JPH04204244A - Complex electrode incorporated in one - Google Patents
Complex electrode incorporated in oneInfo
- Publication number
- JPH04204244A JPH04204244A JP33797390A JP33797390A JPH04204244A JP H04204244 A JPH04204244 A JP H04204244A JP 33797390 A JP33797390 A JP 33797390A JP 33797390 A JP33797390 A JP 33797390A JP H04204244 A JPH04204244 A JP H04204244A
- Authority
- JP
- Japan
- Prior art keywords
- electrodes
- electrode
- insulating layer
- lead wires
- integrated composite
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000002131 composite material Substances 0.000 claims description 24
- 239000000758 substrate Substances 0.000 claims description 6
- 238000005259 measurement Methods 0.000 abstract description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract description 4
- 239000004642 Polyimide Substances 0.000 abstract description 4
- WABPQHHGFIMREM-UHFFFAOYSA-N lead(0) Chemical compound [Pb] WABPQHHGFIMREM-UHFFFAOYSA-N 0.000 abstract description 4
- 229920002120 photoresistant polymer Polymers 0.000 abstract description 4
- 229920001721 polyimide Polymers 0.000 abstract description 4
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract description 2
- 238000005530 etching Methods 0.000 abstract description 2
- 229910017604 nitric acid Inorganic materials 0.000 abstract description 2
- 238000004528 spin coating Methods 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 18
- 210000005056 cell body Anatomy 0.000 description 12
- 210000002569 neuron Anatomy 0.000 description 12
- 239000002609 medium Substances 0.000 description 10
- 239000011521 glass Substances 0.000 description 9
- 239000000463 material Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 239000012981 Hank's balanced salt solution Substances 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 210000000857 visual cortex Anatomy 0.000 description 5
- 238000000034 method Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000007758 minimum essential medium Substances 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Substances [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 230000008054 signal transmission Effects 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000007772 electrode material Substances 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000007261 regionalization Effects 0.000 description 2
- 239000004925 Acrylic resin Substances 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004026 adhesive bonding Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000005388 borosilicate glass Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000003822 epoxy resin Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 150000004687 hexahydrates Chemical class 0.000 description 1
- AMGQUBHHOARCQH-UHFFFAOYSA-N indium;oxotin Chemical compound [In].[Sn]=O AMGQUBHHOARCQH-UHFFFAOYSA-N 0.000 description 1
- 239000011810 insulating material Substances 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229940046892 lead acetate Drugs 0.000 description 1
- 239000005355 lead glass Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 238000001259 photo etching Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 229920006122 polyamide resin Polymers 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- XOLBLPGZBRYERU-UHFFFAOYSA-N tin dioxide Chemical compound O=[Sn]=O XOLBLPGZBRYERU-UHFFFAOYSA-N 0.000 description 1
- 229910001887 tin oxide Inorganic materials 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明（よ 生体活動の電気的計瓢 特に神経細胞の電
気的活動を計測する神経電気生理の分野で用いる、多電
極を有する一体化複合電極に関する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an integrated composite electrode having multiple electrodes used in the field of neuroelectrophysiology, particularly for measuring the electrical activity of nerve cells. .
従来の技術 従来 神経細胞の電気的活動を計測するに（ｉ。Conventional technology Conventionally, to measure the electrical activity of nerve cells (i.
