JP7016150B2 - Method for producing cell adhesion substrate - Google Patents
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Description
本発明は、細胞接着領域と非細胞接着領域とを備えた細胞接着基材の作製方法に関する。 The present invention relates to a method for producing a cell adhesion substrate having a cell adhesion region and a non-cell adhesion region.
カバースリップやシャーレなどの培養基板表面上の指定した微小領域にのみ培養細胞を接着又は配列させる技術が現在世界中で研究が進められている。この技術を使えば、細胞の運動、細胞の増殖、細胞死、細胞の分化制御などの観察が可能となるほか、細胞に対する薬剤の効果確認が容易となる。 Research is currently underway around the world on techniques for adhering or arranging cultured cells only in specified microscopic regions on the surface of a culture substrate such as coverslips and petri dishes. Using this technology, it is possible to observe cell motility, cell proliferation, cell death, cell differentiation control, etc., and it is easy to confirm the effect of the drug on cells.
培養基材表面上に培養細胞を接着又は配列させる技術としては、例えば、基板と、前記基板表面に設けられたポリエチレングリコール層と、前記ポリエチレングリコール層上に配置された細胞外マトリクスを含む、細胞固定化用基板が提案されている（特許文献１参照）。また、光触媒の作用により細胞の接着性が変化する細胞接着性変化材料でカバースリップをコートして、マスクを入れて光照射することでパターン化した接着領域を作製する方法が提案されている（特許文献２参照）。 Techniques for adhering or arranging cultured cells on the surface of a cultured substrate include, for example, cells including a substrate, a polyethylene glycol layer provided on the surface of the substrate, and an extracellular matrix arranged on the polyethylene glycol layer. An immobilization substrate has been proposed (see Patent Document 1). In addition, a method has been proposed in which a coverslip is coated with a cell adhesion changing material whose cell adhesion is changed by the action of a photocatalyst, a mask is put in the mask, and light irradiation is performed to produce a patterned adhesion region (). See Patent Document 2).
一方、親水性表面の作製に関し、基盤材料を低温ガスプラズマ中で処理することにより、前記基盤材料に親水性表面を形成することからなる細胞培養基盤作製方法が提案されている（特許文献３参照）。 On the other hand, regarding the production of a hydrophilic surface, a method for producing a cell culture substrate, which comprises forming a hydrophilic surface on the substrate material by treating the substrate material in a low temperature gas plasma, has been proposed (see Patent Document 3). ).
上記特許文献１に記載の方法では、ポリエチレングリコールを用いており、ポリエチレングリコールでは細胞接着を完全に阻害できないという問題があった。さらに、マイクロパターン形成にはインクジェット印刷技術が必要となり、基板の作製は非常に煩雑で、かつ基板自体が高価になるという問題もあった。 In the method described in Patent Document 1, polyethylene glycol is used, and there is a problem that polyethylene glycol cannot completely inhibit cell adhesion. Further, there is a problem that inkjet printing technology is required for micro-pattern formation, the production of the substrate is very complicated, and the substrate itself becomes expensive.
また、上記特許文献２に記載の方法では、一時的に細胞を基材上に固定することは可能であるが、光触媒の作用により細胞の接着性が変化するという特殊な細胞接着性変化材料を用いる必要があり高価となるほか、長時間細胞が接着領域に維持することが難しいという問題があった。そのため、例えば運動性の細胞を観察する場合に、細胞が顕微鏡視野から出てしまい、同じ細胞を長時間観察し続けることができないという問題があった。 Further, in the method described in Patent Document 2, although it is possible to temporarily fix cells on a substrate, a special cell adhesion changing material in which the adhesion of cells is changed by the action of a photocatalyst is used. In addition to being expensive because it needs to be used, there is a problem that it is difficult for cells to maintain in the adhesion region for a long time. Therefore, for example, when observing motile cells, there is a problem that the cells come out of the field of view under the microscope and the same cells cannot be continuously observed for a long time.
さらに、上記特許文献３に記載の方法は、細胞が接着することは可能であるものの、細胞接着能力が低いという問題があった。 Further, the method described in Patent Document 3 has a problem that cells can adhere to each other, but the cell adhesion ability is low.
そこで、本発明の課題は、簡便かつ低コストで作製でき、基材上の指定した領域にのみ細胞接着が可能な細胞接着基材を提供することにある。 Therefore, an object of the present invention is to provide a cell adhesion substrate that can be easily and inexpensively produced and that can adhere to cells only in a designated region on the substrate.
