JP2011122953A - Kit for bioassay for cell examination - Google Patents
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本発明は、細胞の任意個所での電気刺激及び／又は計測及び計測方法の選択を任意且つ連続的に行うことが可能な細胞バイオアッセイ用キット等に関する。この細胞バイオアッセイ用キットは細胞の各種機能、特に、電気生理学的機能の解明及び／又は細胞に対する薬剤効果の評価を目的とするような各種の細胞検査方法の実施に使用することが出来る。 The present invention relates to a kit for cell bioassay and the like that can arbitrarily and continuously perform electrical stimulation and / or measurement and measurement method selection at arbitrary locations of cells. This kit for cell bioassay can be used for carrying out various cell inspection methods for the purpose of elucidating various functions of cells, in particular, electrophysiological functions and / or evaluating drug effects on cells.
製薬、医療、及び食品等の分野において、生体を模した培養細胞アッセイ系（バイオアッセイ系）は動物実験に代わる簡便かつ低コストな薬効・毒性試験法として注目されており、研究開発が活発化している。更に、最近、人工多能性幹細胞（iPS 細胞）の登場により、バイオアッセイ系の開発が一層加速し始めた。患者から樹立したiPS細胞は患者の病態をin vitroで再現可能であり、将来、各患者に合った薬剤のスクリーニングへのiPS細胞を用いたバイオアッセイ系の応用が期待されている。 In the fields of pharmaceuticals, medical care, foods, etc., cultured cell assay systems (bioassay systems) that mimic living organisms are attracting attention as simple and low-cost medicinal and toxicological test methods that can replace animal experiments, and research and development have become active. ing. Furthermore, recently, with the advent of induced pluripotent stem cells (iPS cells), the development of bioassay systems has begun to accelerate further. IPS cells established from patients can reproduce the pathology of patients in vitro, and in the future, application of bioassay systems using iPS cells to screening for drugs suitable for each patient is expected.
このような培養細胞を用いたバイオアッセイ系の構築には、生体内類似の機能や構造を維持した細胞組織のin vitro培養法及び適切な細胞機能計測技術の開発が求められる。
特に、薬剤の作用部位に応じ細胞への刺激及び／又は活性計測個所及び計測手法を任意に選択し、薬剤効果を部位選択的かつ複合的に解析可能なアッセイ系は有効である。そのためには、活性を保持した細胞が任意形状にデバイス上にパターン化されること、更には、刺激電極及び計測センサも細胞パターンに合わせて任意に配置されることが望まれる。
In order to construct a bioassay system using such cultured cells, it is required to develop an in vitro culture method for cell tissues maintaining similar functions and structures in vivo and appropriate cell function measurement techniques.
In particular, an assay system capable of analyzing a drug effect in a site-selective and complex manner by arbitrarily selecting a site for measuring stimulation and / or activity and a measuring technique according to the site of action of the drug is effective. For this purpose, it is desirable that cells having activity be patterned on the device in an arbitrary shape, and that the stimulation electrode and the measurement sensor are also arbitrarily arranged in accordance with the cell pattern.
これまでに、電極アレイ基板上で培養した細胞パターンに対する局所刺激箇所とその機能を対応付けた評価を目的として、刺激用電極や機能計測用センサアレイなどを配置したデバイス上で筋管細胞（非特許文献１）、心筋細胞（非特許文献２）、又は神経細胞（非特許文献３）をパターン培養する取り組みがなされている。 Up to now, myotube cells (non-synthesizing) have been performed on a device on which stimulation electrodes, sensor arrays for functional measurement, etc. are arranged for the purpose of associating the local stimulation location with the function of the cell pattern cultured on the electrode array substrate. Efforts are being made to perform pattern culture of Patent Document 1), cardiomyocytes (Non-Patent Document 2), or neurons (Non-Patent Document 3).
微小電極を細胞パターンの局所へ近接させて行う局所電気刺激・計測法（非特許文献４）も開発されている。 A local electrical stimulation / measurement method (Non-Patent Document 4) in which a microelectrode is brought close to the local part of a cell pattern has also been developed.
一方、細胞接着性領域をパターン修飾したアクリルアミドゲル上で筋管細胞を培養した例（非特許文献５）があるが、収縮弛緩制御は試みられていない。 On the other hand, there is an example (Non-Patent Document 5) in which myotube cells are cultured on an acrylamide gel in which the cell adhesion region is pattern-modified, but contraction / relaxation control has not been attempted.
又、特許文献１には、実質的にゲルに包埋された細胞パターンにおいて細胞の増殖、運動及び分化からなる群から選択される少なくとも１つに関連する生物学的指標を試験することを特徴とする生物学的試験法、並びにそのためのキットに関する発明が記載されており、上記細胞パターンを、細胞パターンを形成可能な培養表面を有する培養器具上で細胞培養を行い、培養後に培養表面にゲルを被覆することにより形成することが記載されている。 Patent Document 1 is characterized by testing a biological index related to at least one selected from the group consisting of cell proliferation, movement and differentiation in a cell pattern substantially embedded in a gel. The present invention relates to a biological test method and a kit therefor, and the cell pattern is cultured on a culture instrument having a culture surface capable of forming the cell pattern, and the gel is formed on the culture surface after the culture. It is described that it is formed by coating.
培養細胞を電気的に刺激し、それに伴う活性変化などを計測する細胞アッセイは神経系・筋系細胞等を中心にその電気生理学的機能解明に必須である。特に、パターン状に培養した細胞（細胞パターン）の局所に対する刺激及び／又は計測は部位特異的な細胞機能評価に有効である。 Cell assays that electrically stimulate cultured cells and measure changes in activity are essential for elucidating their electrophysiological functions, especially in nervous and muscle cells. In particular, local stimulation and / or measurement of cells cultured in a pattern (cell pattern) is effective for site-specific cell function evaluation.
しかしながら、上記のような従来の電極アレイ基板上で培養した細胞パターンに対する局所刺激・活性計測法においては、デバイス上では培養自体が難しく、また刺激・計測箇所は細胞パターン後に変更できないため細胞の増殖・分化状況及び構造に応じた刺激・計測個所の任意選択が不可能であった。更に、パターン細胞はデバイス上での使用に限定されるために他の評価法を利用した複合的な機能評価は困難であった。 However, in the local stimulation / activity measurement method for the cell pattern cultured on the conventional electrode array substrate as described above, the culture itself is difficult on the device, and the stimulation / measurement location cannot be changed after the cell pattern, so that cell proliferation・ Stimulation / measurement locations could not be arbitrarily selected according to differentiation status and structure. Furthermore, since the pattern cells are limited to use on the device, it has been difficult to perform a complex function evaluation using other evaluation methods.
又、微小電極を細胞パターンの局所へ近接させて行う局所電気刺激・計測法においては、細胞から一定距離にプローブを設置して細胞と電極の位置を合わせるのが困難であり、かつそのシステム全体が煩雑であって再現性が得られない、という問題点があった。 In addition, in the local electrical stimulation / measurement method in which the microelectrode is brought close to the local area of the cell pattern, it is difficult to align the position of the cell and the electrode by installing a probe at a certain distance from the cell, and the entire system. However, there is a problem that reproducibility cannot be obtained.
本発明者は上記課題を解決すべく、筋管細胞をモデルとして用いた研究の結果、基板上で培養した筋管細胞をゲルシートの表面に写し取り作製した筋管細胞ゲルシートを微小電極アレイ基板等の細胞局所刺激計測用デバイス上に任意に配置・再配置することにより局所電気刺激及び／又は計測個所の任意選択の連続操作が可能となることを見出した。また、ゲルシート培養法は基板上に接着した状態で行う従来の培養法と比較し筋管細胞の長期的な収縮弛緩運動の観測・評価を可能とし、デバイス上での利用に最適な培養系であることが示された。更に、筋管細胞と神経細胞のゲルシートを張り合わせるという簡便な操作で神経‐筋共培養系のin-vitro構築に成功し、局所刺激・計測デバイスと組み合わせることで神経‐筋相互作用の部位選択的かつ定量的な評価が可能となることが示された。本発明はこのような知見に基づき完成したものである。 As a result of research using myotube cells as a model, the inventor has developed a myotube cell gel sheet prepared by copying the myotube cells cultured on the substrate onto the surface of the gel sheet, as a microelectrode array substrate, etc. The present inventors have found that local electrical stimulation and / or optional continuous operation of measurement points can be performed by arbitrarily arranging and rearranging on the device for measuring cellular local stimulation. In addition, the gel sheet culturing method enables observation and evaluation of long-term contraction / relaxation movement of myotube cells compared to the conventional culturing method performed on a substrate, and is an optimal culture system for use on a device. It was shown that there is. Furthermore, we succeeded in the in-vitro construction of the nerve-muscle co-culture system with a simple operation of pasting myotube cells and nerve cell gel sheets together, and selecting the site of nerve-muscle interaction by combining with local stimulation / measurement devices. It was shown that a quantitative and quantitative evaluation is possible. The present invention has been completed based on such findings.
