JP2017023049A - Cell culture vessel having observation window part, cell culture apparatus, and method for observing cultured cells from the side thereof - Google Patents
Cell culture vessel having observation window part, cell culture apparatus, and method for observing cultured cells from the side thereof Download PDFInfo
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Abstract
Description
本発明は、培養細胞の観察技術に関する。 The present invention relates to a technique for observing cultured cells.
一般的な細胞培養プレートやシャーレ等で培養する平面的な２次元培養に対して、縦方向の厚みを持たせて細胞を培養する３次元培養が知られており、この３次元培養は２次元培養よりも生体内の組織細胞の状態に近く、より生体に近い反応が観察できるとして、生体内環境を模した実験モデルの確立に適しているとされている。
例えば、３次元培養された細胞は細胞間の刺激伝達が３次元的であり、細胞塊の内部と外部とで培地や薬剤の供給経路が異なるなど生体に近い環境を再現できることや、肝細胞など２次元培養ではすぐに活性が低下してしまう細胞でも、３次元培養では長期間にわたって活性を維持できること、さらには、幹細胞から各種細胞への分化において効率を高めることができるなど、３次元培養を使用した組織モデルに関する研究報告がなされている。
また、このような３次元培養を行う培養容器が開発されている（特許文献１、２参照）。
In contrast to the planar two-dimensional culture that is cultivated in a general cell culture plate or petri dish, three-dimensional culture is known in which cells are cultured with a thickness in the vertical direction. It is said that it is suitable for the establishment of an experimental model that mimics the in vivo environment, since it can be observed to be closer to the state of tissue cells in the living body than in culture and to observe reactions closer to the living body.
For example, cells that are three-dimensionally cultured have three-dimensional stimulation transmission between cells, and can reproduce an environment close to a living body, such as a medium and a drug supply route differing between the inside and outside of a cell mass, hepatocytes, etc. Even if a cell whose activity decreases immediately in two-dimensional culture, the activity can be maintained over a long period of time in three-dimensional culture, and further, the efficiency in differentiation from stem cells to various cells can be increased. Research reports on the tissue model used have been made.
In addition, culture vessels for performing such three-dimensional culture have been developed (see Patent Documents 1 and 2).
一方、生体内環境を模した試験モデルにおいては、生きたままの細胞組織や生体分子を観察することが不可欠である。
一般的な２次元培養による細胞は、細胞培養器の底面部にカバーガラスを配置したガラスボトムデッシュやカバーガラスチャンバーと呼ばれる培養容器を使用して、正立・倒立顕微鏡により観察を行うことができる。
これに対し、３次元培養は、ガラスボトムデッシュの上に透明のポリカーボネート膜等の支持体を有するインサートを載せ、共焦点顕微鏡を使用して観察を行うと、３次元培養の「ＸＹ面」を観察することができる（特許文献１、３を参照）。
また、共焦点顕微鏡を使用して「Ｚ方向」に複数枚スキャンすれば３次元の画像構築が可能であり、３次元培養の側面部の画像を得ることができるものの、分解能は焦点深度の厚みに依存するために著しく低下することに加え、複数回細胞をスキャンすることで光毒性、蛍光標識の褐色が発生するなど問題も多い。
さらに、組織を固定して切片を作成する観察手法は広く知られており、これを利用すれば、３次元培養の側面観察も可能であるが、生きたままの細胞側面を観察することは不可能である。
On the other hand, in a test model simulating an in vivo environment, it is indispensable to observe living cell tissues and biomolecules.
Cells by general two-dimensional culture can be observed with an upright / inverted microscope using a glass bottom dish or a culture vessel called a cover glass chamber in which a cover glass is placed on the bottom of a cell culture device. .
In contrast, in 3D culture, when an insert having a support such as a transparent polycarbonate film is placed on a glass bottom dish and observed using a confocal microscope, the “XY plane” of 3D culture is obtained. It can be observed (see Patent Documents 1 and 3).
In addition, if a plurality of sheets are scanned in the “Z direction” using a confocal microscope, it is possible to construct a three-dimensional image, and an image of the side part of the three-dimensional culture can be obtained, but the resolution is the thickness of the depth of focus. In addition to a significant decrease due to the dependence on the cell, there are many problems such as phototoxicity and fluorescence-labeled brown color generated by scanning a cell several times.
Furthermore, observation techniques for fixing a tissue and preparing a section are widely known. If this technique is used, it is possible to observe the side of three-dimensional culture, but it is not possible to observe the side of a living cell. Is possible.
そこで、本発明は、細胞、多数の細胞の凝集体であるスフェロイド、３次元に積層化させた組織細胞を生きたままの状態で、顕微鏡等を使用して側面から観察する手段を開発することを目的としている。 Therefore, the present invention develops a means for observing a cell, a spheroid that is an aggregate of a large number of cells, and a three-dimensionally laminated tissue cell from the side using a microscope or the like in a living state. It is an object.
