JP4931017B2 - Cell extension stimulation load device - Google Patents

Cell extension stimulation load device Download PDF

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JP4931017B2
JP4931017B2 JP2008003220A JP2008003220A JP4931017B2 JP 4931017 B2 JP4931017 B2 JP 4931017B2 JP 2008003220 A JP2008003220 A JP 2008003220A JP 2008003220 A JP2008003220 A JP 2008003220A JP 4931017 B2 JP4931017 B2 JP 4931017B2
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transparent substrate
extension
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cells
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JP2009159925A (en
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克也 佐藤
和幸 南
晃裕 下野
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NATIONAL UNIVERSITY CORPORATION YAMAGUCHI UNIVERSITY
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Description

本発明は、培養細胞に伸展刺激負荷を与える装置に関する。   The present invention relates to an apparatus for applying an extension stimulation load to cultured cells.

力学的刺激の負荷が細胞の機能調節や分化誘導に重要なシグナルとなっているといわれている。再生医療で所望の組織を細胞群から再形成させるためには、生化学的シグナルによる誘導のみではなく、力学的刺激の適切な負荷による誘導が効果的な補助作用を果たすと考えられている。しかしながら、細胞が力学的刺激を負荷された際の感知・応答機構は未解明な点が多く、力学的刺激を負荷された細胞の応答を観察するためのデバイスが種々提案されている。これらのデバイスの多くは、弾性を有するポリマーシート上に播種し、ポリマーシート上で細胞を培養した後、ポリマーシートを伸展することにより、該シート上に付着している細胞に伸展刺激負荷を与える手段が用いられている。   It is said that the load of mechanical stimulation is an important signal for regulating cell functions and inducing differentiation. In order to regenerate a desired tissue from a cell group in regenerative medicine, it is considered that not only induction by a biochemical signal but also induction by an appropriate load of a mechanical stimulus performs an effective auxiliary action. However, the sensing and response mechanisms when cells are loaded with mechanical stimuli have many unclear points, and various devices for observing the response of cells loaded with mechanical stimuli have been proposed. Many of these devices are seeded on an elastic polymer sheet, and after culturing cells on the polymer sheet, the polymer sheet is stretched, thereby applying a stretching stimulus load to the cells attached on the sheet. Means are used.

例えば、シートの両端をクランプで挟持し、該クランプの一方は基板に固定し、他方のクランプは牽引装置に接続し、伸展する装置（特許文献１）や、シリコンゴム等の弾性を部材で環状にベルトを形成し、２本のロッドを挿入し、一方は基板に固定し、他方は前進、後退可能なアームに固定し、アームの動きによりベルト状弾性部材を延伸したり、元へ戻したりする装置（特許文献２）等が提案されている。   For example, both ends of the sheet are clamped, one of the clamps is fixed to the substrate, the other clamp is connected to a traction device, and stretched (Patent Document 1), or elastic such as silicon rubber is looped with a member A belt is formed, two rods are inserted, one is fixed to the substrate, the other is fixed to an arm that can be advanced and retracted, and the belt-like elastic member is stretched or returned to the original by the movement of the arm. An apparatus (Patent Document 2) or the like that performs the above has been proposed.

しかしながら既存の細胞伸展刺激負荷装置は、一般的に機構部品を組み合わせて製作するため装置が大型化し、数センチ乃至数十センチ以下の小型化は不可能であった。更に顕微鏡による観察下に伸展を行う場合、伸展に伴い、観察すべき細胞が移動して視野からはずれるため、観察対象を移動し視野に戻す必要があり、伸展や移動により焦点も合わなくなることが多く、これらの調整に手間取り、不便であったばかりでなく、伸展時又はその直後の細胞内の変化を観察することができないという問題もあった。
特開２００７−５４０３４号公報 特開平１０−１５５４７５号公報 However, since the existing cell extension stimulation load device is generally manufactured by combining mechanical parts, the size of the device is increased, and it has been impossible to reduce the size to several centimeters to several tens of centimeters. Furthermore, when stretching under observation with a microscope, the cells to be observed move away from the field of view due to the stretching, so it is necessary to move the observation object back to the field of view, and the focus may become out of focus due to stretching or movement. Many of these adjustments are troublesome and inconvenient, and there is also a problem in that changes in the cells at the time of extension or immediately after that cannot be observed.
JP 2007-54034 A JP-A-10-155475

