JP2006333861A - Method for electrically stimulating cultured muscular cell by using transparent electrode - Google Patents

Method for electrically stimulating cultured muscular cell by using transparent electrode Download PDF

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JP2006333861A
JP2006333861A JP2005194349A JP2005194349A JP2006333861A JP 2006333861 A JP2006333861 A JP 2006333861A JP 2005194349 A JP2005194349 A JP 2005194349A JP 2005194349 A JP2005194349 A JP 2005194349A JP 2006333861 A JP2006333861 A JP 2006333861A
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electrical stimulation
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Hideaki Fujita
英明 藤田
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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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Abstract

<P>PROBLEM TO BE SOLVED: To provide apparatus for culturing cells on a transparent conductive electrode and imparts uniform electrical stimulations on the cells and enabling an optical observation on the live cells and easy to achieve a high throughput. <P>SOLUTION: The invention relates to the apparatus for culturing live cells on the transparent conductive plate, uniformly imparting the electrical stimulation to the live cells on a transparent plate by loading a voltage between the transparent conductive plate and an upper electrode, and optically observing the live cells. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、培養筋肉細胞を電気伝導性透明板上で培養する事により、培養筋肉細胞に均質に電気刺激を与える事を可能にする細胞培養法に関する。  The present invention relates to a cell culture method that makes it possible to apply electrical stimulation to cultured muscle cells uniformly by culturing cultured muscle cells on an electrically conductive transparent plate.

筋肉細胞（骨格筋・心筋）は電気的に刺激する事が可能であり、細胞生物学分野における研究ではこれらの細胞の電気刺激に対する応答の解析などが頻繁に行われている。従来、細胞の電気刺激方法としては底面に接着している細胞に平行な電場を与える方法が一般的であり、このような形態の細胞電気刺激装置は例えば非特許文献１にあるようにいくつか販売されている。しかし、このような配置の場合、多くの培養・電気刺激条件のスクリーニングが難しい事や、電場が細胞に均質にかからないなどの欠点がある。  Muscle cells (skeletal muscle / myocardium) can be stimulated electrically, and in the field of cell biology, analysis of the response of these cells to electrical stimulation is frequently performed. Conventionally, as a method for electrically stimulating cells, a method of applying an electric field parallel to cells adhered to the bottom surface is generally used. Sold. However, such an arrangement has drawbacks that it is difficult to screen for many culture and electrical stimulation conditions and that the electric field is not applied to the cells uniformly.

培養筋肉細胞電気刺激方法における上記の問題を解決するため、特許文献１や特許文献２にあるような上下方向から細胞に電気刺激を与える方法が考案されている。しかし、これらの方法は下部電極が不透明なため細胞を光学的に観察するという細胞生物学上最重要な実験を不可能にしている。また、それぞれのチャンバーに対して配線する必要がある事から、電気刺激条件のスクリーニングに適しているとは言い難い。  In order to solve the above-mentioned problems in the cultured muscle cell electrical stimulation method, methods for applying electrical stimulation to cells from the up and down directions as in Patent Document 1 and Patent Document 2 have been devised. However, these methods make the most important experiment in cell biology of optical observation of cells impossible because the lower electrode is opaque. Moreover, since it is necessary to wire each chamber, it is difficult to say that it is suitable for screening of electrical stimulation conditions.

特許公開２００３−２２５Patent Publication 2003-225 特許公開２００２−３３５９４５Patent Publication 2002-335945 Ｔｈｅ Ｃ−Ｐａｃｅ１００−Ｃｕｌｔｕｒｅ Ｐａｃｅｒ ａｎｄ Ｃ−Ｄｉｓｈ１００−Ｃｕｌｔｕｒｅ Ｄｉｓｈ ｅｌｅｃｔｒｏｄｅ；ＩｏｎＯｐｔｉｘ Ｃｏｒｐｏｒａｔｉｏｎ インターネット＜ＵＲＬ：ｈｔｔｐ：／／ｗｗｗ．ｉｏｎｏｐｔｉｘ．ｃｏｍ／ ＞The C-Pace 100-Culture Paper and C-Dish 100-Culture Disc electrode; IonOptix Corporation Internet <URL: http: // www. ionoptix. com />

上記のように、従来の電極が平行に配置されている手法では薬剤や電気刺激条件のスクリーニングに適した方法で培養筋肉細胞に電気刺激を与える事は難しかった。また、上下方向に電極が配置されている方法では細胞が不透明な透過膜上に培養されているため、光学的観察が出来ないという欠点があった。  As described above, it is difficult to apply electrical stimulation to cultured muscle cells by a method suitable for screening of drugs and electrical stimulation conditions by the conventional technique in which electrodes are arranged in parallel. In addition, the method in which the electrodes are arranged in the vertical direction has a drawback that optical observation cannot be performed because cells are cultured on an opaque permeable membrane.