本発明者らは、上記課題を解決するために鋭意検討を行うなかで、基板上に非細胞接着性の２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）の重合体を塗布し、その上面の特定の領域を、微細孔を有するマスクをしたうえでプラズマ処理をすることで、プラズマ処理された領域、すなわちマスクをしていない微細孔領域のみにおいて細胞接着が可能な基板を作製することが可能であることを見出し、本発明を完成した。 The present inventors applied a non-cell adhesion 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer on a substrate in earnest studies to solve the above problems, and applied a specific region on the upper surface thereof. By performing plasma treatment after masking with micropores, it is possible to produce a substrate capable of cell adhesion only in the plasma-treated region, that is, the micropore region without mask. And completed the present invention.
すなわち、本発明は、以下のとおりである。
（１）以下の工程（ａ）～（ｃ）を順次備えることを特徴とする細胞接着基材の作製方法。
（ａ）細胞培養基板上に２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）の重合体を塗布する工程；
（ｂ）ＭＰＣの重合体を塗布した細胞培養基板上に所定の形状の孔を有するマスクを載せる工程；
（ｃ）マスクを載せた細胞培養基板をプラズマ処理する工程；
（２）２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）の重合体が、２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）とブチル（メタ）アクリレートからなる共重合体であることを特徴とする上記（１）記載の細胞接着基材の作製方法。
（３）２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）とメタクリル酸ブチルの共重合体が、以下の式（II）で示される化合物であることを特徴とする上記（１）又は（２）記載の細胞接着基材の作製方法。
（４）細胞培養基板がガラス製であることを特徴とする上記（１）～（３）のいずれか記載の細胞接着基材の作製方法。
（５）細胞培養基板の表面が親水化処理されていることを特徴とする上記（１）～（４）のいずれか記載の細胞接着基材の作製方法。
That is, the present invention is as follows.
(1) A method for producing a cell adhesion substrate, which comprises sequentially comprising the following steps (a) to (c).
(A) A step of applying a polymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) onto a cell culture substrate;
(B) A step of placing a mask having holes having a predetermined shape on a cell culture substrate coated with an MPC polymer;
(C) A step of plasma-treating the cell culture substrate on which the mask is placed;
(2) The above-mentioned (1), wherein the polymer of 2-methacryloxyethyl phosphorylcholine (MPC) is a copolymer composed of 2-methacryloxyethylphosphorylcholine (MPC) and butyl (meth) acrylate. Method for producing cell adhesion substrate.
(3) The cell according to (1) or (2) above, wherein the copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and butyl methacrylate is a compound represented by the following formula (II). Method for producing an adhesive base material.
(4) The method for producing a cell adhesion substrate according to any one of (1) to (3) above, wherein the cell culture substrate is made of glass.
(5) The method for producing a cell adhesion substrate according to any one of (1) to (4) above, wherein the surface of the cell culture substrate is hydrophilized.
本発明により、所望の形状又は大きさの領域のみで細胞が培養可能な基材を簡便かつ低コストで作製可能となる。また、マスクの形状又は大きさは自在に変えることができるため、所望の形状や大きさで細胞を培養して細胞シートを作製することが可能となる。 INDUSTRIAL APPLICABILITY According to the present invention, a substrate on which cells can be cultured only in a region having a desired shape or size can be produced easily and at low cost. Further, since the shape or size of the mask can be freely changed, it is possible to culture cells in a desired shape and size to prepare a cell sheet.
図１は、本発明における細胞接着基材の作製方法の概略を示す図である。カバースリップに所定の形状の孔（図１では四角）を有するマスクを載せ、プラズマ処理を行う。その結果、プラズマ処理を行った領域（マスクの孔の領域）は細胞接着領域、プラズマ処理を行っていない領域（マスク領域）は細胞非接着領域としてパターン化が可能となる。 FIG. 1 is a diagram showing an outline of a method for producing a cell adhesion substrate in the present invention. A mask having a hole (square in FIG. 1) having a predetermined shape is placed on the coverslip, and plasma treatment is performed. As a result, the plasma-treated region (mask pore region) can be patterned as a cell adhesion region, and the plasma-treated region (mask region) can be patterned as a cell non-adhesion region.
また、図２に示すように、ＣｅｌｌＭａｓｋ Ｏｒａｎｇｅのような蛍光を発する色素を用いれば、細胞接着領域と細胞非接着領域を区別することが容易となる。 Further, as shown in FIG. 2, if a dye that emits fluorescence such as CellMask Orange is used, it becomes easy to distinguish between the cell adhesion region and the cell non-adhesion region.