本発明は、上記課題を解決するために鋭意検討した結果、細胞を培養し観察する容器について、細胞を播種する支持体上の培養面に対して６０度から１２０度の範囲の角度をなす様に観察窓部を配置したものである。これにより、細胞、多数の細胞の凝集体であるスフェロイド、３次元に積層化させた組織細胞を生きたままの状態で観察できることとなった。 The present invention has been intensively studied to solve the above-mentioned problems, and as a result, the container for culturing and observing cells has an angle in the range of 60 to 120 degrees with respect to the culture surface on the support on which the cells are seeded. The observation window part is arranged in FIG. As a result, cells, spheroids that are aggregates of a large number of cells, and three-dimensionally laminated tissue cells can be observed as they are.
本発明は、具体的には次の事項を要旨とする。
１．細胞を播種する支持体上の培養面に対して、６０度から１２０度の範囲の角度をなすように配置された観察窓部を有する細胞培養容器。
２．細胞を播種する支持体の素材が、ポリカーボネート、コラーゲン、ポリスチレン、ガラスのいずれかより選択されることを特徴とする１．記載の細胞培養容器。
３．観察窓部が、顕微鏡観察に適した部材により覆われていることを特徴とする、１．または２．記載の細胞培養容器。
４．観察窓部が、顕微鏡観察用のカバーガラスにより覆われていることを特徴とする、１．または２．記載の細胞培養容器。
５．細胞を播種する支持体によって区切られた第１室と第２室とを備えることを特徴とする１．から４．のいずれか一項に記載の細胞培養容器。
６．細胞を播種する支持体が、ポリカーボネート膜またはコラーゲン膜から選択されることを特徴とする５．記載の細胞培養容器。
７．第１室に細胞植え付け用及び培地注入交換用の開口部を、第２室に培地注入交換用の開口部を有することを特徴とする５．または６．記載の細胞培養容器。
８．第１室及び第２室のそれぞれの開口部は、着脱可能に取り付けられるキャップを有し、前記キャップは液密性と気体の流通を許容するガス透過領域とを備えることを特徴とする７．記載の細胞培養容器。
９．１．から８．のいずれか一項に記載の細胞培養容器が連続して隣接していることを特徴とする、細胞培養装置。
１０．細胞培養容器の観察窓部が連続して１つの窓部を構成するように配置されたことを特徴とする、９．記載の細胞培養装置。
１１．１．から８．のいずれか一項に記載の細胞培養容器または、９．または１０．記載の細胞培養装置を用いて、３次元培養した培養細胞の側面方向を、観察窓部を通して側面方向から観察する方法。
１２．観察する対象が３次元皮膚モデルであることを特徴とする、１１．記載の観察方法。
The gist of the present invention is specifically as follows.
1. A cell culture container having an observation window portion arranged to form an angle in a range of 60 degrees to 120 degrees with respect to a culture surface on a support on which cells are seeded.
2. 1. The material of the support on which the cells are seeded is selected from polycarbonate, collagen, polystyrene, and glass. The cell culture container described.
3. 1. The observation window is covered with a member suitable for microscopic observation. Or 2. The cell culture container described.
4). 1. The observation window is covered with a cover glass for microscopic observation. Or 2. The cell culture container described.
5. 1. A first chamber and a second chamber separated by a support for seeding cells are provided. To 4. The cell culture container according to any one of the above.
6). 4. The support on which the cells are seeded is selected from polycarbonate membranes or collagen membranes. The cell culture container described.
7). 4. The first chamber has an opening for cell planting and medium injection exchange, and the second chamber has an opening for medium injection exchange. Or 6. The cell culture container described.
8). 6. Each opening part of a 1st chamber and a 2nd chamber has a cap attached so that attachment or detachment is possible, The said cap is provided with the gas permeation area | region which accept | permits liquid-tightness and gas distribution | circulation. The cell culture container described.
9.1. To 8. A cell culture apparatus according to any one of the above, wherein the cell culture containers are adjacent to each other.
10. 8. The observation window part of the cell culture container is arranged so as to constitute one window part continuously, The cell culture device described.
11.1. To 8. 8. The cell culture container according to any one of Or 10. A method of observing the lateral direction of cultured cells that have been three-dimensionally cultured from the lateral direction through an observation window using the cell culture apparatus described above.
12 10. an object to be observed is a three-dimensional skin model; The observation method described.