本発明は、培養筋肉細胞を電気伝導性透明電極上で培養し、培養筋肉細胞に均質に電気刺激を与え、かつ光学的観察が可能でハイスループット化が容易な装置の提供を目的としている。  An object of the present invention is to provide a device that cultures cultured muscle cells on an electrically conductive transparent electrode, uniformly applies electrical stimulation to the cultured muscle cells, enables optical observation, and facilitates high throughput.

本発明によれば、電気伝導性透明板上に培養筋肉細胞を培養し、電気伝導性透明板と上部電極の間に電圧を負荷することにより透明板上の培養筋肉細胞に均一に電気刺激を与え、かつ細胞が生きたまま光学的観察が可能な装置を提供する。  According to the present invention, cultured muscle cells are cultured on an electrically conductive transparent plate, and a voltage is applied between the electrically conductive transparent plate and the upper electrode to uniformly stimulate the cultured muscle cells on the transparent plate. Provided is a device which can be optically observed while the cells are alive.

また、本発明の細胞電気刺激装置を使用することにより、薬剤や電気刺激条件などのスクリーニングを簡易に行う事が可能となる。  In addition, by using the cellular electrical stimulation device of the present invention, it is possible to easily perform screening for drugs and electrical stimulation conditions.

また、本発明は、底部に光学的に透明な電極を設置してなる所定の容積を有する容器と前記容器の上方から被せて前記容器を密封し、かつ前記透明電極に対向して配設した対向電極を備え、前記容器と上下方向に対向して設置される蓋体とを備えたことを特徴とする電気刺激用キュベットを提供する。  Further, the present invention provides a container having a predetermined volume formed by placing an optically transparent electrode on the bottom, and the container is sealed from above the container, and disposed opposite to the transparent electrode. There is provided an electrical stimulation cuvette comprising a counter electrode, and comprising a lid body disposed opposite to the container in the vertical direction.

以上説明したように本発明によれば、電気伝導性透明板上に培養した培養筋肉細胞に電気刺激を与え、かつ、同時にその細胞を光学的に観察する事が可能となる。培養筋肉細胞研究の分野では細胞の電気刺激依存的な応答を調べる事が重要であり、その際に光学的観察は主要な手段の一つである。また、電極は上下方向に配置されているため、薬剤や電気刺激条件のスクリーニングに適している。さらに、電気伝導性透明板はガラス面に光学的に透明な電気伝導性物質を蒸着したものであるため、ガラス面上に回路を構成する事により同一のガラス面上で部位に応じて同時に様々な条件の電気刺激を与える事を可能にする。また、培養筋肉細胞に電気刺激を与えることによって細胞に擬似的運動効果を与えることができるため、細胞内のエネルギー代謝に対する薬剤の影響を調べる事も可能となる。  As described above, according to the present invention, it is possible to apply electrical stimulation to cultured muscle cells cultured on an electrically conductive transparent plate and simultaneously optically observe the cells. In the field of cultured muscle cell research, it is important to examine the electrical stimulus-dependent response of cells, and optical observation is one of the main means. Further, since the electrodes are arranged in the vertical direction, they are suitable for screening for drugs and electrical stimulation conditions. Furthermore, since the electrically conductive transparent plate is obtained by vapor-depositing an optically transparent electrically conductive material on the glass surface, a circuit is formed on the glass surface so that various types can be simultaneously performed on the same glass surface depending on the part. It is possible to give electrical stimulation under various conditions. In addition, by applying electrical stimulation to the cultured muscle cells, it is possible to give a pseudo-motion effect to the cells, so that it is possible to investigate the influence of the drug on the intracellular energy metabolism.

以下、本発明の実施の形態を、図面を参照しながら説明する。  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

図１は本発明の実施の形態による培養筋肉細胞電気刺激装置の概略構成を示す図、図２はその外観斜視図である。図１を参照すると、ウエル型培養皿（４）の下部に電気伝導性透明板（１）が電気伝導面が上向きになるように設置されており、この電気伝導性透明板上に培養筋肉細胞（３）を培養する。上部電極（５）は培地（２）に接するように蓋体（６）に設置され、下部に設置された電気伝導性透明板（１）間に通電する事によって培養筋肉細胞（３）に電気刺激を与える。  FIG. 1 is a diagram showing a schematic configuration of a cultured muscle cell electrical stimulation device according to an embodiment of the present invention, and FIG. 2 is an external perspective view thereof. Referring to FIG. 1, an electrically conductive transparent plate (1) is placed at the bottom of a well-type culture dish (4) so that the electrically conductive surface faces upward, and cultured muscle cells are placed on the electrically conductive transparent plate. Incubate (3). The upper electrode (5) is placed on the lid (6) so as to be in contact with the culture medium (2), and the cultured muscle cells (3) are electrically connected by energizing between the electrically conductive transparent plates (1) placed on the lower part. Give a stimulus.