本発明における細胞培養基板の材質としては、細胞を培養でき、細胞を顕微鏡で観察できるものであればよく、顕微鏡観察をする上で透過性の観点から、ガラス、石英、セラミック、サファイナなどの透明無機材料や、ポリスチレン、ポリプロピレン、ポリメタクリルアクリルアミドなどの高分子化合物を好適に挙げることができる。また、細胞培養基板の形状としては特に制限されず、カバースリップ形状、シャーレ形状、ボトムディッシュ形状などを挙げることができる。 The material of the cell culture substrate in the present invention may be any material as long as the cells can be cultured and the cells can be observed with a microscope. From the viewpoint of permeability for microscopic observation, transparent glass, quartz, ceramic, sapphire, etc. Inorganic materials and polymer compounds such as polystyrene, polypropylene and polymethacrylic acrylamide can be preferably mentioned. Further, the shape of the cell culture substrate is not particularly limited, and examples thereof include a coverslip shape, a petri dish shape, and a bottom dish shape.
上記細胞培養基板は油などの表面の汚れを除去し、その後のＭＰＣなどの重合体の塗布において細胞培養基板と上記重合体との接着性を高めるために親水化処理されていてもよく、親水化処理としては、プラズマ処理や、酸又はアルカリ処理や、有機溶媒処理を挙げることができる。 The cell culture substrate may be hydrophilized in order to remove stains on the surface such as oil and to enhance the adhesiveness between the cell culture substrate and the polymer in the subsequent application of the polymer such as MPC. Examples of the culture treatment include plasma treatment, acid or alkali treatment, and organic solvent treatment.
本発明における細胞の種類としては原生生物、動物細胞、植物細胞、細菌、酵母を例示することができる。 Examples of cell types in the present invention include prokaryotes, animal cells, plant cells, bacteria, and yeast.
原生生物としては、細胞性粘菌、アメーバ、藻類、繊毛虫を例示することができ、細胞性粘菌としては、キイロタマホコリカビ（Ｄｉｃｔｙｏｓｔｅｌｉｕｍ ｄｉｓｃｏｉｄｅｕｍ）の細胞を好適に例示することがでる。動物細胞を用いる場合、由来としてはヒト、サル、マウス、ラット、ハムスター、ウサギ、ヤギ、ヒツジ、ウマ、ブタ、イヌなどの哺乳動物由来を挙げることができ、細胞の種類としてはプライマリー細胞、細胞株、受精卵、神経細胞などの哺乳動物細胞を例示することができる。動物細胞を用いる場合には、接着性細胞でも浮遊性細胞でもよいが、接着性細胞を好適に例示することができる。また、植物細胞としては、ニコチアナ・タバカム、シロイヌナズナ由来の細胞を例示することができる。細菌としては、大腸菌、古細菌、マイコプラズマを例示することができる。なお、細胞や細菌としては、細胞壁溶解酵素などで細胞壁を分解したプロトプラストであることが好ましい。 As the protozoa, cellular slime molds, amoeba, algae, and ciliates can be exemplified, and as the cellular slime mold, cells of Dictyostelium discoidium can be preferably exemplified. When animal cells are used, the origin can be derived from mammals such as humans, monkeys, mice, rats, hamsters, rabbits, goats, sheep, horses, pigs and dogs, and the cell types include primary cells and cells. Mammalian cells such as strains, fertilized eggs, and nerve cells can be exemplified. When animal cells are used, they may be adhesive cells or floating cells, and adhesive cells can be preferably exemplified. In addition, examples of plant cells include cells derived from Nicotiana tabacum and Arabidopsis thaliana. Examples of the bacterium include Escherichia coli, archaea, and mycoplasma. The cells and bacteria are preferably protoplasts in which the cell wall is decomposed by a cell wall lytic enzyme or the like.
本発明における２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）の重合体としては、次の式（Ｉ）に示すＭＰＣ単量体からなる単重合体であっても、ＭＰＣ単量体とＭＰＣ以外の単量体からなる共同重合体であってもよい。 As the polymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) in the present invention, even if it is a homopolymer composed of the MPC monomer represented by the following formula (I), it is a single amount other than the MPC monomer and the MPC. It may be a copolymer composed of a body.