本発明は、顕微鏡による観察下に伸展を行っても観察すべき細胞が視野からはずれることがない。しかも、サンプル細胞が上下方向にずれ難いため、ピントが固定される細胞への伸展刺激負荷デバイスを提供することを目的とする。   In the present invention, cells to be observed do not deviate from the field of view even when stretched under observation with a microscope. Moreover, since the sample cells are difficult to shift in the vertical direction, an object of the present invention is to provide an extension stimulation load device for cells to which the focus is fixed.

本発明は、上記目的を達成するため、次の構成よりなるデバイスを提案する。   In order to achieve the above object, the present invention proposes a device having the following configuration.

すなわち、本発明の基本的態様は、透明基板上に平行して固定された２個の支点軸に、それぞれ一端が回動可能に取り付けられた一対の伸展用アームの他端側は、弾性部材で構成された細胞観察用チャンバーで連結されており、前記両伸展用アームの支点側の対向する位置に動作用突起部が付設されており、更に透明基板に固定されたガイドに従って摺動する動作桿によって、前記両動作突起部を同時に押す機構を有する細胞伸展刺激負荷デバイスである。   That is, the basic aspect of the present invention is that the other end of a pair of extension arms each having one end rotatably attached to two fulcrum shafts fixed in parallel on a transparent substrate is an elastic member. Are connected by a cell observation chamber composed of the above, and an operation protrusion is attached to the opposing position on the fulcrum side of both the extending arms, and further sliding according to a guide fixed to the transparent substrate It is a cell extension stimulation load device having a mechanism for simultaneously pressing both the movement protrusions with a scissors.

本発明のデバイスにあっては、動作桿を押すことにより、該動作桿の先端は２個の伸展用アームの突起部を同時に押し下げることになり、伸展用アームはそれぞれ支点軸を中心に回転し、弾性部材で構成された細胞観察用チャンバーを引き伸ばすことになるのである。   In the device of the present invention, when the operating rod is pushed, the tip of the operating rod simultaneously pushes down the projections of the two extending arms, and the extending arms rotate around the fulcrum shaft. The cell observation chamber made of an elastic member is stretched.

本発明にあっては、また複数個の細胞伸展刺激負荷デバイスを一枚の透明基板を共通の基板として透明基板上に固定した態様を包含する。この場合、一度に複数の披検細胞をそれぞれの細胞観察用チャンバーにセットし、順次顕微鏡の対物レンズの視野に移動させて伸展刺激負荷を行い、観察できるので、従来の伸展刺激負荷デバイスの如く、一回ずつ顕微鏡に被検体をセットしなくてもよいので、極めて効率的に多くの伸展刺激負荷実験を行うことができるのである。   The present invention also includes a mode in which a plurality of cell extension stimulation load devices are fixed on a transparent substrate using a single transparent substrate as a common substrate. In this case, multiple test cells can be set in each cell observation chamber at the same time, and sequentially moved to the field of view of the objective lens of the microscope to perform extension stimulation loading and observation, so as with conventional extension stimulation loading devices Since it is not necessary to set the subject in the microscope once, many extension stimulation load experiments can be performed very efficiently.

本発明デバイスによると、細胞に伸展変化を負荷したとき、剛体変異が小さく、披検細胞が顕微鏡の視野からずれることがないし、またデバイスが透明基板に固定されているので、対物レンズに対する奥行方向のズレも最小限におさえられ、再度ピントを合わせる必要もほとんどないため、細胞の伸展刺激負荷を与えた直後の状態を観察することが可能となる。   According to the device of the present invention, when the cell is subjected to an extension change, the rigid body variation is small, the test cell is not displaced from the field of view of the microscope, and the device is fixed to the transparent substrate, so the depth direction with respect to the objective lens Therefore, it is possible to observe the state immediately after the cell is applied to the cell with the stimulus for stretching.

更に一枚の透明基板上に複数個のデバイス、例えば４〜３０個のデバイスを形成することにより、透明基板を少し移動するだけで次々と伸展刺激負荷を与えた状態を観察できるので、著しく手間を省き、効率よく観察実験を行うことができる。   Furthermore, by forming a plurality of devices, for example, 4 to 30 devices on a single transparent substrate, it is possible to observe the state of applying an extension stimulus load one after another by moving the transparent substrate a little. The observation experiment can be performed efficiently.

