JPWO2010010837A1 - Cell culture support and cell culture method - Google Patents
Cell culture support and cell culture method Download PDFInfo
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- JPWO2010010837A1 JPWO2010010837A1 JP2010521684A JP2010521684A JPWO2010010837A1 JP WO2010010837 A1 JPWO2010010837 A1 JP WO2010010837A1 JP 2010521684 A JP2010521684 A JP 2010521684A JP 2010521684 A JP2010521684 A JP 2010521684A JP WO2010010837 A1 JPWO2010010837 A1 JP WO2010010837A1
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- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon atom Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
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Abstract
本発明は、多種類の細胞を同一層内で培養を可能にし、かつ細胞を破壊することなく、基材から細胞のみを均等に剥離することにある。本発明の細胞培養支持体また細胞培養方法は、基材上に２種類以上の温度応答性高分子により異なる領域に表面被覆を行った細胞培養支持体であって、該温度応答性高分子の少なくとも一つがアクリル系樹脂を含有することを特徴とする。It is an object of the present invention to allow various types of cells to be cultured in the same layer and to evenly peel only the cells from the substrate without destroying the cells. The cell culture support or cell culture method of the present invention is a cell culture support in which different regions are coated with two or more types of temperature-responsive polymers on a substrate, At least one contains an acrylic resin.
Description
本発明は、人や細胞培養、組織培養等の分野において利用される細胞の培養を生体外で行うための細胞培養支持体、この細胞培養支持体を用いた細胞培養方法に関する。 The present invention relates to a cell culture support for culturing cells used in the fields of humans, cell culture, tissue culture and the like in vitro, and a cell culture method using the cell culture support.
従来、動物細胞の一般的な培養法として広く用いられる単層細胞培養法は、生体内で有していた複雑系の細胞本来の培養環境下におかれないため、生存を継続する分化機能を維持することが困難であり、細胞は生存または増殖するものの、生体の複雑系を精密に再現していないため、分化機能の停止や制御困難を招くことがよく知られている。 Conventionally, the monolayer cell culture method, which is widely used as a general method for culturing animal cells, is not placed in the original culture environment of complex cells that have been in vivo, and therefore has a differentiation function that continues to survive. Although it is difficult to maintain and cells survive or proliferate, it is well known that the complex system of the living body is not accurately reproduced, resulting in termination of differentiation function and difficulty in control.
例えば、初代培養細胞の中でも高度に生体の代謝機能が分化した初代肝細胞では、単層培養期間内にその機能が消失してしまい易い。例えば、マウス初代培養肝細胞は、シャーレ内で単層培養を行っても、肝細胞の重要な機能の一つであるアンモニア代謝能が、通常、培養開始から１０代程度で失われてしまうことが知られている。 For example, among primary cultured cells, primary hepatocytes with highly differentiated metabolic functions in the body tend to lose their functions within a monolayer culture period. For example, even when mouse primary cultured hepatocytes are cultured in a monolayer in a petri dish, the ability to metabolize ammonia, which is one of the important functions of hepatocytes, is usually lost after about 10 generations from the start of culture. It has been known.
肝細胞の長期培養化には、（生体の肝臓の構造を考えて）血管由来の細胞との共培養が有効であると考えられているが、通常の培養皿を用いる方法で、ただ単に肝細胞と線維芽細胞あるいは肝細胞と内皮細胞との混合等では安定に共培養することはできない。 For long-term culture of hepatocytes, co-culture with blood vessel-derived cells is considered effective (in view of the structure of the liver in the living body). It cannot be stably co-cultured by mixing cells and fibroblasts or hepatocytes and endothelial cells.
肝実質細胞とラット血管内皮細胞との共培養の例が知られているが（例えば、特許文献１）、基材にパターニングされている温度応答性高分子が、Ｎ−ｎ−プロピルメタクリルアミドモノマー重合体とＮ−イソプロピルアクリルアミドモノマー重合体とそれぞれの単独ポリマーで構成されている。２つの単独ポリマーは、温度応答性の性能に差があるため、細胞培養後の、低温にして剥離する際に均等に剥がれず、一部細胞が破損する。 An example of co-culture of liver parenchymal cells and rat vascular endothelial cells is known (for example, Patent Document 1), but the temperature-responsive polymer patterned on the substrate is Nn-propylmethacrylamide monomer It is composed of a polymer, an N-isopropylacrylamide monomer polymer, and respective homopolymers. Since the two single polymers have a difference in temperature-responsive performance, they do not peel evenly when peeling at a low temperature after cell culture, and some cells are damaged.
また細胞培養基材への細胞接着層のパターニングには、例えば、特許文献２のようにインクジェット法により細胞接着材料で細胞接着層を基材にパターニングする方法があるが、１つのポリマーで基材に塗布し、一定の線幅を描いているだけであり、複数のポリマーを塗り分ける技術については言及されていない。 In addition, as a method for patterning a cell adhesion layer on a cell culture substrate, for example, as in Patent Document 2, there is a method of patterning a cell adhesion layer on a substrate with a cell adhesion material by an inkjet method. It is merely applied to the film and a certain line width is drawn, and a technique for separately coating a plurality of polymers is not mentioned.
また、細胞接着材料としてポリエチレングリコール（ＰＥＧ）を用いているため、細胞の剥離ができない。また、基材へＰＥＧを固定する手段として超臨界二酸化炭素を使っており、プロセスが複雑な上に、装置が大型化してしまう。 Moreover, since polyethylene glycol (PEG) is used as the cell adhesion material, the cells cannot be detached. In addition, supercritical carbon dioxide is used as a means for fixing PEG to the substrate, which makes the process complicated and increases the size of the apparatus.
多種類の細胞を同一層内で培養を可能にし、かつ細胞を破壊することなく、基材から細胞のみを均等に剥離することにある。 It is to enable culturing of many types of cells in the same layer, and to peel only the cells evenly from the substrate without destroying the cells.
本発明の上記目的は、以下の構成により達成することができる。 The above object of the present invention can be achieved by the following configuration.
１．基材上に２種類以上の温度応答性高分子により異なる領域に表面被覆を行った細胞培養支持体であって、該温度応答性高分子の少なくとも一つがアクリル系樹脂を含有することを特徴とする細胞培養支持体。 1. A cell culture support having a surface coated with two or more kinds of temperature-responsive polymers on a substrate, wherein at least one of the temperature-responsive polymers contains an acrylic resin. A cell culture support.
２．前記アクリル系樹脂を含有する温度応答性高分子の少なくとも一つが下記一般式（１）で表されることを特徴とする前記１に記載の細胞培養支持体。 2. 2. The cell culture support according to 1, wherein at least one of the temperature-responsive polymers containing the acrylic resin is represented by the following general formula (1).
（一般式（１）において、Ｒ１、Ｒ２は水素原子、炭素原子数１〜８のアルキル基、アリール基を表す。Ｒ５、Ｒ６、Ｒ７は水素原子またはメチル基を表し、Ｒ３は水素原子、炭素原子数１〜３０のアルキル基、シクロアルキル基、または、−（ＣＨ２ＣＨ２Ｏ）ｎ−（ＣＨ２ＣＨ（ＣＨ３）Ｏ）ｍ−Ｒ０で表されるポリオキシアルキレン基を表す。ここにおいてｎは１〜３００、ｍは０〜６０の整数を表す。また、Ｒ０は、水素原子、炭素原子数１〜３０のアルキル基を表す。Ｒ４は炭素原子数３以上２２以下のアルキル基を表す。また、ｘ、ｙ、ｚは各成分の質量％を表し、０≦ｘ≦８０、０≦ｙ≦８０、０≦ｘ≦４０、ここでｘ＋ｙ＋ｚ＝１００である。）
３．前記温度応答性高分子の少なくとも一つがＮ−イソプロピルアクリルアミドのホモポリマーもしくは他のモノマー成分との共重合ポリマーであることを特徴とする前記１または２に記載の細胞培養支持体。(In the general formula (1), R 1, R 2 is a hydrogen atom, .R 5, R 6, R 7 represents an alkyl group, an aryl group having 1 to 8 carbon atoms is a hydrogen atom or a methyl radical, R 3 is a hydrogen atom, an alkyl group having 1 to 30 carbon atoms, a cycloalkyl group, or a poly- represented by — (CH 2 CH 2 O) n — (CH 2 CH (CH 3 ) O) m —R 0. Represents an oxyalkylene group, wherein n represents 1 to 300, m represents an integer of 0 to 60, R 0 represents a hydrogen atom or an alkyl group having 1 to 30 carbon atoms, and R 4 represents a carbon atom. Represents an alkyl group having a number of 3 or more and 22 or less, and x, y, and z represent mass% of each component, 0 ≦ x ≦ 80, 0 ≦ y ≦ 80, 0 ≦ x ≦ 40, where x + y + z = 100 .)
3. 3. The cell culture support according to 1 or 2, wherein at least one of the temperature-responsive polymers is a homopolymer of N-isopropylacrylamide or a copolymer with another monomer component.
４．前記温度応答性高分子がインクジェット法によって基材上にパターニングされていることを特徴とする前記１〜３のいずれか１項に記載の細胞培養支持体。 4). 4. The cell culture support according to any one of 1 to 3, wherein the temperature-responsive polymer is patterned on a substrate by an inkjet method.
５．前記１〜４のいずれか１項に記載の細胞培養支持体上で２種類以上の異なる細胞を培養することを特徴とする細胞培養方法。 5. A cell culture method comprising culturing two or more different cells on the cell culture support according to any one of 1 to 4 above.
６．前記５に記載の細胞培養方法であって、２種類以上の異なる細胞を温度応答性高分子の応答温度以下にして剥離することを特徴とする細胞培養方法。 6). 6. The cell culturing method according to 5 above, wherein two or more kinds of different cells are detached at a temperature lower than the response temperature of the temperature-responsive polymer.
７．前記６に記載の細胞培養方法で培養した細胞を重ね合わせることを特徴とする細胞培養方法。 7). 7. A cell culture method comprising superposing cells cultured by the cell culture method described in 6 above.
本発明の細胞培養支持体により、多種類の細胞を同一層内で培養を可能にし、かつ細胞を破壊することなく、基材から細胞のみを剥離する技術が達成できた。 With the cell culture support of the present invention, it was possible to cultivate many types of cells in the same layer, and to achieve a technique for peeling only the cells from the substrate without destroying the cells.
以下本発明を実施するための最良の形態について詳しく説明する。 Hereinafter, the best mode for carrying out the present invention will be described in detail.