１．培養している細胞組織を側面から観察できる細胞培養容器を実現した。
本発明の細胞培養容器は、培養面に対して６０度から１２０度の範囲の角度をなす様に配置された観察窓部を有するので、培養面を下から観察する従来の細胞培養容器では観察することができなかった、細胞の側面や、細胞増殖により形成される組織構造の側面の観察を、細胞が生きたままの状態で容易に行うことを可能とする。
２．本発明の細胞培養容器は、培養面に対して６０度から１２０度の範囲の角度をなす様に配置された観察窓部が、顕微鏡観察に適したカバーガラス等の部材で覆われているので、簡便に解像度の高い３次元培養細胞の側面観察画像を得ることを可能とする。また、培養面を下から観察する従来の細胞培養容器に比べて、スキャン回数を大きく減らすことができるので、光毒性、蛍光標識の褐色化等の問題も無く、時間経過による培養細胞の変化を捉えるビデオ撮影やタイムラプスイメージング撮影を可能とする。
また、本発明の細胞培養容器は、培養細胞の側面方向の経時変化を映像や画像として捉えることを可能とするので、細胞接着の形成過程の解明に有用である。
３．本発明の細胞培養容器は、細胞植え付け用と培地注入交換用の開口部があり、それぞれ着脱可能に取り付けられるキャップを有し、前記キャップは液密性と気体の流通を許容するガス透過領域とを備えているので、培養細胞の観察を継続させながら、培養条件を調整することを可能とする。
４．本発明の細胞培養容器を連続して隣接させた細胞培養装置は、実験条件が異なる培養細胞を同時に並べて観察することを可能とし、実験効果の検証効率を向上させることができる。
５．本発明の細胞培養容器や細胞培養装置は、層状構造を有する上皮系の細胞層の各層または組織構造の側面を同時に観察することを可能とするほか、３次元皮膚モデルを使用して皮膚の分化や浸透性を側面から観察することを可能とするので、将来的に、化粧品や医薬品の効果検証にも応用することが可能である。
1. A cell culture vessel that can observe the cultured cell tissue from the side was realized.
Since the cell culture container of the present invention has an observation window portion arranged so as to form an angle in the range of 60 degrees to 120 degrees with respect to the culture surface, the conventional cell culture container that observes the culture surface from below is observed. Observation of the side surface of the cell and the side surface of the tissue structure formed by cell proliferation, which could not be performed, can be easily performed while the cell remains alive.
2. In the cell culture container of the present invention, the observation window arranged so as to form an angle in the range of 60 to 120 degrees with respect to the culture surface is covered with a member such as a cover glass suitable for microscopic observation. Therefore, it is possible to easily obtain a side observation image of a three-dimensional cultured cell with high resolution. In addition, since the number of scans can be greatly reduced compared to conventional cell culture vessels that observe the culture surface from below, there are no problems such as phototoxicity and browning of fluorescent labels, and changes in cultured cells over time Capturing video and time-lapse imaging is possible.
The cell culture container of the present invention is useful for elucidating the formation process of cell adhesion because it enables the temporal change in the lateral direction of the cultured cell to be captured as an image or an image.
3. The cell culture container of the present invention has openings for cell planting and medium infusion exchange, and has caps that are detachably attached, and the cap has liquid tightness and a gas permeable region that allows gas flow. Therefore, it is possible to adjust the culture conditions while continuing to observe the cultured cells.
4). The cell culture apparatus in which the cell culture containers of the present invention are continuously adjacent to each other makes it possible to simultaneously observe and observe cultured cells having different experimental conditions, and to improve the verification efficiency of the experimental effect.
5. The cell culture container and the cell culture device of the present invention enable simultaneous observation of each layer of the epithelial cell layer having a layered structure or the side of the tissue structure, and differentiation of skin using a three-dimensional skin model. In the future, it can be applied to the verification of the effects of cosmetics and pharmaceuticals.
本発明は、細胞を培養し観察する容器について、細胞を播種する支持体上の培養面に対して６０度から１２０度の範囲の角度をなす様に観察窓部を配置したことを特徴とするものであり、細胞、多数の細胞の凝集体であるスフェロイド、３次元に積層化させた組織細胞を生きたままの状態で、容易に、解像度高く、継続的に、側面からの観察を可能とするものである。
以下、本発明の実施の形態について図面を参照して説明するが、本発明の技術範囲はこれに限定されるものではない。
The present invention is characterized in that the observation window portion is arranged so as to form an angle in the range of 60 degrees to 120 degrees with respect to the culture surface on the support on which the cells are seeded, for the container for culturing and observing the cells. Spheroids, which are aggregates of cells, a large number of cells, and three-dimensionally laminated tissue cells can be observed from the side easily, with high resolution and continuously. To do.
Hereinafter, embodiments of the present invention will be described with reference to the drawings, but the technical scope of the present invention is not limited thereto.
＜基本構造＞
図１は、本発明の一例の細胞培養容器Ａを模式的に示す側面図である。
本発明の細胞培養容器Ａは、図１に示すように、細胞を播種する支持体１上の細胞２の培養面３に対して、角度をなす様に配置された観察窓部４を備える。培養面３に対して観察窓部４を配置する方向は、概ね垂直方向であればよく、具体的には、培養面３と観察窓部４のなす角度が、６０度から１２０度の範囲で配置することができる。顕微鏡の光路によっては、光の反射やエバネッセント波を避けるために、培養面３と観察窓部４のなす角度を厳密な垂直からずらした方が良い場合がある。細胞培養容器Ａは、培養液を入れてもよい皿状容器５に載置されている。
細胞培養容器Ａは、一般に細胞を培養する容器に使用される材質であればよく、細胞接着を極力低下させる材質であれば好ましく、容量は目的に応じて適宜設定できるが、少なくとも顕微鏡観察に適した容量とする。
細胞を播種する支持体１の素材としては、ポリカーボネート、コラーゲン、ポリスチレン、ガラス等が挙げられる。特に、皿状容器５に培養液を入れて使用する場合には、細胞を播種する支持体１は、ポリカーボネート膜、コラーゲン膜等の播種した細胞２の接着性を向上させる素材であって、細胞培養に必要な栄養成分を透過する能力を有する素材であることが好ましい。
<Basic structure>
FIG. 1 is a side view schematically showing a cell culture container A as an example of the present invention.
