JP6413492B2 - Polymer compound, coating material, molded article coated with coating material, and method for producing the same - Google Patents
Polymer compound, coating material, molded article coated with coating material, and method for producing the same Download PDFInfo
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Description
本発明は、高分子化合物、コーティング材、コーティング材を被覆した成形体並びにその製造方法に関する。 The present invention relates to a polymer compound, a coating material, a molded article coated with the coating material, and a method for producing the same.
細胞培養容器は表面を親水性処理して細胞付着を防止する必要があり、これまでに様々な種類の処理がなされている。 Cell culture containers need to be treated with a hydrophilic surface to prevent cell adhesion, and various types of treatment have been performed so far.
例えば、特許文献１には、ホスホリルコリン基を有する親水性メタクリレートにブチルメタクリレートなどの疎水性メタクリレートを共重合したポリマー溶液を容器表面に塗布乾燥することで親水性としている。また、特許文献２、３にはホスホコリン基を有する親水性メタクリレートと側鎖にアジド基を有する光反応性メタクリレート、更に必要な場合は疎水性のメタクリレートなどを共重合したもので容器表面に塗布する前に表面を酸素プラズマ処理してその後ポリマーを塗布した後、ＵＶ照射してアジド基をナイトレンに変え容器表面のラジカルイオンや親水化処理ポリマー内で架橋させて強固に容器表面に結合させているものが開示されている。 For example, in Patent Document 1, a hydrophilic polymer is made hydrophilic by applying a polymer solution obtained by copolymerizing a hydrophilic methacrylate having a phosphorylcholine group to a hydrophobic methacrylate such as butyl methacrylate on the surface of a container. In Patent Documents 2 and 3, a hydrophilic methacrylate having a phosphocholine group and a photoreactive methacrylate having an azide group in the side chain, and if necessary, a copolymer of hydrophobic methacrylate and the like are applied to the surface of the container. Oxygen plasma treatment on the surface before application of the polymer, then UV irradiation to change the azide group to nitrene and crosslink in the radical ion on the vessel surface or in the hydrophilized polymer to firmly bond to the vessel surface Are disclosed.
特許文献１に記載の技術は、ポリマー処理は容器と共有結合で接続されていないため強い処理で容器表面から剥離または染み出しが生じる。特許文献２，３に記載の技術は、アジド基からＵＶ照射で生成するナイトレンで容器表面と共有結合できるものの、剥離、染み出しを十分に防止できない可能性がある。 In the technique described in Patent Document 1, since the polymer treatment is not covalently connected to the container, peeling or exudation occurs from the container surface by a strong treatment. Although the techniques described in Patent Documents 2 and 3 can be covalently bonded to the container surface with nitrene generated from the azide group by UV irradiation, there is a possibility that peeling and bleeding cannot be sufficiently prevented.
本発明は、上記に鑑みてなされたものであり、容器と強固に結合し、かつ分子内でも架橋する高分子化合物、該高分子化合物を用いたコーティング材を提供することにある。
The present invention has been made in view of the above, and it is an object of the present invention to provide a polymer compound that is firmly bonded to a container and also crosslinks in the molecule, and a coating material using the polymer compound.
このような目的は、下記（１）から（１０）に記載の本発明により達成される。
（１）下記一般式（１）で示される構造を有することを特徴とする高分子化合物。
（式中、Ｒ１、Ｒ３及びＲ６は互いに独立して、共重合して得られた状態の重合性原子団
を表わし、下記（３）又は（４）であり、；Ｒ２、Ｒ４及びＲ７
は互いに独立して、置換基を有しても良いフェニル基又は−Ｃ（Ｏ）−、−Ｃ（Ｏ）Ｏ−
、−Ｏ−、もしくは−Ｓ−で示される基を表わし；Ｒ５は置換基を有しても良いフェニル
基又は−ＯＣ（Ｏ）−、−Ｃ（Ｏ）−、−Ｏ−で示される基を表わし；ｌ及びｍは互いに
独立して、２以上の整数を表わし；ｎは１以上の整数を表わし；ａ及びｂは互いに独立し
て２以上の整数を表わし；ｃは０もしくは１以上の整数を表わす。；Ｒ１を含む構造単位
とＲ３を含む構造単位とＲ６を含む構造単位はランダムな順序で結合するか、またはブロ
ック状態で結合している。）
（２）一般式（１）のＲ２、Ｒ４、Ｒ５，またはＲ７がエステル基であり、ｌ, ｍは２以
上１０以下の整数であり、ｎは１以上１０以下の整数である、（１）に記載の高分子化合
物。
（３）一般式（１）で示される構造を有する高分子化合物が下記一般式（２）で示される
構造を有する（１）または（２）に記載の高分子化合物。
（式中、Ｒ１、Ｒ３及びＲ６は互いに独立して、共重合して得られた状態の重合性原子団を表わし、下記（３）又は（４）であり、；ａ、ｂは２以上の整数であり、ｃは０もしくは１以上の整数であり；ｎは１以上１０以下の整数であり；ホスホリルコリン基を含む側鎖を有する構造単位とアジドシンナモイル基を含む構造単位とアルキレート基を含む構造単位とはランダムな順序またはブロックな順序で結合している。）
（４）一般式（１）及び（２）で示される構造の高分子化合物は、ａ／（ａ＋ｂ＋ｃ）の
値が０．３０以上０．９０以下であり、ｂ／（ａ＋ｂ＋ｃ）の値が０．１０以上０．６０
以下であり、ｃ／（ａ＋ｂ＋ｃ）の値が０以上０．５０以下である（１）ないし（３）い
ずれか１つに記載の高分子化合物。
（５）（１）ないし（４）いずれか１つに記載の高分子化合物を含むコーティング材。
（６）コーティング材によって被覆された被覆部を有する成形体であって、前記コーティ
ング材が（５９記載のコーティング材である成形体。
（７）（６）記載の成形体用いて製造される生化学容器。
（８）（７）の生化学容器を用いて製造される細胞培養容器。
（９）成形体に（５）記載のコーティング材を塗布、乾燥する第一の工程と、光照射して前記コーティング材を硬化する第二の工程を有する、成形体の製造方法。
（１０）前記第二の工程において、光照射量が１０mJ/cm 2 以上１０００mJ/cm 2 以下である
、（９）に記載の成形体の製造方法。
Such an object is achieved by the present invention described in the following (1) to (10).
(1) A polymer compound having a structure represented by the following general formula (1).
(In the formula, R1, R3 and R6 each independently represent a polymerizable atomic group obtained by copolymerization, and are the following (3) or (4) ; R2, R4 and R7
Are each independently a phenyl group which may have a substituent or -C (O)-, -C (O) O-
, -O-, or -S-; R5 represents a phenyl group which may have a substituent or a group represented by -OC (O)-, -C (O)-or -O- And l and m each independently represent an integer of 2 or more; n represents an integer of 1 or more; a and b independently of each other represent an integer of 2 or more; c is 0 or 1 or more Represents an integer. The structural unit containing R1, the structural unit containing R3, and the structural unit containing R6 are bonded in a random order or bonded in a block state. )
(2) R2, R4, R5 or R7 in the general formula (1) is an ester group, l and m are integers of 2 or more and 10 or less, and n is an integer of 1 or more and 10 or less, (1) The high molecular compound as described in.
