JP7097800B2 - Electrode holding member - Google Patents

Electrode holding member Download PDF

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JP7097800B2
JP7097800B2 JP2018221404A JP2018221404A JP7097800B2 JP 7097800 B2 JP7097800 B2 JP 7097800B2 JP 2018221404 A JP2018221404 A JP 2018221404A JP 2018221404 A JP2018221404 A JP 2018221404A JP 7097800 B2 JP7097800 B2 JP 7097800B2
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徳幸 中谷
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Screen Holdings Co Ltd
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    • 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
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    • 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

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Description

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 electrical 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, techniques for measuring the electrical resistance value of cells for investigating the properties of cultured cells and the state of culture have been known. For example, in epithelial cell resistance (TEER) measurement, cells themselves cultured in a membrane form are measured by arranging electrodes on one side and the other side of a cell culture membrane in a culture medium and measuring the electrical resistance value. Measure the electrical resistance value of. A technique for measuring the electrical 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 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 vessel equipped with the 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 of the present invention is to provide a technique capable of easily measuring the electrical resistance of cultured cells using a general-purpose cell culture vessel. ..

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電極部を有し、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。
In order to solve the above problems, the 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. It has a flat plate-like and insulating substrate extending in the horizontal direction, and a plurality of electrode portions arranged in the vicinity of each of a part or all of the observation openings, each of which has a plurality of electrode portions. One end thereof is fixed to the substrate and has a first electrode portion and a second electrode portion extending downward from the substrate . The first electrode portion includes an insulating first columnar member and the first columnar member. It has a first working electrode provided on the side surface and a first reference electrode provided on the side surface of the first columnar member, and the second electrode portion has an insulating second columnar member and the second columnar member. It has a second working electrode provided on the side surface of the member and a second reference electrode provided on the side surface of the second columnar member .

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。
The second invention of the present application is the electrode holding member of the first invention, wherein the substrate has a plurality of conductors having at least a part printed on the upper surface of the substrate, the first electrode portion and the second. The upper end of each of the electrode portions is arranged above the upper surface of the substrate, and the upper surface of the conducting wire printed on the upper surface of the substrate, the first working electrode, the first reference electrode, and the second acting. The electrode and any of the second reference electrodes are electrically connected via a substantially rectangular conductive connecting member.

本願の第３発明は、第１発明または第２発明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 third invention of the present application is the electrode holding member of the first invention or the second invention , and the number of the electrode portions is the same as the number of the observation openings.

本願の第４発明は、第１発明または第２発明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 fourth invention of the present application is the electrode holding member of the first invention or the second invention , and the number of the electrode portions is smaller than the number of the observation openings.

本願の第１発明～第４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 fourth inventions of the present application, the electrical resistance of the cultured cells can be easily measured by using a general-purpose cell culture vessel.

細胞培養容器の上面図である。It is a top view of the cell culture vessel. 電極保持部材の上面図である。It is a top view of the 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 the cell culture container and the electrode holding member. 電極保持部材の部分断面図である。It is a partial 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 a schematic diagram which showed the electrical connection of a measuring 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 referred to as "horizontal direction", and the direction orthogonal to the horizontal direction is referred to as "vertical direction". However, the posture of the cell culture vessel and the electrode holding member when used does not have to be such that the substrate of the electrode holding member is in the horizontal direction.

＜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. Configuration of cell culture container and electrode holding member>
FIG. 1 is a top view of a cell culture vessel 2 used together 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 position of each part of the electrode holding member 1 when the electrode holding member 1 is used is shown by a broken line. 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 vessel 2. FIG. 4 is a partial cross-sectional view taken along the line BB of the electrode holding member 1. In FIG. 4, each component is exaggerated and displayed in order to facilitate grasping the positional relationship, and the dimensions thereof 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 to be measured for electrical resistance using the electrode portion 10 of the electrode holding member 1. The cell culture vessel 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 vessel 2. In the cell culture vessel 2 of the present embodiment, a total of 6 wells 21 in 2 rows vertically and 3 columns horizontally are arranged in a grid pattern. The number of wells in the cell culture container used together with the electrode holding member of the present invention is not limited to 6, and may be, for example, 12, 24, 96, 384, or the like.