即ち、本発明は以下に示す各態様に係るものである。
[態様１]
表面領域に任意のパターンで細胞が培養されて成るゲルシート及び細胞局所刺激計測用デバイスを含む、細胞バイオアッセイ用キット。
[態様２]
細胞が筋細胞である、態様１又は２記載の細胞バイオアッセイ用キット。
[態様３]
筋細胞が筋芽細胞株から分化した筋菅細胞である、態様２記載の細胞バイオアッセイ用キット。
[態様４]
筋細胞による任意のパターンが略平行する複数のラインパターンである、態様１〜３のいずれか一項に記載の細胞バイオアッセイ用キット。
[態様５]
表面領域に任意のパターンで筋細胞が培養されて成るゲルシートが基板上で任意のパターンで培養された筋細胞をゲルシートの表面に移し取ることによって作製されたものである、態様１〜４のいずれか一項に記載の細胞バイオアッセイ用キット。
[態様６]
ゲルシートがフィブリンゲルシートである、態様１〜５のいずれか一項に記載の細胞バイオアッセイ用キット。
[態様７]
細胞局所刺激計測用デバイスが微小電極アレイ基板である、態様１〜６のいずれか一項に記載の細胞バイオアッセイ用キット。
[態様８]
細胞局所刺激計測用デバイスが微小電極アレイを有するマルチウェル電極アレイチップである、態様７記載の細胞バイオアッセイ用キット。
[態様９]
細胞局所刺激計測用デバイスがセンサが配列されたチップである、態様１〜６のいずれか一項に記載の細胞バイオアッセイ用キット。
[態様１０]
センサが酸素センサ又はグルコースセンサを含む、態様９記載の細胞バイオアッセイ用キット。
[態様１１]
ゲルシートが表面領域に任意のパターンで夫々異なる複数の種類の細胞が培養された複数のゲルシートが積層されたものである、態様１〜１０のいずれか一項に記載の細胞バイオアッセイ用キット。
[態様１２]
表面領域に任意のパターンで神経細胞及び筋細胞がそれぞれ培養されて成る複数のゲルシートを含む、態様１〜１１のいずれか一項に記載の細胞バイオアッセイ用キット。
[態様１３]
表面領域に任意のパターンで細胞が培養されて成るゲルシートが細胞局所刺激計測用デバイス上に配置させて成る、態様１〜１２のいずれか一項に記載の細胞バイオアッセイ用キット。
[態様１４]
態様１〜１３記載の細胞バイオアッセイ用キット及び刺激発生装置及び計測装置を含むバイオアッセイ系。
[態様１５]
刺激発生装置が電気パルス発生装置である、態様１４記載のバイオアッセイ系。
[態様１６]
態様１５記載のバイオアッセイ系による細胞検査方法。
[態様１７]
表面領域に任意のパターンで筋細胞が培養されて成るゲルシートを微小電極アレイ基板又はマルチウェル電極アレイチップ上に貼り付け、複数のラインパターン中に任意のものを選択的に電気刺激することを含む、態様１６記載の細胞検査方法。
[態様１８]
細胞検査が電気生理学的機能の解明及び／又は薬剤効果の評価を目的とするものである、態様１７記載の方法。
That is, the present invention relates to each aspect shown below.
[Aspect 1]
A kit for cell bioassay comprising a gel sheet in which cells are cultured in an arbitrary pattern on a surface region and a device for measuring local cell stimulation.
[Aspect 2]
The kit for cell bioassay according to embodiment 1 or 2, wherein the cells are muscle cells.
[Aspect 3]
The kit for cell bioassay according to aspect 2, wherein the myocyte is a myoblast differentiated from a myoblast cell line.
[Aspect 4]
The kit for cell bioassay according to any one of aspects 1 to 3, wherein the arbitrary pattern of muscle cells is a plurality of line patterns substantially parallel to each other.
[Aspect 5]
Any one of Embodiments 1 to 4, wherein the gel sheet formed by culturing myocytes in an arbitrary pattern on the surface region is prepared by transferring the myocytes cultured in an arbitrary pattern on the substrate to the surface of the gel sheet. A kit for cell bioassay according to claim 1.
[Aspect 6]
The kit for cell bioassay according to any one of embodiments 1 to 5, wherein the gel sheet is a fibrin gel sheet.
[Aspect 7]
The kit for cell bioassay according to any one of aspects 1 to 6, wherein the device for measuring local cell stimulation is a microelectrode array substrate.
[Aspect 8]
The kit for cell bioassay according to aspect 7, wherein the device for measuring local cell stimulation is a multiwell electrode array chip having a microelectrode array.
[Aspect 9]
The kit for cell bioassay according to any one of aspects 1 to 6, wherein the device for measuring local cellular stimulation is a chip on which sensors are arranged.
[Aspect 10]
The kit for cell bioassay according to aspect 9, wherein the sensor includes an oxygen sensor or a glucose sensor.
[Aspect 11]
The cell bioassay kit according to any one of aspects 1 to 10, wherein the gel sheet is a laminate of a plurality of gel sheets in which a plurality of different types of cells are cultured in an arbitrary pattern on the surface region.
[Aspect 12]
The kit for cell bioassay according to any one of embodiments 1 to 11, comprising a plurality of gel sheets in which nerve cells and muscle cells are cultured in an arbitrary pattern on the surface region.
[Aspect 13]
The kit for cell bioassay according to any one of embodiments 1 to 12, wherein a gel sheet in which cells are cultured in an arbitrary pattern on a surface region is arranged on a cell local stimulation measurement device.
[Aspect 14]
A bioassay system comprising the cell bioassay kit according to aspects 1 to 13, a stimulus generation device, and a measurement device.
[Aspect 15]
The bioassay system according to aspect 14, wherein the stimulus generator is an electrical pulse generator.
[Aspect 16]
A cell inspection method using the bioassay system according to Aspect 15.
[Aspect 17]
Including a gel sheet formed by culturing myocytes in an arbitrary pattern on a surface region on a microelectrode array substrate or a multiwell electrode array chip, and selectively electrically stimulating an arbitrary one in a plurality of line patterns The cell test method according to Aspect 16.
[Aspect 18]
The method according to aspect 17, wherein the cytological examination is for elucidation of electrophysiological function and / or evaluation of drug effect.
本発明の細胞検査用キットでは、ゲルシート及び細胞局所刺激計測用デバイスの相互の配置・再配置が可能である為に、従来技術では困難であった任意個所での刺激及び／又は計測及び計測方法の選択を任意且つ連続的に行うことが可能となる。このように細胞パターンの任意箇所の刺激が可能な細胞アッセイ系は、非常に良質な比較物を隣接させることが出来るため精度の高い評価を可能とする。 In the kit for cytological examination of the present invention, the gel sheet and the device for local cell stimulation measurement can be mutually arranged / rearranged. Therefore, stimulation and / or measurement and measurement method at an arbitrary position, which has been difficult in the prior art. Can be selected arbitrarily and continuously. As described above, the cell assay system capable of stimulating an arbitrary portion of the cell pattern can make a highly accurate evaluation because it can be adjacent to a very high quality comparator.
又、培養細胞パターンはゲルシート表面領域に保持されているため、デバイス外の最適な条件下で培養した後にデバイス上に配置させることで高活性の細胞パターンを刺激・計測することが可能となる。 In addition, since the cultured cell pattern is held in the gel sheet surface region, it is possible to stimulate and measure a highly active cell pattern by placing it on the device after culturing under optimal conditions outside the device.
更に、細胞パターンへの細胞ゲルシートとデバイスの位置合わせは、例えば、顕微鏡観察下で、正確かつ容易に実施することが可能である。このように、細胞パターンとデバイスは分離可能な為に、刺激計測の前・途中・後の何れにおいても別種の計測や評価をすることも可能である。 Furthermore, the alignment of the cell gel sheet and the device to the cell pattern can be performed accurately and easily, for example, under a microscope. As described above, since the cell pattern and the device are separable, it is possible to perform different types of measurement and evaluation before, during, and after the stimulus measurement.