As shown in FIG. 1, the cell culture container A of the present invention includes an observation window portion 4 arranged at an angle with respect to the culture surface 3 of the cell 2 on the support 1 on which the cells are seeded. The direction in which the observation window 4 is arranged with respect to the culture surface 3 may be substantially vertical, and specifically, the angle formed by the culture surface 3 and the observation window 4 is in the range of 60 degrees to 120 degrees. Can be arranged. Depending on the optical path of the microscope, in order to avoid light reflection and evanescent waves, it may be better to shift the angle formed by the culture surface 3 and the observation window 4 from strict vertical. The cell culture container A is placed on a dish-like container 5 in which a culture solution can be placed.
The cell culture vessel A may be any material that is generally used for a cell culturing vessel, and is preferably a material that reduces cell adhesion as much as possible. The volume can be appropriately set according to the purpose, but is suitable for at least microscopic observation. Capacity.
Examples of the material of the support 1 on which the cells are seeded include polycarbonate, collagen, polystyrene, and glass. In particular, when the culture medium is put in the dish-like container 5 and used, the support 1 on which the cells are seeded is a material that improves the adhesion of the seeded cells 2 such as a polycarbonate membrane and a collagen membrane, It is preferable that the material has the ability to permeate nutrient components necessary for culture.
＜実施態様１＞
図２は、本発明の細胞培養容器Ｂを模式的に示す図であり、図２（ａ）は斜視図、図２（ｂ）は正面図、図２（ｃ）は側面図である。
本発明の細胞培養容器Ｂは、図２に示すように、細胞を播種する支持体１１により第１室１２と第２室１３に区切られており、第１室１２は細胞植え付け用の開口部１４を、第２室１３は培地注入交換用の開口部１５を有している。
さらに、細胞を播種する支持体１１に対して概ね垂直に位置する細胞培養容器Ｂの１つの壁面１６にあって、細胞を播種する支持体１１の界面を含む位置に観察窓部１７が設けられている。この観察窓部１７の大きさは、細胞を播種する支持体１１上に培養される細胞組織の側面が見える大きさでなければならないが、その形は、四角い窓部や丸い窓部等観察に適した形状であればよい。
支持体によって区切られた第１室１２と第２室１３を備える細胞培養容器Ｂの支持体１１は、播種した細胞の接着性を向上させる素材であり、細胞培養に必要な栄養成分を透過する能力を有する素材であることが好ましく、それら素材としては、例えば、ポリカーボネート膜、コラーゲン膜等が挙げられる。
また、観察窓部１７は、顕微鏡観察に適した光透過性の高いカバーガラス等の部材１８で覆う。この部材１８の素材としてはガラスが好ましいが、光の透過性が高く、光の屈折率が低いものであれば、アクリル樹脂などの樹脂製であってもよい。さらに、細胞接着を極力低下させる材質であることが好ましい。
２つの開口部１４と１５は、それぞれ着脱可能に取り付けられるキャップ１９を有し、このキャップ１９は、液密性と気体の流通を許容するガス透過領域とを備えていると、細胞を培養する上で好ましい。
<Embodiment 1>
FIG. 2 is a diagram schematically showing a cell culture container B of the present invention, in which FIG. 2 (a) is a perspective view, FIG. 2 (b) is a front view, and FIG. 2 (c) is a side view.
As shown in FIG. 2, the cell culture vessel B of the present invention is divided into a first chamber 12 and a second chamber 13 by a support 11 for seeding cells, and the first chamber 12 is an opening for cell planting. 14, the second chamber 13 has an opening 15 for exchanging and injecting the medium.
Furthermore, an observation window 17 is provided on one wall surface 16 of the cell culture vessel B positioned substantially perpendicular to the support 11 on which the cells are seeded, including the interface of the support 11 on which the cells are seeded. ing. The size of the observation window 17 must be such that the side surface of the cell tissue cultured on the support 11 on which the cells are seeded can be seen, but the shape of the observation window 17 can be used for observation of a square window or a round window. Any suitable shape may be used.
The support 11 of the cell culture vessel B provided with the first chamber 12 and the second chamber 13 separated by the support is a material that improves the adhesiveness of the seeded cells, and permeates nutrients necessary for cell culture. It is preferable that it is a raw material which has capability, and as such a material, a polycarbonate membrane, a collagen membrane etc. are mentioned, for example.
Further, the observation window portion 17 is covered with a member 18 such as a cover glass having high light transmittance suitable for microscopic observation. The material of the member 18 is preferably glass, but may be made of a resin such as an acrylic resin as long as it has a high light transmittance and a low light refractive index. Furthermore, a material that reduces cell adhesion as much as possible is preferable.
