JP7463127B2 - Crosslinked polymer, gel composition, cell culture material, method for producing cell population, and method for separating cell population - Google Patents
Crosslinked polymer, gel composition, cell culture material, method for producing cell population, and method for separating cell population Download PDFInfo
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- JP7463127B2 JP7463127B2 JP2020029545A JP2020029545A JP7463127B2 JP 7463127 B2 JP7463127 B2 JP 7463127B2 JP 2020029545 A JP2020029545 A JP 2020029545A JP 2020029545 A JP2020029545 A JP 2020029545A JP 7463127 B2 JP7463127 B2 JP 7463127B2
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- 229920001567 vinyl ester resin Polymers 0.000 description 1
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- XKMZOFXGLBYJLS-UHFFFAOYSA-L zinc;prop-2-enoate Chemical compound [Zn+2].[O-]C(=O)C=C.[O-]C(=O)C=C XKMZOFXGLBYJLS-UHFFFAOYSA-L 0.000 description 1
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Description
本発明は、ポリマー架橋体、ゲル状組成物、細胞培養材料、細胞群の製造方法、及び細胞群の分離方法に関する。 The present invention relates to a crosslinked polymer, a gel composition, a cell culture material, a method for producing a cell population, and a method for separating a cell population.
インジェクタブルゲル、細胞培養材料等に使用される刺激応答性材料として、ポリビニルアルコール等の水酸基を有する重合体を、ホウ酸等のＢＯＨ基（ボロン酸基）を有するホウ素化合物により架橋したゲル状組成物が知られている（特許文献１及び２）。このような刺激応答性材料上で細胞群を培養し、培養した細胞群を刺激応答性材料から分離する場合、細胞へ悪影響のあるｐＨ、温度、光等の刺激ではなく、細胞群への悪影響を考慮し、細胞への毒性の少ない、糖若しくは糖類縁体、又はポリビニルアルコール等の水酸基を有する化合物（解離剤）の水溶液により刺激応答性材料を溶解させて除去している。 As a stimuli-responsive material used in injectable gels, cell culture materials, etc., a gel-like composition in which a polymer having a hydroxyl group, such as polyvinyl alcohol, is crosslinked with a boron compound having a BOH group (boronic acid group), such as boric acid, is known (Patent Documents 1 and 2). When culturing a cell group on such a stimuli-responsive material and separating the cultured cell group from the stimuli-responsive material, the stimuli-responsive material is dissolved and removed with an aqueous solution of a sugar or sugar analogue, or a compound having a hydroxyl group, such as polyvinyl alcohol, which is less toxic to cells, taking into consideration the adverse effects on the cell group, rather than stimuli such as pH, temperature, and light, which have an adverse effect on the cells.
しかしながら、特許文献１及び２のゲル状組成物は、重合体とホウ素化合物との結合力が強く、解離剤水溶液の濃度を高めなければ溶解させにくいという問題がある。 However, the gel compositions of Patent Documents 1 and 2 have a problem in that the bonding strength between the polymer and the boron compound is strong, and they are difficult to dissolve unless the concentration of the dissociator solution is increased.
本発明は、上述の事情に鑑みてなされたものであり、より温和な条件で解離剤水溶液に溶解することができる架橋体を提供することを目的とする。また、本発明は、上記架橋体を含むゲル状組成物、及び細胞培養材料、並びにそれらを用いた細胞群の製造方法、及び細胞群の分離方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and aims to provide a crosslinked body that can be dissolved in a dissociation agent aqueous solution under milder conditions. The present invention also aims to provide a gel composition and cell culture material containing the crosslinked body, as well as a method for producing a cell population using them, and a method for separating a cell population.
本発明の架橋体は、ＢＯＨ基を含むホウ素化合物又はその塩により下記式（Ｉ）で表される基（Ｉ）を含む重合体が架橋された構造を有する。
（式（Ｉ）中、＊は前記基（Ｉ）の結合位置を表し、Ｘは－Ｏ－又は－ＮＨ－であり、Ｚは水酸基を有する炭素数１～８の有機基である。）
The crosslinked product of the present invention has a structure in which a polymer containing a group (I) represented by the following formula (I) is crosslinked with a boron compound containing a BOH group or a salt thereof.
(In formula (I), * represents the bonding position of the group (I), X is -O- or -NH-, and Z is an organic group having 1 to 8 carbon atoms and a hydroxyl group.)
上記重合体が、下記式（ＩＩ）で表される構造単位（Ａ）を含むと好ましい。
（式（ＩＩ）中、Ｒは水素原子又はメチル基であり、＊は前記構造単位（Ａ）の結合位置を表す。）
The polymer preferably contains a structural unit (A) represented by the following formula (II).
(In formula (II), R is a hydrogen atom or a methyl group, and * represents the bonding position of the structural unit (A).)
Ｚが水酸基により置換された炭化水素基であると好ましい。 It is preferred that Z is a hydrocarbon group substituted with a hydroxyl group.
Ｚが２つ以上の水酸基を有すると好ましい。 It is preferred that Z has two or more hydroxyl groups.
Ｚにおける炭素数に対する水酸基の個数の比が、０．３以上であると好ましい。 It is preferable that the ratio of the number of hydroxyl groups to the number of carbon atoms in Z is 0.3 or more.
Ｚが下記式（ＩＩＩ）で表される基（ＩＩＩ）であると好ましい。
（式（ＩＩＩ）中、Ｒ１は－ＣＨ（ＯＨ）ＣＨ２ＯＨ又は－ＣＨ２ＯＨであり、Ｒ２は、水素原子又は－ＣＨ２ＯＨであり、Ｒ３は、水素原子又は－ＣＨ２ＯＨであり、＊は前記基（ＩＩＩ）の結合位置を表す。）
It is preferable that Z is a group (III) represented by the following formula (III).
(In formula (III), R 1 is -CH(OH)CH 2 OH or -CH 2 OH, R 2 is a hydrogen atom or -CH 2 OH, R 3 is a hydrogen atom or -CH 2 OH, and * indicates the bonding position of the group (III).)
上記架橋体が、ホウ素化合物又はその塩以外の架橋剤により前記重合体が架橋された構造を更に有すると好ましい。 It is preferable that the crosslinked body further has a structure in which the polymer is crosslinked with a crosslinking agent other than a boron compound or a salt thereof.
上記ホウ素化合物又はその塩が、ホウ酸又はその塩、ポリホウ酸又はその塩、メタホウ酸又はその塩、フェニルホウ酸又はその塩、及び１，４－フェニレンジボロン酸又はその塩から選択される少なくとも一種の化合物であると好ましい。 The boron compound or its salt is preferably at least one compound selected from boric acid or its salt, polyboric acid or its salt, metaboric acid or its salt, phenylboric acid or its salt, and 1,4-phenylenediboronic acid or its salt.
本発明のゲル状組成物は、上記架橋体を含む。 The gel composition of the present invention contains the above-mentioned crosslinked body.
本発明の細胞培養材料は、上記架橋体を含む。 The cell culture material of the present invention contains the above-mentioned crosslinked body.
本発明の細胞群の製造方法は、上記架橋体を含む細胞培養基材上で細胞を培養して細胞群を形成する工程と、解離剤水溶液で細胞培養基材を溶解し、細胞群を分離する工程と、を備える。 The method for producing a cell population of the present invention includes a step of culturing cells on a cell culture substrate containing the crosslinked body to form a cell population, and a step of dissolving the cell culture substrate with an aqueous dissociation agent solution to separate the cell population.
本発明の細胞群の分離方法は、上記架橋体を含むゲル状基材を解離剤水溶液で溶解し、当該ゲル状基材に接触している細胞群を分離する工程を含む。 The cell group separation method of the present invention includes a step of dissolving the gel-like substrate containing the crosslinked body with an aqueous dissociation agent, and separating the cell group in contact with the gel-like substrate.
本発明によれば、より温和な条件で解離剤水溶液に溶解することができる架橋体を提供することができる。また、本発明によれば、上記架橋体を含むゲル状組成物、及び細胞培養材料、並びにそれらを用いた細胞群の製造方法、及び細胞群の分離方法を提供することができる。 According to the present invention, it is possible to provide a crosslinked body that can be dissolved in a dissociation agent aqueous solution under milder conditions. In addition, according to the present invention, it is possible to provide a gel composition and a cell culture material containing the crosslinked body, as well as a method for producing a cell group and a method for separating a cell group using them.
＜架橋体＞
本実施形態の架橋体は、上記基（Ｉ）を有する重合体が、ＢＯＨ基を含むホウ素化合物又はその塩により架橋された構造を有する。なお、架橋体は、上記基（Ｉ）を有する重合体（以下、重合体（Ａ）とも呼ぶ。）が、上記ホウ素化合物により架橋された構造を有していればよく、実際に基（Ｉ）を有する重合体を上記ホウ素化合物により架橋して製造されたものに限定されない。
<Crosslinked Body>
The crosslinked body of the present embodiment has a structure in which a polymer having the above group (I) is crosslinked with a boron compound containing a BOH group or a salt thereof. Note that the crosslinked body is not limited to a crosslinked body produced by actually crosslinking a polymer having the above group (I) with the above boron compound, as long as the polymer having the above group (I) (hereinafter also referred to as polymer (A)) has a structure in which the polymer has been crosslinked with the above boron compound.
＜重合体（Ａ）＞
重合体（Ａ）は、上記基（Ｉ）を有する重合体であれば特に限定されない。重合体（Ａ）は、ポリエステル化合物、ポリエーテル化合物、ポリアミド化合物、ポリイミド化合物、ポリアミドイミド化合物、ポリアミド酸化合物、ポリビニル化合物及びこれらの組み合わせであってよい。また、重合体（Ａ）は、ホモポリマーであってもよく、共重合体であってもよい。基（Ｉ）は、重合体（Ａ）の末端に結合していてもよく、末端以外の部分に結合していてもよい。重合体（Ａ）が主鎖とグラフト鎖を有する場合、基（Ｉ）は、主鎖に結合していても、グラフト鎖に結合していてもよい。また、ゼラチン、ヒアルロン酸、アルギン酸等の天然高分子に基（Ｉ）を導入したものも重合体（Ａ）として使用できる。
<Polymer (A)>
The polymer (A) is not particularly limited as long as it is a polymer having the above group (I). The polymer (A) may be a polyester compound, a polyether compound, a polyamide compound, a polyimide compound, a polyamideimide compound, a polyamic acid compound, a polyvinyl compound, or a combination thereof. The polymer (A) may be a homopolymer or a copolymer. The group (I) may be bonded to the end of the polymer (A) or to a portion other than the end. When the polymer (A) has a main chain and a graft chain, the group (I) may be bonded to the main chain or to the graft chain. In addition, a natural polymer such as gelatin, hyaluronic acid, or alginic acid into which the group (I) has been introduced can also be used as the polymer (A).