ガラス電極等からなる記録電極と、金属電極等からなる
刺激電極とを各々細胞内または細胞間に挿入し 刺激電
極より刺激電流（または電圧）を加えた際欧 神経細胞
の電気的活動を記録電極により計測するのが普通であっ
九
これ以外にＬ 例えば細胞体をガラス吸引電極で突き刺
し 細胞体の内部をガラス吸引電極中の液で還流し こ
のガラス吸引電極から電気信号を与えて細胞を観察する
所謂細胞内還流法等多数の変法があム
発明が解決しようとする課題
上述した従来の技術およびその変法においては主に空間
的な制約と操作精度上の制約で、 １つのサンプル中に
一度に２本以上の記録電極を挿入し神経細胞の電気的活
動を記録する多点同時計測は非常に困難であるという課
題があつ九
測定点が増えるに従って、困難さの度合いが増加し 多
細胞間の観測ができ難いという課題があった
本発明（よ 多点同時計測を簡便に行−＼ 多細胞間に
渡る信号伝達観察を可能ならしめる一体化複合電極を提
供することを目的とする。A recording electrode made of a glass electrode or the like and a stimulation electrode made of a metal electrode or the like are inserted into a cell or between the cells, and when a stimulation current (or voltage) is applied from the stimulation electrode, the electrical activity of the neuron is recorded. In addition to this, for example, the cell body is pierced with a glass suction electrode, the inside of the cell body is refluxed with the liquid in the glass suction electrode, and an electrical signal is applied from this glass suction electrode to observe the cell. A number of modified methods, such as the so-called intracellular perfusion method, are used to solve the problem that the invention aims to solve.The conventional techniques and their modified methods described above are mainly subject to spatial constraints and constraints on operational precision, and the problem that Amu Invention aims to solve is Simultaneous multi-point measurement by inserting two or more recording electrodes at once to record the electrical activity of neurons is extremely difficult.As the number of measurement points increases, the degree of difficulty increases. The object of the present invention is to provide an integrated composite electrode that makes it possible to easily perform simultaneous multi-point measurements and observe signal transmission between multiple cells.
課題を解決するための手段
本発明は 絶縁基板上に 最近接の電極間距離が相等し
い複数個の電極をｔｉえ 前記電極からリード線を放射
状に配設した配線部と、前記配線部上の前記電極上に孔
を有した絶縁層とを設けた一体化複合電極のよって、上
記課題を解決したものである。Means for Solving the Problems The present invention includes a plurality of electrodes having equal distances between the nearest electrodes on an insulating substrate, a wiring section in which lead wires are arranged radially from the electrodes, and a wiring section on the wiring section. The above problem is solved by an integrated composite electrode in which an insulating layer having holes is provided on the electrode.
作用
本発明の一体化複合電極上に培養した細胞に信号を与え
同時に細胞間の信号の伝達を計測する際（ミ 最近接
の電極間距離を調整し しかもこの電極間を等間隔で並
ばせることにより、−細胞体が電極上に配置し この細
胞体から伸びた細胞突起を介した細胞体が、隣合う電極
上に位置する確率が高（なム したがって、隣合う細胞
体間の信号の伝達を検知できも
また 電極から伸ばしたリード線を放射状に配設するこ
とにより、 リード線間の容量成分がなくなり、ノイズ
が減少し測定制度が向上する。Function: When applying a signal to cells cultured on the integrated composite electrode of the present invention and simultaneously measuring the transmission of signals between cells (i.e. adjusting the distance between the nearest electrodes and arranging the electrodes at equal intervals) Therefore, there is a high probability that the cell body is placed on the electrode and the cell body is located on the adjacent electrode via the cell protrusion extending from this cell body. By arranging the lead wires extending from the electrodes in a radial pattern, the capacitance component between the lead wires is eliminated, noise is reduced, and measurement accuracy is improved.
実施例
本発明に供される基板材料としては 細胞培養後顕微鏡
観察する必要があるため透明な基板が好ましく、石英ガ
ラ入 鉛ガラ入 ホウ珪酸ガラス等のガラ入 若しくは
石英等の無期物質、また１飄有機ガラス等の有機物質が
挙げられる力丈 機械的強度と透明性とを加味すると無
期物質が好まし１本発明に供される電極材料としては
例えば酸化インジウム錫（ＩＴＯ）、酸化ａｃｒ、Ａｕ
、Ｃｕ、Ｎｉ、Ａ１等が使用可能である。ただしＩＴＯ
若しくは酸化錫を用いると、電極はわずかに黄色を帯び
た透明なものとなり、神経細胞の顕微鏡下での視認性が
良く、実験操作上有利であるが、とりわけＩＴＯが良導
電性であるため望まし鶏
リード線材料にも同様の材料が適応でき、やはり電極材
料と同様の理由でＩＴＯが好ましｌ、％まｔ＆ 本発明
の供される絶縁層材料として（よ例えばポリイミド（Ｐ
Ｉ）樹脂 エポキシ樹脂アクリレート樹脂 ポリエステ
ルＷＵｔ 或はポリアミド樹脂等透明な樹脂が挙げら
れる。EXAMPLE The substrate material used in the present invention is preferably a transparent substrate since it is necessary to perform microscopic observation after cell culture, and it is preferable to use a transparent substrate containing quartz glass, lead glass, glass containing glass such as borosilicate glass, or an indefinite substance such as quartz. In view of mechanical strength and transparency, indefinite materials are preferred as the electrode materials used in the present invention.