（温度応答性高分子）
本発明においては、温度応答性高分子を一般的な細胞培養基材の上に塗布することが好ましい。温度応答性高分子とは、親水性と疎水性とが温度変化により可逆的に変化する高分子材料のことである。また、好ましい温度応答性高分子として、アクリル系樹脂のモノマー及びアクリルアミドモノマーが共重合したポリマーがあげられる。更に好ましい温度応答性高分子としては前記一般式（１）で表される高分子があげられる。温度応答性高分子を表面に備えた細胞培養基材は、疎水性条件下では細胞と優れた接着性を示すため、細胞を好適に培養、増殖させることができ、また親水性条件下では、細胞との接着性を低下させることができるため、タンパク質加水分解酵素や化学薬品を使用せずに細胞を剥離できるため、細胞の破損や、基材の剥離混入を生じることなく、容易に細胞の回収が可能である。(Temperature responsive polymer)
In the present invention, it is preferable to apply a temperature-responsive polymer on a general cell culture substrate. The temperature-responsive polymer is a polymer material in which hydrophilicity and hydrophobicity are reversibly changed by a temperature change. A preferable temperature-responsive polymer is a polymer obtained by copolymerizing an acrylic resin monomer and an acrylamide monomer. A more preferable temperature-responsive polymer is a polymer represented by the general formula (1). A cell culture substrate having a temperature-responsive polymer on the surface exhibits excellent adhesion to cells under hydrophobic conditions, so that the cells can be cultured and proliferated appropriately, and under hydrophilic conditions, Adhesion with cells can be reduced, so cells can be detached without the use of protein hydrolases or chemicals. Recovery is possible.
さらに、疎水性から親水性あるいは親水性から疎水性への変化が迅速であるため、温度をはじめとする外部環境を変化させる際に細胞に与える影響が少ないので好ましい。温度応答性高分子の親水性と疎水性とが変化する温度を臨界温度といい、特に高温で疎水性、低温で親水性になる時の温度を下限臨界温度という。細胞培養に使用される細胞は、多くは恒温動物由来であるため、人間の体温付近の３７度近辺で培養されることが多く、該温度で細胞が細胞培養支持体に接着しやすい方が良く、つまり、３７度近辺で細胞培養支持体表面は疎水性の性質であることが好ましい、細胞の剥離時には、熱によるたんぱく質の変性を起こさないために低温で細胞培養支持体から剥離した方が好ましいい。つまり低温で細胞培養支持体表面は親水性の性質であることが好ましい。 Furthermore, since the change from hydrophobic to hydrophilic or from hydrophilic to hydrophobic is rapid, there is little influence on cells when changing the external environment including temperature, which is preferable. The temperature at which the hydrophilicity and hydrophobicity of the temperature-responsive polymer changes is called the critical temperature, and the temperature at which it becomes hydrophobic at high temperatures and hydrophilic at low temperatures is called the lower critical temperature. Many of the cells used for cell culture are derived from constant temperature animals. Therefore, the cells are often cultured in the vicinity of 37 ° C. near the human body temperature, and it is better that the cells adhere to the cell culture support at that temperature. In other words, it is preferable that the surface of the cell culture support is hydrophobic at around 37 degrees. When the cells are detached, it is preferable that the cell culture support is detached from the cell culture support at a low temperature so as not to cause denaturation of the protein by heat. Better. That is, it is preferable that the cell culture support surface has a hydrophilic property at a low temperature.
さらには、細胞を好適に培養・剥離できることから、該下限臨界温度が２０℃以上４０℃以下程度の温度範囲にあることが好ましい。 Furthermore, the lower critical temperature is preferably in the temperature range of about 20 ° C. or higher and 40 ° C. or lower because cells can be cultured and detached suitably.
本発明においては、温度応答性高分子の少なくとも一つがアクリル系樹脂を含有することを特徴とする。 In the present invention, at least one of the temperature-responsive polymers contains an acrylic resin.
本発明におけるアクリル系樹脂としては、アクリル酸、メタクリル酸、メチルアクリレート、メチルメタクリレート等の一般的なアクリル樹脂でもよく、その誘導体でかまわない。また脂環式炭化水素骨格からなる多環式炭化水素系化合物であるアクリル樹脂でもよく、多環式炭化水素系化合物としては、脂肪族の多環構造を有し、３次元的な架橋構造を含むものでもよい、例えば、トリシクロデカンジメタノールジメタクリレート、トリシクロデカンジメタノールジアクリレートや、アダマンチルメタクリレート、アダマンチルアクリレート等や、イソボルニルメタクリレート、イソボルニルアクリレート、ビニルノルボルネン等が挙げられる。 The acrylic resin in the present invention may be a general acrylic resin such as acrylic acid, methacrylic acid, methyl acrylate, methyl methacrylate, or a derivative thereof. Further, an acrylic resin which is a polycyclic hydrocarbon compound composed of an alicyclic hydrocarbon skeleton may be used. The polycyclic hydrocarbon compound has an aliphatic polycyclic structure and a three-dimensional crosslinked structure. For example, tricyclodecane dimethanol dimethacrylate, tricyclodecane dimethanol diacrylate, adamantyl methacrylate, adamantyl acrylate, etc., isobornyl methacrylate, isobornyl acrylate, vinyl norbornene and the like may be included.
アクリル系樹脂モノマーおよびアクリルアミドモノマーが共重合したポリマーを用いると細胞の剥離性がよくなる。その理由として、アミドは生体内たんぱく質のペプチド結合と化学的に同じ構造であるため、細胞とポリマーの親和性が強くなり、これが剥離時の細胞破損の原因になっていると考えられる。アクリルアミドモノマーとアクリル系樹脂モノマーを共重合したポリマーを細胞培養の支持体とすることで細胞剥離性と温度応答性の両方が満足できる素材が得られた。 When a polymer in which an acrylic resin monomer and an acrylamide monomer are copolymerized is used, cell detachability is improved. The reason is that the amide has the same chemical structure as the peptide bond of the protein in the living body, and thus the affinity between the cell and the polymer is increased, which is considered to cause cell damage at the time of detachment. By using a polymer obtained by copolymerizing an acrylamide monomer and an acrylic resin monomer as a support for cell culture, a material satisfying both cell detachability and temperature responsiveness was obtained.
温度応答性高分子としては、アクリルアミドポリマーがよく知られており、これはアクリルアミドモノマーを重合することにより得られる。 Acrylamide polymers are well known as temperature-responsive polymers, and can be obtained by polymerizing acrylamide monomers.
このような重合体を与えるアクリルアミドモノマーの例としては、Ｎ−置換アクリルアミド誘導体、Ｎ，Ｎ−ジ置換アクリルアミド誘導体、Ｎ−置換メタクリルアミド誘導体、Ｎ，Ｎ−ジ置換メタクリルアミド誘導体などを好ましく使用することができ、具体的にはＮ−イソプロピルアクリルアミド、Ｎ−イソプロピルメタクリルアミド、Ｎ−ｎ−プロピルアクリルアミド、Ｎ−ｎ−プロピルメタクリルアミド、Ｎ−シクロプロピルアクリルアミド、Ｎ−シクロプロピルメタクリルアミド、Ｎ−エトキシエチルアクリルアミド、Ｎ−エトキシエチルメタクリルアミド、Ｎ−テトラヒドロフルフリルアクリルアミド、Ｎ−テトラヒドロフルフリルメタクリルアミド、Ｎ−エチルアクリルアミド、Ｎ−エチル−Ｎ−メチルアクリルアミド、Ｎ，Ｎ−ジエチルアクリルアミド、Ｎ−メチル−Ｎ−ｎ−プロピルアクリルアミド、Ｎ−メチル−Ｎ−イソプロピルアクリルアミド、Ｎ−アクリロイルピペリディン、Ｎ−アクリロイルピロリジンが挙げられる。 Examples of acrylamide monomers that give such polymers are preferably N-substituted acrylamide derivatives, N, N-disubstituted acrylamide derivatives, N-substituted methacrylamide derivatives, N, N-disubstituted methacrylamide derivatives, and the like. Specifically, N-isopropylacrylamide, N-isopropylmethacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxy Ethyl acrylamide, N-ethoxyethyl methacrylamide, N-tetrahydrofurfuryl acrylamide, N-tetrahydrofurfuryl methacrylamide, N-ethyl acrylamide, N-ethyl-N-methyl acrylamide, N, N Diethyl acrylamide, N- methyl -N-n-propyl acrylamide, N- methyl -N- isopropylacrylamide, N- acryloyl Lupi Peri Dinh include N- acryloyl pyrrolidine.
かかるアクリルアミドポリマーとしては、例えば、ポリ（Ｎ−イソプロピルアクリルアミド）、ポリ（Ｎ−ｎ−プロピルアクリルアミド）、ポリ（Ｎ−シクロプロピルメタクリルアミド）、ポリ（Ｎ−イソプロピルメタクリルアミド）、ポリ（Ｎ−ｎ−プロピルメタクリルアミド）、ポリ（Ｎ−エトキシエチルアクリルアミド）、ポリ（Ｎ−エトキシエチルメタクリルアミド）、ポリ（Ｎ−テトラヒドロフルフリルアクリルアミド）、ポリ（Ｎ−テトラフルフリルメタクリルアミド）、ポリ（Ｎ−エチルアクリルアミド）、ポリ（Ｎ，Ｎ−ジエチルアクリルアミド）、ポリ（Ｎ−アクリロイルピペリディン）、ポリ（Ｎ−アクリロイルピロリディン）が挙げられる。 Examples of such acrylamide polymers include poly (N-isopropylacrylamide), poly (Nn-propylacrylamide), poly (N-cyclopropylmethacrylamide), poly (N-isopropylmethacrylamide), and poly (Nn). -Propylmethacrylamide), poly (N-ethoxyethylacrylamide), poly (N-ethoxyethylmethacrylamide), poly (N-tetrahydrofurfurylacrylamide), poly (N-tetrafurfurylmethacrylamide), poly (N- Ethyl acrylamide), poly (N, N-diethylacrylamide), poly (N-acryloylpiperidine), and poly (N-acryloylpyrrolidine).
また水溶性アクリルアミドモノマーの重合体としては、以上のような単一水溶性アクリルアミドモノマーからの重合体の他、これらから選ばれる複数の異なる水溶性アクリルアミドモノマーを重合して得られる共重合体を用いることも有効である。 In addition to the polymer from the single water-soluble acrylamide monomer as described above, a copolymer obtained by polymerizing a plurality of different water-soluble acrylamide monomers selected from these is used as the polymer of the water-soluble acrylamide monomer. It is also effective.
また上記水溶性アクリルアミドモノマーからなる重合体が好ましいが、上記水溶性アクリルアミドモノマーとそれ以外の水溶性アクリルアミドモノマーまたは有機溶媒可溶性アクリルアミドモノマーとの共重合体も、得られた重合体が親水性および疎水性の両方を示すものであれば使用することができる。共重合に用いられるアクリルアミドモノマーとしては、具体的にはＮ−アルキルアクリルアミド、Ｎ，Ｎ−ジアルキルアクリルアミド、アクリルアミド等のアクリルアミド類、または、Ｎ−アルキルメタクリルアミド、Ｎ，Ｎ−ジアルキルメタクリルアミド、メタクリルアミド等のメタクリルアミド類が挙げられる。なお、より好ましくは、Ｎ−アルキルアクリルアミドまたはＮ，Ｎ−ジアルキルアクリルアミドが用いられる。アルキル基としては、炭素数が１〜４のものが好ましく選択される。その他には、アクリロイルモルフォリン、Ｎ，Ｎ−ジメチルアミノプロピルアクリルアミド、Ｎ−アクリロイルメチルホモピペラディン、Ｎ−アクリロイルメチルピペラディン等も用いることができる。 A polymer comprising the water-soluble acrylamide monomer is preferred, but a copolymer of the water-soluble acrylamide monomer and another water-soluble acrylamide monomer or an organic solvent-soluble acrylamide monomer may also be a hydrophilic and hydrophobic polymer. Any material that exhibits both sexes can be used. Specific examples of acrylamide monomers used for copolymerization include acrylamides such as N-alkylacrylamide, N, N-dialkylacrylamide, and acrylamide, or N-alkylmethacrylamide, N, N-dialkylmethacrylamide, and methacrylamide. And other methacrylamides. More preferably, N-alkylacrylamide or N, N-dialkylacrylamide is used. As the alkyl group, those having 1 to 4 carbon atoms are preferably selected. In addition, acryloyl morpholine, N, N-dimethylaminopropylacrylamide, N-acryloylmethyl homopiperazine, N-acryloylmethylpiperazine and the like can be used.