基（Ｉ）は、重合体（Ａ）中に、ホウ素化合物による架橋を十分に確保する観点から、０．００１ｍｏｌ／ｇ以上含まれていると好ましく、０．００２５ｍｏｌ／ｇ以上含まれているとより好ましく、０．００５ｍｏｌ／ｇ以上含まれていると更に好ましい。なお、単位ｍｏｌ／ｇは、重合体（Ａ）１ｇ当たりの基（Ｉ）のモル数である。 From the viewpoint of ensuring sufficient crosslinking by the boron compound, the group (I) is preferably contained in the polymer (A) in an amount of 0.001 mol/g or more, more preferably 0.0025 mol/g or more, and even more preferably 0.005 mol/g or more. The unit mol/g is the number of moles of the group (I) per gram of the polymer (A).
重合体（Ａ）は、基（Ｉ）が有する水酸基以外に水酸基を有していてもよいが、有していなくてもよい。重合体（Ａ）は、水酸基を０．００２ｍｏｌ／ｇ以上含むと好ましく、０．００５ｍｏｌ／ｇ以上含むとより好ましく、０．０１ｍｏｌ／ｇ以上含むと更に好ましい。なお、単位ｍｏｌ／ｇは、重合体１ｇ当たりの水酸基のモル数である。 Polymer (A) may or may not have hydroxyl groups other than the hydroxyl groups in group (I). Polymer (A) preferably contains 0.002 mol/g or more of hydroxyl groups, more preferably 0.005 mol/g or more, and even more preferably 0.01 mol/g or more. The unit mol/g is the number of moles of hydroxyl groups per gram of polymer.
式（Ｉ）中、Ｘは－Ｏ－又は－ＮＨ－であり、Ｚは水酸基を有する炭素数１～８の有機基である。Ｚは、水酸基により置換された炭化水素基であると好ましい。当該炭化水素基は、芳香族炭化水素基又は脂肪族炭化水素基であってよく、脂肪族炭化水素基であってよい。また、Ｚが有する炭素数は、１～６であることが好ましく、２～６であることがより好ましい。式（Ｉ）、＊は基（Ｉ）の結合位置を表す。つまり、基（Ｉ）は、＊の位置でＸに結合する。 In formula (I), X is -O- or -NH-, and Z is an organic group having 1 to 8 carbon atoms and a hydroxyl group. Z is preferably a hydrocarbon group substituted with a hydroxyl group. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group, and may be an aliphatic hydrocarbon group. Furthermore, Z preferably has 1 to 6 carbon atoms, and more preferably 2 to 6 carbon atoms. In formula (I), * represents the bonding position of group (I). In other words, group (I) bonds to X at the * position.
Ｚが有する水酸基の個数としては、１以上であればよいが、２以上であると好ましい。また、Ｚにおける炭素数に対する水酸基の個数の比が、０．３以上であると好ましく、０．４以上であるとより好ましく、０．５以上であると更に好ましく、０．６以上であると特に好ましい。Ｚにおける炭素数に対する水酸基の個数の比は、１以下であってよい。 The number of hydroxyl groups in Z may be 1 or more, but is preferably 2 or more. The ratio of the number of hydroxyl groups to the number of carbon atoms in Z is preferably 0.3 or more, more preferably 0.4 or more, even more preferably 0.5 or more, and particularly preferably 0.6 or more. The ratio of the number of hydroxyl groups to the number of carbon atoms in Z may be 1 or less.
Ｚが有する水酸基が２以上の場合の水酸基同士の間隔は、４原子以下が好ましい、２原子又は３原子がより好ましい。なお、水酸基同士の間隔とは、一の水酸基が直接結合している炭素原子から他の水酸基が結合している炭素とを結ぶ原子鎖に含まれる原子数の最小値を言う。なお、上記原子数の計算には、当該水酸基が結合している炭素も含むものとする。Ｚの化学構造内において、Ｚが有する各水酸基から４原子以下の間隔で別の水酸基が存在すると好ましく、２原子又は３原子の間隔で別の水酸基が存在すると好ましい。 When Z has two or more hydroxyl groups, the distance between the hydroxyl groups is preferably 4 atoms or less, more preferably 2 or 3 atoms. The distance between the hydroxyl groups refers to the minimum number of atoms contained in the atomic chain connecting the carbon atom to which one hydroxyl group is directly bonded and the carbon to which another hydroxyl group is bonded. The calculation of the number of atoms includes the carbon to which the hydroxyl group is bonded. In the chemical structure of Z, it is preferable that another hydroxyl group is present at a distance of 4 atoms or less from each hydroxyl group possessed by Z, and it is preferable that another hydroxyl group is present at a distance of 2 or 3 atoms.
より具体的には、Ｚは、上記式（ＩＩＩ）で表される基（ＩＩＩ）であると好ましい。式（ＩＩＩ）中、Ｒ１は－ＣＨ（ＯＨ）ＣＨ２ＯＨ又は－ＣＨ２ＯＨであり、Ｒ２は、水素原子又は－ＣＨ２ＯＨであり、Ｒ３は、水素原子又は－ＣＨ２ＯＨであり、＊は前記基（ＩＩＩ）の結合位置を表す。 More specifically, Z is preferably a group (III) represented by the above formula (III), in which R 1 is -CH(OH)CH 2 OH or -CH 2 OH, R 2 is a hydrogen atom or -CH 2 OH, R 3 is a hydrogen atom or -CH 2 OH, and * represents the bonding position of the group (III).
重合体（Ａ）は、基（Ｉ）を有するビニル化合物に由来する構造単位を有すると好ましい。そのような構造単位としては、上記式（ＩＩ）で表される構造単位（Ａ）が挙げられる。式（ＩＩ）中、Ｒは、水素原子又はメチル基である。式（ＩＩ）におけるＸ及びＺは、式（Ｉ）のものと同じ意味である。式（ＩＩ）における＊は、構造単位（Ａ）の結合位置を表す。つまり、個々の構造単位（Ａ）は、＊の位置で他の構造単位と結合する。 It is preferable that the polymer (A) has a structural unit derived from a vinyl compound having a group (I). An example of such a structural unit is the structural unit (A) represented by the above formula (II). In formula (II), R is a hydrogen atom or a methyl group. X and Z in formula (II) have the same meanings as those in formula (I). * in formula (II) represents the bonding position of the structural unit (A). In other words, each structural unit (A) bonds to another structural unit at the position marked with *.
重合体（Ａ）が構造単位（ＩＩ）を有する場合、重合体（Ａ）は、エチレン性不飽和基を有する単量体に由来する構造単位を有するものであると好ましい。なお、「エチレン性不飽和基を有する単量体に由来する構造単位」とは、エチレン性不飽和基を有する単量体をラジカル重合したと仮定した場合に得られる構造と同じ構造を指すものとする。つまり、重合体（Ａ）には、実際にエチレン性不飽和基を有する単量体をラジカル重合して製造されたものだけでなく、他の方法で製造されたものであってもエチレン性不飽和基を有する単量体をラジカル重合したと仮定した場合に得られる構造と同じ化学構造を有するものであれば、エチレン性不飽和基を有する単量体に由来する構造単位に含まれる。 When the polymer (A) has the structural unit (II), it is preferable that the polymer (A) has a structural unit derived from a monomer having an ethylenically unsaturated group. The term "structural unit derived from a monomer having an ethylenically unsaturated group" refers to the same structure as the structure obtained when the monomer having an ethylenically unsaturated group is radically polymerized. In other words, the polymer (A) includes not only those actually produced by radical polymerization of a monomer having an ethylenically unsaturated group, but also those produced by other methods, as long as they have the same chemical structure as the structure obtained when the monomer having an ethylenically unsaturated group is radically polymerized.
構造単位（Ａ）は、下記式（ＩＩＡ）で表される単量体Ａに由来する構造単位であってよい。 The structural unit (A) may be a structural unit derived from monomer A represented by the following formula (IIA):
式（ＩＩＡ）中、Ｒ、Ｘ及びＺは、それぞれ式（ＩＩ）のＲ、Ｘ及びＺと同じ意味である。単量体（Ａ）としては、具体的には、グリセロールモノ（メタ）アクリレート、［トリス（ヒドロキシメチル）メチル］（メタ）アクリレート、Ｎ－（２，３－ジヒドロキシプロピル）（メタ）アクリルアミド、Ｎ－（１，３－ジヒドロキシプロパン-２-イル）（メタ）アクリルアミド、Ｎ－［トリス（ヒドロキシメチル）メチル］（メタ）アクリルアミド等が挙げられる。
In formula (IIA), R, X, and Z have the same meanings as R, X, and Z in formula (II), respectively. Specific examples of the monomer (A) include glycerol mono(meth)acrylate, [tris(hydroxymethyl)methyl](meth)acrylate, N-(2,3-dihydroxypropyl)(meth)acrylamide, N-(1,3-dihydroxypropan-2-yl)(meth)acrylamide, N-[tris(hydroxymethyl)methyl](meth)acrylamide, and the like.