更に後述するように、簡単なフォトリングラフィの手段によるモールディングで製作できるため、ディスポーザブルとすることができ、従来の装置のように洗浄や殺菌等再使用のための手間も省くことができる。   Further, as will be described later, since it can be manufactured by molding by means of simple photolinography, it can be made disposable, and it is possible to save time and effort for reuse such as cleaning and sterilization as in the conventional apparatus.

本発明は、細胞に対して単軸の引張り歪を与えるデバイスである。特に単一細胞と同程度の大きさの細胞観察用チャンバーの細胞載置孔（伸展部）とすることにより、一個の細胞レベル、或いはサブセルラーレベルで観察が可能となる。すなわち、伸展部における細胞は図２（ａ）に示す如く、正常の形状を保っている。これに図２（ｂ）に矢印で示す方向に伸展されたとき、細胞は引っ張られて潰れた状態となる。この時の細胞内では、種々の変化が生じる。その変化の動的状態を観察可能とするデバイスである。   The present invention is a device that applies uniaxial tensile strain to cells. In particular, the cell mounting hole (extension part) of the cell observation chamber having the same size as that of a single cell enables observation at a single cell level or a subcellular level. That is, the cells in the extension part maintain a normal shape as shown in FIG. When the cell is stretched in the direction indicated by the arrow in FIG. 2 (b), the cells are pulled and crushed. Various changes occur in the cells at this time. It is a device that makes it possible to observe the dynamic state of the change.

本発明デバイスの基本的構成を図１（ａ）、（ｂ）に示す。   The basic structure of the device of the present invention is shown in FIGS.

図１（ａ）は、本発明のデバイスの伸展前の状態を示す模式図であり、（ｂ）は伸展した状態を示す模式図である。図中１は透明基板であり、２，２’は伸展用アーム、３は細胞観察用チャンバーであり、４は同チャンバーに設けられた細胞載置用孔（伸展部）である。なお、Ａは被検体となる細胞を表す。   Fig.1 (a) is a schematic diagram which shows the state before extension of the device of this invention, (b) is a schematic diagram which shows the extended state. In the figure, 1 is a transparent substrate, 2 and 2 'are extension arms, 3 is a cell observation chamber, and 4 is a cell mounting hole (extension part) provided in the chamber. A represents a cell to be examined.

透明基板は，共焦点レーザー顕微鏡での観察が可能な材料であり，例えば好適には厚さ０．１〜０．２ｍｍ程度の光学顕微鏡用カバーガラスを用いる．
また、５，５’は透明基板に固定された支点軸であり、これに伸展用アーム２，２’が回動可能に取り付けられている。６は、動作桿を摺動可能に保持するためのガイドであり、斜線の部分で透明基板に固定されている。７は動作桿であり、その先端部Ｂが伸展用アームの動作突起部Ｃ，Ｃ’に接触し、これを押し下げることにより一対の伸展用アームは同時に支点軸５，５’を軸として同じ角度だけ回転させられ、それにつれて両アームの先端部に固定されている弾性部材で構成された細胞観察用チャンバー３は、左右同時に同じ長さだけ引き伸ばされるのである。この時チャンバーは細胞載置孔の存在する部分がもっとも断面積が小さいため、強度が小さく、最もよく伸ばされることになり、そこに載置された細胞は、中心位置をほとんど動くことなく左右に等しく引き伸ばされるのである。しかも、両アームは透明基板に固定された支持軸に取り付けられているため、透明基板に平行に引っ張るので、細胞と透明基板の位置、延いては顕微鏡の対物レンズとの距離はほとんど変わることはない。従って顕微鏡のピントがずれることもほとんどないのである。 The transparent substrate is a material that can be observed with a confocal laser microscope. For example, a cover glass for an optical microscope having a thickness of about 0.1 to 0.2 mm is preferably used.
Reference numerals 5 and 5 ′ denote fulcrum shafts fixed to the transparent substrate, and extension arms 2 and 2 ′ are rotatably attached thereto. Reference numeral 6 denotes a guide for slidably holding the operating rod, and is fixed to the transparent substrate at the shaded portion. Reference numeral 7 denotes an operating rod, the tip B of which contacts the operating projections C and C ′ of the extending arm, and by pushing down, the pair of extending arms simultaneously have the same angle about the fulcrum shafts 5 and 5 ′. The cell observation chamber 3, which is made of an elastic member that is rotated only by the rotation and fixed to the distal ends of both arms, is stretched by the same length at the same time on the left and right. At this time, the chamber has the smallest cross-sectional area where the cell mounting hole is present, so the strength is low and the cell is stretched best, and the cell placed there moves left and right with little movement in the center position. It is stretched equally. Moreover, since both arms are attached to the support shaft fixed to the transparent substrate, the arm is pulled in parallel with the transparent substrate, so that the position of the cell and the transparent substrate, and therefore the distance between the objective lens of the microscope, will hardly change. Absent. Therefore, the focus of the microscope is hardly shifted.