またアクリル系樹脂としては、アクリル樹脂モノマーを基本として他のモノマーと共重合してよく、本発明において、温度応答性高分子としては、好ましくは、下記一般式（１）で表される化合物である。 The acrylic resin may be copolymerized with other monomers based on an acrylic resin monomer. In the present invention, the temperature-responsive polymer is preferably a compound represented by the following general formula (1). is there.
ここにおいてＲ１、Ｒ２は水素原子、炭素原子数１〜８のアルキル基、アリール基を表す。Ｒ５、Ｒ６、Ｒ７は水素原子またはメチル基を表し、Ｒ３は水素原子、炭素原子数１〜３０のアルキル基、シクロアルキル基、または、−（ＣＨ２ＣＨ２Ｏ）ｎ−（ＣＨ２ＣＨ（ＣＨ３）Ｏ）ｍ−Ｒ０で表されるポリオキシアルキレン基を表す。ここにおいてｎは１〜３００、ｍは０〜６０の整数を表す。また、Ｒ０は、水素原子、炭素原子数１〜３０のアルキル基を表す。Ｒ４は炭素原子数３以上２２以下のアルキル基を表す。また、ｘ、ｙ、ｚは各成分の質量％を表し、０≦ｘ≦８０、０≦ｙ≦８０、０≦ｘ≦４０、ここでｘ＋ｙ＋ｚ＝１００である。Here, R 1 and R 2 represent a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or an aryl group. R 5 , R 6 and R 7 each represents a hydrogen atom or a methyl group, and R 3 represents a hydrogen atom, an alkyl group having 1 to 30 carbon atoms, a cycloalkyl group, or — (CH 2 CH 2 O) n — ( It represents a CH 2 CH (CH 3) O ) a polyoxyalkylene group represented by m -R 0. Here, n represents an integer of 1 to 300, and m represents an integer of 0 to 60. R 0 represents a hydrogen atom or an alkyl group having 1 to 30 carbon atoms. R 4 represents an alkyl group having 3 to 22 carbon atoms. Moreover, x, y, z represents the mass% of each component, 0 <= x <= 80, 0 <= y <= 80, 0 <= x <= 40, and it is here x + y + z = 100.
Ｒ１、Ｒ２で表される炭素原子数１〜８のアルキル基としては直鎖でもよく分岐してもよい。例えば、メチル基、エチル基、イソプロピル基、イソブチル基、ドデシル基等の基を表し、また、置換されていてもよい。置換基としては、ハロゲン原子、ヒドロキシ基、カルボキシ基、また、アシル基等の基を含み、例えば、ヒドロキシエチル基、ヒドロキシプロピル基等が挙げられる。シクロアルキル基としてはシクロプロピル基、シクロペンチル基、シクロヘキシル基等が挙げられる。The alkyl group having 1 to 8 carbon atoms represented by R 1 and R 2 may be linear or branched. For example, it represents a group such as a methyl group, an ethyl group, an isopropyl group, an isobutyl group, or a dodecyl group, and may be substituted. Examples of the substituent include groups such as a halogen atom, a hydroxy group, a carboxy group, and an acyl group, and examples thereof include a hydroxyethyl group and a hydroxypropyl group. Examples of the cycloalkyl group include a cyclopropyl group, a cyclopentyl group, and a cyclohexyl group.
Ｒ１、Ｒ２で表されるアリール基としてはフェニル基、またトリル基等の置換フェニル基が挙げられる。また、Ｒ１、Ｒ２の少なくとも一つは水素原子であることが好ましい。Examples of the aryl group represented by R 1 and R 2 include a phenyl group and a substituted phenyl group such as a tolyl group. Moreover, it is preferable that at least one of R 1 and R 2 is a hydrogen atom.
Ｒ３で表される炭素原子数１〜３０のアルキル基は、直鎖でもよく分岐してもよい、分岐してもよいアルキル基としては、例えば、メチル基、エチル基、イソプロピル基、イソブチル基、ドデシル基等の基を表す。無置換のアルキル基、置換アルキル基を含み、無置換のアルキル基としては、例えば、メチル基、エチル基、イソプロピル基、ドデシル基等の基を表す。また、置換アルキル基における置換基としてはハロゲン原子、ヒドロキシ基、カルボキシ基等の基を含み、例えば、ヒドロキシエチル基、ヒドロキシプロピル基等が挙げられる。シクロアルキル基としてはシクロプロピル基、シクロペンチル基、シクロヘキシル基等が挙げられるが、置換シクロアルキル基でもよい、また、シクロアルキル基の骨格炭素がヘテロ原子で置換された例えばオキソラニル基、オキサニル基、ピラジニル基等のヘテロ原子を含む飽和炭化水素基でもよい。The alkyl group having 1 to 30 carbon atoms represented by R 3 may be linear or branched. Examples of the branched alkyl group include a methyl group, an ethyl group, an isopropyl group, and an isobutyl group. Represents a group such as a dodecyl group. An unsubstituted alkyl group and a substituted alkyl group are included, and examples of the unsubstituted alkyl group include groups such as a methyl group, an ethyl group, an isopropyl group, and a dodecyl group. In addition, examples of the substituent in the substituted alkyl group include groups such as a halogen atom, a hydroxy group, and a carboxy group, and examples thereof include a hydroxyethyl group and a hydroxypropyl group. Examples of the cycloalkyl group include a cyclopropyl group, a cyclopentyl group, a cyclohexyl group, and the like. A substituted cycloalkyl group may be used. For example, an oxolanyl group, an oxanyl group, or a pyrazinyl group in which the skeleton carbon of the cycloalkyl group is substituted with a heteroatom. It may be a saturated hydrocarbon group containing a hetero atom such as a group.
Ｒ３に含まれてもよい−（ＣＨ２ＣＨ２Ｏ）ｎ−（ＣＨ２ＣＨ（ＣＨ３）Ｏ）ｍ−Ｒ０で表されるポリオキシアルキレン基としては、オキシエチレン基の繰り返し単位（ｎ）としては１〜３００が好ましく、より好ましいのは６〜１００の範囲が好ましく、さらに好ましいのは８〜５０の範囲である。また、またＲ３に含まれてもよいオキシプロピレン基の繰り返し単位（ｍ）としては０〜６０が好ましく、より好ましいのは０〜３０の範囲が好ましく、さらに好ましいのは０〜１５の範囲である。これらオキシエチレン基とオキシプロピレン基は混在してもよい。Ｒ０で表される炭素原子数１〜３０のアルキル基としては、メチル、エチル、プロピル、ブチル、オクチル、デシル、ドデシル、ステアリル等の基が挙げられる。The polyoxyalkylene group represented by — (CH 2 CH 2 O) n — (CH 2 CH (CH 3 ) O) m —R 0, which may be contained in R 3 , is a repeating unit of an oxyethylene group ( n) is preferably 1 to 300, more preferably 6 to 100, and still more preferably 8 to 50. The repeating unit (m) of the oxypropylene group that may be contained in R 3 is preferably 0 to 60, more preferably 0 to 30 and even more preferably 0 to 15. is there. These oxyethylene groups and oxypropylene groups may be mixed. Examples of the alkyl group having 1 to 30 carbon atoms represented by R 0 include groups such as methyl, ethyl, propyl, butyl, octyl, decyl, dodecyl, stearyl.
Ｒ４で表される炭素原子数３以上２２以下のアルキル基としては、直鎖あるいは分岐アルキル基でもよく、プロピル基、イソプロピル基、ブチル基、ｔ−ブチル基、ヘキシル基、ドデシル基、ステアリル基等を表す。The alkyl group having 3 to 22 carbon atoms represented by R 4 may be a linear or branched alkyl group, and is a propyl group, isopropyl group, butyl group, t-butyl group, hexyl group, dodecyl group, stearyl group. Etc.
一般式（１）の共重合体の製造は、各成分モノマーの共重合により得ることができる。 Manufacture of the copolymer of General formula (1) can be obtained by copolymerization of each component monomer.
下記に各成分モノマーの例を挙げるが、一般式（１）の条件を満たしていれば、これに限定されることはない。 Although the example of each component monomer is given to the following, if the conditions of General formula (1) are satisfy | filled, it will not be limited to this.
一般式（１）において、Ｒ１、Ｒ２、Ｒ５で表される基を含むモノマーとしては、代表的にはダイアセトンアクリルアミド、アクリルアミド、Ｎ−イソプロピルアクリルアミド（ＮＩＰＡＭ）、Ｎ−エチルアクリルアミド、Ｎ−ピロリジニルアクリルアミド、Ｎ−シクロプロピルアクリルアミド、Ｎ−ジエチルアクリルアミド、Ｎ−メチル、Ｎ−イソロプロピルアクリルアミド、Ｎ−プロピルアクリルアミド、Ｎ−メチル、Ｎ−イソロプロピルアクリルアミド、Ｎ−ピペリジニルアクリルアミド、Ｎ−プロピルアクリルアミド、Ｎ−シクロプロピルメタクリルアミド、Ｎ−エチルメタリルアミド、Ｎ−イソプロピルメタクリルアミド等のモノマーが挙げられる。In the general formula (1), examples of the monomer containing a group represented by R 1 , R 2 , R 5 typically include diacetone acrylamide, acrylamide, N-isopropyl acrylamide (NIPAM), N-ethyl acrylamide, N -Pyrrolidinyl acrylamide, N-cyclopropyl acrylamide, N-diethyl acrylamide, N-methyl, N-isopropyl acrylamide, N-propyl acrylamide, N-methyl, N-isopropyl acrylamide, N-piperidinyl acrylamide , N-propylacrylamide, N-cyclopropylmethacrylamide, N-ethylmethallylamide, N-isopropylmethacrylamide and the like.
一般式（１）において、Ｒ３、Ｒ６を含むモノマー、また、Ｒ４、Ｒ７で表される基を含むモノマーとしては、下記の中から選択して用いることができる。In the general formula (1), the monomer containing R 3 and R 6 and the monomer containing a group represented by R 4 and R 7 can be selected from the following.
例えば、アクリル酸、メタクリル酸、メチルメタクリレート、ブチルメタクリレート等が挙げられる。 For example, acrylic acid, methacrylic acid, methyl methacrylate, butyl methacrylate and the like can be mentioned.