(3) The polymer compound according to (1) or (2), wherein the polymer compound having a structure represented by the general formula (1) has a structure represented by the following general formula (2).
(Wherein R1, R3 and R6 each independently represent a polymerizable atomic group in a state obtained by copolymerization, and are the following (3) or (4) ; a and b are 2 or more C is an integer of 0 or 1 or more; n is an integer of 1 or more and 10 or less; a structural unit having a side chain containing a phosphorylcholine group, a structural unit containing an azidocinnamoyl group, and an alkylate group. (The structural units are included in a random or block order.)
(4) In the polymer compound having the structure represented by the general formulas (1) and (2), the value of a / (a + b + c) is 0.30 or more and 0.90 or less, and the value of b / (a + b + c) is 0. .10 or more 0.60
The polymer compound according to any one of (1) to (3), wherein c / (a + b + c) is 0 or more and 0.50 or less.
(5) A coating material comprising the polymer compound according to any one of (1) to (4).
(6) A molded body having a coating portion coated with a coating material, wherein the coating material is the coating material according to (59).
(7) A biochemical container manufactured using the molded article according to (6).
(8) A cell culture container manufactured using the biochemical container of (7).
(9) A method for producing a molded body, which includes a first step of applying and drying the coating material according to (5) to the molded body, and a second step of curing the coating material by light irradiation.
(10) The method for producing a molded article according to (9), wherein, in the second step, the light irradiation amount is 10 mJ / cm 2 or more and 1000 mJ / cm 2 or less.
本発明の高分子化合物を使用することにより、頑強性に優れたコーティング材を得ることが可能となり、さらに頑強性に優れた細胞培養容器をはじめとする成形体を得ることが
可能となる。
By using the polymer compound of the present invention, a coating material excellent in robustness can be obtained, and further, a molded body including a cell culture container excellent in robustness can be obtained.
以下、本発明の高分子化合物、該高分子化合物を使用したコーティング材、コーティング材を使用した容器、また容器の製造方法について説明する。 Hereinafter, the polymer compound of the present invention, a coating material using the polymer compound, a container using the coating material, and a method for producing the container will be described.
本発明の高分子化合物は、下記一般式（１）で示される構造を有することを特徴とする
高分子化合物である。
（式中、Ｒ１、Ｒ３及びＲ６は互いに独立して、共重合して得られた状態の重合性原子団を表わし、下記（３）又は（４）であり、；Ｒ２、Ｒ４及びＲ７は互いに独立して、置換基を有しても良いフェニル基又は−Ｃ（Ｏ）−、−Ｃ（Ｏ）Ｏ−、−Ｏ−、もしくは−Ｓ−で示される基を表わし；Ｒ５は置換基を有しても良いフェニル基又は−ＯＣ（Ｏ）−、−Ｃ（Ｏ）−、−Ｏ−で示される基を表わし；ｌ及びｍは互いに独立して、２以上の整数を表わし；ｎは１以上の整数を表わし；ａ及びｂは互いに独立して２以上の整数を表わし；ｃは０もしくは１以上の整数を表わす。；Ｒ１を含む構造単位とＲ３を含む構造単位とＲ６を含む構造単位はランダムな順序で結合するか、またはブロック状態で結合している。）
The polymer compound of the present invention is a polymer compound having a structure represented by the following general formula (1).
(In the formula, R1, R3 and R6 independently represent a polymerizable atomic group obtained by copolymerization, and are the following (3) or (4) ; R2, R4 and R7 are Independently, it represents a phenyl group which may have a substituent or a group represented by -C (O)-, -C (O) O-, -O- or -S-; R5 represents a substituent. An optionally substituted phenyl group or a group represented by —OC (O) —, —C (O) —, —O—; and l and m each independently represent an integer of 2 or more; A and b each independently represent an integer of 2 or more; c represents an integer of 0 or 1; a structural unit containing R1, a structural unit containing R3, and a structure containing R6 Units are combined in a random order or in a block state.)
親水性のホスホコリン基メタクリレートと側鎖にアジド並びに不飽和基を含有するメタクリレートを共重合した本発明の高分子化合物は、容器を酸素プラズマ処理した後ポリマーを塗布してＵＶ照射することで容器表面と共有結合をつくり、さらにアジドにＵＶ照射することにより形成されるナイトレンが分子内の不飽和結合と架橋して十分な強度を有する塗膜を形成することができる。またホスホコリン基を親水性部に用いているので初代細胞の培養も可能である。 The polymer compound of the present invention obtained by copolymerizing a hydrophilic phosphocholine methacrylate and a methacrylate containing an azide and an unsaturated group in the side chain is coated with a polymer after the container is subjected to oxygen plasma treatment, and then irradiated with UV. The nitrene formed by forming a covalent bond with the azide and irradiating the azide with UV can be crosslinked with the unsaturated bond in the molecule to form a coating film having sufficient strength. Moreover, since the phosphocholine group is used for the hydrophilic part, primary cells can be cultured.
本発明の高分子化合物は、一般式（１）のＲ２、Ｒ４、Ｒ５，またはＲ７がエステル基であり、ｌ, ｍは２以上１０以下の整数であり、ｎは１以上１０以下の整数であることが好ましい。上記構造、範囲であることで、容器との強固な結合、分子間の強固な結合、かつ最適な親水性を有することによる細胞付着防止の効果を有する高分子化合物を得ることが可能となる。 In the polymer compound of the present invention, R 2 , R 4 , R 5 , or R 7 in the general formula (1) is an ester group, l and m are integers of 2 to 10, and n is 1 to 10 The following integers are preferred. With the above structure and range, it is possible to obtain a polymer compound having an effect of preventing cell adhesion by having strong bonds with the container, strong bonds between molecules, and optimal hydrophilicity.
さらに、一般式（１）の好ましい形態は、下記一般式（２）記載の高分子化合物である
。
（式中、Ｒ１、Ｒ３及びＲ６は互いに独立して、共重合して得られた状態の重合性原子団を表わし、下記（３）又は（４）であり、；ａ、ｂは２以上の整数であり、ｃは０もしくは１以上の整数であり；ｎは１以上１０以下の整数であり；ホスホコリン基を含む側鎖を有する構造単位とアジドシンナモイル基を含む構造単位とアルキレート基を含む構造単位とはランダムな順序またはブロックな順序で結合している。）
この構造、範囲にあることで、成形体とのより強固な結合、分子間のより強固な結合、かつ最適な親水性を有することによる細胞付着の効果を有する高分子化合物を得ることが可能となる。
Furthermore, the preferable form of General formula (1) is a high molecular compound of following General formula (2) description.