ＭＰＣ以外の単量体としては、メチル（メタ）アクリレート、エチル（メタ）アクリレート、ブチル（メタ）アクリレート、ラウリル（メタ）アクリレート、ステアリル（メタ）アクリレート、２－エチルヘキシル（メタ）アクリレート等のアルキル（メタ）アクリレートや、ベンジル（メタ）アクリレート、フェノキシエチル（メタ）アクリレート、シクロヘキシル（メタ）アクリレート、ポリプロピレングリコールモノ（メタ）アクリレート、ポリテトラメチレングリコールモノ（メタ）アクリレート、ポリプロピレングリコールジ（メタ）アクリレート、ポリテトラメチレングリコールモノ（メタ）アクリレート、ポリプロピレングリコールポリエチレングリコールモノ（メタ）アクリレート、メタクリル酸ナトリウム、２－ヒドロキシ－３－メタクリロイルオキシプロピルトリメチルアンモニウムなどを挙げることができ、ブチル（メタ）アクリレートを好適に挙げることができる。なお、上記「（メタ）アクリレート」とは、アクリレート又はメタクリレートを意味する。２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）とブチル（メタ）アクリレートとの共重合体の例を以下の式（II）に示す。 Examples of monomers other than MPC include alkyls such as methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, lauryl (meth) acrylate, stearyl (meth) acrylate, and 2-ethylhexyl (meth) acrylate. Meta) acrylate, benzyl (meth) acrylate, phenoxyethyl (meth) acrylate, cyclohexyl (meth) acrylate, polypropylene glycol mono (meth) acrylate, polytetramethylene glycol mono (meth) acrylate, polypropylene glycol di (meth) acrylate, Polytetramethylene glycol mono (meth) acrylate, polypropylene glycol polyethylene glycol mono (meth) acrylate, sodium methacrylate, 2-hydroxy-3-methacryloyloxypropyltrimethylammonium and the like can be mentioned, and butyl (meth) acrylate is preferable. Can be mentioned. The above-mentioned "(meth) acrylate" means acrylate or methacrylate. An example of a copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and butyl (meth) acrylate is shown in the following formula (II).
式（II）中、ｍ、ｎは各構成単位のモル比を示すための数字であり、モル比でｍ／ｎ＝７０／３０～９０／１０である。） In the formula (II), m and n are numbers for indicating the molar ratio of each structural unit, and the molar ratio is m / n = 70/30 to 90/10. )
本発明のマスクの材質としては、ポリイミド、アルミニウム、ナイロンなどを挙げることができる。マスクの孔は細胞を培養したい領域に合わせて任意の大きさ、形状とすることができる。 Examples of the material of the mask of the present invention include polyimide, aluminum, nylon and the like. The pores of the mask can be of any size and shape according to the area where the cells are to be cultured.
上記「細胞培養基板上に２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）の重合体を塗布する工程」におけるＭＰＣ重合体の塗布方法としては特に制限されないが、ＭＰＣ重合体をエタノールなどの有機溶媒に溶解し、かかる溶解液を、スピンコーターなどの回転式塗布装置を用いて細胞培養基板上に塗布する方法を挙げることができる。 The method for applying the MPC polymer in the above-mentioned "step of applying the polymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) on the cell culture substrate" is not particularly limited, but the MPC polymer is dissolved in an organic solvent such as ethanol. A method of applying such a lysate onto a cell culture substrate using a rotary coating device such as a spin coater can be mentioned.
上記「マスクを載せた細胞培養基板をプラズマ処理する工程」におけるプラズマ処理時間としては特に制限されないが、１０～６０秒、好ましくは２０～４０秒を挙げることができる。ＭＰＣ上では細胞が接着しないが、上記プラズマ処理によって細胞が接着できるようになる。 The plasma treatment time in the above-mentioned "step of plasma-treating the cell culture substrate on which the mask is placed" is not particularly limited, but may be 10 to 60 seconds, preferably 20 to 40 seconds. Although the cells do not adhere on the MPC, the plasma treatment allows the cells to adhere.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの
例示に限定されるものではない。
Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.