Each of the two openings 14 and 15 has a cap 19 that is detachably attached. The cap 19 is provided with a liquid-tightness and a gas-permeable region that allows a gas flow, and cultures cells. Preferred above.
図３は、本発明の細胞培養容器Ｂの作成方法を示す図であり、図３（ａ）は市販されている細胞培養容器Ｃの切断面を、図３（ｂ）は接着前の各パーツを、図３（ｃ）は接着後の培養容器を模式的に示している。
本発明の細胞培養容器Ｂは、図３に示すように、市販されている細胞培養容器Ｃに穴Ｙをあけ切断面Ｘで切断したパーツを２つ作成し、細胞を播種する支持体１１、観察窓部１７の一部を有する第１室１２及び、観察窓部１７の一部を有する第２室１３の３つのパーツを接着させ、観察窓部１７を覆う部材１８を設置することにより作成できる。
本発明の細胞培養容器の作成方法の一例を説明するが、本発明はこの説明に何ら限定されるものではない。
市販されている細胞培養容器Ｃ（例えば、ファルコン社製の培養面積１２．５ｃｍ２、容量２５ｍｌのもの）の培養面の中央部に、観察窓部１７となる観察用の穴Ｙ（例えば、直径１０ｍｍ程度）を開け、観察用の穴Ｙの直径を含みフラスコ開口部Ｚと水平になる切断面Ｘを持つように容器Ｃを切断する。
切断された容器の開口部Ｚを有するパーツを２つ使用して、ポリカーボネート（例えば、ミリポア社製のもの）等の細胞を播種する支持体１１を挟み、細胞毒性の低い接着剤（例えば、コニシ社製のバスボンドＱ）を使用して接着し、さらに、観察窓部１７を覆うようにカバーガラス（例えば、松浪硝子工業社製のもの）等の部材１８を細胞毒性の低い接着剤で接着することにより、本発明の細胞培養容器Ｂを得ることができる。
カバーガラスの素材としてはガラスが好ましいが、光の透過性が高く、光の屈折率が低いものであれば、アクリル樹脂などの樹脂製であってもよい。
接着剤による接着は、超音波溶接に代えてもよいが、ポリカーボネート等の細胞を播種する支持体１１とカバーガラス等の部材１８が概ね直交するように配置することが重要である。また、液漏れが起こらないようにする必要もある。
3A and 3B are diagrams showing a method for producing the cell culture container B of the present invention. FIG. 3A shows a cut surface of the commercially available cell culture container C, and FIG. 3B shows each part before bonding. FIG. 3 (c) schematically shows the culture vessel after adhesion.
As shown in FIG. 3, the cell culture container B of the present invention is a support 11 for seeding cells by making two parts by making a hole Y in a commercially available cell culture container C and cutting the cut surface X, Created by bonding the three parts of the first chamber 12 having a part of the observation window 17 and the second chamber 13 having a part of the observation window 17 and installing a member 18 covering the observation window 17. it can.
An example of the method for producing the cell culture container of the present invention will be described, but the present invention is not limited to this description.
An observation hole Y (for example, a diameter) serving as an observation window 17 is formed in the center of the culture surface of a commercially available cell culture vessel C (for example, a culture area of 12.5 cm 2 and a volume of 25 ml manufactured by Falcon). About 10 mm), and the container C is cut so as to have a cut surface X that includes the diameter of the observation hole Y and is horizontal to the flask opening Z.
Using two parts having the opening Z of the cut container, the support 11 on which cells such as polycarbonate (for example, manufactured by Millipore) are seeded is sandwiched, and an adhesive having low cytotoxicity (for example, Konishi) And a member 18 such as a cover glass (for example, manufactured by Matsunami Glass Industrial Co., Ltd.) is adhered with an adhesive having low cytotoxicity so as to cover the observation window portion 17. Thus, the cell culture container B of the present invention can be obtained.
The cover glass material is preferably glass, but may be made of a resin such as an acrylic resin as long as it has high light transmission and low light refractive index.
Adhesion with an adhesive may be replaced by ultrasonic welding, but it is important that the support 11 on which cells such as polycarbonate are seeded and the member 18 such as a cover glass are arranged so as to be substantially orthogonal. It is also necessary to prevent liquid leakage.
＜従来例＞
図４は、従来の２次元培養の観察方法を示す図である。
従来の２次元培養の観察方法は、図４に示すように、細胞培養容器の底面部にカバーガラス２１を配置したガラスボトムデッシュやカバーガラスチャンバーと呼ばれる細胞培養容器２２を使用して、培養細胞２３を正立・倒立顕微鏡２４により観察を行うことができる。
図５は、従来の３次元培養の観察方法を示す図である。
従来の３次元培養の観察方法は、図５に示すように、ガラスボトムデッシュ２５の上に透明のポリカーボネート膜等の支持体を有するインサート２６を載せ、３次元培養細胞２７を共焦点顕微鏡２８により観察を行うと、３次元培養細胞２７の底面（「ＸＹ面」）を観察することができる
この状態で、共焦点顕微鏡を使用して「Ｚ方向」に複数枚スキャンすれば３次元の画像構築が可能であることは上述のとおりであるが、解像度の低さや細胞毒性等の問題により、実用性は低いものである。
<Conventional example>
FIG. 4 is a diagram illustrating a conventional two-dimensional culture observation method.