(Wherein R1, R3 and R6 each independently represent a polymerizable atomic group in a state obtained by copolymerization, and are the following (3) or (4) ; a and b are 2 or more C is an integer of 0 or 1 or more; n is an integer of 1 or more and 10 or less; a structural unit having a side chain containing a phosphocholine group, a structural unit containing an azidocinnamoyl group, and an alkylate group. (The structural units are included in a random or block order.)
By being in this structure and range, it is possible to obtain a polymer compound having the effect of cell attachment by having stronger bonds with the molded body, stronger bonds between molecules, and optimal hydrophilicity. Become.
また、一般式（１）及び（２）で示される構造の高分子化合物は、ａ／（ａ＋ｂ＋ｃ）の値が０．３０以上０．９０以下であり、ｂ／（ａ＋ｂ＋ｃ）の値が０．１０以上０．６０以下であり、ｃ／（ａ＋ｂ＋ｃ）の値が０以上０．５０以下である。各数値が上記範囲内にあることで、成形体とのさらなる強固な結合、分子間のさらなる強固な結合、かつ最適な親水性を有することによる細胞付着の効果を有する高分子化合物を得ることが可能となる。 In the polymer compound having the structure represented by the general formulas (1) and (2), the value of a / (a + b + c) is 0.30 or more and 0.90 or less, and the value of b / (a + b + c) is 0.00. It is 10 or more and 0.60 or less, and the value of c / (a + b + c) is 0 or more and 0.50 or less. By having each numerical value within the above range, it is possible to obtain a polymer compound having an effect of cell adhesion due to further strong bonding with the molded body, further strong bonding between molecules, and optimal hydrophilicity. It becomes possible.
前記高分子化合物を使用したコーティング材は、前述のとおり成形体との強固な結合、分子間の強固な結合、かつ親水性を有することによる細胞付着の効果に優れる。
なお、該コーティング材には、前記高分子化合物以外に溶剤、硬化剤、触媒、増感剤、カップリング剤、充填材などを添加してもよい。特に、溶剤としては、メタノール・エタノール・イソプロプルアルコールなどのアルコール系溶媒、酢酸エチルなどのエステル系溶媒、アセトンなどのケトン系溶媒などの極性溶媒、水、またはこれらの混合溶媒が好適に用いられ、その中でも水−エタノールの混合溶媒が溶解性の点で優れる。
As described above, the coating material using the polymer compound is excellent in the effect of cell adhesion due to the strong bond with the molded body, the strong bond between molecules, and the hydrophilicity.
In addition to the polymer compound, a solvent, a curing agent, a catalyst, a sensitizer, a coupling agent, a filler, and the like may be added to the coating material. In particular, polar solvents such as alcohol solvents such as methanol, ethanol and isopropyl alcohol, ester solvents such as ethyl acetate, ketone solvents such as acetone, water, or a mixed solvent thereof are preferably used as the solvent. Of these, a water-ethanol mixed solvent is excellent in terms of solubility.
また、本発明の成形体はコーティング材によって被覆された被覆部を有する。前記のとおり本発明のコーティング材は成形体と強固に結合し、コーティング材を構成する分子間の強固な結合による強度に優れる。また本発明の成形体は細胞吸着を抑制可能なものとなる。本発明の成形体としては、生化学容器、細胞培養容器等が挙げられ、特に細胞培養容器に好適に用いられる。生化学容器としては、遠沈管、試験管、保存用のボトル、チューブ、バッグ、免疫分析用容器などがある。
また細胞培養容器としては、細胞を容器内部に入れて培養するものであれば特に形態は限定しないが、例えば、各種シャーレ、マルチウェルプレート、培養フラスコ、培養バッグが対象となる。
次に、成形体へのコーティング材の接触方法を下記に記す。
Moreover, the molded object of this invention has the coating | coated part coat | covered with the coating material. As described above, the coating material of the present invention is firmly bonded to the molded body, and is excellent in strength due to the strong bonding between molecules constituting the coating material. In addition, the molded article of the present invention can suppress cell adsorption. Examples of the molded body of the present invention include biochemical containers and cell culture containers, and are particularly preferably used for cell culture containers. Biochemical containers include centrifuge tubes, test tubes, storage bottles, tubes, bags, immunoassay containers, and the like.
The cell culture vessel is not particularly limited as long as cells are cultured inside the vessel, and for example, various petri dishes, multiwell plates, culture flasks, and culture bags are targeted.
Next, a method for contacting the coating material with the compact is described below.
まず、成形体に前記コーティング材を塗布する。塗布する方法としては、成形体にコーティング材をピペット、ディスペンサー等により、手動または、自動化ロボットでスポット状に塗布する方法、コーティング溶液の入った漕に浸漬させ塗布する方法、ロールなどで塗布する方法が挙げられる。以下、容器にコーティング材をスポットで接触させる方法について述べる。なお、成形体はあらかじめ、低温プラズマ、コロナ放電や放射線照射処理により表面を酸化処理しておいてもよい。この処理により高分子物質との架橋において成形体側の架橋点を増やすことが出来る。 First, the coating material is applied to the molded body. As a method of coating, a coating material is applied to a molded body manually or with an automated robot in a spot shape using a pipette, dispenser, etc. Is mentioned. Hereinafter, a method of bringing the coating material into contact with the container with a spot will be described. In addition, you may oxidize the surface of a molded object beforehand by low temperature plasma, a corona discharge, or a radiation irradiation process. By this treatment, the number of crosslinking points on the molded body side can be increased in crosslinking with the polymer substance.
成形体に、ピペット、ディスペンサー等により、手動または、自動化ロボットでコーティング材を接触させた後、コーティング材が溶剤を含有する場合は乾燥させる。乾燥させる方法としては、乾燥機中で乾燥させる方法、減圧乾燥機中で乾燥させる方法、熱板上で乾燥させる方法等がある。なお、溶剤を含有しない場合、乾燥工程を省略することができる。 After the coating material is brought into contact with the molded body manually or by an automated robot using a pipette, a dispenser or the like, if the coating material contains a solvent, it is dried. Examples of the drying method include a method of drying in a dryer, a method of drying in a vacuum dryer, and a method of drying on a hot plate. In addition, when not containing a solvent, a drying process can be skipped.
その後、前記コーティング材を塗布済みの成形体に光照射してコーティング材を硬化する。光照射する条件としては、１０mJ/cm 2 以上１０００mJ/cm 2 以下が挙げられ、５０mJ/cm 2 以上８００J/cm 2 以下であることが好ましく、１００mJ/cm 2 以上６００mJ/cm 2 以下であることがさらに好ましい。
この条件により、ナイトレンを発生させてナイトレンと容器との結合、分子中に含まれるビニル基との反応等による架橋をさせるという効果を有する。
前記、高分子化合物と成形体が架橋することにより、成形体の表面から高分子化合物が脱離することがなくなり、安定した表面性状を得ることが出来る。
特に細胞培養容器においては、最終形態において滅菌が必須であるので、各種滅菌操作（放射線滅菌、プラズマ滅菌）によっても表面性状が変化しないという効果が得られる。
Thereafter, the coated material is irradiated with light to cure the coating material. It The conditions of light irradiation, include 10 mJ / cm 2 or more 1000 mJ / cm 2 or less, it is preferably, 100 mJ / cm 2 or more 600 mJ / cm 2 or less at 50 mJ / cm 2 or more 800 J / cm 2 or less Is more preferable.