１．培養基材の作製
ガラスボトムディッシュ（Ｄ１１１３０Ｈ、松浪硝子工業社製）を真空デバイス（ＰＢ－１：真空デバイス社製）により、真空引きを１分、その後プラズマ処理（第一プラズマ処理）を３０秒間行い、基材表面を親水処理した。プラズマ処理後、スピンコーターを用いて、０．５％のＬｉｐｉｄｕｒｅ－ＣＭ５２０６（登録商標）（２－メタクリロイルオキシエチルホスホリルコリンとブチル（メタ）アクリレートの共重合体：日油株式会社）を含むエタノール溶液１０μＬをカバースリップ上に塗布した。乾燥後、塗布面の上から後述の幅の孔を持ち、厚さ５－１５μｍのアルミニウム又はポリイミドからなるマスク（松陽産業社製）を載せた。次いで、マスクを載せた上記ガラスボトムディッシュの上面から真空デバイス（ＰＢ－１０：真空デバイス社製）で３０秒間プラズマ処理（第二プラズマ処理）して培養基材を作製した。このプラズマ処理により、プラズマ処理された領域は細胞接着領域となり、プラズマ処理されていない領域（マスクを載せた領域）は細胞非接着領域とした。なお、コントロールとして、第二プラズマ処理なしの培養基材を作製した。
1. 1. Preparation of culture substrate Glass bottom dish (D11130H, manufactured by Matsunami Glass Industry Co., Ltd.) is evacuated by a vacuum device (PB-1: manufactured by Vacuum Device Co., Ltd.) for 1 minute, and then plasma treatment (first plasma treatment) is performed for 30 seconds. The surface of the substrate was treated with hydrophilicity. After plasma treatment, using a spin coater, 10 μL of an ethanol solution containing 0.5% Lipidure-CM5206 (registered trademark) (copolymer of 2-methacryloyloxyethyl phosphorylcholine and butyl (meth) acrylate: NOF CORPORATION). Was applied on the coverslip. After drying, a mask (manufactured by Matsuyo Sangyo Co., Ltd.) having a width of 5-15 μm and made of aluminum or polyimide having a width described later was placed on the coated surface. Next, a culture substrate was prepared by plasma treatment (second plasma treatment) for 30 seconds with a vacuum device (PB-10: manufactured by Vacuum Device Co., Ltd.) from the upper surface of the glass bottom dish on which the mask was placed. By this plasma treatment, the plasma-treated region became a cell adhesion region, and the non-plasma-treated region (the region on which the mask was placed) was defined as a cell non-adhesion region. As a control, a culture medium without the second plasma treatment was prepared.
２．細胞性粘菌の接着
上記で作製した培養基材上に、細胞性粘菌キイロタマホコリカビ（Ｄｉｃｔｙｏｓｔｅｌｉｕｍ ｄｉｓｃｏｉｄｅｕｍ）１～２×１０６Ｃｅｌｌｓ含むＨＬ５培養液２ｍＬを載せて１０分以上２２℃で静置した。マスクは多数の円形細孔を有するものを用いた。その後培養基材上の培養液を吸引して培養基材に付着しなかった細胞を含む培養液を除去し、さらに１時間、２２℃で培養した。培養液を吸引して除去した直後、又はその後引き続き２４時間ほど培養した培養基材を位相差顕微鏡で観察した。培養液を吸引して除去した直後の画像、及び２４時間培養後の最後の２時間の動画像を平均化した画像をそれぞれ図３（ａ）、（ｂ）に示す。
2. 2. Adhesion of cellular slime mold On the culture substrate prepared above, 2 mL of HL5 culture medium containing Cellular slime mold Dictyostelium discoidium 1 to 2 × 10 6 Cells is placed and allowed to stand at 22 ° C. for 10 minutes or more. Placed. A mask having a large number of circular pores was used. Then, the culture medium on the culture substrate was sucked to remove the culture medium containing the cells that did not adhere to the culture substrate, and the cells were further cultured at 22 ° C. for 1 hour. Immediately after the culture medium was aspirated and removed, or thereafter, the culture substrate cultured for about 24 hours was observed with a phase-contrast microscope. The images immediately after the culture solution is sucked and removed and the images obtained by averaging the moving images of the last 2 hours after the 24-hour culture are shown in FIGS. 3 (a) and 3 (b), respectively.
図３（ａ）、（ｂ）に示すように、細胞性粘菌は細胞接着領域に接着し、培養２４時間培養後もマスクされていない細胞接着領域のみに接着して培養されていることが確認された。したがって、本発明を用いれば培養する細胞が細胞接着領域にとどまることから細胞の運動を制限できる。また、顕微鏡視野の大きさの接着領域を用意すれば，運動性の細胞を観察する場合に運動領域を制限できるので、細胞が顕微鏡視野から出ることなく長時間細胞の観察を続けることや、細胞運動の軌道を人為的に制御したうえで細胞の行動解析を行うことができる。これにより、細胞周期の全過程の細胞内の分子の動態を追跡することが可能となる。なお、図では示していないが、コントロールの培養基材では、細胞性粘菌が接着せず、培養液を吸引すると培養基材に細胞は残らなかった。 As shown in FIGS. 3 (a) and 3 (b), the cellular slime mold adheres to the cell adhesion region and adheres only to the unmasked cell adhesion region even after culturing for 24 hours. confirmed. Therefore, according to the present invention, the cells to be cultured remain in the cell adhesion region, so that the movement of the cells can be restricted. In addition, if an adhesive region of the size of the microscopic field is prepared, the motor area can be restricted when observing motile cells, so that cells can be continuously observed for a long time without leaving the microscopic field, or cells can be observed. It is possible to analyze the behavior of cells after artificially controlling the trajectory of movement. This makes it possible to track the dynamics of intracellular molecules during the entire cell cycle. Although not shown in the figure, the cell viscous bacteria did not adhere to the control culture medium, and no cells remained on the culture medium when the culture medium was aspirated.