For example, indium tin oxide (ITO), ACR oxide, Au
, Cu, Ni, A1, etc. can be used. However, ITO
Alternatively, if tin oxide is used, the electrode becomes transparent with a slight yellow tinge, making it easy to see nerve cells under a microscope, which is advantageous for experimental operations, but ITO is especially desirable because it has good conductivity. A similar material can be applied to the lead wire material, and ITO is preferable for the same reason as the electrode material (for example, polyimide (P)
I) Resin Transparent resins such as epoxy resin, acrylate resin, polyester WUt, or polyamide resin may be used.
これらの樹脂；Ｌ 配線部上に通常の手法によって塗布
して絶縁層が構成される。な耘 絶縁層材料が光照射重
合性であると、パターン形成が可能となるため好ましし
−
特に 絶縁層材料がＰＩであり培養する細胞が培養神経
細胞である場合に（表 良好な生育を示すため望ましｌ
ｖ犯 さらにＰＩＯ中でＬ ネガティブフォトセンシ
ティブポリイミド（ＮＰＩ）力匁 半導体のパターン形
成と同様に 配線部に塗布した後フォトエツチングプロ
セスを用いて孔を形成できるため好ましへ
まｔ二 絶縁層の厚み：よ 絶縁性が付与できる程度
であればよく、特に限定するものではない低通常０．１
〜１０μｍが好ましく、 １〜５μｍ程度がさらに好ま
しへ
本発明の一体化複合電極（主 直接細胞を培養して細胞
の電気活動を計測記録する。培養条件若しくは細胞の種
類によって、細胞体の大きさ若しくは細胞突起の長さが
異なるが、一体化複合電極の最近接の電極間距離（よ
１０〜１０００μｍが好ましく℃ 電極間距離が１０μ
ｍ未満であると、互いに近接し過ぎるため細胞体が細胞
突起を介して相隣合う確率が減り、またリード線の配線
も困難になる。ま？＝ＩＯ００μｍを越えると、 リー
ド線の配線はしやすいが、細胞突起が１０００μｍ程度
も伸びることは稀なため、細胞体が電極上に位置する確
率が減ａ 一般の条件で培養した細胞突起の長さ１よ
平均１００μｍ程度であるたべ電極間距離も１００μｍ
程度が望ましｔ、％電極の形状（飄 最近接の電極間距
離を一定にする要請のた嵌 正方形か円形が好ましく、
細胞体の形状は一般には球状体なので円形形状の電極が
好ましくｌ ま−Ｌ 電極の大きさζ友 培養する細
胞体の大きさ程度が好ましく、正方形状の電極の場合一
辺が１５〜２０μ爪 円形状の電極の場合直径が１５〜
２０μｍが好ましくも
さらに 本発明の一体化複合電極の絶縁層中の孔は 一
体化複合電極上で培養した細胞体に電気信号を与えて刺
激させると同時！ミ 隣合う細胞体から刺激信号を検知
するた玖 電極を露出させる目的で形成し 電極中心部
に位置すム この孔の大きさ（よ 電極以下の大きさを
有すればよく、 ５〜２０μｍ程度が好ましく〜
また 本発明の一体化複合電極の電極中心部力丈同心円
状若しくは６×６の格子状の各交点に位置する構成であ
ると、 リード線を放射状に配線でき、特に６×６の格
子状の場合はリード線の配線の構成が簡単にすることが
できるため好ましくも以下具体的実施例で、本発明の一
体化複合電極をさらに詳細に説明する。These resins are applied onto the L wiring portion by a conventional method to form an insulating layer. It is preferable that the insulating layer material is polymerizable by light irradiation because it enables pattern formation, especially when the insulating layer material is PI and the cells to be cultured are cultured nerve cells (Table 1). Desirable to show
Negative photosensitive polyimide (NPI) is preferable because holes can be formed using a photo-etching process after coating it on the wiring, similar to semiconductor pattern formation.Thickness of the insulating layer ：Yo It is sufficient as long as it can provide insulation properties, and there is no particular limitation. Low usually 0.1
~10 μm is preferred, and approximately 1 ~ 5 μm is more preferred. The distance between the nearest electrodes of the integrated composite electrode (
Preferably 10 to 1000 μm °C Distance between electrodes is 10 μm
If it is less than m, the cell bodies will be too close to each other, reducing the probability that the cell bodies will be adjacent to each other via cell protrusions, and also making it difficult to wire lead wires. Ma? = When the IO exceeds 00 μm, it is easy to wire the lead wire, but it is rare for cell protrusions to extend as much as 1000 μm, so the probability that the cell body will be located on the electrode decreases.a Length of cell protrusions cultured under normal conditions Sa1!