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 vessel 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、筒部３１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 portion 32 is placed on the upper surface of the cell culture vessel 2, the tubular portion 31 and the cell culture portion 33 are arranged inside the well 21 at a position where the cell culture portion 33 does not contact the bottom surface of the well 21. Can be done. In the present 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 predetermined positions. For example, the support portions 32 may be arranged only at two locations in the circumferential direction of the insert cup 20, or may be arranged at four or more locations. Further, the support portion 32 may have a flange shape extending 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 membrane that covers the lower opening of the tube section 31. A membrane having cell adhesion is used for the cell culture unit 33. A plate-shaped member provided with a large number of fine through holes may be used for the cell culture unit 33. When culturing the cells to be measured, the culture solution is injected into the well 21 at least to a position where the cell culture unit 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を行うための各電極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 arranging an electrode in the well 21 of the cell culture vessel 2. Specifically, the electrode holding member 1 is placed on the cell culture vessel 2, and the electrodes 42, 43 for measuring the electric resistance value of the cells cultured on the insert cup 20 in each well 21. , 52, 53 are retained. 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 conductors 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-shaped and insulating substrate extending in the horizontal direction. The substrate 11 is formed of, for example, a 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 so as to match the position of each well 21 of the cell culture vessel 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。 One end of each conductor 12 is an electrode connection terminal 121 that is electrically connected to any of the electrodes 42, 43, 52, and 53 described later of the electrode portion 10. 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 conducting wire 12 including the electrode connecting terminal 121 is formed by being printed on the upper surface of the substrate 11. The other parts of the conductor 12 may be printed on the inner layer of the substrate 11 formed in a plurality of 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 portions 10 is arranged in the vicinity of the observation opening 13. In the present embodiment, the electrode portion 10 is arranged for each of the observation openings 13. However, the electrode portion 10 may be arranged only for a part of the observation openings 13. The electrode portion 10 has a first electrode portion 40 and a second electrode portion 50, respectively.

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 formed of, for example, glass.

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 whole of 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 at intervals. In the present embodiment, the first working electrode 42 and the first reference electrode 43 are metal thin films formed on the side surfaces of the first columnar member 41, respectively. However, the first working electrode 42 and the first reference electrode 43 may each have a metal plate bonded to the first columnar member 41 or other configurations.

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 portion 50 has a second columnar member 51, a second working electrode 52, and a second reference electrode 53. One end of the second electrode portion 50 is fixed to the substrate 11. Further, 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 formed 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 whole of 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 at intervals. In the present embodiment, the second working electrode 52 and the second reference electrode 53 are metal thin films formed on the side surfaces of the second columnar member 51, respectively. However, the second working electrode 52 and the second reference electrode 53 may have a metal plate bonded to the second columnar member 51 or have other configurations, respectively.

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 into which the first electrode portion 40 and the second electrode portion 50 are inserted around the observation opening 13. As shown in FIGS. 3 and 4, the upper end portions of the first electrode portion 40 and the second electrode portion 50 are respectively arranged above the upper surface of the substrate 11.

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 conductors 12 are arranged at positions adjacent to the through holes 14. As shown in FIG. 4, the first working electrode 42 and the first reference electrode 43 are each electrically connected to the electrode connecting terminal 121 of the conducting wire 12 via the conductive connecting 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 cuboid shape. As shown in the enlarged portion in FIG. 2 and FIG. 4, the conductive connecting member 15 is arranged on the electrode connecting terminal 121 of each conducting wire 12. Further, as shown in FIG. 4, the vicinity of the upper end portion of the first working electrode 42 is adhered 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 one conducting wire 12 are adhered to each other by the conductive adhesive 16. As a result, the first working electrode 42 and one conducting wire 12 are electrically connected 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 portion of the first reference electrode 43 is adhered to the side surface of the other 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 other conducting wire 12 are adhered to each other by the conductive adhesive 16. As a result, the first reference electrode 43 and the other conductor 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。 Further, the first working electrode 42 and the first reference electrode 43 are adhered to the substrate 11 via the conductive connecting member 15, so that the upper end portion 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。 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 in the same manner as the first working electrode 42 and the first reference electrode 43.

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 (right side in FIG. 1). ). Then, the electrode portion 10 is inserted into the desired well 21 by placing the electrode holding member 1 on the cell culture vessel 2.

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 vessel 2, each observation opening 13 is arranged substantially in the center of each well 21. Here, as shown in FIG. 2, the first electrode portion 40 is arranged on one side of the observation opening 13 (on the right side in FIG. 2), and the second electrode portion 50 is on the other side of the observation opening 13 (on the right side in FIG. 2). It is arranged on the left side in FIG. 2). As described above, since the insert cup 20 is unevenly arranged on 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. Will be inserted into.

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 portion 50 projects downward from the first electrode portion 40. As a result, when the electrode holding member 1 is placed on the cell culture vessel 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 is arranged. The lower end of the cell culture unit 33 is arranged below the cell culture unit 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>
Subsequently, a method of measuring the electrical resistance of the 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 view showing the electrical connection of the measuring unit 9. Note that FIG. 5 shows the electrical connection of the measurement unit 9 when there is only one set of electrodes 42, 43, 52, 53. When the measurement is performed on the plurality of electrode portions 10 at the same time, the configuration of the measurement unit 9 is different. For example, the connection destination of the power supply device 91 of the measuring device 90 and the voltmeter 92, which will be 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 is composed of 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 includes 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 conducting 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 cultured cells, it is necessary to inject into the well 21 an 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 addition, in order to make the condition of the resistance value in the culture solution uniform, it is preferable to fill the well 21 with the culture solution.