以上に説明した本発明の細胞伸展刺激負荷デバイスは、その上部構造物を図３に示すように一枚の透明基板上に複数個設置することができる。   The cell extension stimulation load device of the present invention described above can have a plurality of superstructures installed on a single transparent substrate as shown in FIG.

本発明に用いられる細胞観察用チャンバーは弾性部材で構成される。一般にはシリコンゴム、特に下側から観察する構造の共焦点レーザー顕微鏡や全反射顕微鏡や下方が光をあてる近接場光顕微鏡などを用いるためには、透明性の高い弾性部材で、少なくとも細胞を載置する部分を構成する必要があり、好ましくは、ダウコーニング社製 ＣＹ５２−２７６（商品名）や、シンエツエラストマＫＥ−１０６（商品名）などが用いられる。   The cell observation chamber used in the present invention is composed of an elastic member. In general, in order to use a silicon rubber, especially a confocal laser microscope or a total reflection microscope having a structure that is observed from the lower side, or a near-field light microscope that illuminates the lower part, at least a cell is mounted with a highly transparent elastic member. The CY52-276 (trade name) manufactured by Dow Corning Co., Ltd., or Shin-Etsu Elastomer KE-106 (trade name) is preferably used.

更に細胞観察用チャンバーは、一軸方向に伸展した場合、これと直交する側が内側に引っ張られ、幅を狭める方向に力が働き、予期しない歪を生ずる恐れがあるため、図４に斜視図を示すごとく、横方向に縮むのを防止する支持部を設け、力の分散を図るのも好ましい態様となる。なお、図４中●部は透明基板に固定しておくのも好ましい。   Furthermore, when the cell observation chamber extends in a uniaxial direction, the side perpendicular to the cell is pulled inward, and a force acts in the direction of narrowing the width, which may cause unexpected distortion. FIG. 4 shows a perspective view. Thus, it is also a preferable aspect to provide a support portion that prevents the lateral contraction and to distribute the force. In FIG. 4, it is also preferable that the portion ● is fixed to a transparent substrate.

以上、本発明の基本的構造を説明したが、本発明のデバイスは一般的に一つの細胞を観察することを目的とするものであり、極めて小型とすることが望ましく、一般には本体部の長手方向（たて方向）が５ｍｍ以下、好ましくは２ｍｍ程度、横方向が３ｍｍ以下、好ましくは２ｍｍ程度、厚さ０．０５ｍｍ〜０．２ｍｍ、好ましくは０．１ｍｍ程度のものであり、細胞観察チャンバーの長さは２ｍｍ以下、好ましくは１ｍｍ程度であり、細胞載置孔は長さ０．３〜０．５ｍｍ、幅０．０５〜０．１５ｍｍ程度のものであり、一般に部品をそれぞれ加工して組み立てることは困難であるため、Ｆｏｕｌｄｓ，Ｉ．Ｇらが提唱している（Ｊｏｕｎａｌ ｏｆ ｍｉｃｒｏｍｅｃｈａｎｉｃｓ ａｎｄ ｍｉｃｒｏｅｎｇｉｎｅｅｒｉｎｇ，ＮＯ．１６，ｐｐ２１０９−２１１５（２００６））フォトリングラフィによって成形される。例えば超厚膜用レジスト（ＳＵ−８ ２０５０フォトレジスト，ＭＩＣＲＯ ＣＨＥＭ社製）等が好適に使用される。   Although the basic structure of the present invention has been described above, the device of the present invention is generally intended for observing one cell, and is desirably extremely small, and generally has a length of the main body. The cell observation chamber has a direction (vertical direction) of 5 mm or less, preferably about 2 mm, a lateral direction of 3 mm or less, preferably about 2 mm, and a thickness of 0.05 mm to 0.2 mm, preferably about 0.1 mm. The length of the cell is 2 mm or less, preferably about 1 mm, and the cell mounting hole has a length of about 0.3 to 0.5 mm and a width of about 0.05 to 0.15 mm. Because it is difficult to assemble, Folds, I. et al. It is molded by photolinography proposed by G et al. (Junal of micromechanics and microengineering, No. 16, pp 2109-2115 (2006)). For example, an ultra-thick film resist (SU-8 2050 photoresist, manufactured by MICRO CHEM) is preferably used.