また、例えば（ポリオキシアルキレン）アクリレートおよびメタクリレートは、市販のヒドロキシポリ（オキシアルキレン）材料、例えば商品名“プルロニック”［Ｐｌｕｒｏｎｉｃ（旭電化工業（株）製）］、アデカポリエーテル（旭電化工業（株）製）、カルボワックス［Ｃａｒｂｏｗａｘ（グリコ・プロダクス）］、トリトン［Ｔｏｒｉｔｏｎ（ローム・アンド・ハース（Ｒｏｈｍ ａｎｄ Ｈａａｓ製））］およびＰ．Ｅ．Ｇ（第一工業製薬（株）製）として販売されているものを公知の方法でアクリル酸、メタクリル酸、アクリルクロリド、メタクリルクロリドまたは無水アクリル酸等と反応させることによって製造できる。 Further, for example, (polyoxyalkylene) acrylate and methacrylate are commercially available hydroxy poly (oxyalkylene) materials such as “Pluronic” (Pluronic (manufactured by Asahi Denka Kogyo Co., Ltd.)), Adeka polyether (Asahi Denka Kogyo ( ), Carbowax [Carbowax (Glyco Products)], Triton [Toriton (Rohm and Haas)] and P.I. E. What is marketed as G (made by Daiichi Kogyo Seiyaku Co., Ltd.) can be manufactured by making it react with acrylic acid, methacrylic acid, acrylic chloride, methacrylic chloride, acrylic anhydride, etc. by a well-known method.
また、上市されているものとして、日本油脂株式会社製のポリアルキレングリコールモノ（メタ）アクリレートとしてブレンマーＰＥ−９０、ブレンマーＰＥ−２００、ブレンマーＰＥ−３５０、ブレンマーＡＥ−９０、ブレンマーＡＥ−２００、ブレンマーＡＥ−４００、ブレンマーＰＰ−１０００、ブレンマーＰＰ−５００、ブレンマーＰＰ−８００、ブレンマーＡＰ−１５０、ブレンマーＡＰ−４００、ブレンマーＡＰ−５５０、ブレンマーＡＰ−８００、ブレンマー５０ＰＥＰ−３００、ブレンマー７０ＰＥＰ−３５０Ｂ、ブレンマーＡＥＰシリーズ、ブレンマー５５ＰＥＴ−４００、ブレンマー３０ＰＥＴ−８００、ブレンマー５５ＰＥＴ−８００、ブレンマーＡＥＴシリーズ、ブレンマー３０ＰＰＴ−８００、ブレンマー５０ＰＰＴ−８００、ブレンマー７０ＰＰＴ−８００、ブレンマーＡＰＴシリーズ、ブレンマー１０ＰＰＢ−５００Ｂ、ブレンマー１０ＡＰＢ−５００Ｂなどがあげられる。同様に日本油脂株式会社製のアルキル末端ポリアルキレングリコールモノ（メタ）アクリレートとしてブレンマーＰＭＥ−１００、ブレンマーＰＭＥ−２００、ブレンマーＰＭＥ−４００、ブレンマーＰＭＥ−１０００、ブレンマーＰＭＥ−４０００、ブレンマーＡＭＥ−４００、ブレンマー５０ＰＯＥＰ−８００Ｂ、ブレンマー５０ＡＯＥＰ−８００Ｂ、ブレンマーＰＬＥ−２００、ブレンマーＡＬＥ−２００、ブレンマーＡＬＥ−８００、ブレンマーＰＳＥ−４００、ブレンマーＰＳＥ−１３００、ブレンマーＡＳＥＰシリーズ、ブレンマーＰＫＥＰシリーズ、ブレンマーＡＫＥＰシリーズ、ブレンマーＡＮＥ−３００、ブレンマーＡＮＥ−１３００、ブレンマーＰＮＥＰシリーズ、ブレンマーＰＮＰＥシリーズ、ブレンマー４３ＡＮＥＰ−５００、ブレンマー７０ＡＮＥＰ−５５０など、また共栄社化学株式会社製ライトエステルＭＣ、ライトエステル１３０ＭＡ、ライトエステル０４１ＭＡ、ライトアクリレートＢＯ−Ａ、ライトアクリレートＥＣ−Ａ、ライトアクリレートＭＴＧ−Ａ、ライトアクリレート１３０Ａ、ライトアクリレートＤＰＭ−Ａ、ライトアクリレートＰ−２００Ａ、ライトアクリレートＮＰ−４ＥＡ、ライトアクリレートＮＰ−８ＥＡなどが挙げられ、これらの中から選択し用いることができる。 Moreover, as what is marketed, it is Blemmer PE-90, Blemmer PE-200, Blemmer PE-350, Blemmer AE-90, Blemmer AE-200, Blemmer as polyalkylene glycol mono (meth) acrylate made by NOF Corporation. AE-400, Blemmer PP-1000, Blemmer PP-500, Blemmer PP-800, Blemmer AP-150, Blemmer AP-400, Blemmer AP-550, Blemmer AP-800, Blemmer 50 PEP-300, Blemmer 70PEP-350B, Blemmer AEP series, Blemmer 55PET-400, Blemmer 30PET-800, Blemmer 55PET-800, Blemmer AET series, Blemmer 30PPT-800, Blemmer 50P T-800, Blemmer 70PPT-800, Blemmer APT series, Brenmer 10PPB-500B, such as Brenmer 10APB-500B and the like. Similarly, Blemmer PME-100, Blemmer PME-200, Blemmer PME-400, Blemmer PME-1000, Blemmer PME-4000, Blemmer AME-400, Blemmer as alkyl-terminated polyalkylene glycol mono (meth) acrylates manufactured by NOF Corporation. 50POEP-800B, Blemmer 50AOEP-800B, Blemmer PLE-200, Blemmer ALE-200, Blemmer ALE-800, Blemmer PSE-400, Blemmer PSE-1300, Blemmer ASE series, Blemmer PKEP series, Blemmer AKEP series, Blemmer AE-300 , Blemmer ANE-1300, Blemmer PNEP series, Blemmer PNPE series, Blemmer 43ANE -500, BLEMMER 70ANEP-550, etc., and Kyoeisha Chemical Co., Ltd. light ester MC, light ester 130MA, light ester 041MA, light acrylate BO-A, light acrylate EC-A, light acrylate MTG-A, light acrylate 130A, light Examples thereof include acrylate DPM-A, light acrylate P-200A, light acrylate NP-4EA, light acrylate NP-8EA, and the like.
これらの共重合体からなるポリマーは親水性と親油性を併せもち下限臨界温度によりその性質が可逆的に変化する。下限臨界温度を変化させるには、一般式（１）のｘ、ｙ、ｚの共重合比を変えることで任意に決めることができる。 Polymers composed of these copolymers have both hydrophilicity and lipophilicity, and their properties reversibly change depending on the lower critical temperature. In order to change the lower critical temperature, it can be arbitrarily determined by changing the copolymerization ratio of x, y, z in the general formula (1).
例えば、オキシエチレン基の繰り返し単位を多くすることで下限臨界温度をあげることができる。またＲ４、Ｒ７の置換基を疎水的にすることで下限臨界温度を下げることができる。For example, the lower critical temperature can be increased by increasing the number of repeating units of the oxyethylene group. The lower critical temperature can be lowered by making the substituents of R 4 and R 7 hydrophobic.
目的の下限臨界温度に応じた混合比率で、モノマーを混合し、例えば溶媒として各モノマー、また共重合体に対し溶解性のよい溶媒、例えばメチルエチルケトン等に溶解し、重合開始剤を加えて、室温あるいは加温、またはＵＶ光で溶液重合させればよい。 Mixing the monomers at a mixing ratio according to the target lower critical temperature, for example, dissolving each monomer as a solvent, or a solvent having good solubility for the copolymer, such as methyl ethyl ketone, adding a polymerization initiator, Alternatively, solution polymerization may be performed by heating or UV light.
共重合の形態としては、ランダム共重合でもよく、ブロック共重合でもよい。下限臨界温度の親水、疎水変化をシャープにするためには、ランダム共重合が好ましい。 The form of copolymerization may be random copolymerization or block copolymerization. Random copolymerization is preferred in order to sharpen the hydrophilic and hydrophobic changes at the lower critical temperature.
これらの、また本発明に係るアクリル系樹脂を含有する温度応答性高分子においては、重合開始剤としては、アクリル系樹脂モノマーとアクリルアミドモノマーの重合であるため、ラジカルを発生する開始剤であることが好ましく、アゾ系開始剤、過酸化物系開始剤を用いることができる。 In these temperature-responsive polymers containing an acrylic resin according to the present invention, the polymerization initiator is a polymerization of an acrylic resin monomer and an acrylamide monomer, and is therefore an initiator that generates radicals. Are preferable, and azo initiators and peroxide initiators can be used.
油溶性の過酸化物系あるいはアゾ系開始剤が好ましく、一例を挙げると、例えば、過酸化ベンゾイル、過酸化ラウロイル、過酸化オクタノイル、オルソクロロ過酸化ベンゾイル、オルソメトキシ過酸化ベンゾイル、メチルエチルケトンパーオキサイド、ジイソプロピルパーオキシジカーボネート、キュメンハイドロパーオキサイド、シクロヘキサノンパーオキサイド、ｔ−ブチルハイドロパーオキサイド、ジイソプロピルベンゼンハイドロパーオキサイド等の過酸化物系開始剤、２，２′−アゾビスイソブチロニトリル、２，２′−アゾビス（２，４−ジメチルバレロニトリル）、２，２′−アゾビス（２，３−ジメチルブチロニトリル）、２，２′−アゾビス（２−メメチルブチロニトリル）、２，２′−アゾビス（２，３，３−トリメチルブチロニトリル）、２，２′−アゾビス（２−イソプロピルブチロニトリル）、１，１′−アゾビス（シクロヘキサン−１−カルボニトリル）、２，２′−アゾビス（４−メチキシ−２，４−ジメチルバレロニトリル）、２−（カルバモイルアゾ）イソブチロニトリル、４，４′−アゾビス（４−シアノバレリン酸）、ジメチル−２，２′−アゾビスイソブチレート等がある。 Oil-soluble peroxide-based or azo-based initiators are preferred. For example, benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, benzoyl peroxide, orthomethoxybenzoyl peroxide, methyl ethyl ketone peroxide, diisopropyl Peroxide initiators such as peroxydicarbonate, cumene hydroperoxide, cyclohexanone peroxide, t-butyl hydroperoxide, diisopropylbenzene hydroperoxide, 2,2'-azobisisobutyronitrile, 2,2 '-Azobis (2,4-dimethylvaleronitrile), 2,2'-azobis (2,3-dimethylbutyronitrile), 2,2'-azobis (2-methylbutyronitrile), 2,2' Azobis (2,3,3-trimethyl Butyronitrile), 2,2'-azobis (2-isopropylbutyronitrile), 1,1'-azobis (cyclohexane-1-carbonitrile), 2,2'-azobis (4-methoxy-2,4-dimethylvalero) Nitrile), 2- (carbamoylazo) isobutyronitrile, 4,4'-azobis (4-cyanovaleric acid), dimethyl-2,2'-azobisisobutyrate and the like.
特に、ターシャリイソブチルハイドロパーオキサイド、クメンハイドロパーオキサイド、パラメンタンハイドロパーオキサイドなどの有機過酸化物類、過酸化水素等が好ましい。 In particular, organic peroxides such as tertiary isobutyl hydroperoxide, cumene hydroperoxide, paramentane hydroperoxide, hydrogen peroxide, and the like are preferable.