Under these conditions, nitrene is generated, and there is an effect that the nitrene is bonded to the container and cross-linked by reaction with a vinyl group contained in the molecule.
By cross-linking the polymer compound and the molded body, the polymer compound is not detached from the surface of the molded body, and a stable surface property can be obtained.
In particular, since sterilization is essential in the final form of the cell culture container, the effect that the surface properties are not changed by various sterilization operations (radiation sterilization, plasma sterilization) can be obtained.
以下に合成例、実施例、比較例を挙げて、本発明をさらに具体的に説明するが、本発明はこれに限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to synthesis examples, examples and comparative examples, but the present invention is not limited thereto.
＜光反応性モノマーの合成＞
合成例１：モノマー（メタクリロイルオキシエチル4-アジド桂皮酸エステル：ＭＥＣＡｚ）の合成
テフロン（登録商標）製撹拌機、ジムロート冷却機、温度計、滴下漏斗を取り付けた四つ口フラスコを十分に乾燥した。この反応器に４−アジド桂皮酸１８．９ｇ（０．１０モル）とN,N-ジメチルホルムアミド１３０ｍｌを仕込み、乾燥窒素を流しながら室温下で撹拌した。４−アジド桂皮酸が完全に溶けた後、反応器を０℃付近まで冷却し、滴下漏斗を用いて塩化チオニル１３．７５ｇ（０．１１５モル）を一時間かけて滴下した。全ての塩化チオニルを滴下後、反応器を６５℃まで加熱して４時間反応を継続させた。その後、約４０℃でＮ，Ｎ−ジメチルホルムアミドを減圧留去し、容器を移して室温で一昼夜、真空乾燥することで１９．８ｇの４−アジド桂皮酸クロライドを得た。
得られた４−アジド桂皮酸クロライド１０ｇ（０．０４８モル）をテフロン（登録商標）製撹拌機、ジムロート冷却器、温度計、滴下漏斗を取り付けた四つ口フラスコにトルエン１９０ｍｌ、２−ヒドロキシエチルメタクリレート（ＨＥＭＡ）７．０ｇ（０．０５４モル）と一緒に仕込み、撹拌しながら５０℃に加熱した。そこにトリエチルアミン１３ｇとトルエン１９０ｍｌの混合液を滴下漏斗を用いて一時間かけて滴下した。滴下終了後更に４時間反応を継続させた。反応終了後塩酸塩を濾紙で濾捌後、濾液を５％ＨＣｌ水溶液で抽出して分液ロート下層のＨＥＭA水溶液を除いた。上層のトルエン溶液には無水の硫
酸ナトリウムを加えて脱水した後トルエンをエバポレートして除去し、真空乾燥機で一昼夜乾燥して１４．６ｇのＭＥＣＡｚモノマーを得た。
<Synthesis of photoreactive monomer>
Synthesis Example 1: Synthesis of monomer (methacryloyloxyethyl 4-azido cinnamate: MECAz) A four-necked flask equipped with a Teflon (registered trademark) stirrer, Dimroth cooler, thermometer, and dropping funnel was sufficiently dried. . The reactor was charged with 18.9 g (0.10 mol) of 4-azido cinnamic acid and 130 ml of N, N-dimethylformamide and stirred at room temperature while flowing dry nitrogen. After 4-azido cinnamic acid was completely dissolved, the reactor was cooled to around 0 ° C., and thionyl chloride (13.75 g, 0.115 mol) was added dropwise over 1 hour using a dropping funnel. After all thionyl chloride was added dropwise, the reactor was heated to 65 ° C. and the reaction was continued for 4 hours. Thereafter, N, N-dimethylformamide was distilled off under reduced pressure at about 40 ° C., and the container was transferred and vacuum dried at room temperature for 24 hours to obtain 19.8 g of 4-azido cinnamic acid chloride.
190 g of toluene and 2-hydroxyethyl were added to 10 g (0.048 mol) of 4-azido cinnamic acid chloride obtained in a four-necked flask equipped with a Teflon (registered trademark) stirrer, Dimroth cooler, thermometer, and dropping funnel. Charged together with 7.0 g (0.054 mol) of methacrylate (HEMA) and heated to 50 ° C. with stirring. A mixed solution of 13 g of triethylamine and 190 ml of toluene was dropped therein over 1 hour using a dropping funnel. The reaction was further continued for 4 hours after the completion of the dropping. After completion of the reaction, the hydrochloride was filtered off with filter paper, and the filtrate was extracted with 5% HCl aqueous solution to remove the HEMA aqueous solution under the separatory funnel. To the upper toluene solution, anhydrous sodium sulfate was added for dehydration, and then toluene was removed by evaporation, followed by drying overnight in a vacuum dryer to obtain 14.6 g of MECAz monomer.