重合体（Ａ）は、構造単位（Ａ）以外の構造単位（構造単位（Ｂ）とも呼ぶ。）を有していてよい。構造単位（Ｂ）としては、不飽和カルボン酸に由来する構造単位、（メタ）アクリル酸エステルに由来する構造単位、ビニル基を有するアミド化合物に由来する構造単位、芳香族ビニル単量体に由来する構造単位、等の構造単位（Ａ）を誘導する単量体以外の単量体（以下、単量体（Ｂとも呼ぶ。））が挙げられる。 The polymer (A) may have a structural unit (also referred to as structural unit (B)) other than the structural unit (A). Examples of the structural unit (B) include a monomer (hereinafter also referred to as monomer (B)) other than the monomer that derives the structural unit (A), such as a structural unit derived from an unsaturated carboxylic acid, a structural unit derived from a (meth)acrylic acid ester, a structural unit derived from an amide compound having a vinyl group, and a structural unit derived from an aromatic vinyl monomer.
不飽和カルボン酸としては、（メタ）アクリル酸、マレイン酸、フマル酸、クロトン酸、けい皮酸、イタコン酸、シトラコン酸、無水マレイン酸、マレイン酸モノメチル、マレイン酸モノブチル、無水イタコン酸、イタコン酸モノメチル、イタコン酸モノブチル、ビニル安息香酸、シュウ酸モノヒドロキシエチル（メタ）アクリレート、アリルオキシメチルアクリル酸等の環化重合性不飽和カルボン酸などを挙げることができる。これらは１種又は２種以上を適宜選択し用いることができる。 Examples of unsaturated carboxylic acids include cyclic polymerizable unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, crotonic acid, cinnamic acid, itaconic acid, citraconic acid, maleic anhydride, monomethyl maleate, monobutyl maleate, itaconic anhydride, monomethyl itaconate, monobutyl itaconate, vinyl benzoic acid, monohydroxyethyl oxalate (meth)acrylate, and allyloxymethylacrylic acid. These can be used alone or in combination of two or more.
芳香族ビニル単量体としては、スチレン及びその誘導体が挙げられ、具体的には、スチレン、α－メチルスチレン、ハロゲン化スチレン等が挙げられる。また、ビニル基を有するアミド化合物としては、（メタ）アクリルアミド、Ｎ－メチロール（メタ）アクリルアミド、ダイアセトン（メタ）アクリルアミド等の（メタ）アクリルアミド及びその誘導体、Ｎ－ビニルピロリドン、Ｎ－ビニル－５－メチルピロリドン、Ｎ－ビニルピペリドン、Ｎ－ビニル－ε－カプロラクタム、１－（２－プロペニル）－２－ピペリドン等のビニル基及び環状アミド基を有する化合物、Ｎ－ビニルホルムアミド、Ｎ－ビニルアセトアミド等のビニル基及び環状アミド基を有する化合物などが挙げられる。ビニルエステル化合物としては、酢酸ビニル、プロピオン酸ビニル等が挙げられる。 Examples of aromatic vinyl monomers include styrene and its derivatives, specifically styrene, α-methylstyrene, and halogenated styrene. Examples of amide compounds having a vinyl group include (meth)acrylamide and its derivatives, such as (meth)acrylamide, N-methylol (meth)acrylamide, and diacetone (meth)acrylamide; compounds having a vinyl group and a cyclic amide group, such as N-vinylpyrrolidone, N-vinyl-5-methylpyrrolidone, N-vinylpiperidone, N-vinyl-ε-caprolactam, and 1-(2-propenyl)-2-piperidone; and compounds having a vinyl group and a cyclic amide group, such as N-vinylformamide and N-vinylacetamide. Examples of vinyl ester compounds include vinyl acetate and vinyl propionate.
（メタ）アクリル酸エステルに由来する構造単位としては、以下の式（ＩＶ）で表される構造単位が挙げられる。 An example of a structural unit derived from a (meth)acrylic acid ester is the structural unit represented by the following formula (IV):
（式（ＩＶ）中、Ｒ３１は、水素原子又はメチル基を表し、Ｒ３２は、炭素数１～３０の炭化水素基を表す。）
(In formula (IV), R 31 represents a hydrogen atom or a methyl group, and R 32 represents a hydrocarbon group having 1 to 30 carbon atoms.)
Ｒ３２の炭化水素基としては、直鎖のアルキル基、分岐鎖のアルキル基、脂環式炭化水素基、芳香族炭化水素基等が挙げられる。Ｒ３２の炭素数は、１～２０であってよく、１～１８であってよい。Ｒ３２としては、より具体的には、メチル基、エチル基、プロピル基、ブチル基、２－エチルヘキシル基、ラウリル基、ステアリル基、シクロヘキシル基、イソボルニル基等が挙げられる。 Examples of the hydrocarbon group of R 32 include a linear alkyl group, a branched alkyl group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, etc. The number of carbon atoms of R 32 may be 1 to 20, or 1 to 18. More specific examples of R 32 include a methyl group, an ethyl group, a propyl group, a butyl group, a 2-ethylhexyl group, a lauryl group, a stearyl group, a cyclohexyl group, an isobornyl group, etc.
重合体（Ａ）における構造単位（Ａ）の含有量は、重合体（Ａ）の総量に対して、５０質量％以上であると好ましく、６０質量％以上であると好ましく、７０質量％以上であると更に好ましく、８０質量％以上であるとより更に好ましく、９０質量％以上であると特に好ましい。 The content of structural unit (A) in polymer (A) is preferably 50% by mass or more, more preferably 60% by mass or more, even more preferably 70% by mass or more, even more preferably 80% by mass or more, and particularly preferably 90% by mass or more, based on the total amount of polymer (A).
重合体（Ａ）における構造単位（Ｂ）の含有量は、重合体（Ａ）の総量に対して、５０質量％未満であると好ましく、３５質量％以下であると好ましく、２５質量％以下であると更に好ましく、１０質量％以下であるとより更に好ましく、５質量％以下であると特に好ましい。重合体（Ａ）は、構造単位（Ｂ）を含まなくてもよい。 The content of the structural unit (B) in the polymer (A) is preferably less than 50% by mass, more preferably 35% by mass or less, even more preferably 25% by mass or less, even more preferably 10% by mass or less, and particularly preferably 5% by mass or less, based on the total amount of the polymer (A). The polymer (A) may not contain the structural unit (B).
重合体（Ａ）の重量平均分子量は、特に制限はないが、例えば、１０，０００～５，０００，０００であると好ましく、１００，０００～３，０００，０００であるとより好ましい。重量平均分子量は、ゲルパーミエイションクロマトグラフィー（ＧＰＣ）等で測定することができる。 The weight average molecular weight of polymer (A) is not particularly limited, but is preferably 10,000 to 5,000,000, and more preferably 100,000 to 3,000,000. The weight average molecular weight can be measured by gel permeation chromatography (GPC) or the like.
＜ホウ素化合物＞
ホウ素化合物は、ＢＯＨ基（ボロン酸基）を含む化合物又はその塩である。ホウ素化合物は、重合体（Ａ）の水酸基と反応して重合体（Ａ）を架橋させる架橋剤として作用する。ホウ素化合物としては、ＢＯＨ基を有する化合物としては、特に制限はなく、無機ホウ素化合物及び有機ホウ素化合物のいずれであってもよい。ホウ素化合物の塩としては特に制限されないが、ナトリウム塩等のアルカリ金属塩などが挙げられる。ホウ素化合物は、一種又は二種以上を使用することができる。
<Boron compounds>
The boron compound is a compound containing a BOH group (boronic acid group) or a salt thereof. The boron compound acts as a crosslinking agent that reacts with the hydroxyl group of the polymer (A) to crosslink the polymer (A). The boron compound is not particularly limited as a compound having a BOH group, and may be either an inorganic boron compound or an organic boron compound. The salt of the boron compound is not particularly limited, and may be an alkali metal salt such as a sodium salt. One or more kinds of boron compounds may be used.
無機ホウ素化合物としては、ホウ酸又はその塩、メタホウ酸又はその塩、ポリホウ酸（硼砂等）又はその塩等が挙げられる。有機ホウ素化合物としては、フェニルホウ酸又はその塩、及び１，４－フェニレンジボロン酸又はその塩、ｍ－アミノフェニルボロン酸又はその塩、ボロン酸基を有する構造単位を有する重合体又はその塩等が挙げられる。 Inorganic boron compounds include boric acid or its salts, metaboric acid or its salts, polyboric acid (borax, etc.) or its salts, etc. Organic boron compounds include phenylboric acid or its salts, 1,4-phenylenediboronic acid or its salts, m-aminophenylboronic acid or its salts, polymers having structural units with boronic acid groups or their salts, etc.
ボロン酸基を有する構造単位を誘導するための単量体としては、４－（アクリロイルアミノ）フェニルボロン酸、３－（アクリロイルアミノ）フェニルボロン酸、３－（メタクリロイルアミノ）フェニルボロン酸、４－ビニルフェニルボロン酸、４－（ビニルカルバモイル）フェニルボロン酸、２－メトキシピリジン－５－ボロン酸、５－カルボキシチオフェン－２－ボロン酸、それらの芳香族水素原子がフッ素原子で単数または複数置換された化合物、及び、それらのアミノフェニルボロン酸構造がＮ－アルキルアミノメチルフェニルボロン酸に置換された化合物等が挙げられる。ボロン酸基を有する構造単位を有する重合体は、これらの単量体の（共）重合体、又は他のビニル化合物との共重合体であってよい。 Examples of monomers for deriving structural units having a boronic acid group include 4-(acryloylamino)phenylboronic acid, 3-(acryloylamino)phenylboronic acid, 3-(methacryloylamino)phenylboronic acid, 4-vinylphenylboronic acid, 4-(vinylcarbamoyl)phenylboronic acid, 2-methoxypyridine-5-boronic acid, 5-carboxythiophene-2-boronic acid, compounds in which one or more of the aromatic hydrogen atoms are replaced with fluorine atoms, and compounds in which the aminophenylboronic acid structure is replaced with N-alkylaminomethylphenylboronic acid. The polymer having a structural unit having a boronic acid group may be a (co)polymer of these monomers, or a copolymer with other vinyl compounds.