The average distance between the pan electrodes is about 100μm, and the distance between them is also 100μm.
The shape of the electrodes (in order to keep the distance between the nearest electrodes constant) is preferably square or circular.
The shape of the cell body is generally spherical, so a circular electrode is preferable.The size of the electrode is preferably about the same size as the cell body to be cultured.In the case of a square electrode, each side is 15 to 20μ. For shaped electrodes, the diameter is 15~
The diameter of the hole in the insulating layer of the integrated composite electrode of the present invention is preferably 20 μm, and the hole in the insulating layer of the integrated composite electrode of the present invention can be simultaneously stimulated by applying an electric signal to the cell bodies cultured on the integrated composite electrode! M. A hole for detecting stimulation signals from adjacent cell bodies. A hole formed for the purpose of exposing the electrode. A hole located at the center of the electrode. The size of this hole (it only needs to be smaller than the electrode, about 5 to 20 μm. It is preferable that the electrode center portion of the integrated composite electrode of the present invention is arranged in concentric circles or at each intersection of a 6×6 grid, so that the lead wires can be wired radially, especially in a 6×6 grid. In the case of a lattice-like structure, the structure of the wiring of the lead wires can be simplified, so it is preferable that the integrated composite electrode of the present invention will be explained in more detail with reference to specific examples below.
実施例１ 先ず、複合電極配線部の作製について述べる。Example 1 First, the production of the composite electrode wiring section will be described.
一体化複合電極の基盤は機械的強度の強い透明な絶縁素
材として、厚さ］、ｍｍの硬質ガラス（ＩＷＡＫＩ
Ｃ０ＤＥ ７７４０ ＧＬＡＳＳ （居城硝子（株
）製）以下同じ）を用い九
配線部外寸（表 リードレスチップキャリアーパッケー
ジ実装用ソケット（７５３ＥＲＩＥＳ （山゛−電機工
業（株）製））に適合するものとし九電極およびリード
線の材料にはＩＴ○を用シＸＩＷＡＫＩ Ｃ０ＤＥ
７７４０ ＧＬＡＳＳ上に約１０００八属にコート
レ その後洗浄し九次（ミ 第１図に示すような６×６
の格子上の各交点に電極の中心部が位置し 各電極の最
近接の電極の中心間距離が等しく、 しかもリード線が
放射状に伸びた形状の電極１およびリード線２のパター
ンになるよう置 フォトレジストを用いて露光し 純水
５０、塩酸５０、硝酸１の体積比で混合した溶液中でＩ
Ｔ○をエツチングした後、フォトレジストを除去し九
電極１の直径は１５μＲリ一ドｗＡ２の幅は１０μへ
電極中心間距離は１００μｍの配線部を形成しな
次いで、絶縁層としてネガティブフォトセンシティブポ
リイミド（以下ＮＰＩと略す）を、乾燥後の厚みが１μ
ｍとなるようにスピンコートレ第２図に示すように配線
部の各電極の中心に直径１０μｍの孔３ができるよう番
！ 絶縁層パターンを露光形成させ九
リードレスチップキャリアーパッケージ実装用ソケット
との接点１よ 金およびニッケルでコートし 耐久性を
向上させな さらぬ 電極１の部分を１％の塩化白金（
ＩＶ）酸六水和物と０．０１％酢酸鉛の混合水溶液に！
極を浸漬り、５０ｍＡ／ｃｍ２の電流を３０ｓ間通電し
電極表面に白金黒を析出させる所謂プラチナイズするこ
とで、インピーダンスを低下させた後、以下の実験に供
し島な杖 本実施例では電極１およびリード２の部分に
ＩＴＯ１絶縁層にＮＰＩを用いたが、用いる材料はこれ
らに限定されないことは既に述べｆ島また 本発明の一
体化複合電極を構成するためのプロセスは本実施例の方
法に限定されな賎実施例２
装置 一体化複合電極上での神経細胞の培養について述
べも
実施例１のようにして構成した一体化複合電極上で、神
経細胞としてラット大脳視覚皮質を培養し九
以下、培養法について詳細に述べる。The base of the integrated composite electrode is a transparent insulating material with strong mechanical strength, made of hard glass (IWAKI) with a thickness of
C0DE 7740 GLASS (manufactured by Ishiro Glass Co., Ltd.) (the same applies hereinafter) shall be used (Table 1). Use IT○ for the material of the electrode and lead wire.XIWAKI C0DE
7740 GLASS was coated with about 1,000 eight genera. After that, it was washed and divided into nine layers (6 x 6 as shown in Figure 1).