本発明の細胞バイオアッセイ用キットは、その構成要素として、表面領域に任意のパターンで細胞が培養されて成るゲルシート及び細胞局所刺激計測用デバイスを含むものである。 The kit for cell bioassay of the present invention includes, as its constituent elements, a gel sheet in which cells are cultured in an arbitrary pattern on the surface region and a device for measuring local cell stimulation.
細胞の種類には特に制限はなく、例えば、平滑筋細胞、心筋細胞、C2C12筋芽細胞のような培養細胞株若しくはこれら細胞株から培養して分化した筋管細胞などの筋細胞、及び神経細胞などを挙げることが出来る。 There are no particular limitations on the type of cells, for example, smooth muscle cells, cardiomyocytes, cultured cell lines such as C2C12 myoblasts, or myocytes such as myotube cells cultured and differentiated from these cell lines, and nerve cells And so on.
本明細書において、「ゲルシートの表面領域」とは、少なくともゲルの表面を含み、表面から比較的浅いゲルの内部も有する三次元の領域を意味し、細胞及びゲルシートの構成成分、培養条件等にもよるが、通常、ゲルシートの表面から５０〜１００μｍ程度の深さまでの領域である。 In the present specification, the “surface region of the gel sheet” means a three-dimensional region including at least the surface of the gel and also having the interior of the gel relatively shallow from the surface. However, it is usually a region from the surface of the gel sheet to a depth of about 50 to 100 μm.
ゲルシートの表面領域に培養細胞により形成される任意のパターンの模様・形状・大きさ等に特に制約はなく、細胞局所刺激計測用デバイスの種類・構造、該細胞バイオアッセイ用キットを用いる細胞検査方法の目的等に応じて、適宜、選択することが出来る。例えば、細胞局所刺激計測用デバイスとして微小電極アレイ基板を用いる場合には、細胞パターンとして、微小電極アレイ基板の電極の大きさ及び距離等に対応した、略平行する複数のラインパターン（各ラインの幅は、例えば、数十〜数百μｍ）及びドットパターン等の中から任意の組み合わせを適宜選択することが出来る。 There are no particular restrictions on the pattern, shape, size, etc. of the arbitrary pattern formed by the cultured cells on the surface region of the gel sheet, the type and structure of the device for measuring local cell stimulation, and the cell inspection method using the cell bioassay kit Depending on the purpose and the like, it can be appropriately selected. For example, when a microelectrode array substrate is used as a cell local stimulation measurement device, a plurality of substantially parallel line patterns (each line corresponding to the size and distance of the electrodes of the microelectrode array substrate) are used as the cell pattern. As for the width, for example, an arbitrary combination can be appropriately selected from among several tens to several hundreds μm) and a dot pattern.
このようなゲルシートは、当業者に公知の任意の方法で作製することが可能である。例えば、基板上で任意のパターンで培養された細胞をゲルシートの表面に移し取ることによって、表面領域に任意のパターンで該細胞が培養されて成るゲルシートを作製することが出来る。 Such a gel sheet can be produced by any method known to those skilled in the art. For example, by transferring cells cultured in an arbitrary pattern on the substrate to the surface of the gel sheet, a gel sheet in which the cells are cultured in an arbitrary pattern on the surface region can be produced.
即ち、ガラス、金属、及び合成高分子等の適当な基板上に、2-methacryloyloxyethyl phosphorylcholineポリマー、ポリエチレングリコール、ポリアクリルアミド、ポリビニルアルコール等の適当な細胞非接着性物質（細胞接着阻害物質）を塗付することによって細胞非接触領域を設け、該基板の表面露出部からなるパターン上で細胞を適当な条件下で一定期間培養することによって基板上に細胞パターンを形成させ、その後、該基板上にゲルシートを形成し、適当時間をかけて基板上の細胞をゲルシートの表面に接着させ、その結果、基板上に形成された細胞パターンをゲルシートの表面に移し取ることが出来る。 That is, an appropriate cell non-adhesive substance (cell adhesion inhibitor) such as 2-methacryloyloxyethyl phosphorylcholine polymer, polyethylene glycol, polyacrylamide, and polyvinyl alcohol is applied on an appropriate substrate such as glass, metal, and synthetic polymer. A cell non-contact region is formed, and a cell pattern is formed on the substrate by culturing the cells for a certain period of time on a pattern composed of the exposed surface portion of the substrate, and then a gel sheet is formed on the substrate. The cells on the substrate are adhered to the surface of the gel sheet over an appropriate time, and as a result, the cell pattern formed on the substrate can be transferred to the surface of the gel sheet.
本発明でゲルシートとして使用できるものには、その表面又は内部で細胞が増殖・分化・運動等の細胞機能を発揮できるようなものである限り、その構成成分・原料等に特に制限はない。その一例として、フィブリノーゲンをゲル化することによって得られるフィブリンゲルシート、コラーゲンゲルシート、Matrigel（商標）シート、及びPuraMatrix（商標）シート等を挙げることが出来る。 There are no particular restrictions on the components, materials, etc. of the gel sheet that can be used in the present invention as long as the cells can exhibit cell functions such as proliferation, differentiation, and movement on the surface or inside thereof. Examples thereof include a fibrin gel sheet obtained by gelling fibrinogen, a collagen gel sheet, a Matrigel ™ sheet, and a PuraMatrix ™ sheet.
ゲルシート自体の大きさ・形状・厚みも細胞局所刺激計測用デバイスの種類・構造、該細胞バイオアッセイ用キットを用いる細胞検査方法の目的等に応じて、適宜、選択することが出来る。通常、ゲルシート自体は縦・横が1 ｃｍ、厚さ：2 ｍｍ程度である。更に、培養細胞パターンは、通常、縦・横が5 ｍｍ程度のゲルシートの表面領域に形成されている。 The size, shape, and thickness of the gel sheet itself can be appropriately selected according to the type and structure of the device for measuring local cell stimulation, the purpose of the cell testing method using the cell bioassay kit, and the like. Usually, the gel sheet itself is about 1 cm in length and width and about 2 mm in thickness. Furthermore, the cultured cell pattern is usually formed in the surface region of a gel sheet having a length and width of about 5 mm.
尚、表面領域に任意のパターンで細胞が培養されて成るゲルシートは、適当な培養液を含む状態で包装して、液体窒素等で凍結保存することも可能である。 The gel sheet in which cells are cultured in an arbitrary pattern on the surface region can be packaged in a state containing an appropriate culture solution and stored frozen with liquid nitrogen or the like.
本発明の細胞バイオアッセイ用キットに含まれる細胞局所刺激計測用デバイスは細胞に局所的刺激を与えるための手段及び/又は該刺激に対する細胞の応答を計測するための手段を含むデバイスであって、該キットを用いる本発明の細胞検査方法の目的等に応じて、当業者に公知の任意の構成をとることが可能である。ここで「刺激」とは、例えば、電気的刺激、化学物質による刺激及び伸展刺激のように、細胞の各種性質、特性及び/又は機能等に影響を与え得るような細胞外からの物理的、化学的又は機械的な刺激全般を意味する。従って、電気的刺激を細胞の局所に与える場合には、例えば、微小電極アレイ基板、微小電極アレイを有するマルチウェル電極アレイチップが挙げられる。更に、刺激に伴う細胞活性を計測する目的のため，例えば各種センサ（酸素センサ、グルコースセンサ等）を該デバイスであるチップ上に配列させることも出来る。 The device for measuring cellular local stimulation contained in the kit for cell bioassay of the present invention is a device comprising means for giving a local stimulation to a cell and / or means for measuring the response of the cell to the stimulation, Depending on the purpose of the cell test method of the present invention using the kit, any configuration known to those skilled in the art can be employed. Here, “stimulation” refers to, for example, physical from the outside that can affect various properties, characteristics, and / or functions of cells, such as electrical stimulation, stimulation by chemical substances, and extension stimulation, Means any chemical or mechanical stimulus in general. Therefore, when applying electrical stimulation locally to a cell, for example, a microelectrode array substrate and a multiwell electrode array chip having a microelectrode array can be used. Furthermore, for the purpose of measuring cell activity accompanying stimulation, for example, various sensors (oxygen sensor, glucose sensor, etc.) can be arranged on the chip as the device.