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 unit 33 and the cells cultured on the cell culture unit 33 (hereinafter, referred to as “cell unit”). The resistance Rw1 is the electrical resistance of the culture solution between the first working electrode 42 and the cell portion. The resistance Rw2 is the electrical resistance of the culture solution between the second working electrode 52 and the cell portion. The resistance Rr1 is the electrical resistance of the culture medium between the first reference electrode 43 and the cell portion. The resistance Rr2 is the electrical resistance of the culture medium between the second reference electrode 53 and the cell portion.

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 unit and the resistance Rm of the cell culture unit 33 in the state where the cells are not cultured are predetermined. 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 applied by the voltmeter 92. Measure the value. The electric 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 unit 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 using the power supply device 91, an oxidation reaction of the culture solution and an 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. As a result, 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 in the vicinity of the first working electrode 42 and the second working electrode 52, respectively, and the potential between the reference electrodes 43 and 53 is measured. By calculating using the measured potential, the resistance Rm of the cell portion can be measured more accurately.

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. Further, by arranging the first electrode portion 40 and the second electrode portion 50 of the electrode portion 10 at positions that do not overlap with the observation opening 13 of the observation opening 13 in the vertical direction, the electrical resistance of the cultured cells can be measured. , Cell observation 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 example>
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 portions included in the electrode holding member is the same as the number of observation openings. That is, the electrode holding member had an 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 a 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。 Further, in the above embodiment, an example of the conducting wire 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. Moreover, the arrangement of the conducting wire 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 embodiment, the first electrode portion and the second electrode portion are arranged in a straight line in a 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 vessel, the second electrode portion may be arranged at the corner portion 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 embodiment, the shape of the well of the cell culture container on which the electrode holding member is placed is substantially quadrangular in top view, but the present invention is not limited to this. The shape of the wells of the cell culture vessel 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。 Further, the detailed configuration of the electrode holding member may be different from each drawing of the present application. Further, the elements appearing in the above-described embodiments and modifications may be appropriately combined as long as there is no contradiction.

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 1 Electrode holding member 2 Cell culture container 9 Measuring unit 10 Electrode part 11 Substrate 12 Lead wire 13 Observation opening 14 Through hole 15 Conductive connection member 16 Conductive adhesive 20 Insert cup 21 Well 31 Cylinder part 32 Support part 33 Cell culture part 40 1st electrode part 41 1st column member 42 1st working electrode 43 1st reference electrode 50 2nd electrode part 51 2nd column member 52 2nd working electrode 53 2nd reference electrode

Claims (4)

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
を有し、
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。 An electrode holding member for arranging an electrode in the recess of a well plate having a plurality of recesses.
A flat plate-like and insulating substrate that has multiple observation openings arranged in a grid pattern and extends in the horizontal direction.
A plurality of electrode portions arranged in the vicinity of each of a part or all of the observation openings, and
Have,
Each of the electrode portions
One end thereof is fixed to the substrate and has a first electrode portion and a second electrode portion extending downward from the substrate.
The first electrode portion is
Insulating first columnar member and
A first working electrode provided on the side surface of the first columnar member and
A first reference electrode provided on the side surface of the first columnar member and
Have,
The second electrode portion is
Insulating second columnar member and
A second working electrode provided on the side surface of the second columnar member and
A second reference electrode provided on the side surface of the second columnar member,
The electrode holding member having . 請求項１に記載の電極保持部材であって、The electrode holding member according to claim 1.
前記基板は、 The substrate is
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 Multiple conductors, at least partially printed on the top surface of the substrate
を有し、Have,
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、 The upper end of each of the first electrode portion and the second electrode portion is arranged above the upper surface of the substrate.
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 upper surface of the conducting 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 conductive connecting members. An electrode holding member that is electrically connected via. 請求項１または請求項２に記載の電極保持部材であって、The electrode holding member according to claim 1 or 2.
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 number of the electrode portions is the same as the number of the observation openings, the electrode holding member. 請求項１または請求項２に記載の電極保持部材であって、The electrode holding member according to claim 1 or 2.
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 number of the electrode portions is smaller than the number of the observation openings, the electrode holding member.
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Citations (5)

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US20050221274A1 (en) 2004-03-31 2005-10-06 Negulescu Paul A Multiwell plate assembly for use in high throughput assays
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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JPS63157971A (en) * 1986-12-19 1988-06-30 Shimadzu Corp Electrode holding mechanism for well

Patent Citations (5)

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Publication number Priority date Publication date Assignee Title
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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