またＵＶ硬化性開始剤も同様に重合開始剤として好ましく、例えば、アセトフェノン、アセトフェノンベンジルケタール、１−ヒドロキシシクロヘキシルフェニルケトン、２，２−ジメトキシ−２−フェニルアセトフェノン、キサントン、フルオレノン、ベンズアルデヒド、フルオレン、アントラキノン、トリフェニルアミン、カルバゾール、３−メチルアセトフェノン、４−クロロベンゾフェノン、４，４′−ジメトキシベンゾフェノン、４，４′−ジアミノベンゾフェノン、ミヒラーケトン、ベンゾインプロピルエーテル、ベンゾインエチルエーテル、ベンジルジメチルケタール、１−（４−イソプロピルフェニル）−２−ヒドロキシ−２−メチルプロパン−１−オン、２−ヒドロキシ−２−メチル−１−フェニルプロパン−１−オン等の光ラジカル開始剤等が挙げられる。 Similarly, UV curable initiators are also preferred as polymerization initiators, such as acetophenone, acetophenone benzyl ketal, 1-hydroxycyclohexyl phenyl ketone, 2,2-dimethoxy-2-phenylacetophenone, xanthone, fluorenone, benzaldehyde, fluorene, anthraquinone. , Triphenylamine, carbazole, 3-methylacetophenone, 4-chlorobenzophenone, 4,4'-dimethoxybenzophenone, 4,4'-diaminobenzophenone, Michler's ketone, benzoin propyl ether, benzoin ethyl ether, benzyldimethyl ketal, 1- ( 4-Isopropylphenyl) -2-hydroxy-2-methylpropan-1-one, 2-hydroxy-2-methyl-1-phenylpropan-1-one, etc. Photoradical initiators, and the like.
これら重合開始剤は、モノマーに対して、０．０１〜２０質量％、特に、０．１〜１０質量％使用されるのが好ましい。 These polymerization initiators are preferably used in an amount of 0.01 to 20% by mass, particularly 0.1 to 10% by mass, based on the monomer.
本発明に係る共重合体の製造において反応の場としては、有機溶媒または水を使ってもよく、または使わなくてもよい。後工程で溶媒を除く必要があるため、好ましくは、溶媒を使用しない方がよい。有機溶媒としては、メタノール、エタノール、イソプロピルアルコール等のアルコール類、酢酸エチル、酢酸メチル等のエステル類、メチルエチルケトン、アセトン等のケトン類、エーテル、イソプロピルエーテル等エーテル類、またテトラヒドロフラン、ジオキサン等の環状エーテル類、あるいは芳香族炭化水素であるトルエン等特に制限はないが、原料となるモノマーに対しまた生成する共重合体に対し、溶解性の高い溶媒を選択して用いることが好ましい。 In the production of the copolymer according to the present invention, an organic solvent or water may or may not be used as a reaction field. Since it is necessary to remove the solvent in a later step, it is preferable not to use a solvent. Examples of the organic solvent include alcohols such as methanol, ethanol and isopropyl alcohol, esters such as ethyl acetate and methyl acetate, ketones such as methyl ethyl ketone and acetone, ethers such as ether and isopropyl ether, and cyclic ethers such as tetrahydrofuran and dioxane. Although there is no particular limitation such as toluene, which is an aromatic hydrocarbon, or the like, it is preferable to select and use a highly soluble solvent for the monomer as a raw material and for the copolymer to be formed.
重合温度が余り低くならない様に、溶媒の沸点としては５０℃以上が好ましく、７０℃以上がさらに好ましい。しかしながら、１５０℃以上と高くなると、その後の取り扱いに工数を要するので、１５０℃以下であることが好ましい。 The boiling point of the solvent is preferably 50 ° C. or higher and more preferably 70 ° C. or higher so that the polymerization temperature does not become too low. However, if it becomes as high as 150 ° C. or higher, man-hours are required for subsequent handling.
前記共重合反応において、重合後、最終的には、固形分濃度が１０質量％以上４０質量％以下であることが好ましく、また、最終的な共重合体を含む溶液の粘度が固形分３０質量％換算で１０ｍＰａ・ｓ以上５００ｍＰａ・ｓ以下となる重合度であることが好ましい。 In the copolymerization reaction, after polymerization, the solid content concentration is preferably 10% by mass or more and 40% by mass or less, and the viscosity of the final solution containing the copolymer is 30% by mass. The degree of polymerization is preferably 10 mPa · s or more and 500 mPa · s or less in terms of%.
本発明に係る共重合反応においては、残モノマー量を１質量％以下とし、反応を終了させる。この測定は、ガスクロマトグラフにて行う。 In the copolymerization reaction according to the present invention, the residual monomer amount is set to 1% by mass or less, and the reaction is terminated. This measurement is performed with a gas chromatograph.
共重合ポリマーを含有する反応液は、貧溶媒と混合し、析出させ、さらに、溶解、析出を繰り返し、固形分として、単離することができる。 The reaction liquid containing the copolymer can be mixed with a poor solvent and precipitated, and further, dissolution and precipitation can be repeated and isolated as a solid content.
本発明に用いられる温度応答性高分子の具体例としては実施例中、Ｎｏ．Ｅ〜Ｌ等で表されるポリマーが挙げられる。 Specific examples of the temperature-responsive polymer used in the present invention include No. 1 in the Examples. Examples thereof include polymers represented by E to L and the like.
本発明の細胞培養基材に上記温度応答性高分子を塗布する際には、その特性を改良する目的で、重合時に公知慣用の有機架橋剤を使用してもよい。使用する有機架橋剤濃度は特に限定されず、目的に応じて選択できる。使用できる有機架橋剤としては、従来から公知のＮ，Ｎ′−メチレンビスアクリルアミド、Ｎ，Ｎ′−プロピレンビスアクリルアミド、ジ（アクリルアミドメチル）エーテル、１，２−ジアクリルアミドエチレングリコール、１，３−ジアクリロイルエチレンウレア、エチレンジアクリレート、Ｎ，Ｎ′−ジアリルタータルジアミド、Ｎ，Ｎ′−ビスアクリリルシスタミンなどの二官能性化合物や、トリアリルシアヌレート、トリアリルイソシアヌレートなどの三官能性化合物が例示できる。 When the temperature-responsive polymer is applied to the cell culture substrate of the present invention, a known and commonly used organic crosslinking agent may be used during polymerization for the purpose of improving the characteristics. The concentration of the organic crosslinking agent to be used is not particularly limited and can be selected according to the purpose. Examples of the organic crosslinking agent that can be used include conventionally known N, N′-methylenebisacrylamide, N, N′-propylenebisacrylamide, di (acrylamidomethyl) ether, 1,2-diacrylamide ethylene glycol, 1,3- Bifunctional compounds such as diacryloylethyleneurea, ethylene diacrylate, N, N'-diallyl tartaramide, N, N'-bisacrylylcystamine, and trifunctional compounds such as triallyl cyanurate and triallyl isocyanurate Can be exemplified.
（細胞培養支持体について）
本発明にかかわる細胞培養支持体の基材としては各種高分子材料、ガラス、改質ガラス、ウール布、コットン布、紙、金属（例えばアルミニウム）等が挙げられる。取り扱い上の観点より本発明の細胞培養支持体における基材としては、プラスチック材料（例えば、セルロースアセテート、ポリエステル、ポリエチレンテレフタレート、ポリエチレンナフタレート、ポリアミド、ポリイミド、セルローストリアセテートまたはポリカーボネート、ポリスチレン、ポリメチルメタクリレート等）が好ましく、本発明においてはポリスチレンが特に好ましい。支持体の厚みとしては５０〜３０００μｍ程度、好ましくは７０〜１８００μｍである。(About cell culture supports)
Examples of the base material for the cell culture support according to the present invention include various polymer materials, glass, modified glass, wool cloth, cotton cloth, paper, metal (for example, aluminum) and the like. From the viewpoint of handling, as a substrate in the cell culture support of the present invention, a plastic material (for example, cellulose acetate, polyester, polyethylene terephthalate, polyethylene naphthalate, polyamide, polyimide, cellulose triacetate or polycarbonate, polystyrene, polymethyl methacrylate, etc. And polystyrene is particularly preferred in the present invention. The thickness of the support is about 50 to 3000 μm, preferably 70 to 1800 μm.
細胞培養用の基材は、温度応答性高分子を付着し易くさせるため基材表面にグロー放電、コロナ放電、真空プラズマ処理、大気圧プラズマ処理またはシランカップリング処理などで表面処理を施されていてもよい。 The base material for cell culture is surface-treated by glow discharge, corona discharge, vacuum plasma treatment, atmospheric pressure plasma treatment or silane coupling treatment on the surface of the base material in order to make the temperature-responsive polymer easy to adhere. May be.
（温度応答性高分子の被覆方法）
本発明の温度応答性高分子を基材の表面に被覆する方法としては、基材にモノマーで塗布して、重合する方法と、あらかじめモノマーを重合して温度応答性高分子にしてから塗布する方法がある。どちらの方法でもよいが、パターニングの観点からモノマーで基材へ塗布後、重合する方が好ましい。(Temperature-responsive polymer coating method)
As a method of coating the surface of the substrate with the temperature-responsive polymer of the present invention, a method in which the substrate is coated with a monomer and polymerized, and a method in which the monomer is polymerized in advance to form a temperature-responsive polymer are applied. There is a way. Either method may be used, but from the viewpoint of patterning, it is preferable to polymerize after coating on a substrate with a monomer.
基材上に温度応答性高分子、またはそのモノマーを塗布する方法には特に制限はなく、例えばバーコーター法、カーテンコート法、浸漬法、エアーナイフ法、ホッパー塗布法、リバースロール塗布法、グラビア塗布法、エクストリュージョン塗布法、真空蒸着法（スパッタリング法）等の公知の方法を用いることができる。また２種類の温度応答性高分子を細胞培養基材の支持体上にパターニングする際には、リソグラフィー法、インクジェット方法、スーパーインクジェット方法で塗布することが好ましい。 There is no particular limitation on the method for applying the temperature-responsive polymer or its monomer on the substrate, for example, bar coater method, curtain coating method, dipping method, air knife method, hopper coating method, reverse roll coating method, gravure. Known methods such as a coating method, an extrusion coating method, and a vacuum deposition method (sputtering method) can be used. Moreover, when patterning two types of temperature-responsive polymers on the support of the cell culture substrate, it is preferable to apply them by a lithography method, an inkjet method, or a super inkjet method.
温度応答性高分子を基材上へ固定化する技術としては、熱による硬化、またはＵＶ照射による硬化のどちらを採用してもよい。 As a technique for fixing the temperature-responsive polymer on the substrate, either heat curing or UV irradiation curing may be employed.
反応が早いという観点でＵＶ照射による硬化が好ましく、ＵＶ光を用いる場合は、光源として、例えば、０．１ｋＰａ〜１ＭＰａまでの動作圧力を有する低圧、中圧、高圧水銀ランプ、メタルハライドランプや紫外域の発光波長を持つキセノンランプ、冷陰極管、熱陰極管、ＬＥＤ等従来公知のものが用いられる。 Curing by UV irradiation is preferable from the viewpoint of fast reaction, and when UV light is used, the light source is, for example, a low pressure, medium pressure, high pressure mercury lamp, metal halide lamp or ultraviolet region having an operating pressure of 0.1 kPa to 1 MPa. Conventionally known lamps such as xenon lamps, cold cathode fluorescent lamps, hot cathode fluorescent lamps, and LEDs having a light emission wavelength of 2 are used.