このような細胞局所刺激計測用デバイスは当業者に公知の任意の方法で作製することが出来る。例えば、微小電極アレイ基板は、一般的な半導体デバイス作製技術（フォトリソグラフィ法、スパッタリング蒸着法，及びリフトオフ法）を用いて作製することが出来る。 Such a cell local stimulation measurement device can be produced by any method known to those skilled in the art. For example, the microelectrode array substrate can be manufactured using a general semiconductor device manufacturing technique (a photolithography method, a sputtering deposition method, and a lift-off method).
更に、本発明の細胞バイオアッセイ用キットは、表面領域に任意のパターンで夫々異なる複数の種類の細胞が培養された複数のゲルシートを含むことが出来る。このようにして作製したある種類の細胞が表面領域に任意のパターンで培養されて成るゲルシートを別の細胞が表面領域に任意のパターンで培養されて成るゲルシートに積層することによって異種細胞の共培養系を簡便に構築することが出来る。 Furthermore, the cell bioassay kit of the present invention can include a plurality of gel sheets in which a plurality of different types of cells are cultured in an arbitrary pattern on the surface region. Co-culture of heterogeneous cells by laminating a gel sheet formed by culturing a certain type of cells on the surface region in an arbitrary pattern on the gel sheet formed by culturing another cell on the surface region in an arbitrary pattern The system can be constructed easily.
以上のようにして作製された表面領域に任意のパターンで細胞が培養されて成るゲルシートを、細胞局所刺激計測用デバイス上に配置・再配置させることで、高活性を保持した細胞パターンに対する刺激及び／又は計測箇所、並びに計測方法を任意に選択することができる細胞バイオアッセイ用キットが構成される。このように細胞パターンの任意箇所の刺激可能な本発明の細胞バイオアッセイキットは、非常に良質な比較物を隣接させることが出来、精度の高い評価を実施することが可能となる。 The gel sheet formed by culturing cells in an arbitrary pattern on the surface region prepared as described above is arranged and rearranged on the cell local stimulation measurement device, thereby stimulating the cell pattern retaining high activity and A cell bioassay kit capable of arbitrarily selecting a measurement location and a measurement method is configured. As described above, the cell bioassay kit of the present invention that can stimulate any part of the cell pattern can be adjacent to a very good quality comparison product, and can be evaluated with high accuracy.
例えば、神経細胞及び筋細胞が表面領域に任意のパターンで培養されて成る夫々のゲルシートを積層して細胞局所刺激計測用デバイス上に配置・再配置させることによって、細胞局所刺激計測用デバイス上で神経・筋細胞共培養系を構築することが出来、このような細胞バイオアッセイ用キットを用いることによって、神経・筋接合を介した筋管細胞の機能制御、例えば、グルコース代謝制御等を検査・評価することが出来る。 For example, by laminating each gel sheet in which nerve cells and muscle cells are cultured in an arbitrary pattern on the surface area, and arranging and rearranging them on the cell local stimulation measurement device, A nerve / muscle cell co-culture system can be constructed. By using such a cell bioassay kit, functional control of myotube cells via nerve / muscular junction, for example, glucose metabolism control, etc. Can be evaluated.
本発明の細胞バイオアッセイ用キットは、例えば、当業者に公知の任意の適当な電気パルス発生装置等の刺激発生装置及び/又は顕微鏡及びカメラ等の適当な計測装置と適宜組み合わせることによってバイオアッセイ系を構成することが出来る。尚、本発明の細胞バイオアッセイ用キット及びバイオアッセイ系は、上記の各要素に加えて、その使用目的等に応じて、適宜、必要なその他の物質・器具・装置類を含むことが出来る。 The kit for cell bioassay of the present invention is a bioassay system, for example, by appropriately combining it with a stimulus generator such as any appropriate electric pulse generator known to those skilled in the art and / or a suitable measuring device such as a microscope and a camera. Can be configured. The cell bioassay kit and bioassay system of the present invention can contain other necessary substances, instruments, and devices as appropriate depending on the purpose of use in addition to the above-described elements.
こうして構成されたバイオアッセイ系によって、例えば、細胞の各種性質、特性及び/又は機能（例えば、運動、増殖、及び、分化）等、特に、筋細胞の電気生理学的機能の解明及び／又は筋細胞に対する薬剤効果の評価を目的とするような各種の細胞検査方法を実施することが出来る。 With the bioassay system thus configured, e.g. elucidation of the electrophysiological function of muscle cells and / or muscle cells, such as various properties, characteristics and / or functions of cells (e.g. movement, proliferation and differentiation). Various cell inspection methods for the purpose of evaluating the drug effect on the cell can be carried out.
具体的には、例えば、表面領域に任意のパターンで細胞が培養されて成るゲルシートを微小電極アレイ基板等の細胞局所刺激計測用デバイス上に貼り付け、細胞パターン中の任意個所へ電気等で選択的に刺激し、その時の細胞応答を計測することを含むような方法を挙げることが出来る。 Specifically, for example, a gel sheet formed by culturing cells in an arbitrary pattern on the surface region is pasted on a cell local stimulation measurement device such as a microelectrode array substrate, and selected to an arbitrary location in the cell pattern by electricity or the like And a method that includes stimulating and measuring the cellular response at that time.
以下に実施例を参照して本発明を具体的に説明するが、これらは単に本発明の説明のために提供されているものである。従って、これらの実施例は、本願で開示する発明の範囲を限定し、又は制限するものではない。本発明では、特許請求の範囲の請求項に記載された技術的思想に基づく様々な実施形態が可能であることは当業者には容易に理解される。 The present invention will be described in detail with reference to the following examples, which are merely provided for explaining the present invention. Accordingly, these examples do not limit or limit the scope of the invention disclosed herein. It is easily understood by those skilled in the art that various embodiments based on the technical idea described in the claims can be made in the present invention.
筋管細胞ラインパターンを転写したフィブリンゲルシートの作製
（１）細胞パターニングに用いるMPC ポリマパターンガラス基板作製（図１）
細胞非接着性ポリマである2-methacryloyloxyethyl phosphorylcholine (MPC)ポリマーパターンの作製は、過去に実施例が報告されているマイクロモールド法により行った（Kaji H, Kawashima T, Nishizawa M. 2006. Patterning cellular motility using an electrochemical technique and a geometrically confined environment. Langmuir 22(25):10784-10787. ）。まず、poly(dimethylsiloxane) (PDMS)からなる流路をスライドガラス表面に貼付けた。1% wt MPCポリマを含むエタノール溶液を流路チャネルに導入し、室温で１晩エタノール溶媒を蒸発させることでガラス基板上にMPCポリマを固定化した。その後PDMSを剥がし、細胞接着領域となるガラス表面がライン形状に露出されたMPCポリマパターニングガラス基板を得た。最後に、基板にUVを15分照射し滅菌処理を施した。
Preparation of fibrin gel sheet transferred myotube cell line pattern (1) Preparation of MPC polymer pattern glass substrate used for cell patterning (Figure 1)
Preparation of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer pattern, which is a cell non-adhesive polymer, was performed by the micromold method reported in the past (Kaji H, Kawashima T, Nishizawa M. 2006. Patterning cellular motility using an electrochemical technique and a geometrically confined environment. Langmuir 22 (25): 10784-10787. First, a flow path made of poly (dimethylsiloxane) (PDMS) was attached to the surface of the slide glass. An ethanol solution containing 1% wt MPC polymer was introduced into the flow channel and the ethanol solvent was evaporated overnight at room temperature to immobilize the MPC polymer on the glass substrate. Thereafter, PDMS was peeled off to obtain an MPC polymer patterning glass substrate in which the glass surface serving as a cell adhesion region was exposed in a line shape. Finally, the substrate was sterilized by UV irradiation for 15 minutes.