The centers of the electrodes are located at each intersection on the grid, the distance between the centers of the nearest electrodes is equal, and the electrodes 1 and 2 are arranged in a pattern in which the lead wires extend radially. I was exposed using a photoresist in a solution containing 50 parts of pure water, 50 parts of hydrochloric acid, and 1 part of nitric acid by volume.
After etching T○, remove the photoresist and
The diameter of electrode 1 is 15μR, and the width of lead wA2 is 10μ.
A wiring part with a distance between electrode centers of 100 μm was formed, and then negative photosensitive polyimide (hereinafter abbreviated as NPI) was formed as an insulating layer to a thickness of 1 μm after drying.
Spin coating so that a hole 3 with a diameter of 10 μm is formed at the center of each electrode in the wiring part as shown in Figure 2. An insulating layer pattern is formed by exposure, and contact points 1 with the leadless chip carrier package mounting socket are coated with gold and nickel to improve durability.
IV) Mixed aqueous solution of acid hexahydrate and 0.01% lead acetate!
After lowering the impedance by immersing the electrode and applying a current of 50 mA/cm2 for 30 seconds to deposit platinum black on the electrode surface, the impedance was lowered. Although NPI was used for the insulating layer of ITO 1 and lead 2, it has already been mentioned that the materials used are not limited to these. EXAMPLE 2 Apparatus: Culture of neurons on an integrated composite electrode is described. Rat cerebral visual cortex was cultured as a neuron on an integrated composite electrode constructed as in Example 1. The culture method will be described in detail below.
（イ）生後２、３日を経過したＳＤプラット脳を摘出し
氷冷したハンクス液に浸たす。(b) Remove the SD Pratt brain after 2 or 3 days of age and immerse it in ice-cold Hank's solution.
（ロ）水冷ハンクス液中の脳から視覚皮質を切り出し
イーグルの最小必須培地（以下ＭＥＭと略す〉液中に移
す。(b) Cutting out the visual cortex from the brain in water-cooled Hank's solution
Transfer to Eagle's minimum essential medium (hereinafter abbreviated as MEM).
（ハ）ＭＥＭ液中で、視覚皮質をできるだけ細かく、最
大でも０．２ｍｍ角となるように 切断する。(c) In MEM solution, cut the visual cortex as finely as possible, to a maximum of 0.2 mm square.
（ニ）細かく切断した視覚皮質を遠沈管に入れカルシウ
ムおよびマグネシウムを含まないハンクス液（ＣＭＦハ
ンクス液）で３回洗浄した後、適量の同液中に分散する
。(d) The finely cut visual cortex is placed in a centrifuge tube, washed three times with Hank's solution (CMF Hank's solution) that does not contain calcium and magnesium, and then dispersed in an appropriate amount of the same solution.
（ホ）上記（ニ）の遠沈管中に トリプシンのＣＭＦハ
ンクス溶液（０，２５重量パーセント）を加え 全量を
倍にすム 緩やかに撹はんしなが収 ３７℃で１５分か
ら２０分間恒温状態に保ち酵素反応をおこなわせた（即
ちインキュベート）。(e) Add CMF Hanks solution of trypsin (0.25% by weight) into the centrifuge tube (d) above and double the total volume. Gently stir and collect. Keep at constant temperature at 37℃ for 15 to 20 minutes. The enzymatic reaction was carried out (ie, incubated).