以下に実施例を示す。
（実施例）
図５に示すような、シリコーンエラストマー構造体の長軸方向に対称な引張りひずみを負荷できるリンク機構をカバーガラス上に製作する。 Examples are shown below.
(Example)
As shown in FIG. 5, a link mechanism capable of applying a tensile strain symmetrical to the long axis direction of the silicone elastomer structure is manufactured on the cover glass.

このリンク機構を用いることで、動作桿の一点をマイクロマニピュレータで押し込むことにより、細胞観察用チャンバーに接続された左右のリンク先端に対称の変位を生じさせることができる。リンク機構の寸法は、全幅約１．５ｍｍ、全長約２ｍｍの大きさとする。作動変位としては、押し込み部をマイクロマニピュレータで１０〜２０μｍ押し込んだ際に、リンク先端が片側３０〜６０μｍ変位し、微小細胞観察用チャンバーに１０〜２０％のひずみを生じさせることが可能であるように設計した。
製作プロセス
デバイスの製作プロセスを図６に示し、その概要について述べる。なお、リンク機構のガイド部、軸受け部の製作プロセスは、Ｆｏｕｌｄｓらが報告している多重露光による立体構造製作プロセスを参考とした。ここで、図６に示すプロセスチャートは右図に示すＡ−Ａ断面を示している。まず、図６のプロセス（１）で、超厚膜用レジスト（ＳＵ−８ ２０５０フォトレジスト）を用いて構造体の原型を製作し、離型剤としてフッ素樹脂（Ｄｕｒａｓｕｒｆ ＤＳ−３３０４Ｚ、ハーベス社：商品名）をコーティングする。次に、（２）、（３）のプロセスでシリコーンエラストマーをキャスティングし、原形を転写した微小鋳型を製作する。次に、（４）で犠牲層Ｇｅをパターニングする。さらに、（５）、（６）のプロセスでモールディングによりシリコーンエラストマー構造体を成形する。 By using this link mechanism, a symmetric displacement can be generated at the left and right link tips connected to the cell observation chamber by pushing one point of the operating rod with a micromanipulator. The dimensions of the link mechanism are a total width of about 1.5 mm and a total length of about 2 mm. As the operating displacement, when the pushing portion is pushed by 10 to 20 μm with a micromanipulator, the tip of the link is displaced by 30 to 60 μm on one side, and it is possible to cause 10 to 20% strain in the micro cell observation chamber. Designed.
Manufacturing Process The device manufacturing process is shown in FIG. In addition, the manufacturing process of the guide part of a link mechanism and a bearing part referred the three-dimensional structure manufacturing process by multiple exposure reported by Fouls et al. Here, the process chart shown in FIG. 6 shows an AA cross section shown in the right figure. First, in the process (1) of FIG. 6, a prototype of the structure is manufactured using an ultra-thick film resist (SU-8 2050 photoresist), and a fluororesin (Durasurf DS-3304Z, Harves: (Product name) is coated. Next, a silicone elastomer is cast by the processes of (2) and (3), and a micro mold in which the original shape is transferred is manufactured. Next, the sacrificial layer Ge is patterned in (4). Further, a silicone elastomer structure is formed by molding in the processes (5) and (6).