ホウ素化合物の使用量は、重合体（Ａ）１００質量部に対して、ホウ素化合物は０．１～１０００質量部が好ましく、１～５００質量部がより好ましく、１０～２００質量部であると更に好ましい。また、ホウ素化合物の使用量は、重合体（Ａ）における水酸基１モルに対して０．０２５～２５０モルが好ましく、０．２５～１２５モルがより好ましく、２．５～５０モルが更に好ましい。なお、ホウ素化合物のモル数は、ホウ素化合物に含まれるホウ素原子のモル数とする。 The amount of the boron compound used is preferably 0.1 to 1000 parts by mass, more preferably 1 to 500 parts by mass, and even more preferably 10 to 200 parts by mass, per 100 parts by mass of polymer (A). The amount of the boron compound used is preferably 0.025 to 250 moles, more preferably 0.25 to 125 moles, and even more preferably 2.5 to 50 moles, per mole of hydroxyl groups in polymer (A). The number of moles of the boron compound refers to the number of moles of boron atoms contained in the boron compound.
＜その他の架橋剤＞
架橋体は、ＢＯＨ基を含むホウ素化合物又はその塩以外の架橋剤（その他の架橋剤）により架橋された構造を有していてもよい。その他の架橋剤としては、重合体（Ａ）に構造単位として組み込まれる内部架橋剤と、重合体（Ａ）が有する官能基と反応して直接又はホウ素化合物を介して間接的に複数の重合体（Ａ）の分子同士を架橋する外部架橋剤とが挙げられる。また、その他の架橋剤は、化学構造内に重合体（Ａ）に構造単位として組み込まれる部分と、重合体（Ａ）が有する官能基又はホウ素化合物と反応する部分を有するもの（つまり、内部架橋剤と外部架橋剤の両方の機能を有するもの）であってもよい。架橋構造としては、例えば、多価金属イオンによる架橋構造、多価アルコールによる架橋構造、分子性の内部架橋剤による架橋構造が挙げられる。
<Other crosslinking agents>
The crosslinked body may have a structure crosslinked by a crosslinking agent (other crosslinking agent) other than the boron compound containing a BOH group or its salt. Examples of the other crosslinking agent include an internal crosslinking agent incorporated as a structural unit into the polymer (A) and an external crosslinking agent that reacts with a functional group possessed by the polymer (A) to crosslink a plurality of molecules of the polymer (A) directly or indirectly via a boron compound. In addition, the other crosslinking agent may have a portion incorporated as a structural unit into the polymer (A) in the chemical structure and a portion that reacts with a functional group possessed by the polymer (A) or a boron compound (that is, a crosslinking agent having both functions of an internal crosslinking agent and an external crosslinking agent). Examples of the crosslinking structure include a crosslinking structure by a polyvalent metal ion, a crosslinking structure by a polyhydric alcohol, and a crosslinking structure by a molecular internal crosslinking agent.
内部架橋剤としては、化学構造内に重合反応の反応点を複数有する化合物が挙げられる。例えば、重合体（Ａ）がビニル化合物に由来する構造単位を有するものである場合、内部架橋剤としては、多官能ビニル化合物が挙げられる。言い換えれば、重合体（Ａ）は、多官能ビニル化合物に由来する構造単位を有するものであってもよい。 An example of an internal crosslinking agent is a compound having multiple reactive sites for polymerization reaction within its chemical structure. For example, when the polymer (A) has a structural unit derived from a vinyl compound, an example of an internal crosslinking agent is a polyfunctional vinyl compound. In other words, the polymer (A) may have a structural unit derived from a polyfunctional vinyl compound.
多官能ビニル化合物としては、分子性の多官能ビニル化合物が挙げられる。分子性の多官能ビニル化合物としては、分子内に二つ以上のエチレン性不飽和基を有する化合物であれば、特に限定されないが、具体的には、多官能（メタ）アクリル酸アミド、多官能（メタ）アクリル酸エステル、芳香族ポリビニル化合物等が挙げられる。多官能ビニル化合物は、１種又は２種以上を使用できる。 Examples of polyfunctional vinyl compounds include molecular polyfunctional vinyl compounds. Molecular polyfunctional vinyl compounds are not particularly limited as long as they have two or more ethylenically unsaturated groups in the molecule, but specific examples include polyfunctional (meth)acrylic acid amides, polyfunctional (meth)acrylic acid esters, aromatic polyvinyl compounds, and the like. One or more types of polyfunctional vinyl compounds can be used.
多官能（メタ）アクリル酸エステルとしては、エチレングリコールジアクリレート、１，４－ブタンジオールジアクリレート、１，６－ヘキサンジオールジアクリレート、１，９－ノナンジオールジアクリレート、シクロヘキサンジメタノールジアクリレート、エトキシ化ビスフェノールＡジアクリレート、トリシクロデカンジメタノールジアクリレート、トリメチロールプロパントリアクリレート、ペンタエリスリトールトリアクリレート、ペンタエリスリトールテトラアクリレート、デンドリマーアクリレート、エチレングリコールジメタクリレート、１，４－ブタンジオールジメタクリレート、１，６－ヘキサンジオールジメタクリレート、エトキシ化ビスフェノールＡジメタクリレート、トリメチロールプロパントリメタクリレート等が挙げられる。 Examples of polyfunctional (meth)acrylic acid esters include ethylene glycol diacrylate, 1,4-butanediol diacrylate, 1,6-hexanediol diacrylate, 1,9-nonanediol diacrylate, cyclohexanedimethanol diacrylate, ethoxylated bisphenol A diacrylate, tricyclodecane dimethanol diacrylate, trimethylolpropane triacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, dendrimer acrylate, ethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, 1,6-hexanediol dimethacrylate, ethoxylated bisphenol A dimethacrylate, and trimethylolpropane trimethacrylate.
また、多官能（メタ）アクリル酸エステルとしては、上記の化合物以外にも下記式（Ｖ）の化合物も挙げることができる。
（式（Ｖ）中、Ｒ２１及びＲ２２は、それぞれ独立に水素原子又はメチル基を表し、ｒは、２～１０の整数を表し、Ｒ２３は、炭素数２又は３のアルキレン基を表す。）
In addition to the above compounds, examples of the polyfunctional (meth)acrylic acid ester include the compounds of the following formula (V).
(In formula (V), R 21 and R 22 each independently represent a hydrogen atom or a methyl group, r represents an integer of 2 to 10, and R 23 represents an alkylene group having 2 or 3 carbon atoms.)
式（Ｖ）中、ｒは、２～７が好ましく、３～５がより好ましい。 In formula (V), r is preferably 2 to 7, and more preferably 3 to 5.
式（Ｖ）の化合物としては、ジエチレングリコールジ（メタ）アクリレート、トリエチレングリコールジ（メタ）アクリレート、テトラエチレングリコールジ（メタ）アクリレート、ジプロピレングリコールジ（メタ）アクリレート、トリプロピレングリコールジ（メタ）アクリレート、テトラプロピレングリコールジ（メタ）アクリレート等が挙げられる。 Examples of compounds of formula (V) include diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, and tetrapropylene glycol di(meth)acrylate.
多官能（メタ）アクリル酸アミドとしては、Ｎ，Ｎ’－メチレンビスアクリルアミド等が挙げられる。 Examples of polyfunctional (meth)acrylic acid amides include N,N'-methylenebisacrylamide.
芳香族ポリビニル化合物としては、ジビニルベンゼン等が挙げられる。 Examples of aromatic polyvinyl compounds include divinylbenzene.
上記重合体（Ａ）における、架橋剤に由来する構造単位の含有量は、重合体（Ａ）１００質量部に対して、０．５～２０質量部であると好ましく、１～１０質量部であるとより好ましい。 The content of the structural unit derived from the crosslinking agent in the above polymer (A) is preferably 0.5 to 20 parts by mass, and more preferably 1 to 10 parts by mass, per 100 parts by mass of polymer (A).
また、多官能ビニル化合物としては、不飽和カルボン酸と多価金属イオンとの塩も挙げられる。不飽和カルボン酸と多価金属イオンとの塩に由来する構造単位を重合体（Ａ）に導入すると、複数の重合体（Ａ）の分子同士を多価金属イオンにより架橋した構造を形成することができる。多価金属イオンとしては、２価以上の金属イオンであれば特に制限はないが、２価又は３価の金属イオンが好ましく、亜鉛イオン、カルシウムイオン、マグネシウムイオン、アルミニウムイオン、ネオジムイオンが挙げられる。不飽和カルボン酸としては、上述の単量体（Ｂ）として例示したものが挙げられるが、（メタ）アクリル酸が好ましい。不飽和カルボン酸と多価金属イオンとの塩としては、（メタ）アクリル酸亜鉛、（メタ）アクリル酸カルシウム、（メタ）アクリル酸マグネシウム、（メタ）アクリル酸アルミニウム、（メタ）アクリル酸ネオジム等が好ましい。 In addition, examples of polyfunctional vinyl compounds include salts of unsaturated carboxylic acids and polyvalent metal ions. When a structural unit derived from a salt of an unsaturated carboxylic acid and a polyvalent metal ion is introduced into the polymer (A), a structure in which multiple molecules of the polymer (A) are crosslinked with each other by the polyvalent metal ion can be formed. The polyvalent metal ion is not particularly limited as long as it is a divalent or higher metal ion, but divalent or trivalent metal ions are preferred, such as zinc ions, calcium ions, magnesium ions, aluminum ions, and neodymium ions. Examples of unsaturated carboxylic acids include those exemplified as the monomer (B) above, but (meth)acrylic acid is preferred. Examples of salts of unsaturated carboxylic acids and polyvalent metal ions include zinc (meth)acrylate, calcium (meth)acrylate, magnesium (meth)acrylate, aluminum (meth)acrylate, and neodymium (meth)acrylate.
上記重合体（Ａ）における、不飽和カルボン酸と多価金属イオンとの塩に由来する構造単位の含有量は、重合体（Ａ）１００質量部に対して、０．５～２０質量部であると好ましく、１～１０質量部であるとより好ましい。 The content of structural units derived from a salt of an unsaturated carboxylic acid and a polyvalent metal ion in the above polymer (A) is preferably 0.5 to 20 parts by mass, and more preferably 1 to 10 parts by mass, per 100 parts by mass of polymer (A).