（へ）牛胎児血清（ＦＣＳ）１０％を含むダルベツコ変
更イーグル培地とＦ−１２培地とを１対１の体積比で混
合したＤＭＥＭ／Ｆ １２混合培地を、上記（ホ）を経
た遠沈管中に加え 全量をさらに倍にすム 先端をバー
ナーであぶり口径を小さくしたパスツールピペットて
緩やかにピペッティングを繰り返しく最大２０回程度）
、細胞をほぐす。(f) DMEM/F 12 mixed medium, which is a mixture of Dulbecco's modified Eagle medium containing 10% fetal calf serum (FCS) and F-12 medium at a volume ratio of 1:1, is placed in a centrifuge tube after passing through (e) above. In addition, double the total volume using a Pasteur pipette with a smaller diameter and burnt the tip with a burner.
(Repeat gentle pipetting up to 20 times)
, loosen cells.
（ト）９８０６． ６５ｍ／ｓ２（即ち１０００ ｇ）
）で約５分間遠心分離を行う。遠心分離終了後、上溝を
捨て、沈澱をＦＣ３５％を含むＤＭＥＭ／Ｆ１２混合培
地に混濁する。(g)9806. 65m/s2 (i.e. 1000g)
) for about 5 minutes. After centrifugation, the upper groove is discarded and the precipitate is suspended in a DMEM/F12 mixed medium containing 35% FC.
（チ）上記（ト）の混濁液を、再び１０００ｇで５分間
遠心分離する。(H) Centrifuge the turbid liquid from (G) above again at 1000g for 5 minutes.
（す）上記操作（ト）および（チ）をあと２回（計３回
）繰り返す。(S) Repeat the above operations (g) and (h) two more times (total 3 times).
（ヌ）最終的に得られた沈澱を、 １ｒｎｌの５％ＦＣ
３を含むＤＭＥＭ／Ｆ １２混合培地に混濁し混濁液中
の細胞濃度を赤血球勘定板を用いて計測すム 同様の培
地を用いて細胞濃度を２〜４Ｘ１０６個／ｍｌになるよ
うに調整すも
（ル）一体化複合電極上に直径１２ｍ爪 高さ７ｍｍの
プラスティック製円筒を、複合電極の中心と円筒の中心
を合わせて接着することにより構成した細胞培養用ウェ
ル中ζへ あらかじめ５％ＦＣ８を含むＤＭＥＭ／Ｆ
１２混合培地５００μｌを加え ＣＯ２インキユベータ
内（０２濃度９５瓢ＣＯｅ濃度５販 湿度９７９Ａ 温
度３７℃）で暖めておく。(J) The finally obtained precipitate was added to 1rnl of 5% FC.
DMEM/F12 mixed medium containing 3 is suspended, and the cell concentration in the turbid liquid is measured using a red blood cell counting plate.Adjust the cell concentration to 2 to 4 x 106 cells/ml using the same medium. (l) Add 5% FC8 in advance to a cell culture well constructed by gluing a 7 mm high plastic cylinder with a nail of 12 m in diameter onto the integrated composite electrode, aligning the center of the composite electrode with the center of the cylinder. Contains DMEM/F
Add 500 μl of 12 mixed medium and warm it in a CO2 incubator (02 concentration: 95% COe concentration: 5%, humidity: 979A, temperature: 37°C).
（ヲ）上記（ル）のウェル中に細胞濃度を調整した混濁
液１００μｍを静かに加え 再びＣＯｅインキュベータ
内に静置する。(2) Gently add 100 μm of the turbid solution with adjusted cell concentration into the well in (1) above, and leave it in the COe incubator again.
（ヨ）上記（ル）の操作より３日後（ミ 培地の半量を
新しいものと交換すム 交換培地はＦＣＳを含まないＤ
ＭＥＭ／Ｆ ｌ ２混合培地を用いも（り）以１！！、
４〜５日毎に上記と同様の培地交換を行う。(Y) Three days after the operation in (L) above (Mi) Replace half of the medium with a new one.D The replacement medium does not contain FCS.
Also using MEM/Fl2 mixed medium (1)! ! ,
The medium is replaced every 4 to 5 days in the same manner as above.
