接続される。また、電圧計９２の２つの入力端子は、導線１２を介して第１参照電極４３および第２参照電極５３に接続される。 The measuring device 90 has a power supply device 91 and a voltmeter 92. The two output terminals of the power supply device 91 are connected to the first working electrode 42 and the second working electrode 52 via the lead wire 12. Further, the two input terminals of the voltmeter 92 are connected to the first reference electrode 43 and the second reference electrode 53 via the lead wire 12.
　培養細胞の電気抵抗計測を行う場合、ウェル２１には、第１作用電極４２および第１参照電極４３の少なくとも先端部が浸る量の培養液を注入する必要がある。なお、培養液中の抵抗値の条件を均一化するためには、ウェル２１内を培養液で満たすことが好ましい。 When measuring the electrical resistance of the cultured cells, it is necessary to inject into the well 21 a sufficient amount of the culture solution in which at least the tips of the first working electrode 42 and the first reference electrode 43 are immersed. In order to equalize the resistance value conditions in the culture medium, it is preferable to fill the well 21 with the culture medium.
　図５において、抵抗Ｒｍは、細胞培養部３３および細胞培養部３３上に培養された細胞（以下、「細胞部」と称する）による電気抵抗である。抵抗Ｒｗ１は、第１作用電極４２と細胞部との間における培養液の電気抵抗である。抵抗Ｒｗ２は、第２作用電極５２と細胞部との間における培養液の電気抵抗である。抵抗Ｒｒ１は、第１参照電極４３と細胞部との間における培養液の電気抵抗である。抵抗Ｒｒ２は、第２参照電極５３と細胞部との間における培養液の電気抵抗である。 In FIG. 5, the resistance Rm is the electrical resistance of the cell culture section 33 and the cells cultured on the cell culture section 33 (hereinafter, referred to as “cell section”). The resistance Rw1 is the electric resistance of the culture solution between the first working electrode 42 and the cell part. The resistance Rw2 is the electric resistance of the culture solution between the second working electrode 52 and the cell part. The resistance Rr1 is the electric resistance of the culture solution between the first reference electrode 43 and the cell part. The resistance Rr2 is the electrical resistance of the culture solution between the second reference electrode 53 and the cell part.
　各電極４２，４３，５２，５３と細胞部との間における各抵抗Ｒｗ１，Ｒｒ１，Ｒｗ２，Ｒｒ２と、細胞が培養されていない状態の細胞培養部３３の抵抗Ｒｍとの抵抗値はそれぞれ、予めコントロールとして測定しておく。 The resistance values of the resistances Rw1, Rr1, Rw2, Rr2 between the electrodes 42, 43, 52, 53 and the cell part and the resistance Rm of the cell culture part 33 in the state where the cells are not cultured are respectively set in advance. Measure as a control.
　そして、電源装置９１を駆動させて第１作用電極４２と第２作用電極５２との間に電位をかけ、同時に、電圧計９２によって第１参照電極４３と第２参照電極５３との間の電圧値を計測する。計測した電圧値から、作用電極４２，５２間の正確な電圧値を推測することにより、作用電極４２，５２間の電気抵抗値を算出する。さらに、作用電極４２，５２間の電気抵抗値から細胞部の抵抗Ｒｍを算出する。これにより、細胞培養部３３上に培養された細胞の電気的特性を得ることができる。 Then, the power supply device 91 is driven to apply a potential between the first working electrode 42 and the second working electrode 52, and at the same time, the voltage between the first reference electrode 43 and the second reference electrode 53 is measured by the voltmeter 92. Measure the value. The electrical resistance value between the working electrodes 42 and 52 is calculated by estimating the accurate voltage value between the working electrodes 42 and 52 from the measured voltage value. Further, the resistance Rm of the cell portion is calculated from the electric resistance value between the working electrodes 42 and 52. Thereby, the electrical characteristics of the cells cultured on the cell culture section 33 can be obtained.
　なお、電源装置９１を用いて第１作用電極４２と第２作用電極５２との間に電位をかけると、第１作用電極４２および第２作用電極５２の電極表面において、培養液の酸化反応および還元反応が起きる。これにより、第１作用電極４２および第２作用電極５２の電極表面には、電気二重層が形成される。このため、電源装置９１による出力電位が、作用電極４２，５２間の正確な電圧値とはならない。そこで、第１作用電極４２および第２作用電極５２のそれぞれの近傍に第１参照電極４３および第２参照電極５３を配置し、参照電極４３，５３間の電位を計測する。その計測電位を利用して計算することにより、より正確に細胞部の抵抗Ｒｍを計測することができる。 When a potential is applied between the first working electrode 42 and the second working electrode 52 by using the power supply device 91, the oxidation reaction of the culture solution and the oxidation reaction of the culture solution occur on the electrode surfaces of the first working electrode 42 and the second working electrode 52. A reduction reaction occurs. Thereby, an electric double layer is formed on the electrode surfaces of the first working electrode 42 and the second working electrode 52. Therefore, the output potential of the power supply device 91 does not become an accurate voltage value between the working electrodes 42 and 52. Therefore, the first reference electrode 43 and the second reference electrode 53 are arranged near the first working electrode 42 and the second working electrode 52, respectively, and the potential between the reference electrodes 43 and 53 is measured. The resistance Rm of the cell part can be more accurately measured by performing calculation using the measured potential.