３．市販の細胞接着基板との比較
市販の線状の細胞接着性制御パターンを有する細胞接着基板「ＣｙｔｏＧｒａｐｈ（登録商標）」（大日本印刷社製）上にＨＬ５培養液で培養した細胞性粘菌（１×１０６個を載せて１０分以上２２℃で静置した。制御パターンは幅１０μｍであった。その後、培養基材上の培養液を吸引して除去し、さらに１時間、２２℃で培養した。培養液を吸引して除去した直後、又はその後引き続き２時間培養した培養基材を位相差顕微鏡で観察した。培養液を吸引して除去した直後の画像、及び２時間培養した状況の動画像を平均化した画像をそれぞれ図４（ａ）、（ｂ）に示す。
3. 3. Comparison with commercially available cell adhesion substrate Cell adhesion bacteria cultured in HL5 culture medium on a commercially available cell adhesion substrate "CytoGraph (registered trademark)" (manufactured by Dainippon Printing Co., Ltd.) having a linear cell adhesion control pattern. 6 pieces of 1 × 10 were placed and allowed to stand at 22 ° C. for 10 minutes or more. The control pattern was 10 μm in width. After that, the culture solution on the culture substrate was aspirated and removed, and the cells were further removed at 22 ° C. for 1 hour. The cultured substrate was observed with a phase difference microscope immediately after the culture solution was aspirated and removed, or after that, the culture substrate that had been cultured for 2 hours was observed with a phase difference microscope. Images obtained by averaging moving images are shown in FIGS. 4 (a) and 4 (b), respectively.
図４（ａ）、（ｂ）に示すように、細胞性粘菌を載せた直後は細胞性粘菌が細胞接着性制御パターン領域に接着しているが、その後２時間経過後に、細胞接着性制御パターン以外の領域にも移動していることが確認された。 As shown in FIGS. 4 (a) and 4 (b), the cell-adhesive bacteria adhered to the cell adhesion control pattern region immediately after the cell-adhesive bacteria were placed, but after 2 hours, the cell adhesion was achieved. It was confirmed that the cells were moved to areas other than the control pattern.
４．Ｃｏｓ１細胞の培養
細胞性粘菌と同様にアフリカミドリザルの培養細胞であるＣｏｓ１細胞でも培養を行った。マスクは直径１００μｍの多数の円形細孔を有するもの（松陽産業社製）を用いた以外、上記細胞性粘菌の場合と同様の方法でプラズマ処理及び細胞培養（２４時間培養）を行った。結果を図５（ａ）、（ｂ）に示す。
4. Culturing of Cos1 cells Cos1 cells, which are cultured cells of African green monkeys, were also cultured in the same manner as the cellular slime mold. The mask was subjected to plasma treatment and cell culture (24-hour culture) in the same manner as in the case of the above-mentioned cellular slime mold, except that a mask having a large number of circular pores having a diameter of 100 μm (manufactured by Matsuyo Sangyo Co., Ltd.) was used. The results are shown in FIGS. 5 (a) and 5 (b).
図５（ａ）、（ｂ）に示すように、細胞性粘菌だけでなく動物細胞においても細胞接着領域のみに接着して培養していることが確認された。また、マスクの孔を任意の形状とすることで細胞接着領域の形状を自由に設計し、任意の領域で細胞を培養できることが確認された。かかる結果から、例えば、図５（ａ）のように円形の大きな孔を有するマスクを用いれば，この円形の接着領域内に限定して，細胞の運動などを長時間調べることが可能となる。 As shown in FIGS. 5 (a) and 5 (b), it was confirmed that not only the cellular slime mold but also the animal cells adhered to and cultured only in the cell adhesion region. It was also confirmed that the shape of the cell adhesion region can be freely designed by making the hole of the mask an arbitrary shape, and the cells can be cultured in the arbitrary region. From such a result, for example, if a mask having a large circular hole is used as shown in FIG. 5A, it is possible to investigate the movement of cells for a long time only in the circular adhesive region.