これら一連の操作により、一体化複合電極上でラット大
脳視覚皮質の神経細胞を培養することができ通
細胞は絶縁層（ＰＩ）上でもプラチナイズされた電極上
でも良好に生育した
したがって、適当な位置にある電極を刺激電極または記
録電極として用いれば 神経細胞電気活動の同時多点計
測が可能であっ九
な耘 神経細胞の培養法は本実施例以外にも多数の変法
があり、本実施例に限定されるものではなし−
発明の効果
本発明は 絶縁基板上に 最近接の電極間距離が相等し
い複数個の電極を備え 前言己電極からり−ド線を放射
状に配設した配線部と、前記配線部上の前記電極上に孔
を有した絶縁層とを設けた一体化複合電極を用℃（この
上での神経細胞の培養が可能であった
さらにこの電極を用いれは 従来不可能または非常に困
難であった神経細胞電気活動の同時多点計測及び多細胞
に渡る信号伝達観察が実現できる第１図は本発明の一体
化複合電極の一実施例の配線部のパターン上面＠ 第２
図は本発明の一体化複合電極の一実施例の絶縁層のパタ
ーン上面図である。Through these series of operations, it was possible to culture neurons in the rat cerebral visual cortex on the integrated composite electrode, and the cells grew well both on the insulating layer (PI) and on the platinized electrode. Simultaneous multi-point measurement of neuron electrical activity is possible by using the electrodes at different locations as stimulation electrodes or recording electrodes. Effects of the Invention The present invention provides a wiring section in which a plurality of electrodes having the same distance between nearest electrodes are provided on an insulating substrate, and lead wires from the electrodes are arranged radially. and an insulating layer with a hole on the electrode on the wiring part.Culture of nerve cells on this electrode was possible. Simultaneous multi-point measurement of nerve cell electrical activity and observation of signal transmission across multiple cells, which have been extremely difficult, are now possible. Figure 1 shows the upper surface of the pattern of the wiring section of an embodiment of the integrated composite electrode of the present invention. Second
The figure is a top view of the pattern of the insulating layer of one embodiment of the integrated composite electrode of the present invention.
Claims (5)
数個の電極を備え、前記電極からリード線を放射状に配
設した配線部と、前記配線部上の前記電極上に孔を有し
た絶縁層とを設けたことを特徴とする一体化複合電極。(1) A wiring section including a plurality of electrodes having the same distance between nearest electrodes on an insulating substrate, and a wiring section in which lead wires are arranged radially from the electrodes, and a hole on the electrodes on the wiring section. An integrated composite electrode characterized in that it is provided with an insulating layer.
る請求項１記載の一体化複合電極。(2) The integrated composite electrode according to claim 1, wherein the distance between the nearest electrodes is 10 to 1000 μm.
一辺の長さ若しくは直径が１５〜２０μｍである請求項
１記載の一体化複合電極。(3) The size of the electrode is square or circular,
The integrated composite electrode according to claim 1, wherein the length of one side or the diameter is 15 to 20 μm.
若しくは３何れかに記載の一体化複合電極。(4) Claim 1, wherein the size of the hole is less than or equal to the size of the electrode.
or the integrated composite electrode according to any one of 3.