（２）MPC ポリマーパターン基板を用いた筋管細胞のパターン培養（図２）
1x105 cells/ mL 濃度のC2C12 筋芽細胞をMPC ポリマーパターニング基板上に播種し、37℃，5% CO2 環境下10 分間静置し、細胞をガラス表面露出部に接着させた。余分な細胞を洗い流した後、C2C12 筋芽細胞がガラス表面露出領域内でコンフルエントとなるまでダルベッコ改変イーグル培地＋10 % 牛胎児血清を用い37℃，5% CO2 環境下で培養した（約１週間）。その後、ダルベッコ改変イーグル培地＋2 % 牛血清+1 nM インスリンに培地を置換し、筋管細胞へと分化させた（約1 週間）。栄養源の供給及び老廃物除去の目的から、培地交換は分化前は１日おき、分化誘導後は毎日行った。以上の操作により基板上にライン形状の筋管細胞パターンを作製した。
(2) Pattern culture of myotube cells using MPC polymer pattern substrate (Figure 2)
C2C12 myoblasts at a concentration of 1x10 5 cells / mL were seeded on an MPC polymer patterning substrate and allowed to stand in a 37 ° C, 5% CO2 environment for 10 minutes to adhere the cells to the exposed glass surface. After washing away excess cells, the cells were cultured in Dulbecco's modified Eagle's medium + 10% fetal calf serum in a 37 ° C, 5% CO2 environment until C2C12 myoblasts became confluent within the exposed glass surface (approximately 1 week) . Thereafter, the medium was replaced with Dulbecco's modified Eagle medium + 2% bovine serum + 1 nM insulin and differentiated into myotube cells (about 1 week). For the purpose of supplying nutrient sources and removing waste products, medium exchange was performed every other day before differentiation and daily after differentiation induction. The line-shaped myotube cell pattern was produced on the board | substrate by the above operation.
図３(A)にMPC ポリマラインパターニング基板（ライン幅800 μm）で形成した筋管細胞の位相差顕微鏡写真を示す。写真の白点線はMPC ポリマとガラス露出部の境界線であり、点線で囲まれたラインの中で繊維状に見えるのが筋管細胞である。筋管細胞は約２週間の培養期間中、MPC ポリマで囲まれたガラス領域からはみ出ることなくライン形状を維持した。図３（B）-(D)は個々の筋管細胞を蛍光色素Cell Tracker Green で染めた後の蛍光イメージであり、それぞれライン幅が 800 μm(B),250 μm(C)，及び100 μm(D)のパターンである。 FIG. 3 (A) shows a phase contrast micrograph of myotube cells formed on an MPC polymer line patterning substrate (line width 800 μm). The white dotted line in the photograph is the boundary line between the MPC polymer and the exposed glass part, and the myotubes appear to be fibrous in the line surrounded by the dotted line. Myotube cells maintained a line shape during the culture period of about 2 weeks without protruding from the glass area surrounded by the MPC polymer. Figures 3 (B)-(D) are fluorescence images after dyeing individual myotubes with the fluorescent dye Cell Tracker Green, with line widths of 800 μm (B), 250 μm (C), and 100 μm, respectively. This is the pattern (D).
MPC ラインに対する筋管細胞の配向角はライン幅の減少とともに減少し、幅800 μmでは12.6±4.7°，250 μm、100 μmではそれぞれ3.1±2.6°, 3.6±3.4°となった（0°に近づくほど筋管細胞がMPC ライン方向に沿って並んでいることを表す）。以上より、MPC ポリマパターン幅を狭くすることでより配向性の高い筋管細胞ラインパターンを形成可能であることが示された。 The orientation angle of myotubes with respect to the MPC line decreased as the line width decreased and became 12.6 ± 4.7 °, 250 μm, and 100 μm at the width of 800 μm, 3.1 ± 2.6 ° and 3.6 ± 3.4 °, respectively (at 0 ° The closer it is, the more myotubes are lined up along the MPC line). From the above, it was shown that a highly oriented myotube cell line pattern can be formed by narrowing the MPC polymer pattern width.
（３）筋管細胞ラインパターンのフィブリンゲルへの転写（図４）
筋管細胞パターン基板表面にフィブリノーゲン混合溶液を塗布し、37℃、5% CO2 環境下4 時間静置しフィブリノーゲンのゲル化及びゲルへの筋管細胞パターンの接着を促した。その後、丁寧にゲルを基板から剥離し、筋管細胞パターンが転写されたフィブリンゲルシート（フィブリノーゲンがゲル化したものの名称）を得た．フィブリノーゲン混合溶液の組成は15 mg/mL フィブリノーゲン，0.5 mg/mL アプロチニン、10 U/mL トロンビンを含むダルベッコ改変イーグル培地＋2 % 牛血清＋2% MEM アミノ酸，1% MEM 非必須アミノ酸である。
(3) Transfer of myotube cell line pattern to fibrin gel (Fig. 4)
A fibrinogen mixed solution was applied to the surface of the myotube cell pattern substrate and allowed to stand for 4 hours in an environment of 37 ° C. and 5% CO2 to promote gelation of fibrinogen and adhesion of the myotube cell pattern to the gel. Thereafter, the gel was carefully peeled from the substrate to obtain a fibrin gel sheet (name of fibrinogen gelled) on which the myotube cell pattern was transferred. The composition of the fibrinogen mixed solution is 15 mg / mL fibrinogen, 0.5 mg / mL aprotinin, Dulbecco's modified Eagle medium containing 10 U / mL thrombin + 2% bovine serum + 2% MEM amino acids, 1% MEM non-essential amino acids.
図５A にフィブリンゲルへの転写前のガラス基板上(a)及びフィブリンゲルへの転写後(b)の筋管細胞ラインパターンの蛍光イメージを示す。基板からゲルへの細胞の転写効率はライン幅に関係なくほぼ100％%であった（幅100μm から800μm の範囲で評価）。このような筋管細胞のゲルへの転写はガラス基板と比較しフィブリンゲルへの細胞の接着力が強いために起こる現象と考えられる。図５A から分かるように、転写された筋管細胞はガラス基板上での形状、位置、配向性を正確に維持していた。図５B にフィブリノーゲン混合溶液のゲル化直後及びゲル化4 時間後のゲルシート断面の蛍光イメージを示す。白く見える領域が筋管細胞ラインパターンの断面、白点線はガラス基板表面を表す。ゲル化直後、ゲル化４時間後で筋管細胞の位置はほとんど変化していないことから、筋管細胞パターンはゲルの表面に転写されていることが確認できた。 FIG. 5A shows fluorescence images of myotube cell line patterns on a glass substrate before transfer to fibrin gel (a) and after transfer to fibrin gel (b). The transfer efficiency of cells from the substrate to the gel was almost 100% regardless of the line width (evaluated in the range of 100 μm to 800 μm in width). Such transfer of myotube cells to the gel is considered to be a phenomenon that occurs because the adhesion of the cells to the fibrin gel is stronger than that of the glass substrate. As can be seen from FIG. 5A, the transferred myotube cells accurately maintained the shape, position, and orientation on the glass substrate. FIG. 5B shows fluorescence images of the gel sheet cross section immediately after gelation of the fibrinogen mixed solution and after 4 hours of gelation. The area that appears white is the cross section of the myotube cell line pattern, and the white dotted line represents the glass substrate surface. Immediately after gelation, 4 hours after gelation, the position of myotube cells was hardly changed, so that it was confirmed that the myotube cell pattern was transferred to the surface of the gel.
電気パルス印加による筋管細胞の収縮運動能誘導と収縮変移量計測
ゲルに転写した筋管細胞への電気刺激印加は、電圧パルス発生装置に接続した電極チャンバを用いて行った（図６）。電極チャンバはプラスティックシャーレ及びその両端に60mm 離して配置された2 枚の平板炭素電極から構成される．電極チャンバ内に筋管細胞ゲルシートを配置し、37℃，5% CO2 環境下、電圧パルス（パルス電圧, 0.7 V/mm; パルス幅,2.0 ms; 周波数, 1.0 Hz）を持続して印加することで筋管細胞の収縮運動能を誘導した。筋管細胞の収縮変移量は収縮運動時の動画を撮影・保存し、筋管細胞パターン上の特徴的構造を表す輝度の軌跡をμm 単位に変換することで評価した（図７）。筋管細胞の観察には共焦点顕微鏡を用い、その観察画像をデジタルCCD カメラで撮影しパソコン上で保存処理した。動画表示・解析ソフトはMetaMorph software (ver. 7.1.7.0)を用い、変異量解析にはフリーソフトのImageJ を用いた。
Induction of contractile motility of myotube cells and measurement of contraction displacement by electric pulse application Electric stimulation was applied to myotube cells transferred to gel using an electrode chamber connected to a voltage pulse generator ( FIG. 6). The electrode chamber consists of a plastic petri dish and two flat carbon electrodes placed 60 mm apart on both ends. Place the myotube cell gel sheet in the electrode chamber and apply a voltage pulse (pulse voltage, 0.7 V / mm; pulse width, 2.0 ms; frequency, 1.0 Hz) continuously in an environment of 37 ° C and 5% CO2. Induced contractile motility of myotube cells. The amount of contraction and displacement of myotube cells was evaluated by photographing and storing a moving image during contraction, and converting the luminance locus representing the characteristic structure on the myotube cell pattern into μm units (FIG. 7). A confocal microscope was used for observation of myotube cells, and the observation image was taken with a digital CCD camera and stored on a personal computer. The video display / analysis software was MetaMorph software (ver. 7.1.7.0), and the mutation amount analysis was free software ImageJ.