As shown in FIG. 4, a conventional two-dimensional culture observation method uses a glass bottom dish in which a cover glass 21 is arranged on the bottom surface of a cell culture container or a cell culture container 22 called a cover glass chamber. 23 can be observed with an upright / inverted microscope 24.
FIG. 5 is a diagram illustrating a conventional three-dimensional culture observation method.
As shown in FIG. 5, a conventional three-dimensional culture observation method is such that an insert 26 having a support such as a transparent polycarbonate film is placed on a glass bottom dish 25 and a three-dimensional cultured cell 27 is placed by a confocal microscope 28. When observed, the bottom surface (“XY plane”) of the three-dimensional cultured cell 27 can be observed. In this state, if a plurality of sheets are scanned in the “Z direction” using a confocal microscope, a three-dimensional image is constructed. As described above, the practicality is low due to problems such as low resolution and cytotoxicity.
＜観察方法＞
図６は、本発明の３次元培養の側面観察容器の概念図である。
本発明の３次元培養の側面観察容器３１は、図６に示すように、ポリカーボネート膜やコラーゲン膜等の支持体３２の上に、３次元培養された組織細胞３３が積層されており、支持体３２と直交して配置されたカバーガラス３４を介して顕微鏡３５により観察を行うと、３次元に積層化させた組織細胞３３を生きたままの状態で、容易に、解像度高く、継続的に、側面から観察できる。
また、培養細胞の側面方向の経時変化を映像や画像として捉えることができるので、細胞接着の形成過程の解明や、層状構造を有する上皮系の細胞層の各層を同時に観察することが可能である。
図７は、本発明の３次元培養の側面観察の概念図であり、図７（ａ）は細胞培養容器３１を立てた状態で観察する場合であり、図７（ｂ）は細胞培養容器３１を横にした状態で観察する場合である。
図７（ａ）、（ｂ）に示すように、使用する顕微鏡３５に応じて本発明の細胞培養容器３１の載置面を変えて観察することが可能である。
<Observation method>
FIG. 6 is a conceptual diagram of the three-dimensional culture side observation container of the present invention.
As shown in FIG. 6, the three-dimensional culture side observation container 31 of the present invention has a three-dimensionally cultured tissue cell 33 laminated on a support 32 such as a polycarbonate membrane or a collagen membrane. When observing with a microscope 35 through a cover glass 34 arranged orthogonal to 32, the tissue cells 33 laminated in a three-dimensional manner can be easily, with high resolution, continuously, It can be observed from the side.
In addition, the temporal changes in the lateral direction of cultured cells can be captured as images and images, so it is possible to elucidate the formation process of cell adhesion and simultaneously observe each layer of epithelial cell layers with a layered structure. .
FIG. 7 is a conceptual diagram of the side view of the three-dimensional culture of the present invention. FIG. 7 (a) shows a case where the cell culture container 31 is erected, and FIG. 7 (b) shows the cell culture container 31. This is a case of observing in a state where is placed sideways.
As shown in FIGS. 7A and 7B, it is possible to observe by changing the mounting surface of the cell culture container 31 of the present invention according to the microscope 35 to be used.
＜実施態様２＞
図８は、本発明の多室型細胞培養装置Ｄを模式的に示す図である。
本発明の多室型細胞培養装置Ｄは、図８に示すように、細胞を播種する支持体４２により区切られた第１室４３、第２室４４を有する本発明の細胞培養容器が連続して隣接しており、支持体４２に対して概ね直交して配置されたカバーガラス４５が、全ての第１室４３と第２室４４を覆うように配置されているために、支持体４２上に３次元に積層化させた組織細胞それぞれを同時に並べて観察することが可能であるので、実験条件が異なる組織モデルを比較しやすいという利点を有する。
本発明の細胞培養装置Ｄは、細胞植え付け用の開口部４６と培地注入交換用の開口部（図は省略）を有しており、細胞培養に適した条件を設定しながら、生きたままの細胞組織の観察を継続することを可能とする。
<Embodiment 2>
FIG. 8 is a diagram schematically showing the multi-chamber cell culture apparatus D of the present invention.
As shown in FIG. 8, the multi-chamber cell culture apparatus D of the present invention has a continuous cell culture container of the present invention having a first chamber 43 and a second chamber 44 separated by a support 42 for seeding cells. Since the cover glass 45 disposed adjacent to each other and substantially orthogonal to the support 42 is disposed so as to cover all the first chambers 43 and the second chambers 44, In addition, since it is possible to observe the tissue cells stacked three-dimensionally at the same time, there is an advantage that tissue models with different experimental conditions can be easily compared.
The cell culture device D of the present invention has an opening 46 for planting cells and an opening (not shown) for exchanging and injecting a medium, and is alive while setting conditions suitable for cell culture. It is possible to continue observation of cellular tissue.