なお、本発明において、温度応答性高分子の塗布量は、細胞培養の目的に応じた適量を選ぶことが好ましいが、細胞を培養し、その後、細胞を基材から剥離するという目的の場合には、０．１μｇ／ｃｍ２以上、５．０μｇ／ｃｍ２以下が好ましく、０．５μｇ／ｃｍ２以上、３．０μｇ／ｃｍ２以下がより好ましい。In the present invention, the application amount of the temperature-responsive polymer is preferably selected in accordance with the purpose of cell culture, but for the purpose of culturing cells and then peeling the cells from the substrate. is, 0.1 [mu] g / cm 2 or more, preferably 5.0 [mu] g / cm 2 or less, 0.5 [mu] g / cm 2 or more, 3.0 [mu] g / cm 2 or less being more preferred.
（温度応答性高分子の細胞培養基材へのパターニングについて）
本発明は、同一層内に異なる領域を被覆した２種類以上の温度応答性高分子上で２種類以上の細胞を異なる領域で培養できればよいが、細胞を積層する際には、各層間の細胞パターンが同じであった方が組織的な機能発現という点で好ましい。そのためには、温度応答性高分子が基材へパターニングされた細胞培養支持体の方が好ましい。(Patterning of temperature-responsive polymer to cell culture substrate)
In the present invention, it is sufficient that two or more types of cells can be cultured in different regions on two or more types of temperature-responsive polymers in which different regions are coated in the same layer. The same pattern is preferable in terms of systematic function expression. For this purpose, a cell culture support in which a temperature-responsive polymer is patterned on a substrate is preferred.
本発明に係る温度応答性高分子を基材の表面に所定パターンで付加する際には、温度応答性高分子を構成する材料を液状化して付加することが好ましい。液状化した温度応答性高分子を構成する材料を所定のパターンで付加する方法としては、スクリーン印刷法やインクジェット法などの印刷方法を用いることが好ましい。このような印刷方法を用いると、より簡単に温度応答性高分子を構成する材料を細胞培養する基材の表面に任意のパターンで付加することができる。また、所定のパターンに穿孔したマスクを作製し、これを基材表面上に設置した後、温度応答性高分子を構成する材料を含有した溶液をスプレー塗布する方法を採用することもできる。 When the temperature-responsive polymer according to the present invention is added to the surface of the substrate in a predetermined pattern, it is preferable to add the material constituting the temperature-responsive polymer in a liquefied state. As a method for adding the material constituting the liquefied temperature-responsive polymer in a predetermined pattern, it is preferable to use a printing method such as a screen printing method or an ink jet method. By using such a printing method, the material constituting the temperature-responsive polymer can be more easily added in an arbitrary pattern to the surface of the substrate for cell culture. Alternatively, a method may be employed in which a mask perforated in a predetermined pattern is prepared and placed on the substrate surface, and then a solution containing a material constituting the temperature-responsive polymer is spray applied.
なお、本発明の細胞培養支持体を用いて血管や神経系などの複雑で個体差のあるパターンを形成する場合、特に、個別対応性の高いインクジェット法が好ましい。それ以外の方法としては、スクリーン印刷方法、スプレー塗布方法でも可能であるが、複雑なパターンを形成しようとした場合、隣接する異種の温度応答性高分子間でにじみによる混合、重なり合い、境界線ですきまが開いてしまうなどの問題を起こしやすい。この観点からも解像度の高いインクジェット方法がより好ましい。 In addition, when a complicated and individual pattern such as a blood vessel or nervous system is formed using the cell culture support of the present invention, an inkjet method with high individual correspondence is particularly preferable. As other methods, screen printing method and spray coating method are also possible, but when complex patterns are to be formed, mixing, overlapping, and boundary lines due to blurring between adjacent different types of temperature-responsive polymers Prone to problems such as opening gaps. From this point of view, an inkjet method having a high resolution is more preferable.
また、温度応答性高分子を構成する材料を液状にする方法としては、温度応答性高分子を構成する材料を加熱して軟化させる方法や、温度応答性高分子を構成する材料を所定の溶媒に溶解する方法などが挙げられるが、温度調整を行う必要のない点から溶媒に溶解する方法が好適である。 In addition, as a method for making the material constituting the temperature-responsive polymer into a liquid state, the material constituting the temperature-responsive polymer is heated and softened, or the material constituting the temperature-responsive polymer is changed to a predetermined solvent. Although the method etc. which melt | dissolve are mentioned, the method of melt | dissolving in a solvent from the point which does not need to perform temperature adjustment is suitable.
またパターニングできる線幅としては、１μｍ以上１０００μｍ以下が好ましく、さらに好ましくは、３μｍ以上１００μｍ以下が好ましい。 The line width that can be patterned is preferably 1 μm or more and 1000 μm or less, more preferably 3 μm or more and 100 μm or less.
（細胞培養支持体上で培養される細胞とその積層化について）
本発明の細胞培養支持体上で培養される細胞は温度応答性高分子の下限臨界温度以下にし、支持体表面を親水化することで培養された細胞を剥離する。この剥離された細胞を重ね合わせるにあたり、細胞が概ね２〜２００層程度、特に５〜１００層程度重なり合った積層体が得られるように、細胞数を調整することが望ましい。積層体において細胞が多数重なりすぎている場合には積層体の中央層部の細胞が栄養不足、ガス交換不足になり、細胞の重なりが少なすぎる場合は細胞数が少なく十分な機能を発揮する積層体が形成され難い。上記範囲であればこのような問題は生じない。(About cells cultured on cell culture supports and their lamination)
The cells cultured on the cell culture support of the present invention are brought to a temperature lower than the lower critical temperature of the temperature-responsive polymer, and the cultured cells are detached by hydrophilizing the support surface. In stacking the peeled cells, it is desirable to adjust the number of cells so that a laminated body in which the cells are overlapped by about 2 to 200 layers, particularly 5 to 100 layers is obtained. If there are too many cells in the laminate, the cells in the central layer of the laminate will be undernutrition and gas exchange will be insufficient. The body is difficult to form. If it is the said range, such a problem will not arise.
本発明の細胞培養支持体は、２種類以上の下限臨界温度をもった温度応答性高分子で表面を加工しているため、それに合わせる形で２種類以上の異なった細胞を同一層内に共培養できる。この細胞のうち少なくとも１つを血管内皮細胞等の血管再生能力を秘めた細胞にすることで、積層数を増やしても細胞が栄養不足、ガス交換不足になりにくく、本発明の目的である多種類の細胞の分化機能を精密且つ厳密に発現、さらに組織としての機能を維持するこのできる細胞の培養方法が確立できることとなった。 Since the surface of the cell culture support of the present invention is processed with a temperature-responsive polymer having two or more kinds of lower critical temperatures, two or more kinds of different cells are co-located in the same layer. Can be cultured. By making at least one of these cells a cell having an ability to regenerate blood vessels such as vascular endothelial cells, the cells are less likely to be insufficient in nutrients and gas exchange even if the number of layers is increased, which is the object of the present invention. It has become possible to establish a cell culture method capable of precisely and precisely expressing the differentiation function of various types of cells and maintaining the tissue function.
積層体の細胞数は、例えば積層体の載った透過性膜を切り出し、これをホルマリン固定後、パラフィン包埋切片を作製して顕微鏡観察することにより確認することができる。 The number of cells in the laminate can be confirmed by, for example, cutting out a permeable membrane on which the laminate is mounted, fixing this with formalin, preparing a paraffin-embedded section, and observing under a microscope.
本発明方法の対象となる細胞としては、特に限定されるものではないが、接着性の動物細胞が好適である。細胞の由来も特に限定されず、ヒト、マウス、ラット等のいずれの動物由来のものも使用できる。また、接着性の動物細胞は、初代培養細胞および株化細胞の双方を対象とすることができる。 Although it does not specifically limit as a cell used as the object of this invention method, Adhesive animal cell is suitable. The origin of the cell is not particularly limited, and those derived from any animal such as human, mouse, rat and the like can be used. Adhesive animal cells can target both primary cultured cells and established cells.
本発明方法は、特に、細胞の機能維持が困難な初代培養細胞の培養に適する。初代培養細胞は、軟骨、骨、皮膚、神経、口腔、消化管、肝臓、膵臓、腎臓、腺組織、副腎、心臓、筋肉、腱、脂肪組織、結合組織、生殖器、眼球、血管、骨髄または血液のいずれの組織に由来するものであってもよい。細胞培養支持体表面の温度応答性高分子の種類１つに対して１つの細胞を藩種することが適しており、細胞は、単一組織に由来する単一種類の細胞を用いることもできる。複数の温度応答性高分子が表面に存在している場合は、その数だけ異なる複数種の細胞を用いることもできる。 The method of the present invention is particularly suitable for culturing primary cultured cells in which cell function maintenance is difficult. Primary cultured cells are cartilage, bone, skin, nerve, oral cavity, digestive tract, liver, pancreas, kidney, glandular tissue, adrenal gland, heart, muscle, tendon, adipose tissue, connective tissue, genital organ, eyeball, blood vessel, bone marrow or blood It may be derived from any of these tissues. It is suitable to seed one cell for one kind of temperature-responsive polymer on the surface of the cell culture support, and a single kind of cell derived from a single tissue can be used as the cell. . When a plurality of temperature-responsive polymers are present on the surface, a plurality of different types of cells can be used.
具体的には、例えば、軟骨細胞、骨芽細胞、表皮角化細胞、メラニン細胞、神経細胞、神経幹細胞、グリア細胞、肝細胞、腸上皮細胞、膵β細胞、膵外分泌細胞、腎糸球体内皮細胞、尿細管上皮細胞、乳腺細胞、甲状腺細胞、唾液腺細胞、副腎皮質細胞、副腎髄質細胞、心筋細胞、骨格筋細胞、平滑筋細胞、脂肪細胞、脂肪前駆細胞、水晶体細胞、角膜細胞、血管内皮細胞、骨髄間質細胞またはリンパ球などを使用できる。細胞は、一種を単独でまたは二種以上を組み合わせて用いることができる。 Specifically, for example, chondrocytes, osteoblasts, epidermal keratinocytes, melanocytes, neurons, neural stem cells, glial cells, hepatocytes, intestinal epithelial cells, pancreatic β cells, pancreatic exocrine cells, renal glomerular endothelium Cells, tubular epithelial cells, breast cells, thyroid cells, salivary gland cells, adrenal cortex cells, adrenal medullary cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, adipocytes, adipose precursor cells, lens cells, corneal cells, vascular endothelium Cells, bone marrow stromal cells or lymphocytes can be used. Cells can be used singly or in combination of two or more.