なお、さらに２４時間培養し、増殖が進んで細胞が接着領域に接着しきれないほどの数になった場合には、これらの細胞は基材には接着できずに浮遊した状態となった。しかしながら、浮遊細胞は吸引して除去することで接着領域に接着した細胞を観察可能であった。 When the cells were further cultured for 24 hours and the number of cells increased to the extent that they could not adhere to the adhesive region, these cells could not adhere to the substrate and were in a floating state. However, it was possible to observe the cells adhering to the adhesive region by sucking and removing the floating cells.
５．様々な形状のＣｏｓ１細胞シートの作製
マスクにおいて、幅６００μｍのダイヤ形やスペード形の孔を有する以外は、上記と同様の方法でプラズマ処理及び細胞培養を行った。結果を図６（ａ）、（ｂ）に示す。
5. Preparation of Cos1 cell sheets of various shapes Plasma treatment and cell culture were performed in the same manner as described above except that the mask had diamond-shaped or spade-shaped pores having a width of 600 μm. The results are shown in FIGS. 6 (a) and 6 (b).
図６（ａ）、（ｂ）に示すとおり、孔の形状に従って、すなわち孔の形状に沿った接着領域内に細胞が増殖し、孔の形状に沿った細胞シートを形成していること、及び非細胞接着領域には細胞が移動していないことが明らかとなった。このように、本発明を用いれば、任意の形状の細胞シートを作製することが可能となることが確認された。 As shown in FIGS. 6A and 6B, cells proliferate according to the shape of the pore, that is, in the adhesion region along the shape of the pore, and form a cell sheet along the shape of the pore. It was revealed that cells did not move to the non-cell adhesion region. As described above, it was confirmed that it is possible to produce a cell sheet having an arbitrary shape by using the present invention.
６．非接着領域の染色
上記で用いたＬｉｐｉｄｕｒｅはリン脂質類似構造を有するので、リン脂質に結合すると赤い蛍光を発する蛍光色素で染色することができる。上記「１．培養基材の作製」と同様の方法で培養基材を作製し、かかる培養基材上にＣｅｌｌＭａｓｋ Ｏｒａｎｇｅ（ＭｏｌｅｃｕｌａｒＰｒｏｂｅ社製）を加え得て１分、２２℃経過後に蛍光顕微鏡で観察した結果を図７に示す。なお、ＣｅｌｌＭａｓｋ Ｏｒａｎｇｅはリン脂質に結合すると赤い蛍光を発する色素である。
6. Staining of non-adhesive regions Since the Lipidure used above has a phospholipid-like structure, it can be stained with a fluorescent dye that emits red fluorescence when bound to phospholipids. A culture substrate is prepared by the same method as in "1. Preparation of culture substrate", CellMask Orange (manufactured by MolecularProbe) can be added to the culture substrate, and observed with a fluorescence microscope after 1 minute and 22 ° C. The results are shown in FIG. CellMask Orange is a dye that emits red fluorescence when bound to phospholipids.
図７から明らかなように、Ｌｉｐｉｄｕｒｅにおけるプラズマ処理されていな非接着領域のみが蛍光顕微鏡で観察すると赤く染色されていた。かかる方法により、細胞接着領域と非接着領域の区別がつきやすく、細胞観察をより視認しやすくすることが可能となる。また、細胞のトラッキングを行う場合に、観察対象の細胞や接着領域を探しやすくすることが可能となる。 As is clear from FIG. 7, only the non-plasma-treated non-adhesive region in Lipidure was stained red when observed with a fluorescence microscope. By such a method, it becomes easy to distinguish between the cell adhesion region and the non-adhesion region, and it becomes possible to make the cell observation more visible. In addition, when tracking cells, it becomes possible to easily find cells and adhesive regions to be observed.