に位置する請求項１記載の一体化複合電極。(5) The integrated composite electrode according to claim 1, wherein the plurality of electrode centers are located at each intersection of a 6×6 grid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33797390A JP2949845B2 (en) | 1990-11-30 | 1990-11-30 | Electrodes for measuring cell electrical activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33797390A JP2949845B2 (en) | 1990-11-30 | 1990-11-30 | Electrodes for measuring cell electrical activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04204244A true JPH04204244A (en) | 1992-07-24 |
| JP2949845B2 JP2949845B2 (en) | 1999-09-20 |
Family
ID=18313740
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33797390A Expired - Lifetime JP2949845B2 (en) | 1990-11-30 | 1990-11-30 | Electrodes for measuring cell electrical activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2949845B2 (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0585933A2 (en) * | 1992-09-04 | 1994-03-09 | Matsushita Electric Industrial Co., Ltd. | Planar electrode |
| US5563067A (en) * | 1994-06-13 | 1996-10-08 | Matsushita Electric Industrial Co., Ltd. | Cell potential measurement apparatus having a plurality of microelectrodes |
| US5810725A (en) * | 1993-04-16 | 1998-09-22 | Matsushita Electric Industrial Co., Ltd. | Planar electrode |
| US6297025B1 (en) | 1994-06-13 | 2001-10-02 | Matsushita Electric Industrial Co., Ltd | Measurement of complete electrical waveforms of tissue or cells |
| WO2002006809A1 (en) * | 2000-07-13 | 2002-01-24 | Matsushita Electric Industrial Co., Ltd. | Extracellular recording integrated composite electrode |
| WO2003001194A1 (en) * | 2001-06-20 | 2003-01-03 | Matsushita Electric Industrial Co., Ltd. | Extracellular recording electrode |
| USRE40209E1 (en) | 1994-06-13 | 2008-04-01 | Matsushita Electric Industrial Co., Ltd. | Cell potential measurement apparatus having a plurality of microelectrodes |
| CN102247137A (en) * | 2010-05-19 | 2011-11-23 | 中国科学院电子学研究所 | Microelectrode array-based multichannel neural information detection system |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3801617B2 (en) | 2003-06-27 | 2006-07-26 | 松下電器産業株式会社 | Pharmacological measurement apparatus and system and well container used therefor |
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1990
- 1990-11-30 JP JP33797390A patent/JP2949845B2/en not_active Expired - Lifetime
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0585933A3 (en) * | 1992-09-04 | 1995-09-13 | Matsushita Electric Ind Co Ltd | Planar electrode |
| EP0585933A2 (en) * | 1992-09-04 | 1994-03-09 | Matsushita Electric Industrial Co., Ltd. | Planar electrode |
| US5810725A (en) * | 1993-04-16 | 1998-09-22 | Matsushita Electric Industrial Co., Ltd. | Planar electrode |
| USRE38323E1 (en) | 1994-06-13 | 2003-11-18 | Matsushita Electric Industrial Co., Ltd. | Cell potential measurement apparatus having a plurality of microelectrodes |
| US5563067A (en) * | 1994-06-13 | 1996-10-08 | Matsushita Electric Industrial Co., Ltd. | Cell potential measurement apparatus having a plurality of microelectrodes |
| US6297025B1 (en) | 1994-06-13 | 2001-10-02 | Matsushita Electric Industrial Co., Ltd | Measurement of complete electrical waveforms of tissue or cells |
| USRE40209E1 (en) | 1994-06-13 | 2008-04-01 | Matsushita Electric Industrial Co., Ltd. | Cell potential measurement apparatus having a plurality of microelectrodes |
| US6894511B2 (en) | 2000-07-13 | 2005-05-17 | Matsushita Electric Industrial Co., Ltd. | Extracellular recording multiple electrode |
| WO2002006809A1 (en) * | 2000-07-13 | 2002-01-24 | Matsushita Electric Industrial Co., Ltd. | Extracellular recording integrated composite electrode |
| WO2003001194A1 (en) * | 2001-06-20 | 2003-01-03 | Matsushita Electric Industrial Co., Ltd. | Extracellular recording electrode |
| US7041492B2 (en) | 2001-06-20 | 2006-05-09 | Matsushita Electric Industrial Co., Ltd. | Extracellular recording electrode |
| CN102247137A (en) * | 2010-05-19 | 2011-11-23 | 中国科学院电子学研究所 | Microelectrode array-based multichannel neural information detection system |
| WO2015151395A1 (en) * | 2014-03-31 | 2015-10-08 | パナソニックＩｐマネジメント株式会社 | Electrochemical measurement device |
| CN106164243A (en) * | 2014-03-31 | 2016-11-23 | 松下知识产权经营株式会社 | Electrochemical gaging device |
| JP6074874B2 (en) * | 2014-03-31 | 2017-02-08 | パナソニックＩｐマネジメント株式会社 | Electrochemical measurement device |
| JPWO2015151395A1 (en) * | 2014-03-31 | 2017-04-13 | パナソニックＩｐマネジメント株式会社 | Electrochemical measurement device |
| JP2017083462A (en) * | 2014-03-31 | 2017-05-18 | パナソニックＩｐマネジメント株式会社 | Electrochemical measurement device |
| CN106164243B (en) * | 2014-03-31 | 2018-04-27 | 松下知识产权经营株式会社 | Electrochemical gaging device |
| US10457907B2 (en) | 2014-03-31 | 2019-10-29 | Panasonic Intellectual Property Management Co., Ltd. | Electrochemical measurement device |
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