なお、多価金属イオンは、外部架橋剤として導入することもできる。例えば、不飽和カルボン酸に由来する構造単位を有する重合体（Ａ）を合成後、多価金属イオン添加して後架橋することにより架橋構造を形成してもよい。 The polyvalent metal ion can also be introduced as an external crosslinking agent. For example, after synthesizing a polymer (A) having structural units derived from an unsaturated carboxylic acid, a polyvalent metal ion can be added to perform post-crosslinking to form a crosslinked structure.
外部架橋剤としては、多価金属イオン以外に、多価アルコールが挙げられる。多価アルコールは、ホウ素化合物と反応して間接的に重合体（Ａ）を架橋することができる。これにより、架橋体内に多価アルコールに由来する構造を導入することができる。多価アルコールとしては、ポリビニルアルコール、変性ポリビニルアルコール（カチオン性、アニオン性、反応型のいずれであってもよい）、ポリグリセリン等が挙げられる。カチオン性の変性ポリビニルアルコールとしては、第四級アンモニウム基等のカチオン性の官能基を導入したポリビニルアルコールが挙げられ、具体的には、三菱ケミカル株式会社製の「ゴーセネックス（登録商標）Ｋ－４３４」等のゴーセネックス（登録商標）Ｋシリーズが挙げられる。アニオン性の変性ポリビニルアルコールとしては、スルホン酸基、カルボキシル基等のアニオン性の官能基を導入したポリビニルアルコールが挙げられ、具体的には、三菱ケミカル株式会社製の「ゴーセネックス（登録商標）Ｌ－３２６６」等のゴーセネックス（登録商標）Ｌシリーズ、「ゴーセネックス（登録商標）Ｔ－３３０」等のゴーセネックス（登録商標）Ｔシリーズなどが挙げられる。反応型の変性ポリビニルアルコールとしては、アセトアセチル基等の反応性の官能基を導入したポリビニルアルコールが挙げられ、具体的には、三菱ケミカル株式会社製の「ゴーセネックス（登録商標）Ｚ－１００」等のゴーセネックス（登録商標）Ｚシリーズが挙げられる。多価アルコールの使用量は、重合体（Ａ）１００質量部に対して、好ましくは１～２００質量部、より好ましくは１０～１５０質量部であると好ましく、更に好ましくは５０～１２０質量部の多価アルコールに由来する構造を有する。 In addition to polyvalent metal ions, examples of external crosslinking agents include polyhydric alcohols. The polyhydric alcohols can react with boron compounds to indirectly crosslink the polymer (A). This allows the introduction of a structure derived from the polyhydric alcohol into the crosslinked body. Examples of polyhydric alcohols include polyvinyl alcohol, modified polyvinyl alcohol (which may be cationic, anionic, or reactive), polyglycerin, and the like. Examples of cationic modified polyvinyl alcohols include polyvinyl alcohols into which cationic functional groups such as quaternary ammonium groups have been introduced, specifically the Gohsenx K series, such as "Gohsenx K-434" manufactured by Mitsubishi Chemical Corporation. Examples of anionic modified polyvinyl alcohols include polyvinyl alcohols into which anionic functional groups such as sulfonic acid groups and carboxyl groups have been introduced, specifically the Gohsenx L series, such as "Gohsenx L-3266" manufactured by Mitsubishi Chemical Corporation, and the Gohsenx T series, such as "Gohsenx T-330" manufactured by Mitsubishi Chemical Corporation. Examples of reactive modified polyvinyl alcohol include polyvinyl alcohols into which reactive functional groups such as acetoacetyl groups have been introduced, and specific examples include the Gosenex (registered trademark) Z series, such as "Gosenex (registered trademark) Z-100" manufactured by Mitsubishi Chemical Corporation. The amount of polyhydric alcohol used is preferably 1 to 200 parts by mass, more preferably 10 to 150 parts by mass, and even more preferably 50 to 120 parts by mass of a structure derived from the polyhydric alcohol, relative to 100 parts by mass of polymer (A).
＜重合体（Ａ）の製造方法＞
本実施形態の架橋体は、重合体（Ａ）をホウ素化合物により架橋することによって製造することができる。重合体（Ａ）の製造方法としては、特に限定されないが、例えば、基（Ｉ）を有する単量体を重合することによって製造することができる。
<Method for producing polymer (A)>
The crosslinked body of the present embodiment can be produced by crosslinking the polymer (A) with a boron compound. The method for producing the polymer (A) is not particularly limited, but the polymer can be produced, for example, by polymerizing a monomer having a group (I).
重合体（Ａ）の製造方法としては、例えば、重合体（Ａ）が構造単位（Ａ）を有する場合、重合体（Ａ）は、単量体（Ａ）、任意成分として単量体（Ｂ）又は多官能ビニル化合物を含む単量体組成物を重合する方法が挙げられる。 As a method for producing polymer (A), for example, when polymer (A) has structural unit (A), polymer (A) can be produced by polymerizing a monomer composition containing monomer (A) and, as an optional component, monomer (B) or a polyfunctional vinyl compound.
単量体組成物を重合させる際には、重合開始剤を用いることができる。 A polymerization initiator can be used when polymerizing the monomer composition.
重合開始剤としては、ラジカル重合開始剤（特に熱重合開始剤）が好ましく、ベンゾイルパーオキシド、ラウロイルパーオキシド、オクタノイルパーオキシド、オルトクロロベンゾイルパーオキシド、オルトメトキシベンゾイルパーオキシド、ジイソプロピルパーオキシジカーボネート、シクロヘキサノンパーオキサイド、ｔ－ヘキシルパーオキシ－２－エチルヘキサノエート（商品名：パーヘキシルＯ（登録商標））、１，１－ジ（ｔ－ヘキシルパーオキシ）シクロヘキサン（商品名：パーヘキサＨＣ（登録商標））、クメンヒドロパーオキシド、ｔ－ブチルヒドロパーオキシド、メチルエチルケトンパーオキシド、ジイソプロピルベンゼンヒドロパーオキシド等の過酸化物系重合開始剤；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’－４－メトキシ－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のアゾ化合物系重合開始剤；が挙げられる。
これらは、それぞれ単独で用いてもよく、２種類以上を併用いてもよい。
As the polymerization initiator, a radical polymerization initiator (particularly a thermal polymerization initiator) is preferable, and examples of the peroxide-based initiator include benzoyl peroxide, lauroyl peroxide, octanoyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, diisopropyl peroxydicarbonate, cyclohexanone peroxide, t-hexylperoxy-2-ethylhexanoate (trade name: Perhexyl O (registered trademark)), 1,1-di(t-hexylperoxy)cyclohexane (trade name: Perhexa HC (registered trademark)), cumene hydroperoxide, t-butyl hydroperoxide, methyl ethyl ketone peroxide, and diisopropylbenzene hydroperoxide. Polymerization initiators: azo compound-based polymerization initiators such as 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-trimethylbutyronitrile), 2,2'-azobis(2-isopropylbutyronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile), 2,2'-4-methoxy-2,4-dimethylvaleronitrile), 2-(carbamoylazo)isobutyronitrile, 4,4'-azobis(4-cyanovaleric acid), and dimethyl-2,2'-azobisisobutyrate.
These may be used alone or in combination of two or more kinds.
重合開始剤の使用量は、特に限定されないが、通常、重合体（Ａ）の原料モノマー１００質量部あたり、好ましくは０．００１～２０質量部、より好ましくは０．０１～１０質量部である。 The amount of the polymerization initiator used is not particularly limited, but is usually preferably 0.001 to 20 parts by mass, more preferably 0.01 to 10 parts by mass, per 100 parts by mass of the raw material monomers of polymer (A).
単量体組成物は、溶媒を含んでいてもよい。つまり、単量体組成物は、溶媒の存在下で、重合してもよい。溶媒としては、特に限定されず、水及び有機溶媒が挙げられるが、水が好ましい。 The monomer composition may contain a solvent. That is, the monomer composition may be polymerized in the presence of a solvent. The solvent is not particularly limited, and may be water or an organic solvent, with water being preferred.
重合体（Ａ）をホウ素化合物により架橋させる方法は、特に制限されないが、水中で重合体（Ａ）とホウ素化合物を架橋反応させる方法が挙げられる。例えば、重合体（Ａ）の水溶液にホウ素化合物又はその水溶液を添加することによって重合体（Ａ）を架橋させることができる。水中で重合体（Ａ）をホウ素化合物により架橋させた場合、架橋体は、水溶液又はゲル状組成物の形態で得ることができる。得られた架橋体の水溶液又はゲル状組成物は、凍結乾燥等により乾燥させて乾燥体としてもよい。乾燥体は、例えば、紐状、シート状等の形状であってよいが、粉砕して粉末としてもよい。 The method for crosslinking the polymer (A) with a boron compound is not particularly limited, but may include a method of crosslinking the polymer (A) with a boron compound in water. For example, the polymer (A) can be crosslinked by adding a boron compound or an aqueous solution thereof to an aqueous solution of the polymer (A). When the polymer (A) is crosslinked with a boron compound in water, the crosslinked body can be obtained in the form of an aqueous solution or a gel composition. The aqueous solution or gel composition of the crosslinked body obtained may be dried by freeze-drying or the like to obtain a dry body. The dry body may be in the form of, for example, a string or sheet, or may be pulverized to obtain a powder.
なお、ゲル状組成物は、完全に流動性のなくなったゲルの状態のものでなくてもよく、高粘度の水溶液（粘性水溶液）であってもよい。粘性水溶液の粘度は、特に限定されないが、１０Ｐａ・ｓ以上であってよい。また、ゲル状組成物は、紐状、シート状、粉末状等の固形物であってもよいが、ハイドロゲルであってもよい。 The gel composition does not have to be in a gel state with no complete fluidity, but may be a highly viscous aqueous solution (viscous aqueous solution). The viscosity of the viscous aqueous solution is not particularly limited, but may be 10 Pa·s or more. The gel composition may be a solid material in the form of a string, sheet, powder, or the like, or may be a hydrogel.