本発明の細胞培養容器の支持体上に、所望の細胞を播種し、好適な条件下で播種した細胞を単層または多層培養することにより、組織モデルを構築することができる。
組織モデルの形態は限定されないが、例えば、被蓋上皮細胞や腺上皮細胞などを培養して構築できる上皮組織モデル、線維芽細胞や脂肪細胞などを培養して構築できる結合組織モデル、筋芽細胞や心筋細胞や平滑筋細胞などを培養して構築できる筋組織モデル、および神経細胞やグリア細胞などを培養して構築できる神経組織モデル、真皮由来の線維芽細胞をコラーゲンゲル内に包埋培養して真皮様組織を再構築し、さらに表皮角化細胞を重層培養することで得られる皮膚モデルなどが挙げられる。特に、医薬品、生理活性物質、化粧品あるいは洗剤等の化学物質の生体に対する作用を評価できる組織モデルの形態としては、生体に暴露あるいは投与された化学物質の移行経路の反映できる組織モデルの構築が重要となる。この視点からは、化学物質が最初に暴露される上皮細胞あるいは内皮細胞のみで構成される「組織シート（１種類細胞）」モデル、上皮細胞あるいは内皮細胞の次に化学物質に暴露される間充織細胞まで含めた上皮細胞と間充織細胞あるいは内皮細胞と間充織細胞の２種類の細胞で構成される「器官様プレート（２種類細胞）」モデル、さらに化学物質の移行に伴い暴露が進行する上皮細胞と間充織細胞と内皮細胞の３種類の細胞で構成される「器官様プレート（３種類細胞）」モデル等を例示することができる。
A tissue model can be constructed by seeding desired cells on the support of the cell culture container of the present invention and culturing the seeded cells under a suitable condition in a monolayer or multilayer.
Although the form of the tissue model is not limited, for example, an epithelial tissue model that can be constructed by culturing capped epithelial cells or glandular epithelial cells, a connective tissue model that can be constructed by culturing fibroblasts, adipocytes, etc., myoblasts Tissue model that can be constructed by culturing cells, cardiomyocytes, smooth muscle cells, etc., nerve tissue model that can be constructed by culturing nerve cells or glial cells, and dermal fibroblasts embedded in collagen gel And a skin model obtained by reconstructing a dermis-like tissue and further culturing epidermal keratinocytes. In particular, as a form of tissue model that can evaluate the action of chemical substances such as pharmaceuticals, physiologically active substances, cosmetics, and detergents on the living body, it is important to construct a tissue model that can reflect the migration path of the chemical substances exposed to the living body It becomes. From this point of view, the “tissue sheet (one type of cell)” model composed of only epithelial cells or endothelial cells to which chemical substances are first exposed, and the interval between epithelial cells or endothelial cells that are exposed to chemical substances. An “organ-like plate (two types of cells)” model composed of two types of cells, epithelial cells including mesenchymal cells and mesenchymal cells or endothelial cells and mesenchymal cells. An “organ-like plate (three types of cells)” model composed of three types of cells, ie, epithelial cells that progress, mesenchymal cells, and endothelial cells can be exemplified.
以下、実施例により本発明の図２に示す細胞培養容器Ｂを使用して細胞を培養し、観察する方法について詳しく説明するが、本発明はこれらの実施例に何ら限定されるものではない。 Hereinafter, the method for culturing and observing cells using the cell culture vessel B shown in FIG. 2 of the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
＜実施例１＞単層細胞培養とその観察方法
新生児ヒト表皮ケラチノサイト(NHEKneo)を1×105cells/cm2の濃度となるよう、細胞植え付け用の開口部１４から細胞懸濁液を播種し、第２室１３に気泡が入らないように１２mL、第１室１２に３mLのEpilife（Life technologies/#M-EPI-500-CA）を用いて２日間培養後、細胞核と細胞膜を汎用の蛍光プローブにより染色した。その後、共焦点顕微鏡（オリンパス社/FV1000）を用いてカバーガラス面１８より観察を行った。
図９、１０は、１日培養後の単層細胞の側面観察像を示す図であり、図９（ａ）は核を、（ｂ）は細胞膜を、（ｃ）はmerge像の固定細胞の観察図であり、図１０は細胞が移動する様子の観察図である。
<Example 1> Monolayer cell culture and its observation method A cell suspension is seeded from an opening 14 for planting neonatal human epidermal keratinocytes (NHEKneo) to a concentration of 1 × 10 5 cells / cm 2. After culturing for 2 days using 12 mL of Epilife (Life technologies / # M-EPI-500-CA) in the first chamber 12 to prevent bubbles from entering the second chamber 13, the cell nucleus and the cell membrane are generally fluorescent. Stained with a probe. Then, it observed from the cover glass surface 18 using the confocal microscope (Olympus / FV1000).
FIGS. 9 and 10 are diagrams showing a side-view image of a monolayer cell after one-day culture. FIG. 9A is a nucleus, FIG. 9B is a cell membrane, and FIG. 9C is a merge image of a fixed cell. FIG. 10 is an observation diagram showing how cells move.