７．まとめ
上記結果により、本発明を用いれば、培養基材上に細胞接着領域を多数配置することで「細胞マイクロアレイ」を作製することも可能となる。薬剤の効果を調べるために、従来は９６ｗｅｌｌ培養器を用い、それぞれのｗｅｌｌに細胞を接種し、そこに薬剤を投入していた。しかしながら、９６ｗｅｌｌの培養器に細胞を接種する操作は時間がかかり煩雑であった。一方、シャーレなどの培養基材上に「細胞マイクロアレイ」を形成することができれば、９６ｗｅｌｌ培養器を使うことなく一度に薬剤の効果を調べることが可能となる。
7. Summary Based on the above results, according to the present invention, it is possible to prepare a "cell microarray" by arranging a large number of cell adhesion regions on a culture substrate. In order to investigate the effect of the drug, conventionally, a 96-well incubator was used, cells were inoculated into each well, and the drug was put into the incubator. However, the operation of inoculating cells into a 96-well incubator was time-consuming and complicated. On the other hand, if a "cell microarray" can be formed on a culture substrate such as a petri dish, the effect of the drug can be investigated at once without using a 96-well incubator.
また、本発明を用いれば、「細胞マイクロアレイ」間の細胞のシグナル伝達を細胞間の接着なしで調べることが出来る。細胞同士が接着している場合、細胞接着を通した細胞間コミュニケーションと外液を介した拡散性のサイトカインのような細胞間コミュニケーションとを区別するのは困難である。一方で、本発明では非接着領域には細胞が無いために、非接着領域で細胞を隔てた場合には、確実に細胞同士が非接着となり、細胞接着の影響を除外して細胞間コミュニケーションを調べることができる。 In addition, according to the present invention, cell signal transduction between "cell microarrays" can be investigated without cell-cell adhesion. When cells are adhered to each other, it is difficult to distinguish between cell-cell communication through cell adhesion and cell-cell communication such as diffusible cytokines via external fluid. On the other hand, in the present invention, since there are no cells in the non-adhesive region, when the cells are separated by the non-adhesive region, the cells are surely non-adhesive, and the influence of cell adhesion is excluded to promote cell-cell communication. You can look it up.
このほか、本発明を用いれば任意の形状、大きさで細胞シートを作製可能であることから、細胞をバイオセンサー、バイオリアクター、人工臓器、再生医療としても利用可能になる。加えて、接着領域のみに細胞が接着することから、実験に必要な細胞数（特に幹細胞等の貴重な細胞種）を最小限とすることや、投与する薬剤を必要最小限の量で足りるというメリットを有するほか、細胞に薬剤が作用しやすくなるため、薬剤の効果検証やスクリーニングとしても有用である。 In addition, since the cell sheet can be produced in any shape and size by using the present invention, the cells can be used as biosensors, bioreactors, artificial organs, and regenerative medicine. In addition, since cells adhere only to the adhesive region, the number of cells required for the experiment (especially valuable cell types such as stem cells) can be minimized, and the minimum amount of drug to be administered is sufficient. In addition to having merits, it is also useful for verification and screening of drug effects because it facilitates the action of drugs on cells.
Claims (3)
（ａ）表面が親水化処理された細胞培養基板上に２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）とメタクリル酸ブチルからなる共重合体を塗布する工程；
（ｂ）ＭＰＣとメタクリル酸ブチルからなる共重合体を塗布した前記細胞培養基板上に所定の形状の孔を有するマスクを載せる工程；
（ｃ）マスクを載せた前記細胞培養基板をプラズマ処理する工程； A method for producing a cell adhesion substrate, which comprises sequentially providing the following steps (a) to (c).
(A) A step of applying a copolymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) and butyl methacrylate on a cell culture substrate whose surface has been hydrophilized ;
(B) A step of placing a mask having holes having a predetermined shape on the cell culture substrate coated with a copolymer composed of MPC and butyl methacrylate ;
(C) A step of plasma-treating the cell culture substrate on which the mask is placed;
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| US20050255594A1 (en) | 2004-01-28 | 2005-11-17 | Dai Nippon Printing Co., Ltd. | Patterning substrate and cell culture substrate |
| JP2006067987A (en) | 2004-09-01 | 2006-03-16 | Yukio Nagasaki | Method for recovering cell spheroid and cell spheroid |
| WO2018182044A1 (en) | 2017-03-31 | 2018-10-04 | 国立大学法人北海道大学 | Cell culture substrate, cancer cell aggregate and method for manufacturing same using said substrate, and drug screening method using said cancer cell aggregate |
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| JP2006067987A (en) | 2004-09-01 | 2006-03-16 | Yukio Nagasaki | Method for recovering cell spheroid and cell spheroid |
| WO2018182044A1 (en) | 2017-03-31 | 2018-10-04 | 国立大学法人北海道大学 | Cell culture substrate, cancer cell aggregate and method for manufacturing same using said substrate, and drug screening method using said cancer cell aggregate |
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