＜光反応性ポリマーの重合例＞
重合例１：（ＭＰＣ0.6−ＭＥＣAz0.3−ＢＭＡ0.1）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）１２．７ｇ、メタクリロイルオキシエチル４-アジド桂皮酸エステル（ＭＥＣＡｚ）９．０６ｇ、ブチルメタクリ
レート（ＢＭA）１．４２ｇをエタノール１００ｇに溶解し、四つ口フラスコに入れ、３
０分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液１１０ｇを得た。この溶液を４００ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末１６．１ｇを得た。
重合例２：（ＭＰＣ0.6−ＭＥＣAz0.2−ＢＭＡ0.2）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）１２．７ｇ、メタクリロイルオキシエチル４-アジド桂皮酸エステル（ＭＥＣＡｚ）６．０４ｇ、ブチルメタクリ
レート（ＢＭA）２．８４ｇをエタノール９０ｇに溶解し、四つ口フラスコに入れ、３０
分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液１０５ｇを得た。この溶液を４００ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析
出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末１５．３ｇを得た。
重合例３：（ＭＰＣ0.6−ＭＥＣAz0.1−ＢＭＡ0.3）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）１２．７ｇ、メタクリロイルオキシエチル４-アジド桂皮酸エステル（ＭＥＣＡｚ）３．０２ｇ、ブチルメタクリ
レート（ＢＭA）４．２６ｇをエタノール９０ｇに溶解し、四つ口フラスコに入れ、３０
分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液９８ｇを得た。この溶液を４００ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末１４．２ｇを得た。
重合例４：（ＭＰＣ0.7−ＭＥＣAz0.3）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）２０．６５ｇ、メタクリロイルオキシエチル４-アジド桂皮酸エステル（ＭＥＣＡｚ）９．０６ｇをエタノール１
３０ｇに溶解し、四つ口フラスコに入れ、３０分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液１３５ｇを得た。この溶液を６００ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末２２．３ｇを得た。
重合例５：（ＭＰＣ0.5−ＭＥＣAz0.2−ＨｘＭＡ0.3）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）１４．２５ｇ、メタクリロイルオキシエチル４-アジド桂皮酸エステル（ＭＥＣＡｚ）６．０４ｇ、ｎ−ヘキシル
メタクリレート（ＨｘＭA）５．１ｇをエタノール１００ｇに溶解し、四つ口フラスコに
入れ、３０分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液１１５ｇを得た。この溶液を５２０ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末１９．１ｇを得た。
重合例６：（ＭＰＣ0.2−ＭＥＣＡｚ0.4−ＢＭＡ0.4）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）５．９ｇ、メタクリロイルオキシエチル４-アジド桂皮酸エステル（ＭＥＣＡｚ）１２．０８ｇ、ブチルメタクリ
レート（ＢＭA）５．６８ｇをエタノール１００ｇに溶解し、四つ口フラスコに入れ、３
０分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液１０９ｇを得た。この溶液を４００ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末１７．１ｇを得た。
重合例７：（ＭＰＣ0.2−ＭＥＣAz0.2−ＢＭＡ0.6）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）５．９ｇ、メタクリロイルオキシエチル４-アジド桂皮酸エステル（ＭＥＣＡｚ）６．０４ｇ、ブチルメタクリレ
ート（ＢＭA）８．５２ｇをエタノール１００ｇに溶解し、四つ口フラスコに入れ、３０
分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液１０３ｇを得た。この溶液を４００ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末１６．１ｇを得た。
比較重合例１：（ＭＰＣ0.8−ＢＭＡ0.2）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）２３．６ｇ、ブチルメタ
クリレート（ＢＭA）２．８４ｇをエタノール１００ｇに溶解し、四つ口フラスコに入れ
、３０分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液１１８ｇを得た。この溶液を５３０ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末２１．８ｇを得た。
比較重合例２：（ＭＰＣ0.5−ＢＭＡ0.5）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）１４．７５ｇ、ブチルメタクリレート（ＢＭA）７．１ｇをエタノール９０ｇに溶解し、四つ口フラスコに入れ、
３０分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液９５ｇを得た。この溶液を４００ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末１５．６ｇを得た。
比較重合例３：（ＭＰＣ0.3−ＢＭＡ0.7）の重合
２−メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）８．８５ｇ、ブチルメタクリレート（ＢＭA）２１．１４ｇをエタノール１１０ｇに溶解し、四つ口フラスコに入
れ、３０分間窒素を吹き込んだ後、５０℃でｔ−ブチルパーオキシネオデカノエート０．３５ｇを加えて３時間反応させた後、６０℃に上げ更に２時間反応を継続し、ポリマー溶液１２２ｇを得た。この溶液を５００ｍｌのエチルエーテル中にかき混ぜながら徐々に滴下し、析出した沈殿をろ過し、４８時間真空乾燥を実施してポリマー粉末２２．９ｇを得た。
<Example of polymerization of photoreactive polymer>
Polymerization Example 1: Polymerization of (MPC0.6-MECAz0.3-BMA0.1) 2-methacryloyloxyethyl phosphorylcholine (MPC) 12.7 g, methacryloyloxyethyl 4-azido cinnamic acid ester (MECAz) 9.06 g, butyl methacrylate (BMA) 1.42 g is dissolved in 100 g of ethanol and placed in a four-necked flask.
After blowing nitrogen for 0 minutes, 0.35 g of t-butylperoxyneodecanoate was added at 50 ° C. and reacted for 3 hours, then the temperature was raised to 60 ° C. and the reaction was further continued for 2 hours to obtain 110 g of a polymer solution. It was. The solution was gradually added dropwise to 400 ml of ethyl ether while stirring, the deposited precipitate was filtered, and vacuum dried for 48 hours to obtain 16.1 g of polymer powder.
Polymerization Example 2: Polymerization of (MPC0.6-MECAz0.2-BMA0.2) 2-Methacryloyloxyethyl phosphorylcholine (MPC) 12.7 g, methacryloyloxyethyl 4-azido cinnamate (MECAz) 6.04 g, butyl methacrylate (BMA) 2.84 g is dissolved in 90 g of ethanol and placed in a four-necked flask.
After blowing nitrogen for 30 minutes, 0.35 g of t-butylperoxyneodecanoate was added at 50 ° C. and reacted for 3 hours, then the temperature was raised to 60 ° C. and the reaction was further continued for 2 hours to obtain 105 g of a polymer solution. . The solution was gradually added dropwise to 400 ml of ethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried for 48 hours to obtain 15.3 g of polymer powder.
Polymerization Example 3: Polymerization of (MPC0.6-MECAz0.1-BMA0.3) 2-methacryloyloxyethyl phosphorylcholine (MPC) 12.7 g, methacryloyloxyethyl 4-azido cinnamate (MECAz) 3.02 g, butyl methacrylate (BMA) 4.26 g was dissolved in 90 g of ethanol and placed in a four-necked flask.
After blowing nitrogen for 30 minutes, 0.35 g of t-butylperoxyneodecanoate was added at 50 ° C. and reacted for 3 hours, then the temperature was raised to 60 ° C. and the reaction was further continued for 2 hours to obtain 98 g of polymer solution. . This solution was gradually added dropwise to 400 ml of ethyl ether while stirring, the deposited precipitate was filtered, and vacuum dried for 48 hours to obtain 14.2 g of polymer powder.
Polymerization Example 4: Polymerization of (MPC0.7-MECAz0.3) 2-methacryloyloxyethyl phosphorylcholine (MPC) 20.65 g, methacryloyloxyethyl 4-azido cinnamic acid ester (MECAz) 9.06 g of ethanol 1
Dissolve in 30 g, put into a four-necked flask, blow nitrogen for 30 minutes, add 0.35 g of t-butylperoxyneodecanoate at 50 ° C. and react for 3 hours, then raise to 60 ° C. The reaction was continued for 2 hours to obtain 135 g of a polymer solution. This solution was gradually added dropwise to 600 ml of ethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried for 48 hours to obtain 22.3 g of polymer powder.
Polymerization Example 5: Polymerization of (MPC0.5-MECAz0.2-HxMA0.3) 2-Methacryloyloxyethyl phosphorylcholine (MPC) 14.25 g, methacryloyloxyethyl 4-azido cinnamate (MECAz) 6.04 g, n- Dissolve 5.1 g of hexyl methacrylate (HxMA) in 100 g of ethanol, put into a four-necked flask, blow nitrogen for 30 minutes, add 0.35 g of t-butylperoxyneodecanoate at 50 ° C. for 3 hours. After the reaction, the temperature was raised to 60 ° C. and the reaction was continued for another 2 hours to obtain 115 g of a polymer solution. This solution was gradually added dropwise to 520 ml of ethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried for 48 hours to obtain 19.1 g of polymer powder.