次に、図６のプロセス（７）で膜厚１００μｍのＳＵ−８に対して、リンクや軸受部およびガイド部のパターンを露光する。ここでＳＵ−８の現像を行わずそのままプロセス（８）で膜厚５０μｍで上に重ねてコーティングしたＳＵ−８を二重露光する。最後に、プロセス（９）でＳＵ−８構造体全体を現像した後に、犠牲層のＧｅをエッチングして除去し、デバイス構造が完成する。   Next, the pattern of a link, a bearing part, and a guide part is exposed with respect to SU-8 with a film thickness of 100 micrometers by the process (7) of FIG. Here, without developing SU-8, double exposure is applied to SU-8 coated with a film thickness of 50 [mu] m overlaid in the process (8) as it is. Finally, after developing the entire SU-8 structure in process (9), the sacrificial layer Ge is etched away to complete the device structure.

なお、ＳＵ−８フォトレジストの露光条件は、ＭＩＣＲＯ ＣＨＥＭ社のデータシートを参考にして設定した。   The exposure conditions for the SU-8 photoresist were set with reference to the data sheet of MICRO CHEM.

（ａ）、（ｂ）は本発明の基本的構造を示す模式図である。（ａ）は細胞を伸展する前、（ｂ）は伸展した状態を示す。(A), (b) is a schematic diagram which shows the basic structure of this invention. (A) shows the stretched state before the cell is stretched, and (b) shows the stretched state. （ａ）、（ｂ）は、細胞観察チャンバー中の細胞載置孔に細胞を置いた図であり、（ａ）は伸展する前、（ｂ）は伸展した後を示す。(A), (b) is the figure which put the cell in the cell mounting hole in a cell observation chamber, (a) shows before extending, (b) shows after extending. 本発明の細胞伸展刺激負荷デバイスを透明基板上に複数設置した場合の模式図である。It is a schematic diagram when a plurality of cell extension stimulation load devices of the present invention are installed on a transparent substrate. 本発明において、好適に用いられる細胞観察用チャンバーの構造を示す斜視図である。It is a perspective view which shows the structure of the chamber for cell observation used suitably in this invention. 実施例により作製された本発明の細胞伸展刺激負荷デバイス（透明基板を除く）の図である。It is a figure of the cell extension stimulus loading device (except for a transparent substrate) of the present invention produced by the example. 実施例における本発明の細胞伸展刺激負荷デバイスの作製工程図である。It is a manufacturing process figure of the cell expansion stimulation load device of this invention in an Example.

符号の説明Explanation of symbols

１は透明基板
２，２’は伸展アーム
３は細胞伸展用チャンバー
４は細胞載置用孔
５，５’は支点軸
６はガイド
７は動作桿
Ａは細胞
Ｂは動作桿先端部
Ｃは伸展用アームの動作用突起部 1 is transparent substrate 2, 2 'is extension arm 3, cell extension chamber 4 is cell mounting hole 5, 5' is fulcrum shaft 6, guide 7 is moving rod A is cell B is moving rod tip C is extended Projections for arm movement

Claims (2)

透明基板上に、平行して固定された２個の支点軸に、それぞれ一端が回動可能に取り付けられた一対の伸展用アームの他端は、弾性部材で構成された細胞観察用チャンバーで連結されており、前記両伸展アームの支点側の対向する位置に動作用突起部が付設されており、更に透明基板に固定されたガイドに従って摺動する動作桿によって前記両動作突起部を同時に押す機構を有する細胞伸展刺激負荷デバイス。   The other ends of a pair of extension arms each having one end rotatably attached to two fulcrum shafts fixed in parallel on a transparent substrate are connected by a cell observation chamber made of an elastic member. And a mechanism in which operating protrusions are attached at opposing positions on the fulcrum side of the extension arms, and the operating protrusions are simultaneously pressed by an operating rod that slides according to a guide fixed to the transparent substrate. A cell extension stimulation load device comprising: 請求項１記載の細胞伸展刺激負荷デバイスであって、一枚の透明基板上に複数のデバイスが設置されていることを特徴とする細胞伸展刺激負荷デバイス。   The cell extension stimulation load device according to claim 1, wherein a plurality of devices are installed on a single transparent substrate.
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