本実施形態の架橋体を含むゲル状組成物は、細胞培養材料、癒着防止剤、薬剤担体等の医用材料、（農業用）保水材、物質吸着剤、玩具、化粧料、空洞形成材料などに使用できる。 The gel composition containing the crosslinked body of this embodiment can be used as a cell culture material, an adhesion inhibitor, a medical material such as a drug carrier, a water-retaining material (for agricultural use), a substance adsorbent, a toy, a cosmetic material, a cavity-forming material, etc.
ゲル状組成物を細胞培養材料に使用する場合、ゲル状組成物は、細胞培養に使用する添加剤を含んでいてもよい。添加剤としては、Ｌ－グルタミン、アラニルグルタミン等のアミノ酸又はアミノ酸誘導体；アルブミン、インスリン、トランスフェリン、フィブロネクチン、ラミニン、ビトロネクチン（ＥＧＦ、ＦＧＦ等のホルモン、成長因子、サイトカイン、など）、コラーゲン、ゼラチン等のタンパク質；ウシ血清、ウマ血清、ブタ血清、ウサギ血清、ヤギ血清、ヒト血清等の血清；炭酸水素ナトリウム、亜セレン酸ナトリウム等の無機塩；ピルビン酸ナトリウム、ＨＥＰＥＳ（Ｎ’－２－ヒドロキシエチルピペラジン－Ｎ’－２－エタンスルホン酸）等の有機塩などが挙げられる。 When the gel composition is used as a cell culture material, the gel composition may contain additives used in cell culture. Examples of additives include amino acids or amino acid derivatives such as L-glutamine and alanyl glutamine; proteins such as albumin, insulin, transferrin, fibronectin, laminin, vitronectin (hormones such as EGF and FGF, growth factors, cytokines, etc.), collagen, and gelatin; serum such as bovine serum, horse serum, porcine serum, rabbit serum, goat serum, and human serum; inorganic salts such as sodium bicarbonate and sodium selenite; and organic salts such as sodium pyruvate and HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid).
また、細胞培養材料を、シート状、チューブ状、円筒状、粒子状等の所望の形状として、細胞培養基材として使用することができる。細胞培養基材の形状は特に限定されず、目的に応じて適宜変更できる。 The cell culture material can be used as a cell culture substrate in any desired shape, such as a sheet, tube, cylinder, or particle shape. The shape of the cell culture substrate is not particularly limited and can be changed as appropriate depending on the purpose.
＜細胞群の製造方法＞
上記細胞培養基材を用いて、細胞群を製造することができる。すなわち、本実施形態の細胞群の製造方法は、上記細胞培養基材上で細胞を培養して細胞群を形成する工程（培養工程）と、解離剤水溶液で上記細胞培養基材を溶解し、細胞群を分離する工程（分離工程）と、を備えるものであってよい。
<Method of producing cell groups>
A cell group can be produced using the cell culture substrate. That is, the method for producing a cell group of the present embodiment may include a step of culturing cells on the cell culture substrate to form a cell group (culture step), and a step of dissolving the cell culture substrate with an aqueous dissociation agent solution to separate the cell group (separation step).
本実施形態の細胞培養基材は、上記架橋体を含むため、解離剤水溶液への溶解性に優れ、容易に解離剤水溶液に溶解させて細胞群と分離できる。また、解離剤は、糖、糖の類縁体など、細胞毒性の低いものであるものの、解離剤水溶液に高濃度に含まれていると、解離剤水溶液の浸透圧が高まり、分離工程において、分離すべき細胞の脱水などにより悪影響を及ぼす場合がある。しかしながら、本実施形態の細胞培養基材は、解離剤水溶液への溶解性に優れるため、低濃度の解離剤水溶液を使用できる。 The cell culture substrate of this embodiment contains the crosslinked body, and therefore has excellent solubility in an aqueous dissociation agent solution, and can be easily dissolved in the aqueous dissociation agent solution to separate the cell population. In addition, although the dissociation agent is a substance with low cytotoxicity, such as sugar or sugar analogues, if it is contained in a high concentration in the aqueous dissociation agent solution, the osmotic pressure of the aqueous dissociation agent increases, which may have adverse effects on the cells to be separated during the separation process, such as dehydration. However, since the cell culture substrate of this embodiment has excellent solubility in an aqueous dissociation agent solution, a low concentration aqueous dissociation agent solution can be used.
解離剤としては、特に限定されず、架橋体におけるホウ素化合物と重合体（Ａ）との間の結合を解離させ、架橋体の溶解を促す作用のあるものであれば、特に制限はないが、ゼラチン、糖、糖類縁体、多価アルコール、ポリフェノール、ヌクレオチド又はその誘導体等が挙げられ、糖又は糖類縁体が好ましい。 The dissociating agent is not particularly limited as long as it dissociates the bond between the boron compound and the polymer (A) in the crosslinked body and promotes dissolution of the crosslinked body. Examples of the dissociating agent include gelatin, sugar, sugar analogues, polyhydric alcohols, polyphenols, nucleotides or derivatives thereof, and sugar or sugar analogues are preferred.
糖としては、単糖類、二糖類、オリゴ糖、多糖類のいずれであってもよいが、単糖類が好ましく、五炭糖又は六炭糖が好ましく、リボース又はグルコールが更に好ましい。糖類縁体としては、ソルビトール等の炭素数５～６の糖アルコールが好ましい。多価アルコールとしては、カテコール等の低分子化合物、ポリビニルアルコール等の高分子化合物が挙げられる。ヌクレオチドとしては、ＡＴＰ（アデノシン三リン酸）、アデノシン二リン酸（ＡＤＰ）、アデノシン一リン酸（ＡＭＰ）等が挙げられる。ヌクレオチド誘導体としては、ニコチンアミドアデニンジヌクレオチド（ＮＡＤ＋、又はその還元型のＮＡＤＨ）等が挙げられる。 The sugar may be any of monosaccharides, disaccharides, oligosaccharides, and polysaccharides, but is preferably a monosaccharide, more preferably a pentose or hexose, and even more preferably ribose or glucose. The sugar analog is preferably a sugar alcohol having 5 to 6 carbon atoms, such as sorbitol. The polyhydric alcohol may be a low molecular weight compound, such as catechol, or a high molecular weight compound, such as polyvinyl alcohol. The nucleotide may be ATP (adenosine triphosphate), adenosine diphosphate (ADP), adenosine monophosphate (AMP), or the like. The nucleotide derivative may be nicotinamide adenine dinucleotide (NAD+, or its reduced form, NADH), or the like.
解離剤水溶液における解離剤の濃度は特に限定されないが、解離剤水溶液の全量に対して０．１～１０質量％であると好ましく、０．１～５質量％であるとより好ましく、０．１～３質量％であると更に好ましく、０．１～２質量％であると特に好ましい。 The concentration of the dissociator in the aqueous dissociator solution is not particularly limited, but is preferably 0.1 to 10% by mass, more preferably 0.1 to 5% by mass, even more preferably 0.1 to 3% by mass, and particularly preferably 0.1 to 2% by mass, based on the total amount of the aqueous dissociator solution.
製造される細胞群の形状としては、特に限定されず、塊状、チューブ状、円筒状、シート状のいずれであってもよい。細胞群の形状は、例えば、使用する細胞培養基材の形状を変更することにより適宜調節できる。 The shape of the cell mass produced is not particularly limited and may be any of a block, tube, cylinder, and sheet shape. The shape of the cell mass can be appropriately adjusted, for example, by changing the shape of the cell culture substrate used.
分離工程において、細胞培養基材を溶解させる方法としては、特に限定されず、例えば、細胞群が形成された細胞培養基材を解離剤水溶液に浸漬する方法等が挙げられる。浸漬時間は、１０分～２０時間が好ましく、３０分～４時間が好ましい。また、細胞培養基材を浸漬している間、解離剤水溶液の温度は、２０～４０℃であると好ましい。 In the separation step, the method for dissolving the cell culture substrate is not particularly limited, and examples include a method of immersing the cell culture substrate on which the cell groups have been formed in an aqueous dissociation agent. The immersion time is preferably 10 minutes to 20 hours, and more preferably 30 minutes to 4 hours. In addition, while the cell culture substrate is immersed in the aqueous dissociation agent, the temperature of the aqueous dissociation agent is preferably 20 to 40°C.
また、本実施形態の細胞群の製造方法により製造することのできる細胞群は、特に限定されず、あらゆる細胞（例えば、表皮細胞、上皮細胞、内皮細胞、繊維芽細胞、脂肪細胞、免疫細胞、筋細胞、軟骨細胞、骨髄細胞、骨細胞、骨芽細胞、破骨細胞、血球系細胞、神経細胞、肝細胞、膵細胞、腎細胞などの細胞種もしくはこれら細胞の前駆細胞、幹細胞、癌細胞など）、又はそれらの細胞が存在するあらゆる組織・器官（例えば、皮膚、筋肉、骨、関節、骨格筋、血管、脊髄、心臓、胸腺、脾臓、肺、膵臓、腎臓、肝臓、生殖腺、消化管など）の培養に使用することができる。 The cell group that can be produced by the cell group production method of this embodiment is not particularly limited, and can be used for culturing any cell (e.g., epidermal cells, epithelial cells, endothelial cells, fibroblasts, adipocytes, immune cells, muscle cells, chondrocytes, bone marrow cells, bone cells, osteoblasts, osteoclasts, blood cells, nerve cells, hepatic cells, pancreatic cells, kidney cells, or precursor cells of these cells, stem cells, cancer cells, etc.), or any tissue or organ in which such cells exist (e.g., skin, muscle, bone, joint, skeletal muscle, blood vessels, spinal cord, heart, thymus, spleen, lung, pancreas, kidney, liver, gonads, digestive tract, etc.).