Polymerization Example 6: Polymerization of (MPC0.2-MECAz0.4-BMA0.4) 2-Methacryloyloxyethyl phosphorylcholine (MPC) 5.9 g, methacryloyloxyethyl 4-azido cinnamate (MECAz) 12.08 g, butyl methacrylate (BMA) 5.68 g was dissolved in 100 g of ethanol and placed in a four-necked flask.
After blowing nitrogen for 0 minutes, 0.35 g of t-butylperoxyneodecanoate was added at 50 ° C. and reacted for 3 hours, then the temperature was raised to 60 ° C. and the reaction was continued for another 2 hours to obtain 109 g of polymer solution. It was. The solution was gradually added dropwise to 400 ml of ethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried for 48 hours to obtain 17.1 g of polymer powder.
Polymerization Example 7: Polymerization of (MPC0.2-MECAz0.2-BMA0.6) 2-methacryloyloxyethyl phosphorylcholine (MPC) 5.9 g, methacryloyloxyethyl 4-azido cinnamic acid ester (MECAz) 6.04 g, butyl methacrylate (BMA) 8.52 g was dissolved in 100 g of ethanol and placed in a four-necked flask.
After blowing nitrogen for 30 minutes, 0.35 g of t-butylperoxyneodecanoate was added at 50 ° C. and reacted for 3 hours, then the temperature was raised to 60 ° C. and the reaction was continued for another 2 hours to obtain 103 g of polymer solution. . The solution was gradually added dropwise to 400 ml of ethyl ether while stirring, the deposited precipitate was filtered, and vacuum dried for 48 hours to obtain 16.1 g of polymer powder.
Comparative Polymerization Example 1: Polymerization of (MPC0.8-BMA0.2) 23.6 g of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2.84 g of butyl methacrylate (BMA) were dissolved in 100 g of ethanol and put in a four-necked flask. After blowing nitrogen for 30 minutes, 0.35 g of t-butylperoxyneodecanoate was added at 50 ° C. and reacted for 3 hours, then the temperature was raised to 60 ° C. and the reaction was continued for another 2 hours. Obtained. This solution was gradually added dropwise to 530 ml of ethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried for 48 hours to obtain 21.8 g of polymer powder.
Comparative Polymerization Example 2: Polymerization of (MPC0.5-BMA0.5) 2-Methacryloyloxyethyl phosphorylcholine (MPC) 14.75 g and butyl methacrylate (BMA) 7.1 g were dissolved in ethanol 90 g and placed in a four-necked flask. ,
After nitrogen was blown for 30 minutes, 0.35 g of t-butylperoxyneodecanoate was added at 50 ° C. and reacted for 3 hours, then the temperature was raised to 60 ° C. and the reaction was continued for another 2 hours to obtain 95 g of a polymer solution. It was. The solution was gradually added dropwise to 400 ml of ethyl ether while stirring, the deposited precipitate was filtered, and vacuum dried for 48 hours to obtain 15.6 g of polymer powder.
Comparative Polymerization Example 3: Polymerization of (MPC0.3-BMA0.7) 2-Methacryloyloxyethyl phosphorylcholine (MPC) 8.85 g and butyl methacrylate (BMA) 21.14 g were dissolved in 110 g of ethanol and placed in a four-necked flask. After blowing nitrogen for 30 minutes, 0.35 g of t-butylperoxyneodecanoate was added at 50 ° C. and reacted for 3 hours, then the temperature was raised to 60 ° C. and the reaction was continued for another 2 hours. Obtained. The solution was gradually added dropwise to 500 ml of ethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried for 48 hours to obtain 22.9 g of polymer powder.
・ 実施例１−１〜７−１、比較例１−１〜２−１
［コート処理生化学容器］
前記重合により得られたポリマー粉末を、超純水／メタノール＝５０／５０に溶解し、０．５重量％のコート液を調製した。
生化学容器として、１５ｍＬポリプロピレン製遠沈管（住友ベークライト製、ＭＳ− ）を用いた。前処理としてチューブにプラズマ処理装置 （ＢＲＡＮＳＯＮ／ＩＰＣ社製 ＳＥＲＩＥＳ７０００）を用いてプラズマ処理（酸素プラズマ５分間）を行った。
遠沈管に１５ｍＬの先に調製したポリマー溶液を加えて１時間浸漬した後、遠沈管を裏返して溶液を充分廃棄した。ついで、２５℃で１２時間一次乾燥した後、ＵＶランプで２５０ｎｍのＵＶ光を１．０ｍＷ／ｃｍ2×３０秒間照射して水溶性樹脂を硬化させた。
硬化後、超純水で３回繰り返し洗浄し、乾燥させ本発明の生化学用容器（チューブ）を得た。
［生体由来物質吸着性］
（１）ウシ血清アルブミン吸着性
タンパク質吸着測定の一例として、ウシ血清アルブミンの吸着性を次のように評価した。
まず、コート材が容器に密着しているかどうかを確認するため、各実施例及び各比較例で得られたチューブに純水を各々５．０ｍＬずつ分注し、３７℃で１時間振とうさせた。
その後、純水を排出し、２７℃１２時間条件下で乾燥させ、ウシ血清アルブミン（２３２０９ ＰＩＥＲＣＥ社製）を０．５μｇ／ｍＬに希釈した溶液を、実施例及び各比較例で得られたチューブに各々５．０ｍＬずつ分注し、３７℃で１時間インキュベートした後０．０５容量％ｔｗｅｅｎ２０入りリン酸緩衝液ｐＨ７．４で３回洗浄した。
次にブロッキングとして３．０重量％スキムミルク入りリン酸緩衝液ｐＨ７．４溶液を５．５ｍＬずつ分注し、３７℃で１時間インキュベートした後０．０５容量％ｔｗｅｅｎ２０入りリン酸緩衝液ｐＨ７．４で３回洗浄した。次にペルオキシターゼ標識坑ウシ血清
アルブミン抗体（５５２８５ ＣＡＰＰＥＬ社製）をリン酸緩衝液ｐＨ７．４で１．０μｇ／ｍＬに希釈した溶液を各チューブに５．０ｍＬずつ分注し室温で３０分インキュベートした後０．０５容量％ｔｗｅｅｎ２０入りリン酸緩衝液ｐＨ７．４で３回洗浄し、ぺルオキシターゼ用発色キット（ＳＵＭＩＬＯＮ ＭＬ−１１２０Ｔ 住友ベークライト社製）を使用して発色させた後プレートリーダーを使用して４５０／６３０ｎｍの吸光度を測定した。
別途作成した検量線から各チューブ本体に残留したペルオキシターゼ標識坑ウシ血清アルブミン抗体＝ウシ血清アルブミンの重量を求め、吸着率を算出した。
また、コート材中の分子間架橋については次のように評価した。
各実施例及び各比較例で得られたチューブに純水：エタノール＝５０wt%：５０wt%の混合溶液を各々５．０ｍＬずつ分注し、２７℃で１時間振とうさせた。その後、純水を排出し、２７℃１２時間条件下で乾燥させ、上記同様ウシ血清アルブミンの吸着率を算出した。
結果を表１に示す。
なお、表1中の◎、○、×は以下を表す。
生体由来物質密着性１ ◎：吸着率が０．１％未満（アルブミン吸着は極めて少ない＝コート材の容器への密着性は非常に良好）、○：吸着率が０．１％以上1％未満（アルブ
ミン吸着はほとんど起こっていない＝コート材の容器への密着性は良好） ×：吸着率が１％以上（アルブミンの吸着が多数発生＝コート材の容器への密着性は悪い）
生体由来物質密着性２ ◎：吸着率が０．１％未満（アルブミン吸着は極めて少ない＝コート材中の分子間架橋性は非常に良好）、○：吸着率が０．１％以上1％未満（アルブミ
ン吸着はほとんど起こっていない＝コート材中の分子間架橋性は良好） ×：吸着率が１％以上（アルブミンの吸着が多数発生＝コート材中の分子間架橋性は悪い）
-Examples 1-1 to 7-1 and Comparative Examples 1-1 to 2-1
[Coated biochemical container]
The polymer powder obtained by the polymerization was dissolved in ultrapure water / methanol = 50/50 to prepare a 0.5 wt% coating solution.