図８A に、筋管細胞／フィブリンゲルシート（ライン幅250 μm）への電圧パルスの持続印加（パルス電圧, 0.7 V/mm; パルス幅, 2.0 ms; 周波数, 1.0 Hz）に伴う収縮変移量の時間変化を示す。刺激印加１時間後に筋管細胞の収縮運動が観察され、その後、時間とともに収縮変移量が徐々に増加した。刺激印加20 時間後における、筋管細胞の収縮運動のパルス周波数依存性（図８B ）及びパルス幅依存性（図８C ）を示す。図８Bより筋管細胞が1Hz, 2Hzの印加周波数に同調した単収縮運動を示すことが確認できた。また、図８C よりパルス幅の増加とともに収縮変位量が増加した。これは従来の文献で報告されているように（Fujita H, Nedachi T, Kanzaki M. 2007. Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12 myotubes. Exp Cell Res 313(9):1853-1865;Melzer W, Rios E, Schneider MF.1984. Time course of calcium release and removal in skeletal-muscle fibers. Biophys J 45(3):637-641）、脱分極時間の増加により細胞質内Ca イオン濃度が増加したことで収縮変移量が増加したと考えられる。以上より、フィブリンゲル内の筋管細胞の収縮運動は外部から印加した電気刺激により人為的に制御可能であることが示された。 Fig. 8A shows the time of contraction change with continuous application of pulse voltage (pulse voltage, 0.7 V / mm; pulse width, 2.0 ms; frequency, 1.0 Hz) to myotube cells / fibrin gel sheet (line width 250 μm). Showing change. The contraction movement of myotube cells was observed 1 hour after application of the stimulus, and then the contraction displacement gradually increased with time. The pulse frequency dependency (FIG. 8B) and pulse width dependency (FIG. 8C) of contraction movement of myotube cells 20 hours after application of the stimulus are shown. From FIG. 8B, it was confirmed that the myotube cells showed a twitch movement synchronized with the applied frequency of 1 Hz and 2 Hz. Further, as shown in FIG. 8C, the amount of contraction displacement increased as the pulse width increased. As reported in previous literature (Fujita H, Nedachi T, Kanzaki M. 2007. Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12 myotubes. Exp Cell Res 313 (9): 1853-1865; Melzer W, Rios E, Schneider MF. 1984. Time course of calcium release and removal in skeletal-muscle fibers. Biophys J 45 (3): 637-641). Increased depolarization time increased cytosolic Ca ion concentration. It is thought that the amount of contraction shift increased. From the above, it was shown that the contraction movement of myotube cells in the fibrin gel can be artificially controlled by electrical stimulation applied from the outside.
図９A に筋管細胞ゲルシート（ライン幅250 μm）への長期的（1 週間）な電圧パルス持続印加（パルス電圧、0.7 V/mm; パルス幅, 2.0 ms; 周波数, 1.0 Hz）に伴う収縮変移量の時間変化を示す（図９A 中の●プロット）。収縮変移量は時間とともに増加し、印加４日後で最大値を示した。図９B に刺激印加１日後，及び７日後におけるゲル中の筋管細胞の収縮運動を示す。図に示すように、ゲル中の筋管細胞は印加周波数に同調した収縮運動を１週間持続した。また図９C（位相差顕微鏡写真）より、１週間の持続した収縮運動後においても筋管細胞パターンの形状変化及びパターンのゲルからの剥離は見られなかった。 Figure 9A shows the contraction transition of long-term (one week) voltage pulse applied to myotube cell gel sheet (line width 250 μm) (pulse voltage, 0.7 V / mm; pulse width, 2.0 ms; frequency, 1.0 Hz). The change with time is shown (● plot in FIG. 9A). The amount of contraction shift increased with time, and showed a maximum value 4 days after application. FIG. 9B shows the contraction movement of myotube cells in the gel 1 day after the application of the stimulus and 7 days after. As shown in the figure, myotube cells in the gel sustained contraction movement synchronized with the applied frequency for 1 week. Further, from FIG. 9C (phase contrast micrograph), the shape change of the myotube cell pattern and the peeling of the pattern from the gel were not observed even after the continuous contraction movement for one week.
一方、図９A（◆プロット）に，従来のディッシュ培養系における筋管細胞の収縮変移量の時間変化を示す。従来ディッシュ培養系とは、筋管細胞をプラスティックシャーレ底面に接着させ、増殖・分化させたものであり、そのシャーレ両端に設置したカーボン電極で電圧パルスを印加することで収縮運動能を誘導した。筋管細胞はディッシュ底面に強く接着しているため収縮運動量が低く、また収縮運動量が大きい細胞が存在しても培養３日後あたりから次々と剥離した。 On the other hand, FIG. 9A (♦ plot) shows changes over time in the amount of contraction of myotube cells in a conventional dish culture system. The conventional dish culture system is a culture in which myotube cells are adhered to the bottom of a plastic petri dish and proliferated and differentiated, and contractile motility was induced by applying a voltage pulse with carbon electrodes installed at both ends of the petri dish. Myotube cells were strongly adhered to the bottom of the dish, so the contraction momentum was low, and even if cells with a large contraction momentum were present, they separated one after another after about 3 days of culture.
筋管細胞ゲルシートにおける長期的な収縮運動及びパターン構造の持続は、フィブリンゲルの柔軟性により達成されている。柔軟なフィブリンゲルは筋管細胞の活発な収縮運動をサポートするが、従来の堅い培養ディッシュは、その表面に接着した筋管細胞の収縮運動を抑制し、また活発に収縮するほど剥離していく。以上の結果から、フィブリンゲルシートを用いた本発明の培養系は、従来のディッシュ培養系と比較し筋管細胞の収縮運動及びパターン構造の長期維持に有効であることが示された。 Long-term contractile motion and sustained pattern structure in the myotube cell gel sheet are achieved by the flexibility of the fibrin gel. Flexible fibrin gel supports active contraction of myotube cells, but traditional hard culture dishes suppress the contraction of myotubes attached to the surface and detach as active contraction occurs. . From the above results, it was shown that the culture system of the present invention using a fibrin gel sheet is more effective for contraction movement of myotube cells and long-term maintenance of the pattern structure than the conventional dish culture system.
ゲルに転写した C2C12 筋管細胞パターンへの選択的電気刺激
（１）選択的電気刺激用の微小電極アレイ基板作製
まず、スライドガラス表面に感光性ポジ型フォトレジストを塗布し、フォトマスクを介した露光及び現像により微小電極パターン領域に相当するガラス基板表面が露出したフォトレジストパターンを形成した。基板表面に白金薄膜をスパッタ蒸着後、アセトン溶液に浸漬してフォトレジスト及びその表面に蒸着された白金薄膜を溶解除去し（リフトオフ）、微小白金電極アレイを得た（図１０）。
Selective electrical stimulation to C2C12 myotube cell pattern transferred to gel (1) Fabrication of microelectrode array substrate for selective electrical stimulation First, a photosensitive positive photoresist was applied to the surface of the slide glass and passed through a photomask. A photoresist pattern in which the glass substrate surface corresponding to the microelectrode pattern region was exposed by exposure and development was formed. A platinum thin film was sputter-deposited on the substrate surface, then immersed in an acetone solution to dissolve and remove the photoresist and the platinum thin film deposited on the surface (lift-off), thereby obtaining a micro platinum electrode array (FIG. 10).
こうして作製した微小白金電極アレイの上下に配置された、向い合う半円弧状の電極を１対の選択的電気刺激用電極対として用い、一方の電極を作用電極、他方を対極とした。半円弧状電極間の距離は400μm、円弧電極幅は50μm、円弧の半径は150μm とした。 The opposing semicircular arc-shaped electrodes arranged above and below the microplatinum electrode array thus produced were used as a pair of selective electrical stimulation electrodes, with one electrode as the working electrode and the other as the counter electrode. The distance between the semicircular arc electrodes was 400 μm, the arc electrode width was 50 μm, and the radius of the arc was 150 μm.