＜細胞群の分離方法Ｉ＞
本実施形態の細胞群の分離方法は、上記架橋体を含むゲル状基材を解離剤水溶液で溶解し、当該ゲル状基材に接触している細胞群を分離する工程を含むものであってよい。上述のとおり、本実施形態のゲル状基材は、上記架橋体を含むため、解離剤水溶液に容易に溶解させることができるため、当該ゲル状基材に接触（付着）する細胞群と容易に分離できる。ゲル状基材としては、特に限定されないが、ハイドロゲルであってよく、細胞培養基材であってもよい。ゲル状基材を解離剤水溶液で溶解する方法としては、特に制限はないが、上記細胞培養基材を溶解させる方法と同様の方法を採用できる。
<Cell group separation method I>
The method for separating a cell group of this embodiment may include a step of dissolving the gel-like substrate containing the crosslinked body in an aqueous solution of a dissociating agent, and separating a cell group in contact with the gel-like substrate. As described above, since the gel-like substrate of this embodiment contains the crosslinked body, it can be easily dissolved in an aqueous solution of a dissociating agent, and therefore it can be easily separated from a cell group in contact (attached) with the gel-like substrate. The gel-like substrate is not particularly limited, but may be a hydrogel or a cell culture substrate. The method for dissolving the gel-like substrate with an aqueous solution of a dissociating agent is not particularly limited, but a method similar to the method for dissolving the cell culture substrate may be adopted.
＜細胞群の分離方法ＩＩ＞
また、本実施形態の細胞群の分離方法は、ゲル状基材を０．１～５質量％（０．２～３質量％であると好ましく、０．３～２質量％であるとより好ましい。また、０．５～５質量％であってもよい。）の解離剤水溶液で溶解し、当該ゲル状基材に接触している細胞群を分離する工程を含むものであってもよい。ゲル状基材としては、特に限定されないが、上記架橋体を含むものであってよい。また、ゲル状基材としては、特に限定されないが、ハイドロゲルであってよく、細胞培養基材であってもよい。ゲル状基材を解離剤水溶液で溶解する方法としては、特に制限はないが、上記細胞培養基材を溶解させる方法と同様の方法を採用できる。
<Cell group separation method II>
The method for separating a cell group of this embodiment may include a step of dissolving the gel-like substrate with 0.1 to 5% by mass (preferably 0.2 to 3% by mass, more preferably 0.3 to 2% by mass, and may also be 0.5 to 5% by mass) of an aqueous solution of a dissociating agent, and separating the cell group in contact with the gel-like substrate. The gel-like substrate is not particularly limited, but may include the crosslinked body. The gel-like substrate is not particularly limited, but may be a hydrogel or a cell culture substrate. The method for dissolving the gel-like substrate with an aqueous solution of a dissociating agent is not particularly limited, but a method similar to the method for dissolving the cell culture substrate may be used.
＜空洞形成材料＞
本実施形態の架橋体は、解離剤水溶液により容易に溶出することができるため、他の材料に空洞を形成するための材料（空洞形成材料）として使用することができる。例えば、架橋体を含まない基材中に、架橋体を含むゲル構造物（ハイドロゲル等）を封入してゲル封入基材を作製し、次いで、解離剤水溶液でゲルを溶出することにより、空洞が形成された基材を作製することができる。空洞が形成された基材としては特に制限はなく、細胞培養基材等であってよい。空洞が形成された基材が細胞培養基材である場合、基材に空洞が形成された後に細胞培養基材として用いてもよいが、細胞培養中、又は細胞培養後に解離剤水溶液で架橋体を含むゲルを溶出してもよい。基材の材質としては、架橋体を含むゲルよりも解離剤水溶液に対する溶解度が高いものであれば特に制限はないが、ゼラチン等が挙げられる。
<Cavity forming material>
The crosslinked body of the present embodiment can be easily dissolved by a dissociating agent aqueous solution, and therefore can be used as a material for forming cavities in other materials (cavity-forming material). For example, a gel-encapsulated substrate is prepared by encapsulating a gel structure (hydrogel, etc.) containing the crosslinked body in a substrate not containing the crosslinked body, and then dissolving the gel with a dissociating agent aqueous solution, thereby preparing a substrate having a cavity formed therein. The substrate having a cavity formed therein is not particularly limited, and may be a cell culture substrate or the like. When the substrate having a cavity formed therein is a cell culture substrate, it may be used as a cell culture substrate after the cavity is formed in the substrate, or the gel containing the crosslinked body may be dissolved with a dissociating agent aqueous solution during or after cell culture. The material of the substrate is not particularly limited as long as it has a higher solubility in a dissociating agent aqueous solution than the gel containing the crosslinked body, and examples of the material include gelatin.
基材に形成される空洞は、架橋体を含むゲル構造物の形状に相補的な形状である。例えば、ゲル構造物として紐状のものを用いれば、円筒状、チューブ状等の形状の空洞を形成できる。また、ゲル構造物の形状が粒子状であれば、基材に細孔を形成することができ、多孔質基材を得ることもできる。本実施形態の架橋体は、解離剤水溶液で容易に溶出できるため、溶出中に基材に溶出液による悪影響を及ぼすことが少ない。 The cavity formed in the substrate has a shape complementary to the shape of the gel structure containing the crosslinked body. For example, if a string-shaped gel structure is used, a cavity having a cylindrical, tubular, or other shape can be formed. Furthermore, if the gel structure is particulate, pores can be formed in the substrate, and a porous substrate can be obtained. The crosslinked body of this embodiment can be easily dissolved in an aqueous dissociation agent solution, so that the substrate is less adversely affected by the dissolution solution during dissolution.
合成例１（Ｐ（ＧＬＭＡ）の合成）
攪拌子を入れた反応容器にガス導入管、温度計、冷却管を付し、単量体としてグリセロールモノアクリレート（ＧＬＭＡ）１０ｇ、溶媒として精製水９０ｇ、アゾ系ラジカル重合開始剤０．０５ｇ（和光純薬社製、商品名：Ｖ－５０）を仕込み、窒素ガスを流しながら攪拌と昇温を開始した。内温６５℃で重合を開始し、６時間反応を行った。
得られた反応液を大量のアセトン中に撹拌しながら投入することで再沈した。沈殿物を真空乾燥機によって、減圧下、４０℃で１２時間減圧乾燥し、固体の重合体Ｐ（ＧＬＭＡ）を得た。
得られた重合体Ｐ（ＧＬＭＡ）の重量平均分子量は５８２０００であった。なお、重量平均分子量は、ＧＰＣにより測定した値である。
Synthesis Example 1 (Synthesis of P(GLMA))
A gas inlet tube, a thermometer, and a cooling tube were attached to a reaction vessel containing a stirrer, and 10 g of glycerol monoacrylate (GLMA) as a monomer, 90 g of purified water as a solvent, and 0.05 g of an azo radical polymerization initiator (manufactured by Wako Pure Chemical Industries, Ltd., product name: V-50) were charged, and stirring and heating were started while flowing nitrogen gas. Polymerization was started at an internal temperature of 65°C, and the reaction was carried out for 6 hours.
The reaction solution was poured into a large amount of acetone with stirring to cause reprecipitation. The precipitate was dried under reduced pressure at 40° C. for 12 hours using a vacuum dryer to obtain a solid polymer P(GLMA).
The weight average molecular weight of the obtained polymer P(GLMA) was 582000. The weight average molecular weight was a value measured by GPC.
合成例２（Ｐ（１，３－ＤＨＰＭＡ）の合成）
単量体としてＮ－（１，３－ジヒドロキシプロパン２－イル）メタクリルアミド（１，３－ＤＨＰＭＡ）１０ｇを用いた以外は合成例１と同様にして、固体の重合体Ｐ（１，３－ＤＨＰＭＡ）を得た。
得られた重合体Ｐ（１，３－ＤＨＰＭＡ）の重量平均分子量は５２５０００であった。
Synthesis Example 2 (Synthesis of P(1,3-DHPMA))
A solid polymer P (1,3-DHPMA) was obtained in the same manner as in Synthesis Example 1, except that 10 g of N-(1,3-dihydroxypropan-2-yl) methacrylamide (1,3-DHPMA) was used as the monomer.
The weight average molecular weight of the resulting polymer P (1,3-DHPMA) was 525,000.
合成例３（Ｐ（ＧＡｚ０５）の合成
単量体としてグリセロールモノアクリレート（ＧＬＭＡ）９．５ｇ、アクリル酸亜鉛（ＡＡ－Ｚｎ）０．５を用いた以外は合成例１と同様にして、固体の重合体Ｐ（ＧＡｚ０５）を得た。
得られた重合体Ｐ（ＧＡｚ０５）の重量平均分子量は８３９０００であった。
Synthesis Example 3 (Synthesis of P(GAz05)) A solid polymer P(GAz05) was obtained in the same manner as in Synthesis Example 1, except that 9.5 g of glycerol monoacrylate (GLMA) and 0.5 g of zinc acrylate (AA-Zn) were used as monomers.
The weight average molecular weight of the obtained polymer P (GAz05) was 839,000.
実施例１
合成例１で得られたＰ（ＧＬＭＡ）の１０質量％水溶液２００μｌと硼砂の飽和水溶液１００μｌを混合し激しく攪拌することで、ハイドロゲルが得られた。
Example 1
200 μl of the 10% by mass aqueous solution of P(GLMA) obtained in Synthesis Example 1 and 100 μl of a saturated aqueous solution of borax were mixed and vigorously stirred to obtain a hydrogel.
＜溶解試験＞
得られたハイドロゲル５ｍｇに２質量％グルコース水溶液１０００μｌを加え３７℃で静置した。また、得られたハイドロゲル５ｍｇに２質量％ソルビトール水溶液１０００μｌを加え３７℃で静置した。
グルコース水溶液及びソルビトール水溶液のいずれを用いた場合であっても、６０分後には完全にハイドロゲルが溶解及び消失した。
<Dissolution test>
1000 μl of a 2% by mass glucose aqueous solution was added to 5 mg of the obtained hydrogel, and the mixture was allowed to stand at 37° C. Also, 1000 μl of a 2% by mass sorbitol aqueous solution was added to 5 mg of the obtained hydrogel, and the mixture was allowed to stand at 37° C.
Whether the glucose aqueous solution or the sorbitol aqueous solution was used, the hydrogel was completely dissolved and disappeared after 60 minutes.