本実施例では低栄養培地で筋肉様組織へと分化誘導されるＣ２Ｃ１２筋芽細胞を用いた。この細胞は分化誘導後に電位依存性チャンネルを発現し、電気刺激によって収縮運動が起きる事から本発明による刺激が最適な細胞である。Ｃ２Ｃ１２筋芽細胞をＺｎＯでコートしたガラス基板上にてダルベッコ改変イーゲル培地＋１０％牛胎児血清において培養した（図３）。３日間上記条件で培養後、ダルベッコ改変イーゲル培地＋２％ウシ血清に置換し、１週間培養することによって分化したＣ２Ｃ１２筋管細胞を得た（図４）。細胞どうしが融合し、チューブ上の構造を示していることから分化していることがわかる。  In this example, C2C12 myoblasts induced to differentiate into muscle-like tissue in a low nutrient medium were used. This cell expresses a voltage-dependent channel after differentiation induction, and contraction movement occurs by electrical stimulation. Therefore, the stimulation according to the present invention is the optimal cell. C2C12 myoblasts were cultured in Dulbecco's modified Egel medium + 10% fetal bovine serum on a glass substrate coated with ZnO (FIG. 3). After culturing under the above conditions for 3 days, the C2C12 myotube cells differentiated were obtained by substituting Dulbecco's modified Egel medium + 2% bovine serum and culturing for 1 week (FIG. 4). It can be seen that the cells are differentiated from each other because the cells are fused and show the structure on the tube.

ＺｎＯでコートしたガラス基板上にて培養した分化誘導後のＣ２Ｃ１２細胞に本発明による装置によって電気刺激を与えた。Ｃ２Ｃ１２細胞は筋肉様組織に分化する細胞なため、電気刺激によって収縮を誘導する事ができる。電気刺激発生装置としてＩｏｎＯｐｔｉｘ社のＣ−Ｐａｃｅを用い、電圧５Ｖ、周期１Ｈｚ、パルス幅２４ｍｓで電気刺激を与えたところ、細胞の収縮が観察された（図５）。位相差像の特定部位の明るさの変化を調べたところ、１Ｈｚで周期的に変化している事から細胞が与えられた電気刺激に反応して収縮している事がわかる（図６）。  Electrical stimulation was applied to the differentiated C2C12 cells cultured on a glass substrate coated with ZnO by the apparatus according to the present invention. Since C2C12 cells differentiate into muscle-like tissues, contraction can be induced by electrical stimulation. When C-Path of IonOptix was used as the electrical stimulation generator and electrical stimulation was applied at a voltage of 5 V, a period of 1 Hz, and a pulse width of 24 ms, cell contraction was observed (FIG. 5). When the change in the brightness of a specific part of the phase contrast image was examined, it can be seen that the cells contracted in response to the applied electrical stimulation since they periodically change at 1 Hz (FIG. 6).

本発明に係わる筋肉細胞電気刺激装置は、筋肉細胞における分子生物学、細胞生物学、遺伝子工学、再生医療など様々な分野に適用できる。  The muscle cell electrical stimulation apparatus according to the present invention can be applied to various fields such as molecular biology, cell biology, genetic engineering, and regenerative medicine in muscle cells.

本発明の実施の形態による電気刺激装置の概略構成である。1 is a schematic configuration of an electrical stimulation device according to an embodiment of the present invention. 本発明の実施の形態による電気刺激装置の外観斜視図である。1 is an external perspective view of an electrical stimulation device according to an embodiment of the present invention. ＺｎＯコートされたガラス基板上で培養された分化誘導前のＣ２Ｃ１２細胞。C2C12 cells before differentiation induction cultured on a glass substrate coated with ZnO. ＺｎＯコートされたガラス基板上で培養された分化誘導後のＣ２Ｃ１２細胞。C2C12 cells after differentiation induction cultured on a glass substrate coated with ZnO. ＺｎＯコートされたガラス基板上で培養された分化誘導後のＣ２Ｃ１２細胞に本発明による方法で電気刺激を与えた。右図は左図の白い四角で囲まれた部分の拡大図。右上は電気刺激前。右下は電気刺激後。電気刺激によって細胞が収縮し、縞模様が動いている事がわかる（矢印参照）。Electrical stimulation was applied to the differentiated C2C12 cells cultured on a ZnO-coated glass substrate by the method of the present invention. The right figure is an enlarged view of the part surrounded by the white square in the left figure. The upper right is before electrical stimulation. The lower right is after electrical stimulation. It can be seen that the cells are contracted by electrical stimulation and the striped pattern moves (see arrow). 周期的電気刺激（電圧５Ｖ、周期１Ｈｚ、パルス幅２４ｍｓ）に応答する細胞。左図の白い四角で囲まれた部分の明るさの経時変化を調べた（右図）。細胞が動く事によって明るさが電気刺激に応答して変化している事がわかる。A cell that responds to periodic electrical stimulation (voltage 5 V, period 1 Hz, pulse width 24 ms). The change over time of the brightness of the part surrounded by the white square in the left figure was examined (right figure). It can be seen that the brightness changes in response to electrical stimulation as the cells move.