（２）微小電極アレイ基板へのC2C12 筋管細胞ゲルシートの配置・再配置，及び選択的電気刺激による収縮運動制御
図６で示した電極チャンバを用い、あらかじめ収縮運動能を誘導した筋管細胞ゲルシートを図１０の微小電極アレイ基板に貼付け、選択的電気刺激を実施した（図１１）。ゲルシートの貼り付けは顕微鏡観察下で行い、ピンセットでゲルシートを動かしながら筋管細胞ラインパターンを微小電極位置に合わせた。その後、電圧パルス発生装置を用いて目的の電極対に電圧パルス（パルス電圧, 1.8 V/400μm; パルス幅, 10.0 ms; 周波数, 1.0 Hz）を印加し、そこに配置された筋管細胞ラインパターンを選択的に刺激した。尚、選択的電気刺激後の筋管細胞ゲルシートの再配置は、ゲルシートを剥がした後に再度任意の箇所に貼付け、細胞パターンと微小電極アレイ基板の位置を合わせることで行った。
(2) Placement / rearrangement of C2C12 myotube cell gel sheet on microelectrode array substrate and contraction movement control by selective electrical stimulation Myotube cell gel sheet whose contraction motility was previously induced using the electrode chamber shown in FIG. Was affixed to the microelectrode array substrate of FIG. 10, and selective electrical stimulation was performed (FIG. 11). The gel sheet was attached under a microscope, and the myotube cell line pattern was adjusted to the position of the microelectrode while moving the gel sheet with tweezers. Then, a voltage pulse (pulse voltage, 1.8 V / 400μm; pulse width, 10.0 ms; frequency, 1.0 Hz) is applied to the target electrode pair using a voltage pulse generator, and the myotube cell line pattern placed there Selectively stimulated. In addition, the rearrangement of the myotube cell gel sheet after selective electrical stimulation was performed by peeling the gel sheet and pasting it again to an arbitrary position, and aligning the cell pattern and the position of the microelectrode array substrate.
図１２A に、微小電極アレイ基板へ配置したゲルシート内筋管細胞ラインパターン（ライン幅250 μm）の顕微鏡写真を示す。顕微鏡観察下での操作により３本の筋管細胞ラインパターンを微小電極対へ正確に配置させることができた。図１２B に、図１２A の３対の微小電極に対し電圧パルス（パルス電圧,1.8 V/400μm; 周波数, 1Hz; パルス幅, 10 ms）を印加した時の各筋管細胞パターンの収縮運動を示す。縦軸は収縮変移量[μm]、横軸は時間 [s]を表す。最初に３対の電極全てに刺激を印加したところ、全ての筋管細胞が収縮運動を開始した。次に、一旦刺激を止め，中央の電極(2)にのみ刺激を再印加したところ、中央の筋管細胞パターンのみが収縮運動を再開した。これより、微小電極アレイと組み合わせることでゲル中の任意の筋管細胞パターンへ選択的に電気刺激を印加可能であることが示された。また、その後ゲルシートを剥離し、別の筋管細胞パターンへ微小電極アレイを再配置したところ、同様の選択的電気刺激を実行できた。これより、微小電極アレイへの筋管細胞ゲルシートの配置・再配置を繰り返すことで、任意に選択した筋管細胞パターンへの電気刺激の連続操作が可能であることが示された。 FIG. 12A shows a micrograph of the myotube cell line pattern (line width 250 μm) in the gel sheet placed on the microelectrode array substrate. Three myotube cell line patterns could be accurately placed on the microelectrode pair by operation under a microscope. Fig. 12B shows the contraction movement of each myotube pattern when voltage pulses (pulse voltage, 1.8 V / 400μm; frequency, 1Hz; pulse width, 10 ms) are applied to the three pairs of microelectrodes in Fig. 12A. . The vertical axis represents contraction displacement [μm], and the horizontal axis represents time [s]. When stimulation was initially applied to all three pairs of electrodes, all myotubes started to contract. Next, when the stimulus was stopped once and the stimulus was reapplied only to the central electrode (2), only the central myotube pattern resumed contraction. From this, it was shown that electrical stimulation can be selectively applied to an arbitrary myotube cell pattern in a gel by combining with a microelectrode array. Moreover, when the gel sheet was peeled off and the microelectrode array was rearranged to another myotube cell pattern, the same selective electrical stimulation could be performed. From this, it was shown that continuous operation of electrical stimulation to an arbitrarily selected myotube cell pattern is possible by repeatedly arranging and rearranging the myotube cell gel sheet on the microelectrode array.
細胞ゲルシートの積層による神経・筋共培養系の構築
（１）神経細胞ゲルシートの作製
神経細胞ゲルシートは筋管細胞と同様、図１、２及び４で示した手順で作製した。MPC ポリマをパターニングする基板には、神経細胞の接着性向上の目的からアミノシランコートガラス（松波硝子製）を用いた。アミノシランコートガラスへのMPC ポリマのパターニング手順は図１と同様である。1x105 cells/ mL 濃度のNG108-15 神経細胞をMPC ポリマパターニング基板上に播種し、37℃，5% CO2 環境下1 晩静置し、細胞をアミノシランコートガラス表面露出部に接着させた。余分な細胞を洗い流した後、ダルベッコ改変イーグル培地＋10 % 牛胎児血清+2 mM L-グルタミン＋1 mM N6,O2’-dibutyryl-adenosine 3’:5’-cyclic monophosphate を用い37℃，5% CO2 環境下で培養、分化させた。培地交換は2 日おきに行った。分化による神経突起の伸長を確認した後、図４の手順で神経細胞をフィブリンゲルへ転写し、神経細胞ゲルシートを得た。フィブリノーゲン混合溶液には，15 mg/ mL フィブリノーゲン、0.5 mg/ mL アプロチニン、10 U /mL トロンビンを含むダルベッコ改変イーグル培地＋10 % 牛胎児血清+2 mM L-グルタミン＋1 mM N6,O2’-dibutyryl-adenosine 3’:5’-cyclic monophosphate を用いた。
Construction of nerve / muscle co-culture system by laminating cell gel sheets (1) Preparation of nerve cell gel sheet The nerve cell gel sheet was prepared by the procedure shown in FIGS. Aminosilane-coated glass (manufactured by Matsunami Glass) was used as a substrate for patterning the MPC polymer for the purpose of improving the adhesion of nerve cells. The patterning procedure of MPC polymer on aminosilane-coated glass is the same as that shown in FIG. NG108-15 neurons at a concentration of 1x10 5 cells / mL were seeded on an MPC polymer patterning substrate and allowed to stand overnight at 37 ° C in a 5% CO2 environment to attach the cells to the exposed surface of the aminosilane-coated glass. After washing away excess cells, Dulbecco's modified Eagle medium + 10% fetal bovine serum + 2 mM L-glutamine + 1 mM N6, O2'-dibutyryl-adenosine 3 ': 5'-cyclic monophosphate at 37 ° C, 5% CO2 environment Under culture and differentiation. The medium was changed every 2 days. After confirming the extension of neurites due to differentiation, nerve cells were transferred to fibrin gel by the procedure of FIG. 4 to obtain a nerve cell gel sheet. The fibrinogen mixed solution contains 15 mg / mL fibrinogen, 0.5 mg / mL aprotinin, Dulbecco's modified Eagle medium containing 10 U / mL thrombin + 10% fetal calf serum + 2 mM L-glutamine + 1 mM N6, O2'-dibutyryl-adenosine 3 ': 5'-cyclic monophosphate was used.
（２）ゲルシート貼り合わせによる神経・筋共培養系の構築
神経・筋共培養系は、筋管細胞及び神経細胞ゲルシートを個別に作製し、各々の細胞転写面を向かい合わせて張り合わせることで構築した（図１３）。細胞パターンの位置合わせは、顕微鏡観察下、ピンセットで片方のゲルシートを動かしながら行った。位置合わせ後、ゲルシート接触面へフィブリンゲル混合溶液を流し込み、37℃、5% CO2 環境下10 分間静置し、ゲル化させることで2 枚のゲルシートを一体化させた。ゲルシート接着用フィブリノーゲン混合溶液には、15 mg/mL フィブリノーゲン、0.5 mg/mL アプロチニン、10 U/mL トロンビンを含むダルベッコ改変イーグル培地を用いた。
(2) Construction of nerve / muscle co-culture system by pasting gel sheets The nerve / muscle co-culture system is constructed by individually producing myotube cells and nerve cell gel sheets and pasting each cell transfer surface facing each other. (FIG. 13). The alignment of the cell pattern was performed while moving one gel sheet with tweezers under a microscope. After alignment, the fibrin gel mixed solution was poured into the contact surface of the gel sheet, and left to stand for 10 minutes at 37 ° C. in a 5% CO2 environment, and gelled to integrate the two gel sheets. Dulbecco's modified Eagle medium containing 15 mg / mL fibrinogen, 0.5 mg / mL aprotinin and 10 U / mL thrombin was used as the fibrinogen mixed solution for gel sheet adhesion.