このようにして得られた細胞培養物は、例えば医用生体材料用の細胞として使用できる。本発明において、再生医用生体材料とは、ヒト等の動物の組織の代替物として使用される材料をいう。再生医用生体材料としては、培養された細胞の種類に応じて、人工膵臓、人工脾臓、人工腎臓、人工心臓のような人工臓器、人工消化管、人工血管、人工皮膚、人工神経、人工骨、人工軟骨、人工内耳、人工水晶体、人工角膜など、またはこれらの一部分が挙げられる。 The cell culture thus obtained can be used as cells for medical biomaterials, for example. In the present invention, the regenerative medical biomaterial refers to a material used as a substitute for tissue of animals such as humans. Regenerative medical biomaterials include artificial pancreas, artificial spleen, artificial kidney, artificial organs like artificial heart, artificial digestive tract, artificial blood vessel, artificial skin, artificial nerve, artificial bone, depending on the type of cultured cells Examples include artificial cartilage, cochlear implant, artificial lens, artificial cornea, etc., or a part thereof.
またヒト等の動物の組織の代替物として、実験用動物代替細胞、抗癌剤感受性試験、創薬支援等に使用される場合も再生医用生体材料に含まれる。 In addition, as a substitute for tissue of animals such as humans, biomedical materials for regenerative medicine are also used for experimental animal substitute cells, anticancer drug sensitivity tests, drug discovery support, and the like.
以下、実施例を挙げて本発明を詳細に説明するが、本発明はこれらに限定されない。なお、特に断りない限り、実施例中の「％」は「質量％」を示す。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these. Unless otherwise specified, “%” in the examples represents “mass%”.
（１）試料の作製
（１．１）温度応答性高分子Ａ〜Ｌの合成（比較例、本発明）
０．５リットルの四つ口セパラブルフラスコに滴下装置、温度計、窒素ガス導入管、攪拌装置および還流冷却管を付し、メチルエチルケトン（ＭＥＫ）５０ｇ、および表１に記載の組成割合でＮＩＰＡＭ以外のモノマー（単位ｇ）、を仕込み、８０℃に加熱した。さらに表１に記載のＮＩＰＡＭモノマー（単位ｇ）をメチルエチルケトン４３ｇに溶解しさらにラウリルパーオキサイド０．１２ｇを溶解した液を、フラスコ中に２時間かけて滴下した。その後１時間かけて昇温し還流状態になった時点で、ラウリルパーオキサイド０．１７ｇをメチルエチルケトン３３ｇに溶解した液をフラスコ中に２時間かけて滴下し、同温度にてさらに３時間反応させた。その後メチルハイドロキノン０．３３ｇをメチルエチルケトン１０７ｇに溶解した液を添加し冷却後、ポリマー含有量として３０質量％のポリマー溶液Ａ〜Ｌをそれぞれ得た。分子量は、ＧＰＣでポリスチレン換算の重量平均分子量として求めた。(1) Preparation of sample (1.1) Synthesis of temperature-responsive polymers A to L (comparative example, the present invention)
A 0.5 liter four-necked separable flask is equipped with a dropping device, a thermometer, a nitrogen gas introduction tube, a stirring device and a reflux condenser, and 50 g of methyl ethyl ketone (MEK) and other than NIPAM at the composition ratio shown in Table 1 The monomer (unit g) was charged and heated to 80 ° C. Further, a solution obtained by dissolving NIPAM monomer (unit g) shown in Table 1 in 43 g of methyl ethyl ketone and further dissolving 0.12 g of lauryl peroxide was dropped into the flask over 2 hours. Thereafter, when the temperature was raised over 1 hour and the mixture was refluxed, a solution obtained by dissolving 0.17 g of lauryl peroxide in 33 g of methyl ethyl ketone was dropped into the flask over 2 hours and reacted at the same temperature for further 3 hours. . Thereafter, a solution obtained by dissolving 0.33 g of methyl hydroquinone in 107 g of methyl ethyl ketone was added and cooled, and polymer solutions A to L having a polymer content of 30% by mass were obtained. Molecular weight was calculated | required as a weight average molecular weight of polystyrene conversion by GPC.
以下において、
ブレンマーＰＭＥ−４００：−（ＥＯ）ｍ−ＣＨ３（ｍ≒９）を有するメタアクリレートブレンマーＰＳＥ−４００：−（ＥＯ）ｍ−Ｃ１８Ｈ３７（ｍ≒９）を有するメタアクリレート
（ＥＯ；エチレンオキシ基）
上記はすべて日本油脂製。
ＮＩＰＡＭ：Ｎ−イソプロピルアクリルアミド（興人製）
ＤＥＡＡ：Ｎ−ジエチルアクリルアミド（興人製）
ＤＡＡＭ：ダイアセトンアクリルアミド（協和発酵製）
ＢＭＡ：ブチルメタクリレート（東京化成品）
（１．２）温度応答性高分子Ａ〜Ｌの下限臨界温度評価
上記（１．１）で作製されたポリマー／ＭＥＫ溶液の３００ｇを室温で蒸留水１０００ｇに徐々に添加したのち（ポリマー溶液Ｃ及びＤ以外は均一溶液を構成している）、温度を４５℃にあげると、析出するので、これを分離し、４０℃の温水で充分に洗浄し、残モノマーを除いた。その後、単離したポリマーを２５℃の純水に溶解させ１０質量％濃度のポリマー溶液を作製した。その後溶液の温度を上げてゆき、ポリマーが析出した温度を下限臨界温度とした。結果を表１に記載した。ただし、ポリマー溶液Ｃ及びＤは２５℃の純水に溶解せず、既にポリマーが析出しているため、下限臨界温度はなしとした。In the following,
Blemmer PME-400 :-( EO) m -CH 3 (m methacrylate BLEMMER PSE-400 having ≒ 9) :-( EO) m -C 18 H 37 (m ≒ 9) methacrylate having an (EO; Ethyleneoxy group)
The above are all made from Japanese fats and oils.
NIPAM: N-isopropylacrylamide (manufactured by Kojin)
DEAA: N-diethylacrylamide (manufactured by Kojin)
DAAM: Diacetone acrylamide (Kyowa Hakko)
BMA: Butyl methacrylate (Tokyo Chemicals)
(1.2) Evaluation of lower limit critical temperature of temperature-responsive polymers A to L After gradually adding 300 g of the polymer / MEK solution prepared in (1.1) above to 1000 g of distilled water at room temperature (polymer solution C A uniform solution is formed except for D and D), and when the temperature is raised to 45 ° C., it precipitates. This was separated and washed thoroughly with warm water of 40 ° C. to remove residual monomers. Thereafter, the isolated polymer was dissolved in pure water at 25 ° C. to prepare a polymer solution having a concentration of 10% by mass. Thereafter, the temperature of the solution was raised, and the temperature at which the polymer was precipitated was defined as the lower critical temperature. The results are shown in Table 1. However, since the polymer solutions C and D were not dissolved in pure water at 25 ° C. and the polymer had already precipitated, the lower critical temperature was set to none.
表１より、アクリル樹脂の共重合組成比を変化させることで、細胞が培養しやすい体温付近で下限臨界温度を生じるポリマーの下限臨界温度を変化させることが達成できた。 From Table 1, by changing the copolymer composition ratio of the acrylic resin, it was possible to change the lower critical temperature of the polymer that generates the lower critical temperature around the body temperature at which cells are easy to culture.
（１．３）モノマーのインクジェット塗布と重合方法
上記（１．１）で作製したポリマー／ＭＥＫ（３０質量％溶液）と同じ組成になるように、各モノマーを以下表２記載の組成比にして同じく固形分３０質量％になるようにＭＥＫ溶液に溶解させた。また、（１．１）記載のラウリルパーオキサイドの代わりにＵＶ重合開始剤としてＩＲＧＡＣＵＲＥ１８４（チバ・ジャパン製）をモノマーに対して１質量％含有させた。(1.3) Inkjet Application of Monomers and Polymerization Method Each monomer is made to have the composition ratio shown in Table 2 below so as to have the same composition as the polymer / MEK (30% by mass solution) prepared in (1.1) above. Similarly, it was dissolved in the MEK solution so as to have a solid content of 30% by mass. Further, instead of lauryl peroxide described in (1.1), IRGACURE 184 (manufactured by Ciba Japan) as a UV polymerization initiator was contained in an amount of 1% by mass based on the monomer.
さらに表３記載の種類のモノマー／ＭＥＫ溶液（表２記載のＭ〜Ｘ）をインクの代わりに表３記載のインクタンク−１、インクタンク−２に充填し、ノズル口径２５μｍ、駆動周波数１２ｋＨｚ、ノズル数１２８、ノズル密度１８０ｄｐｉ（本発明でいうｄｐｉとは、２．５４ｃｍ当たりのドット数を表す）であるピエゾ型記録ヘッドを用い、最大記録密度７２０×７２０ｄｐｉのオンデマンド型のインクジェットプリンタを使用し、市販のポリスチレン製細胞培養皿（ベクトン・ディッキンソン・ラブウェア（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ Ｌａｂｗａｒｅ）社製 ファルコン（ＦＡＬＣＯＮ）３００１ペトリディッシュ（直径３．５ｃｍ））上に、表３記載のＭ〜Ｘ溶液のうち片方が幅１００μｍの細線になるようそれぞれプリントした。 Further, the monomer / MEK solution of the type shown in Table 3 (M to X shown in Table 2) is filled into the ink tank-1 and ink tank-2 shown in Table 3 instead of the ink, and the nozzle diameter is 25 μm, the driving frequency is 12 kHz, Using an on-demand type ink jet printer with a maximum recording density of 720 × 720 dpi using a piezo type recording head having 128 nozzles and a nozzle density of 180 dpi (dpi in the present invention represents the number of dots per 2.54 cm). On the commercially available polystyrene cell culture dish (Falcon 3001 Petri dish (diameter: 3.5 cm) manufactured by Becton Dickinson Labware) Each one is printed so that it becomes a thin line with a width of 100μm .
各インクを連続吐出し、着弾した後０．１秒後に、１２０Ｗ／ｃｍメタルハライドランプ（日本電池社製 ＭＡＬ ４００ＮＬ、電源電力３ｋＷ・ｈｒ）を照射し、モノマーを重合させた。 Each ink was continuously ejected, and 0.1 seconds after landing, a 120 W / cm metal halide lamp (MAL 400NL manufactured by Nippon Battery Co., Ltd., power supply power 3 kW · hr) was irradiated to polymerize the monomer.
これにより温度応答性高分子をポリスチレン製細胞培養皿表面に吸着させた。 As a result, the temperature-responsive polymer was adsorbed on the surface of the polystyrene cell culture dish.
その後、残モノマーと溶媒であるＭＥＫを取り除くために、大量の水で洗浄し、細胞培養支持体とした。 Thereafter, in order to remove residual monomers and MEK as a solvent, the cells were washed with a large amount of water to obtain a cell culture support.
（１．４）コントロールの重合
表３記載のＮｏ．１８は、コントロールとして、インクジェット塗布の代わりに、表２記載Ｍ溶液を０．１ｍｌディッシュに滴下し、塗布液が水平になるのをまって（１．３）同様の方法で重合させた。(1.4) Control polymerization No. As a control, instead of inkjet coating, the M solution described in Table 2 was dropped into a 0.1 ml dish, and the coating solution was leveled (1.3) and polymerized in the same manner.
（１．５）重合済みポリマーをインクジェットで塗布
表３記載のＮｏ．１９〜２５には、表１記載のポリマー／ＭＥＫ溶液（表３記載の種類）を用いて、（１．３）のモノマー／ＭＥＫ溶液に置き換えて用いた以外は同様の方法で吸着させた。(1.5) Application of polymerized polymer by ink jet No. 1 in Table 3 19 to 25 were adsorbed in the same manner except that the polymer / MEK solution shown in Table 1 (type shown in Table 3) was used instead of the monomer / MEK solution of (1.3).