As a biochemical container, a 15 mL polypropylene centrifuge tube (manufactured by Sumitomo Bakelite, MS-) was used. As a pretreatment, plasma treatment (oxygen plasma for 5 minutes) was performed on the tube using a plasma treatment apparatus (SERIES7000 manufactured by BRANSON / IPC).
After adding 15 mL of the previously prepared polymer solution to the centrifuge tube and dipping for 1 hour, the centrifuge tube was turned over and the solution was sufficiently discarded. Next, after primary drying at 25 ° C. for 12 hours, the water-soluble resin was cured by irradiating UV light of 250 nm with a UV lamp at 1.0 mW / cm 2 × 30 seconds.
After curing, it was washed repeatedly with ultrapure water three times and dried to obtain the biochemical container (tube) of the present invention.
[Adsorption of biological substances]
(1) Bovine serum albumin adsorptivity As an example of protein adsorption measurement, bovine serum albumin adsorptivity was evaluated as follows.
First, in order to confirm whether or not the coating material is in close contact with the container, each of the tubes obtained in each Example and each Comparative Example was dispensed with 5.0 mL of pure water and shaken at 37 ° C. for 1 hour. It was.
Thereafter, pure water was discharged, dried under conditions of 27 ° C. for 12 hours, and a solution obtained by diluting bovine serum albumin (manufactured by 23209 PIERCE) to 0.5 μg / mL was obtained in the tubes obtained in Examples and Comparative Examples. 5.0 mL of each was dispensed, incubated at 37 ° C. for 1 hour, and then washed three times with a phosphate buffer solution pH 7.4 containing 0.05% by volume tween20.
Next, as a blocking, 5.5 mL of a 3.0 wt% skim milk-containing phosphate buffer solution 7.4 was dispensed, incubated at 37 ° C. for 1 hour, and then 0.05 vol% tween 20-containing phosphate buffer solution pH 7.4. And washed 3 times. Next, a solution obtained by diluting peroxidase-labeled anti-bovine serum albumin antibody (55285 CAPPEL) to 1.0 μg / mL with phosphate buffer pH 7.4 was dispensed at 5.0 mL in each tube and incubated at room temperature for 30 minutes. After washing with 0.05 volume% tween 20 phosphate buffer pH 7.4 three times, color was developed using a peroxidase color development kit (SUMILON ML-1120T manufactured by Sumitomo Bakelite Co., Ltd.) and a plate reader was used. The absorbance at 450/630 nm was measured.
The weight of peroxidase-labeled anti-bovine serum albumin antibody = bovine serum albumin remaining in each tube body was determined from a separately prepared calibration curve, and the adsorption rate was calculated.
The intermolecular crosslinking in the coating material was evaluated as follows.
To the tubes obtained in each example and each comparative example, 5.0 mL each of a mixed solution of pure water: ethanol = 50 wt%: 50 wt% was dispensed and shaken at 27 ° C. for 1 hour. Thereafter, the pure water was discharged and dried under the condition of 27 ° C. for 12 hours, and the adsorption rate of bovine serum albumin was calculated as described above.
The results are shown in Table 1.
In Table 1, ◎, ○, × represent the following.
Biomaterial-derived substance adhesion 1 ◎: Adsorption rate is less than 0.1% (albumin adsorption is very low = adhesion of coating material to container is very good), ○: Adsorption rate is 0.1% or more and less than 1% (Album adsorption hardly occurs = Good adhesion of coating material to container) x: Adsorption rate of 1% or more (Many albumin adsorption occurs = Adhesion of coating material to container is poor)
Biological substance adhesion 2 ◎: Adsorption rate is less than 0.1% (albumin adsorption is very low = intermolecular crosslinkability in the coating material is very good), ○: Adsorption rate is 0.1% or more and less than 1% (Album adsorption hardly occurs = Intermolecular crosslinkability in the coating material is good) x: Adsorption rate is 1% or more (Many albumin adsorption occurs = Intermolecular crosslinkability in the coating material is poor)
・ 実施例１−２〜７−２、比較例１−２〜２−２
［コート済み細胞培養容器］
コートする細胞培養容器として、ポリスチレン製２４ウェルプレート成形品（住友ベークライト製）を準備し、酸素プラズマ処理を実施した（プラズマ装置：ＢＲＡＮＳＯＮ／ＩＰＣ社製 ＳＥＲＩＥＳ７０００）。
１ウェルにつき５００μＬの先に調製したポリマー溶液を加えて１時間浸漬した後、プレートを裏返して溶液を充分廃棄した。ついで、２５℃で１２時間一次乾燥した後、ＵＶランプで２５０ｎｍのＵＶ光を１．０ｍＷ／ｃｍ2×３０秒間照射して水溶性樹脂を硬化
させた。硬化後、超純水で３回繰り返し洗浄し、乾燥させた後、γ線を吸収線量６．０ｋＧｙで照射（ラジエ工業株式会社）して滅菌済の本発明の細胞培養容器を得た。
［細胞培養］
（１）マウスＥＳ細胞を用いた胚様体形成細胞培養の一例として、マウスＥＳ細胞を次のように評価した。
まず、コート材が容器に密着しているかどうかを確認するため、各実施例及び各比較例で得られたチューブに純水を各々５．０ｍＬずつ分注し、３７℃で１時間振とうさせた。その後、純水を排出し、２７℃１２時間条件下で乾燥させ、マウスＥＳ細胞を培養液（ダルベッコ改変ＭＥＭ＋１５％ウシ胎児血清）に７，５００ｃｅｌｌｓ／ｍＬの濃度で分散させた細胞懸濁液を調製し、前述の細胞培養容器に２００μＬ／ウェルずつ、２４ウェルに分注し、３７℃、５％ＣＯ２雰囲気下にて５日間培養した。
５日後各ウェルについて、胚様体の形成を顕微鏡下で確認した。
（２）マウスＥＳ細胞胚葉体の心筋への分化の確認
次いで、培養された胚様体を２４ウェルマルチプレート（住友ベークライト社製、ＭＳ−
８０２４０）に１個／ウェルずつ移し替えた後、培養液（ダルベッコ改変ＭＥＭ＋１５％ウシ胎児血清）を５００μＬ／ウェルずつ分注し、５％ＣＯ２雰囲気下にて５日間培養した。
５日後、各ウェルについて、心筋細胞特有の拍動を顕微鏡下で確認した。また、コート材中の分子間架橋については次のように評価した。