図１３C,D に、C2C12 筋管細胞ゲルシート(A)及びNG108-15 神経細胞ゲルシート(B)の積層により構築した神経・筋共培養系の位相差顕微鏡写真(C)及び蛍光イメージ(D)を示す。各細胞の識別を容易にするため、筋管細胞はCell Tracker Green、神経細胞はCell Trackr Red で蛍光染色した。図１３C，D より、神経細胞が筋管細胞パターンに向かって神経突起を伸長しているのが確認できた。このように、細胞ゲルシートを積層させることで任意に細胞が配置された共培養系を容易に構築可能であることが示された。 FIGS. 13C and D show a phase contrast micrograph (C) and a fluorescence image (D) of a nerve / muscle co-culture system constructed by laminating C2C12 myotube cell gel sheet (A) and NG108-15 nerve cell gel sheet (B). Show. In order to facilitate identification of each cell, myotube cells were fluorescently stained with Cell Tracker Green, and neurons were stained with Cell Trackr Red. From FIG. 13C and D, it has confirmed that the nerve cell was extending the neurite toward the myotube cell pattern. Thus, it was shown that a co-culture system in which cells are arbitrarily arranged can be easily constructed by laminating cell gel sheets.
従来の共培養系構築法では異種細胞を培養シャーレ中で同時に混在させ同じ環境下で培養していたため、各細胞にとっての最適条件での培養ができず十分分化した筋、神経細胞からなる共培養系を構築することは困難であった。しかしながら，ゲルシートを用いた本発明による手法では、個別に最適条件下で増殖・分化させてから貼り合わせることができるため、従来と比較し、より機能の発達した細胞同士の共培養が可能となる。 In the conventional co-culture system construction method, heterogeneous cells are mixed together in a culture dish and cultured in the same environment, so co-culture consisting of well-differentiated muscles and nerve cells that cannot be cultured under the optimum conditions for each cell It was difficult to construct a system. However, in the method according to the present invention using a gel sheet, since the cells can be bonded together after being proliferated and differentiated under optimum conditions, it becomes possible to co-cultivate cells with more advanced functions than in the past. .
本発明を利用することによって、細胞パターンに対し使用するデバイス種、あるいはデバイスによる刺激・計測位置等の任意の組み合わせが選択可能な自由度の高いバイオアッセイ系を構築することができる。更に、その応用として、例えば、特定の細胞構造をターゲットとした薬剤効果の評価などが可能となる。開発する薬剤の作用部位に応じ刺激・活性計測個所及び計測手法を任意に選択し、薬剤効果を部位選択的に、かつ複合的に解析可能となる。 By utilizing the present invention, it is possible to construct a bioassay system having a high degree of freedom in which any combination of the device type used for the cell pattern or the stimulation / measurement position by the device can be selected. Furthermore, as an application thereof, for example, it is possible to evaluate drug effects targeting a specific cell structure. The stimulus / activity measurement location and measurement method can be arbitrarily selected according to the site of action of the drug to be developed, and the drug effect can be analyzed site-selectively and in combination.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2017500047A (en) * | 2013-12-18 | 2017-01-05 | シィトーＣｙｔｏｏ | Apparatus and method for normalizing myoblast differentiation into myotubes |
| JPWO2017077985A1 (en) * | 2015-11-05 | 2018-08-23 | 学校法人東京女子医科大学 | Method for producing laminated cell sheet composition, laminated cell sheet composition produced thereby and apparatus for producing the same |
| US10163203B2 (en) | 2013-11-08 | 2018-12-25 | Sony Corporation | Cell analysis system, cell analysis program and cell analysis method |
| US10209236B2 (en) | 2012-12-27 | 2019-02-19 | Sony Corporation | Cell analyzer system, cell analyzer program, and cell analyzing method |
| JP2019200087A (en) * | 2018-05-15 | 2019-11-21 | 学校法人早稲田大学 | Sensor film and laminated body |
| WO2020013270A1 (en) * | 2018-07-12 | 2020-01-16 | 学校法人東北工業大学 | Measurement method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0678889A (en) * | 1992-09-04 | 1994-03-22 | Matsushita Electric Ind Co Ltd | Integrated composite electrode |
| JPH06296595A (en) * | 1993-04-16 | 1994-10-25 | Matsushita Electric Ind Co Ltd | Unified complex electrode |
| JPH0862209A (en) * | 1994-06-13 | 1996-03-08 | Matsushita Electric Ind Co Ltd | Cell potential measuring apparatus |
| WO1997027318A1 (en) * | 1996-01-24 | 1997-07-31 | Matsushita Electric Industrial Co., Ltd. | Method for measuring physicochemical properties of tissues or cells, method for examining chemicals, and apparatus therefor |
| JP2007532103A (en) * | 2004-04-07 | 2007-11-15 | アキシオジェネシス エージー | Non-invasive in vitro functional tissue verification system |
| JP2008118900A (en) * | 2006-11-10 | 2008-05-29 | Dainippon Printing Co Ltd | Test method using cell and testing kit therefor |
| JP2011030574A (en) * | 2010-11-04 | 2011-02-17 | Toyota Central R&D Labs Inc | Output device for myocyte and output evaluating method for myocyte |
-
2009
- 2009-12-11 JP JP2009281158A patent/JP5544474B2/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0678889A (en) * | 1992-09-04 | 1994-03-22 | Matsushita Electric Ind Co Ltd | Integrated composite electrode |
| JPH06296595A (en) * | 1993-04-16 | 1994-10-25 | Matsushita Electric Ind Co Ltd | Unified complex electrode |
| JPH0862209A (en) * | 1994-06-13 | 1996-03-08 | Matsushita Electric Ind Co Ltd | Cell potential measuring apparatus |
| WO1997027318A1 (en) * | 1996-01-24 | 1997-07-31 | Matsushita Electric Industrial Co., Ltd. | Method for measuring physicochemical properties of tissues or cells, method for examining chemicals, and apparatus therefor |
| JP2007532103A (en) * | 2004-04-07 | 2007-11-15 | アキシオジェネシス エージー | Non-invasive in vitro functional tissue verification system |
| JP2008118900A (en) * | 2006-11-10 | 2008-05-29 | Dainippon Printing Co Ltd | Test method using cell and testing kit therefor |
| JP2011030574A (en) * | 2010-11-04 | 2011-02-17 | Toyota Central R&D Labs Inc | Output device for myocyte and output evaluating method for myocyte |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10209236B2 (en) | 2012-12-27 | 2019-02-19 | Sony Corporation | Cell analyzer system, cell analyzer program, and cell analyzing method |
| US10163203B2 (en) | 2013-11-08 | 2018-12-25 | Sony Corporation | Cell analysis system, cell analysis program and cell analysis method |
| US10482598B2 (en) | 2013-11-08 | 2019-11-19 | Sony Corporation | Cell analysis system, cell analysis program and cell analysis method |
| US10861154B2 (en) | 2013-11-08 | 2020-12-08 | Sony Corporation | Cell analysis system, cell analysis program and cell analysis method |
| JP2017500047A (en) * | 2013-12-18 | 2017-01-05 | シィトーＣｙｔｏｏ | Apparatus and method for normalizing myoblast differentiation into myotubes |
| US10711248B2 (en) | 2013-12-18 | 2020-07-14 | Cytoo | Device and method for standardizing myoblast differentiation into myotubes |
| JPWO2017077985A1 (en) * | 2015-11-05 | 2018-08-23 | 学校法人東京女子医科大学 | Method for producing laminated cell sheet composition, laminated cell sheet composition produced thereby and apparatus for producing the same |
| JP2019200087A (en) * | 2018-05-15 | 2019-11-21 | 学校法人早稲田大学 | Sensor film and laminated body |
| JP7070898B2 (en) | 2018-05-15 | 2022-05-18 | 学校法人早稲田大学 | Sensor film and laminate |
| WO2020013270A1 (en) * | 2018-07-12 | 2020-01-16 | 学校法人東北工業大学 | Measurement method |
| JPWO2020013270A1 (en) * | 2018-07-12 | 2021-08-26 | 学校法人東北工業大学 | Measurement method |
| JP7374422B2 (en) | 2018-07-12 | 2023-11-07 | 学校法人東北工業大学 | Measurement method |
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