（１．６）スクリーン印刷方法による塗布、重合
表３記載のＮｏ．２６〜３１には、（１．３）記載のインクジェット塗布と同様のパターンになるように幅１００μｍの細線のマスクを用いて、表３記載の種類のモノマー／ＭＥＫ溶液（表２記載のＭ〜Ｘで作成）を塗りわけ、（１．３）記載のインクジェット塗布と同様のパターンニングを行った。その後０．１秒後に、１２０Ｗ／ｃｍメタルハライドランプ（日本電池社製 ＭＡＬ ４００ＮＬ、電源電力３ｋＷ・ｈｒ）を照射し、モノマーを重合させた。(1.6) Application and polymerization by screen printing method Nos. 26 to 31 were prepared by using a mask of a fine line having a width of 100 μm so as to have the same pattern as that of the ink jet coating described in (1.3). (Prepared with X), and the same patterning as the inkjet coating described in (1.3) was performed. 0.1 seconds later, a monomer was polymerized by irradiation with a 120 W / cm metal halide lamp (MAL 400NL, manufactured by Nippon Battery Co., Ltd., power supply power 3 kW · hr).
これにより温度応答性高分子をポリスチレン製細胞培養皿表面に吸着させた。 As a result, the temperature-responsive polymer was adsorbed on the surface of the polystyrene cell culture dish.
その後、残モノマーと溶媒であるＭＥＫを取り除くために、大量の水で洗浄し、細胞培養支持体とした。 Thereafter, in order to remove residual monomers and MEK as a solvent, the cells were washed with a large amount of water to obtain a cell culture support.
（１．７）細胞の培養
上記作製した細胞培養支持体に培養細胞を播種して細胞の培養を行った。培養する細胞は、パターニングされた温度応答性高分子表面の下限臨界温度違いを利用して、表３記載の細胞をパターニングさせた。培養は、ウシ胎児血清（ＩＣＮ製）を１０％含有するミニマム・エッセンシャル・イーグル培地（ＳＩＧＭＡ製）（ピルビン酸（ＩＣＮ製）および非必須アミノ酸（ＩＣＮ製）を添加剤として含有）使用し、５％炭酸ガス充填３７℃恒温器内で行った。(1.7) Cell Culture The cultured cells were seeded on the cell culture support prepared above, and the cells were cultured. The cells to be cultured were patterned using the difference in the lower critical temperature on the surface of the patterned temperature-responsive polymer surface. The culture is performed using minimum essential eagle medium (manufactured by SIGMA) containing 10% fetal bovine serum (manufactured by ICN) (containing pyruvic acid (manufactured by ICN) and non-essential amino acids (manufactured by ICN) as additives). It was performed in a 37 ° C. incubator filled with% carbon dioxide gas.
播種してから１週間後、この細胞培養アレイを、２０℃恒温槽内に５分間静置してから、表面を光学顕微鏡にて観察したところ、細胞が細胞培養アレイ上にパターニングされて接着して、また十分に増殖していたことが確認された。この取り出した細胞についてトリプシン−ＥＤＴＡ処理を行い、各細胞を個々の状態に分離した後、トリパンブルー染色を行うことによって、生細胞数を計測したところ、表３記載Ｎｏ．１８の培養開始時には８．２×１０２個であった細胞数が、培養後は５．３×１０３個に増加したことが確認された。この細胞培養後の値を１００％として、以下の結果を表３に記載した。本発明は、比較に対して培養効率がよいことが確認できた。One week after seeding, the cell culture array was allowed to stand in a 20 ° C. constant temperature bath for 5 minutes and then the surface was observed with an optical microscope. As a result, the cells were patterned on the cell culture array and adhered. In addition, it was confirmed that the cells had proliferated sufficiently. The extracted cells were treated with trypsin-EDTA, and each cell was separated into individual states, followed by trypan blue staining to measure the number of viable cells. It was confirmed that the number of cells, which was 8.2 × 10 2 at the start of 18 cultures, increased to 5.3 × 10 3 after the culture. The following results are shown in Table 3 with the value after the cell culture as 100%. In the present invention, it was confirmed that the culture efficiency was good for comparison.
（１．８）ブリード耐性
上記細胞を目視観察し、下記の基準に従って細胞のブリード耐性の評価を行った。
◎：細線とベタの境界線がはっきりしている
○：わずかに境界がにじんでいる箇所があるが、実用上問題のない品質である
△：境界部ににじみが認められるが、実用上許容限界内の品質である
×：境界部で明らかなにじみの発生が認められ、線幅が１．５倍ほどとなり、実用上問題となる品質である
××：細線とベタ部の境界が不明瞭な品質であり、ブリード耐性が極めて乏しい
（１．９）細胞の積層
上記（１．７）で作成した細胞で培養１４日後、培養した細胞の上に直径３．５ｃｍのポリビニリデンジフルオライド（ＰＶＤＦ）膜をかぶせ、培地を静かに吸引し、細胞培養支持体材料ごと２０℃で３０分インキュベートし冷却することで、いずれの細胞培養支持体材料上の細胞もそのかぶせた膜と共に剥離させられた。かぶせた膜と細胞を同様に作製した細胞培養支持体材料上で正常に増殖した同一の培養細胞の上にかぶせて、５％炭酸ガス充填３７℃恒温器内で２枚を接着させた。２枚の細胞シートが接着した後、ＰＶＤＦ膜を剥離した。同様の操作を繰り返すことで２０層の細胞シートを作成した。(1.8) Bleed resistance The cells were visually observed, and the bleed resistance of the cells was evaluated according to the following criteria.
◎: The boundary between the fine line and the solid is clear. ○: There is a part where the boundary is slightly blurred, but there is no problem in practical use. △: The boundary is blurred but is practically acceptable. X: The occurrence of clear bleeding at the boundary is recognized, the line width is about 1.5 times, and this is a quality that is a problem in practice. XX: The boundary between the thin line and the solid part is unclear (1.9) Lamination of cells 14 days after culturing with the cells prepared in (1.7) above, the polyvinylidene difluoride (PVDF with a diameter of 3.5 cm) was cultured on the cultured cells. ) Covering the membrane, gently aspirating the medium, incubating with the cell culture support material at 20 ° C. for 30 minutes and cooling, the cells on any cell culture support material were detached with the covered membrane. . The covered membrane and cells were placed on the same cultured cells that were normally grown on the same cell culture support material, and the two were adhered in a 37 ° C. incubator filled with 5% carbon dioxide gas. After the two cell sheets adhered, the PVDF membrane was peeled off. By repeating the same operation, a 20-layer cell sheet was prepared.
かぶせた膜はいずれの細胞シートからも容易に剥がすことができた。２０枚の積層された細胞シートは、細胞、細胞間のデスモソーム構造、および細胞、基材間の基底膜様蛋白質が保持されていた。 The covered membrane could be easily peeled from any cell sheet. The 20 stacked cell sheets retained the cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate.
（１．１０）細胞の剥離性の評価
上記（１．９）で細胞培養支持体上から細胞剥離した際の細胞の状態を目視で観察し、下記の基準に従って、評価を行った。表３記載の結果は、１枚目から２０層目重ね合わせるまでの平均をとった。
◎：問題なく完全に剥離できた
○：わずかに細胞が支持体に残るが、問題ないレベル
△：一部の細胞が支持体に残り、シートに穴が確認されるレベル
×：支持体から剥がれるが、シートがぼろぼろになる
××：支持体から細胞が剥がれない
（１．１１）積層された機能性の評価
肝臓細胞で重要な機能はアンモニア代謝である。よってアンモニア代謝速度の評価を行った。ブタ生体内より採取した細胞を１００％として比率で計算し表３に結果を記載した。本発明の方法は、比較例に対して、生体から直接採取したものと同等かそれ以上であった。(1.10) Evaluation of cell detachability The cell state when the cells were detached from the cell culture support in (1.9) above was visually observed and evaluated according to the following criteria. The results shown in Table 3 were averaged from the first sheet to the 20th layer.
◎: Completely peeled without problems ○: Slightly cells remained on the support, but no problem level △: Level at which some cells remained on the support and holes were confirmed in the sheet ×: Peeled from the support However, the sheet is crumpled. XX: Cells are not peeled off from the support. (1.11) Evaluation of stacked functionality An important function in liver cells is ammonia metabolism. Therefore, the rate of ammonia metabolism was evaluated. The ratio of cells collected from the swine in vivo was calculated as 100%, and the results are shown in Table 3. The method of the present invention was equivalent to or better than that obtained directly from the living body with respect to the comparative example.
本発明の細胞培養支持体を用いることによりブリード耐性が良好であり、細胞の支持体からの剥離が格段に向上し、かつ積層された細胞の組織機能化が発現できた。今後の複雑な組織の細胞培養方法の進展に大きく貢献できる技術であることがわかった。 By using the cell culture support of the present invention, the bleed resistance was good, the detachment of the cell from the support was remarkably improved, and the tissue functionalization of the stacked cells could be expressed. It was found that this technology can greatly contribute to the progress of cell culture methods for complex tissues in the future.
Claims (7)
（一般式（１）において、Ｒ１、Ｒ２は水素原子、炭素原子数１〜８のアルキル基、アリール基を表す。Ｒ５、Ｒ６、Ｒ７は水素原子またはメチル基を表し、Ｒ３は水素原子、炭素原子数１〜３０のアルキル基、シクロアルキル基、または、−（ＣＨ２ＣＨ２Ｏ）ｎ−（ＣＨ２ＣＨ（ＣＨ３）Ｏ）ｍ−Ｒ０で表されるポリオキシアルキレン基を表す。ここにおいてｎは１〜３００、ｍは０〜６０の整数を表す。また、Ｒ０は、水素原子、炭素原子数１〜３０のアルキル基を表す。Ｒ４は炭素原子数３以上２２以下のアルキル基を表す。また、ｘ、ｙ、ｚは各成分の質量％を表し、０≦ｘ≦８０、０≦ｙ≦８０、０≦ｚ≦４０、ここでｘ＋ｙ＋ｚ＝１００である。）2. The cell culture support according to claim 1, wherein at least one of the temperature-responsive polymers containing the acrylic resin is represented by the following general formula (1).
(In the general formula (1), R 1, R 2 is a hydrogen atom, .R 5, R 6, R 7 represents an alkyl group, an aryl group having 1 to 8 carbon atoms is a hydrogen atom or a methyl radical, R 3 is a hydrogen atom, an alkyl group having 1 to 30 carbon atoms, a cycloalkyl group, or a poly- represented by — (CH 2 CH 2 O) n — (CH 2 CH (CH 3 ) O) m —R 0. Represents an oxyalkylene group, wherein n represents 1 to 300, m represents an integer of 0 to 60, R 0 represents a hydrogen atom or an alkyl group having 1 to 30 carbon atoms, and R 4 represents a carbon atom. Represents an alkyl group having a number of 3 or more and 22 or less, and x, y, and z represent mass% of each component, 0 ≦ x ≦ 80, 0 ≦ y ≦ 80, 0 ≦ z ≦ 40, where x + y + z = 100 .)
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