＜実施例２＞ヒト表皮３次元積層化組織の培養とその観察方法
HPEKp細胞(CELLnTEC社/#HPEKp)を3×106cells/cm2の濃度となるよう、細胞植え付け用の開口部から細胞懸濁液を播種し、CnT-PR培地(CELLnTEC社/#CnT-PR)を第２室１３に１２mL、第１室１２に３mLを加え３日間培養後、第１室１２の培地を除去し、３次元化のための培地CnT-PR-3D（CELLnTEC社/#CnT-PR-3D)に交換して、７日間積層化培養した。細胞核と細胞膜を汎用の蛍光プローブにより染色した。その後、共焦点顕微鏡（オリンパス社/FV1000）を用いてカバーガラス面１８より観察を行った。
図１１は、積層化人工皮膚モデルの側面観察像を示す図であり、（ａ）は核を、（ｂ）は細胞膜を、（ｃ）はmerge像の固定細胞の観察図である。
図１１に示すように、細胞が４層程度に積層化されている様子が確認できた。
<Example 2> Culture of human epidermis three-dimensional laminated tissue and its observation method
HPEKp cells (CELLnTEC / # HPEKp) are seeded with the cell suspension from the opening for cell implantation so that the concentration becomes 3 × 10 6 cells / cm 2 and CnT-PR medium (CELLnTEC / # CnT- PR) in the second chamber 13 and 3 mL in the first chamber 12 and cultured for 3 days, the medium in the first chamber 12 is removed, and the medium CnT-PR-3D (CELLnTEC / # CnT-PR-3D) was replaced and cultured for 7 days. Cell nuclei and cell membranes were stained with a general-purpose fluorescent probe. Then, it observed from the cover glass surface 18 using the confocal microscope (Olympus / FV1000).
FIG. 11 is a diagram showing a side-view image of a laminated artificial skin model, where (a) is a nucleus, (b) is a cell membrane, and (c) is an observation view of fixed cells in a merge image.
As shown in FIG. 11, it was confirmed that the cells were laminated in about 4 layers.
Ａ：細胞培養容器
１：細胞を播種する支持体
２：細胞
３：培養面
４：観察窓部
５：皿状容器
６：窓部を覆う部材
Ｂ：細胞培養容器
１１：細胞を播種する支持体
１２：第１室
１３：第２室
１４：細胞植え付け用の開口部
１５：培地注入交換用の開口部
１６：壁面
１７：観察窓部
１８：部材
１９：キャップ
Ｃ：市販の細胞培養容器
Ｘ：切断面
Ｙ：穴
Ｚ：開口部
２１：カバーガラス
２２：従来の細胞培養容器
２３：培養細胞
２４：顕微鏡
２５：ガラスボトムデッシュ
２６：インサート
２７：３次元培養細胞
２８：共焦点顕微鏡
２９：ポリカーボネート膜
３１：側面観察容器
３２：細胞を播種する支持体
３３：３次元培養組織細胞
３４：カバーガラス
３５：顕微鏡
Ｄ：多室型細胞培養装置
４２：細胞を播種する支持体
４３：第１室
４４：第２室
４５：カバーガラス
４６：細胞植え付け用の開口部
A: Cell culture vessel 1: Support for seeding cells 2: Cell 3: Culture surface 4: Observation window portion 5: Dish container 6: Member covering the window portion B: Cell culture vessel 11: Support for seeding cells 12: first chamber 13: second chamber 14: cell planting opening 15: medium injection opening 16: wall surface 17: observation window 18: member 19: cap C: commercially available cell culture container X: Cut surface Y: Hole Z: Opening 21: Cover glass 22: Conventional cell culture vessel 23: Cultured cell 24: Microscope 25: Glass bottom dish 26: Insert 27: Three-dimensional cultured cell 28: Confocal microscope 29: Polycarbonate film 31: Side observation container 32: Support for seeding cells 33: Three-dimensional cultured tissue cells 34: Cover glass 35: Microscope D: Multi-chamber cell culture device 42: Support for seeding cells 43: First chamber 44: Second chamber 45: Bar Glass 46: the opening for the cell planting
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| WO2019012622A1 (en) * | 2017-07-12 | 2019-01-17 | 次郎 大野 | Device for producing three-dimensional cell structure having arbitrary shape, and method for producing same |
| JPWO2018163687A1 (en) * | 2017-03-10 | 2019-03-14 | 日本精工株式会社 | Manipulation system |
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| US11795424B2 (en) * | 2017-02-15 | 2023-10-24 | University Public Corporation Osaka | Cell culture vessel, sample observation cell, and cell culture method |
| JPWO2018163687A1 (en) * | 2017-03-10 | 2019-03-14 | 日本精工株式会社 | Manipulation system |
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| WO2019012622A1 (en) * | 2017-07-12 | 2019-01-17 | 次郎 大野 | Device for producing three-dimensional cell structure having arbitrary shape, and method for producing same |
| JP2019058156A (en) * | 2017-09-28 | 2019-04-18 | オリンパス株式会社 | Image processing device and cell observation system |
| JP2019154285A (en) * | 2018-03-09 | 2019-09-19 | 大日本印刷株式会社 | Container for observation, and observation method of biological sample |
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