符号の説明Explanation of symbols

１ 電気伝導性透明板
２ 培地
３ 培養筋肉細胞
４ ウエル型培養皿
５ 上部電極
６ 蓋体DESCRIPTION OF SYMBOLS 1 Electrically conductive transparent plate 2 Medium 3 Cultured muscle cell 4 Well-type culture dish 5 Upper electrode 6 Lid

Claims (4)

電気伝導性透明板上に培養筋肉細胞を培養する事により、電気伝導性透明板と電極の間に電圧を加えることにより透明板上の培養筋肉細胞に電気刺激を与える事を特徴とした細胞培養法。Cell culture characterized by culturing cultured muscle cells on an electrically conductive transparent plate and applying electrical stimulation to the cultured muscle cells on the transparent plate by applying a voltage between the electrically conductive transparent plate and the electrode Law. 前期の電気伝導性透明板はガラス面上にＺｎＯ膜を蒸着した物である事を特徴とする細胞培養法。A cell culture method characterized in that the electrically conductive transparent plate of the previous period is a product obtained by depositing a ZnO film on a glass surface. 前期の電気伝導性透明板上のＺｎＯ膜にパターン回路を作ることにより様々な電気刺激パターンを培養筋肉細胞に与える事を特徴とする細胞培養法。A cell culture method characterized in that various electrical stimulation patterns are given to cultured muscle cells by creating a pattern circuit on a ZnO film on an electrically conductive transparent plate in the previous period. 底部に光学的に透明な電極を設置してなる所定の容積を有する容器と前記容器の上方から被せて前記容器を密封し、かつ前記透明電極に対向して配設した対向電極を備え、前記容器と上下方向に対向して設置される蓋体とを備えたことを特徴とする請求項３記載の電気刺激用キュベット。A container having a predetermined volume formed by installing an optically transparent electrode at the bottom, and a counter electrode disposed over the container to seal the container and to face the transparent electrode, The cuvette for electrical stimulation according to claim 3, further comprising a container and a lid body disposed to face the container in the vertical direction.
JP2005194349A 2005-06-06 2005-06-06 Method for electrically stimulating cultured muscular cell by using transparent electrode Pending JP2006333861A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090053813A1 (en) * 2007-08-24 2009-02-26 David Mark Evans Multiplexed electroporation apparatus
JP2010119370A (en) * 2008-11-21 2010-06-03 Dainippon Printing Co Ltd Device for pattern cell culture
JP2012120452A (en) * 2010-12-06 2012-06-28 Dainippon Printing Co Ltd Container for cell test, and cell test method using the same
EP3413114A1 (en) * 2017-06-06 2018-12-12 United Arab Emirates University Functionalized optical lens and method of manufacturing
CN113122448A (en) * 2021-04-12 2021-07-16 深圳市第二人民医院(深圳市转化医学研究院) Non-contact electric field device for cell culture and cell culture method thereof

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090053813A1 (en) * 2007-08-24 2009-02-26 David Mark Evans Multiplexed electroporation apparatus
WO2009029519A2 (en) * 2007-08-24 2009-03-05 David Mark Evans Multiplexed electroporation apparatus
WO2009029519A3 (en) * 2007-08-24 2009-04-30 David Mark Evans Multiplexed electroporation apparatus
JP2010119370A (en) * 2008-11-21 2010-06-03 Dainippon Printing Co Ltd Device for pattern cell culture
JP2012120452A (en) * 2010-12-06 2012-06-28 Dainippon Printing Co Ltd Container for cell test, and cell test method using the same
EP3413114A1 (en) * 2017-06-06 2018-12-12 United Arab Emirates University Functionalized optical lens and method of manufacturing
CN109001113A (en) * 2017-06-06 2018-12-14 阿拉伯联合酋长国大学 Functionalization optical lens and manufacturing method
CN113122448A (en) * 2021-04-12 2021-07-16 深圳市第二人民医院(深圳市转化医学研究院) Non-contact electric field device for cell culture and cell culture method thereof

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