実施例２
合成例１で得られたＰ（ＧＬＭＡ）の１０質量％と水溶液１００μｌとポリビニルアルコール（クラレ社製、商品名：「ＰＶＡ２１７」）の１０質量％水溶液１００μｌとを混合し、さらに硼砂の飽和水溶液１００μｌを混合し激しく攪拌することで、ハイドロゲルが得られた。得られたハイドロゲルに対して実施例１と同様に溶解試験を行った。結果を表１に示す。
Example 2
A hydrogel was obtained by mixing 100 μl of a 10% by mass aqueous solution of P(GLMA) obtained in Synthesis Example 1 with 100 μl of a 10% by mass aqueous solution of polyvinyl alcohol (manufactured by Kuraray Co., Ltd., product name: "PVA217"), and further mixing with 100 μl of a saturated aqueous solution of borax and vigorously stirring. A dissolution test was performed on the obtained hydrogel in the same manner as in Example 1. The results are shown in Table 1.
実施例３
Ｐ（ＧＬＭＡ）の１０質量％水溶液に代えて合成例３で得られたＰ（ＧＡｚ０５）の１０質量％水溶液を用いた以外は実施例１と同様にしてハイドロゲルを得た。得られたハイドロゲルに対して実施例１と同様に溶解試験を行った。結果を表１に示す。
Example 3
A hydrogel was obtained in the same manner as in Example 1, except that the 10% by mass aqueous solution of P(GAz05) obtained in Synthesis Example 3 was used instead of the 10% by mass aqueous solution of P(GLMA). The resulting hydrogel was subjected to a dissolution test in the same manner as in Example 1. The results are shown in Table 1.
実施例４
Ｐ（ＧＬＭＡ）の１０質量％水溶液に代えて合成例２で得られたＰ（１，３－ＤＨＰＭＡ）の１０質量％水溶液を用いた以外は実施例１と同様にしてハイドロゲルを得た。得られたハイドロゲルに対して実施例１と同様に溶解試験を行った。結果を表１に示す。
Example 4
A hydrogel was obtained in the same manner as in Example 1, except that the 10% by mass aqueous solution of P(GLMA) was replaced with the 10% by mass aqueous solution of P(1,3-DHPMA) obtained in Synthesis Example 2. The resulting hydrogel was subjected to a dissolution test in the same manner as in Example 1. The results are shown in Table 1.
実施例５
ホウ砂の飽和水溶液に代えてメタホウ酸ナトリウムの３質量％水溶液を用いた以外は実施例３と同様にしてハイドロゲルを得た。得られたハイドロゲルに対して実施例１と同様に溶解試験を行った。結果を表１に示す。
Example 5
A hydrogel was obtained in the same manner as in Example 3, except that a 3% by mass aqueous solution of sodium metaborate was used instead of the saturated aqueous solution of borax. The resulting hydrogel was subjected to a dissolution test in the same manner as in Example 1. The results are shown in Table 1.
比較例１
Ｐ（ＧＬＭＡ）の１０質量％水溶液に代えてポリビニルアルコール（クラレ社製、商品名：「ＰＶＡ２１７」）の１０質量％水溶液を用いた以外は実施例１と同様にしてハイドロゲルを得た。得られたハイドロゲルに対して実施例１と同様に溶解試験を行った。結果を表１に示す。
Comparative Example 1
A hydrogel was obtained in the same manner as in Example 1, except that a 10% by mass aqueous solution of polyvinyl alcohol (manufactured by Kuraray Co., Ltd., product name: "PVA217") was used instead of the 10% by mass aqueous solution of P(GLMA). The resulting hydrogel was subjected to a dissolution test in the same manner as in Example 1. The results are shown in Table 1.
実施例１～５及び比較例１の結果を表１にまとめる。なお、表１における溶解試験の評価基準は以下のとおりである。
Ａ：ハイドロゲルが６０分後に完全に溶解及び消失した。
Ｂ：ハイドロゲルが６０分後には残存していたものの、２４時間後に完全に溶解及び消失した。
Ｃ：ハイドロゲルが２４時間後に残存した。
The results of Examples 1 to 5 and Comparative Example 1 are summarized in Table 1. The evaluation criteria for the dissolution test in Table 1 are as follows.
A: The hydrogel completely dissolved and disappeared after 60 minutes.
B: The hydrogel remained after 60 minutes, but was completely dissolved and disappeared after 24 hours.
C: The hydrogel remained after 24 hours.
実施例６
合成例３で得られたＰ（ＧＡｚ０５）の１０質量％水溶液２００μｌをＣｏｓｔａｒ（登録商標）細胞培養プレート２４ウェル（コーニング社製）に加え、そこにメタホウ酸ナトリウムの３質量％水溶液１００μｌを添加し、オービタルシェイカーで一晩攪拌することで、ゲル化させた。そこに３７℃に温めたゼラチンの１０質量％水溶液を加え１時間かけて浸透させた後、紫外線照射装置で滅菌処理を行い、細胞培養基材を作製した。
Example 6
200 μl of the 10% by mass aqueous solution of P(GAz05) obtained in Synthesis Example 3 was added to a 24-well Costar (registered trademark) cell culture plate (manufactured by Corning), 100 μl of a 3% by mass aqueous solution of sodium metaborate was added thereto, and gelation was performed by stirring overnight with an orbital shaker. A 10% by mass aqueous solution of gelatin warmed to 37° C. was added thereto and allowed to penetrate for 1 hour, followed by sterilization treatment with an ultraviolet irradiation device to prepare a cell culture substrate.
作製した細胞培養基材に、ＭＣ３Ｔ３－Ｅ１細胞を懸濁した培地を１×１０６ｃｅｌｌｓ／ｗｅｌｌで播種し、５体積％ＣＯ２、３７℃の条件で培養した。培養細胞が増殖してシート状になったのを確認したのち、２質量％ソルビトール水溶液１０００μｌを加え３７℃で１時間処理したところ、ゲルが溶解し、剥離した細胞シートを回収することができた。なお、培地には約０．１質量％のグルコースが含まれていた。 The prepared cell culture substrate was seeded with a medium containing a suspension of MC3T3-E1 cells at 1 x 106 cells/well, and cultured under conditions of 5% by volume CO2 and 37°C. After confirming that the cultured cells had proliferated and formed a sheet, 1000 μl of a 2% by mass sorbitol aqueous solution was added and treated at 37°C for 1 hour, whereupon the gel dissolved and the detached cell sheet could be collected. The medium contained approximately 0.1% by mass of glucose.
実施例７
実施例１で得られたＰ（ＧＬＭＡ）１０質量％水溶液と硼砂飽和水溶液で作製したハイドロゲルを、ポジティブディスプレイスメント方式のマイクロピペットを用いて押し出して紐状にした。液体窒素で凍結後、減圧乾燥を行い、紐状構造物を得た。
ゼラチン溶液をホモジナイザーを用いて攪拌して泡立てた後、グルタルアルデヒド溶液を加えて攪拌し、ポリプロピレン製の型に注ぎ、その中に紐状構造物を封入した。ゼラチンがグルタルアルデヒドで架橋されたのを確認後、凍結乾燥して紐状構造物封入ゼラチンスポンジを得た。
ゼラチンスポンジを２質量％グルコース水溶液に６０分間浸漬した後、紐状構造物が溶解して連通した空洞が形成していることが確認された。
Example 7
The hydrogel prepared from the 10% by mass aqueous solution of P(GLMA) obtained in Example 1 and the saturated aqueous solution of borax was extruded into a string shape using a positive displacement micropipette. After freezing with liquid nitrogen, it was dried under reduced pressure to obtain a string-like structure.
The gelatin solution was stirred and foamed using a homogenizer, and then glutaraldehyde solution was added and stirred, and the mixture was poured into a polypropylene mold, in which the string-like structures were encapsulated. After confirming that the gelatin was crosslinked with glutaraldehyde, the mixture was freeze-dried to obtain a gelatin sponge encapsulating the string-like structures.
It was confirmed that after the gelatin sponge was immersed in a 2% by mass glucose aqueous solution for 60 minutes, the string-like structures were dissolved and interconnected cavities were formed.
Claims (10)
前記重合体における前記構造単位（Ａ）の含有量は、重合体の総量に対して、５０質量％以上である、架橋体。
（式（ＩＩ）中、＊は前記構造単位（Ａ）の結合位置を表し、Ｒは水素原子又はメチル基であり、Ｘは－Ｏ－又は－ＮＨ－であり、Ｚは２つ以上の水酸基を有する炭素数１～８の有機基である。） The polymer has a structure in which a polymer including a structural unit (A) represented by the following formula (II) is crosslinked by a boron compound including a BOH group or a salt thereof,
The content of the structural unit (A) in the polymer is 50 mass% or more based on the total amount of the polymer .
( In formula (II), * represents the bonding position of the structural unit (A) , R is a hydrogen atom or a methyl group, X is -O- or -NH-, and Z is an organic group having 1 to 8 carbon atoms and two or more hydroxyl groups.)
（式（ＩＩＩ）中、Ｒ１は－ＣＨ（ＯＨ）ＣＨ２ＯＨ又は－ＣＨ２ＯＨであり、Ｒ２は、水素原子又は－ＣＨ２ＯＨであり、Ｒ３は、水素原子又は－ＣＨ２ＯＨであり、＊は前記基（ＩＩＩ）の結合位置を表す。） The crosslinked body according to any one of claims 1 to 3 , wherein Z is a group (III) represented by the following formula (III):
(In formula (III), R 1 is -CH(OH)CH 2 OH or -CH 2 OH, R 2 is a hydrogen atom or -CH 2 OH, R 3 is a hydrogen atom or -CH 2 OH, and * indicates the bonding position of the group (III).)
解離剤水溶液で前記細胞培養基材を溶解し、前記細胞群を分離する工程と、を備える、細胞群の製造方法。 A step of culturing cells on a cell culture substrate comprising the crosslinked body according to any one of claims 1 to 6 to form a cell population;
dissolving the cell culture substrate with an aqueous dissociation agent to separate the cell population.
A method comprising the steps of dissolving a gel-like substrate containing the crosslinked body according to any one of claims 1 to 6 with an aqueous dissociating agent solution, and separating a cell group in contact with the gel-like substrate.
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