　この電極保持部材１では、各ウェル２１に対応する観察用開口１３を設けている。これにより、細胞培養部３３上で培養される細胞の観察を行いやすい。さらに、電極部１０の第１電極部４０および第２電極部５０を観察用開口１３の観察用開口１３と上下方向に重ならない位置に配置することにより、培養された細胞の電気抵抗の測定と、細胞の観察とを同時に行うことができる。 The electrode holding member 1 is provided with an observation opening 13 corresponding to each well 21. This makes it easy to observe the cells cultured on the cell culture unit 33. Furthermore, by arranging the first electrode portion 40 and the second electrode portion 50 of the electrode portion 10 at positions that do not vertically overlap the observation opening 13 of the observation opening 13, it is possible to measure the electric resistance of the cultured cells. , And observation of cells can be performed at the same time.
　＜３．変形例＞
　以上、本発明の主たる実施形態について説明したが、本発明は、上記の実施形態に限定されるものではない。 <3. Modification>
Although the main embodiments of the present invention have been described above, the present invention is not limited to the above embodiments.
　上記の実施形態では、電極保持部材の有する電極部の数が観察用開口の数と同一であった。すなわち、電極保持部材が、全ての観察用開口のそれぞれに対応する電極部を有していた。しかしながら、本発明の電極保持部材の有する電極部の数は、観察用開口の数よりも少なくてもよい。すなわち、電極保持部材が、複数の観察用開口の一部のみに対応する電極部を有していてもよい。 In the above embodiment, the number of electrode parts of the electrode holding member is the same as the number of observation openings. That is, the electrode holding member had the electrode portion corresponding to each of all the observation openings. However, the number of electrode portions included in the electrode holding member of the present invention may be smaller than the number of observation openings. That is, the electrode holding member may have an electrode portion corresponding to only a part of the plurality of observation openings.
　また、上記の実施形態では、導線部の一例が示されているが、本発明はこれに限られない。導線部は、基板にプリントされたものに限られない。また、導線部の配置は、適宜変更しうる。 Also, in the above embodiment, an example of the conductor portion is shown, but the present invention is not limited to this. The conductor portion is not limited to the one printed on the substrate. Further, the arrangement of the conductor portion can be changed as appropriate.
　また、上記の実施形態では、同じ列に並ぶ複数の電極部において、第１電極部と第２電極部とが一直線上に配置されたが、本発明はこれに限られない。例えば、電極保持部材が細胞培養容器に載置された際に、第２電極部は、ウェルの角部に配置されてもよい。 Further, in the above-described embodiment, the first electrode portion and the second electrode portion are arranged in a straight line in the plurality of electrode portions arranged in the same row, but the present invention is not limited to this. For example, when the electrode holding member is placed on the cell culture container, the second electrode portion may be arranged at the corner of the well.
　また、上記の実施形態では、電極保持部材が載置される細胞培養容器のウェルの形状が上面視で略四角形であったが、本発明はこれに限られない。細胞培養容器のウェルの形状は、上面視で丸形であってもよい。 Further, in the above-described embodiment, the shape of the well of the cell culture container on which the electrode holding member is placed is substantially quadrangular in a top view, but the present invention is not limited to this. The shape of the well of the cell culture container may be round when viewed from above.
　また、電極保持部材の細部の構成については、本願の各図と相違していてもよい。また、上記の実施形態や変形例に登場した各要素を、矛盾が生じない範囲で、適宜に組み合わせてもよい。 Also, the detailed configuration of the electrode holding member may be different from the drawings of the present application. Further, the respective elements appearing in the above-described embodiments and modifications may be appropriately combined within a range where no contradiction occurs.
　１　電極保持部材
　２　細胞培養容器
　９　計測ユニット
　１０　電極部
　１１　基板
　１２　導線
　１３　観察用開口
　１４　貫通孔
　１５　導電性接続部材
　１６　導電性接着剤
　２０　インサートカップ
　２１　ウェル
　３１　筒部
　３２　支持部
　３３　細胞培養部
　４０　第１電極部
　４１　第１柱状部材
　４２　第１作用電極
　４３　第１参照電極
　５０　第２電極部
　５１　第２柱状部材
　５２　第２作用電極
　５３　第２参照電極 DESCRIPTION OF SYMBOLS 1 Electrode holding member 2 Cell culture container 9 Measurement unit 10 Electrode part 11 Substrate 12 Conductive wire 13 Observation opening 14 Through hole 15 Conductive connection member 16 Conductive adhesive 20 Insert cup 21 Well 31 Tube part 32 Support part 33 Cell culture part 40 1st electrode part 41 1st columnar member 42 1st working electrode 43 1st reference electrode 50 2nd electrode part 51 2nd columnar member 52 2nd working electrode 53 2nd reference electrode

Claims (5)

  1. 　複数の凹部を有するウェルプレートの前記凹部内に電極を配置するための電極保持部材であって、
    　格子状に配置された複数の観察用開口を有し、水平方向に延びる平板状かつ絶縁性の基板と、
    　前記観察用開口の一部または全てのそれぞれの近傍に配置される、複数の電極部と、
    を有し、
    　前記電極部はそれぞれ、
    　　一端を前記基板に固定され、前記基板から下方へ延びる第１電極部および第２電極部
    を有する、電極保持部材。     An electrode holding member for disposing an electrode in the recess of a well plate having a plurality of recesses,
    A flat plate-like insulating substrate having a plurality of observation openings arranged in a lattice and extending in the horizontal direction;
    A plurality of electrode portions arranged in the vicinity of each or all of the observation openings,
    Have
    Each of the electrode parts is
    An electrode holding member having one end fixed to the substrate and having a first electrode portion and a second electrode portion extending downward from the substrate.
  2. 　請求項１に記載の電極保持部材であって、
    　前記第１電極部は、
    　　絶縁性の第１柱状部材と、
    　　前記第１柱状部材の側面に設けられる第１作用電極と、
    　　前記第１柱状部材の側面に設けられる第１参照電極と、
    を有し、
    　前記第２電極部は、
    　　絶縁性の第２柱状部材と、
    　　前記第２柱状部材の側面に設けられる第２作用電極と、
    　　前記第２柱状部材の側面に設けられる第２参照電極と、
    を有する、電極保持部材。     The electrode holding member according to claim 1, wherein
    The first electrode portion,
    An insulating first columnar member,
    A first working electrode provided on a side surface of the first columnar member,
    A first reference electrode provided on a side surface of the first columnar member,
    Have
    The second electrode portion,
    An insulating second columnar member,
    A second working electrode provided on a side surface of the second columnar member,
    A second reference electrode provided on a side surface of the second columnar member,
    An electrode holding member having.
  3. 　請求項２に記載の電極保持部材であって、
    　前記基板は、
    　　少なくとも一部が前記基板の上面にプリントされた複数の導線
    を有し、
    　前記第１電極部および前記第２電極部はそれぞれ、その上端部が前記基板の上面よりも上方に配置され、
    　前記基板の上面にプリントされた前記導線の上面と、前記第１作用電極、前記第１参照電極、前記第２作用電極、および前記第２参照電極のいずれかとは、略直方体の導電性接続部材を介して電気的に接続される、電極保持部材。     The electrode holding member according to claim 2, wherein
    The substrate is
    At least a portion having a plurality of conductors printed on the upper surface of the substrate,
    The upper ends of the first electrode portion and the second electrode portion are arranged above the upper surface of the substrate,
    The upper surface of the conductive wire printed on the upper surface of the substrate and any one of the first working electrode, the first reference electrode, the second working electrode, and the second reference electrode are substantially rectangular parallelepiped conductive connecting members. An electrode holding member that is electrically connected via.
  4. 　請求項１ないし請求項３のいずれかに記載の電極保持部材であって、
    　前記電極部の数は、前記観察用開口の数と同一である、電極保持部材。     The electrode holding member according to any one of claims 1 to 3,
    The electrode holding member, wherein the number of the electrode portions is the same as the number of the observation openings.
  5. 　請求項１ないし請求項３のいずれかに記載の電極保持部材であって、
    　前記電極部の数は、前記観察用開口の数よりも少ない、電極保持部材。     The electrode holding member according to any one of claims 1 to 3,
    An electrode holding member in which the number of the electrode portions is smaller than the number of the observation openings.
PCT/JP2019/044577 2018-11-27 2019-11-13 Electrode retention member WO2020110735A1 (en)

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Citations (6)

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Publication number Priority date Publication date Assignee Title
JPS63157971A (en) * 1986-12-19 1988-06-30 Shimadzu Corp Electrode holding mechanism for well
US20030070917A1 (en) * 2001-10-11 2003-04-17 Symyx Technologies, Inc. Synthesis and characterization of materials for electrochemical cells
US20050221274A1 (en) * 2004-03-31 2005-10-06 Negulescu Paul A Multiwell plate assembly for use in high throughput assays
JP2005010180A (en) * 2004-10-07 2005-01-13 Hitachi Ltd Electrophoresis apparatus
JP2012037435A (en) * 2010-08-09 2012-02-23 Hioki Ee Corp Method and device for detecting cell behavior
JP2015163082A (en) * 2012-10-25 2015-09-10 浜松ホトニクス株式会社 cell observation apparatus and cell observation method

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