各実施例及び各比較例で得られた細胞培養器に純水：エタノール＝５０wt%：５０wt%の混合溶液を各々２００μＬ／ウェルずつ、２４ウェルに分注し、２７℃で１時間振とうさせた。その後、純水を排出し、２７℃１２時間条件下で乾燥させ、上記同様心筋細胞特有の拍動を顕微鏡下で確認した。
結果を表２に示す。
なお、表２中の◎、○、×は以下を表す。
分化の確認１ ◎：２４ウェル中、２０ウェル以上で胚様体の形成が認められ、かつ心筋細胞特有の拍動が確認された＝コート材の容器への密着性は非常に良好、○：２４ウェル中、１２ウェル以上２０ウェル未満で胚様体の形成が認められ、かつ心筋細胞特有の拍動が確認された＝コート材の容器への密着性は良好、×：２４ウェル中、１２ウェル未満で胚様体の形成が認められ、かつ心筋細胞特有の拍動が確認された＝コート材の容器への密着性は悪い
分化の確認２ ◎：２４ウェル中、２０ウェル以上で胚様体の形成が認められ、かつ心筋細胞特有の拍動が確認された＝コート材中の分子間架橋性は非常に良好、○：２４ウェル中、１２ウェル以上２０ウェル未満で胚様体の形成が認められ、かつ心筋細胞特有の拍動が確認された＝コート材中の分子間架橋性は良好、×：２４ウェル中、１２ウェル未満で胚様体の形成が認められ、かつ心筋細胞特有の拍動が確認された＝コート材中の分子間架橋性は悪い
[Coated cell culture vessel]
A polystyrene 24-well plate molded product (manufactured by Sumitomo Bakelite) was prepared as a cell culture container to be coated, and oxygen plasma treatment was performed (plasma apparatus: SERIES7000, manufactured by BRANSON / IPC).
After adding 500 μL of the previously prepared polymer solution per well and soaking for 1 hour, the plate was turned over and the solution was discarded. Next, after primary drying at 25 ° C. for 12 hours, the water-soluble resin was cured by irradiating UV light of 250 nm with a UV lamp at 1.0 mW / cm 2 × 30 seconds. After curing, washing with ultrapure water three times and drying, γ rays were irradiated with an absorbed dose of 6.0 kGy (Radie Industries, Ltd.) to obtain a sterilized cell culture container of the present invention.
[Cell culture]
(1) As an example of embryoid body-forming cell culture using mouse ES cells, mouse ES cells were evaluated as follows.
First, in order to confirm whether or not the coating material is in close contact with the container, each of the tubes obtained in each Example and each Comparative Example was dispensed with 5.0 mL of pure water and shaken at 37 ° C. for 1 hour. It was. Thereafter, pure water was discharged, dried at 27 ° C. for 12 hours, and a cell suspension in which mouse ES cells were dispersed in a culture solution (Dulbecco's modified MEM + 15% fetal bovine serum) at a concentration of 7,500 cells / mL. Prepared and dispensed into the above-mentioned cell culture vessel at 200 μL / well in 24 wells and cultured at 37 ° C. in a 5% CO 2 atmosphere for 5 days.
After 5 days, the formation of embryoid bodies was confirmed under a microscope for each well.
(2) Confirmation of mouse ES cell embryoid body differentiation into myocardium Next, the cultured embryoid body was transformed into a 24-well multiplate (Sumitomo Bakelite, MS-
Then, the culture solution (Dulbecco's modified MEM + 15% fetal bovine serum) was dispensed at 500 μL / well and cultured in a 5% CO 2 atmosphere for 5 days.
After 5 days, for each well, pulsations peculiar to cardiomyocytes were confirmed under a microscope. The intermolecular crosslinking in the coating material was evaluated as follows.
In each cell culture vessel obtained in each example and each comparative example, 200 μL / well of pure water: ethanol = 50 wt%: 50 wt% mixed solution was dispensed into 24 wells and shaken at 27 ° C. for 1 hour. It was. Thereafter, pure water was discharged and dried under conditions of 27 ° C. for 12 hours, and the pulsation peculiar to cardiomyocytes was confirmed under a microscope as described above.
The results are shown in Table 2.
In Table 2, “◎”, “◯”, and “x” represent the following.
Confirmation of differentiation 1 ◎: In 24 wells, formation of embryoid bodies was observed in 20 or more wells, and pulsation peculiar to cardiomyocytes was confirmed = the adhesion of the coating material to the container was very good, ○: In 24 wells, formation of embryoid bodies was observed in 12 or more well but less than 20 wells, and the pulsation peculiar to cardiomyocytes was confirmed = adhesion to the container of the coating material was good, x: in 24 wells, 12 Formation of embryoid bodies was observed in less than wells, and pulsation peculiar to cardiomyocytes was confirmed = adhesion of the coating material to the container was confirmed to be poor 2 ◎: Embryoid-like in 20 wells or more in 24 wells The formation of the body was confirmed and the pulsation peculiar to the myocardial cell was confirmed = the intermolecular cross-linking property in the coating material was very good, ○: the formation of the embryoid body in 12 to 20 wells in 24 wells And pulsations peculiar to cardiomyocytes were confirmed = coat Intermolecular crosslinkability in the material is good, x: formation of embryoid bodies was observed in less than 12 wells in 24 wells, and pulsation peculiar to cardiomyocytes was confirmed = intermolecular crosslinkability in the coating material was bad
実施例の生化学容器は、コート材が十分に容器に密着しているため、純水処理を行ってもウシ血清アルブミンの密着が起こらず、かつコート材中の分子間の架橋度が十分高いた
め、水−エタノール混合溶液でも溶解しなかった。一方、比較例の生化学容器は、コート材が十分に容器に密着しておらず、純水処理を行うことでコート材が剥離し、それが原因でウシ血清アルブミンの密着が発生した。また、コート材中の分子間の架橋度が不十分であったため、水−エタノール混合溶液で溶解した。
また、実施例の細胞培養容器は、コート材が十分に容器に密着しているため、純水処理を行っても細胞の分化が発生し、かつコート材中の分子間の架橋度が十分高いため、水−エタノール混合溶液でも溶解しなかった。一方、比較例の細胞培養容器は、コート材が十分に容器に密着しておらず、純水処理を行うことでコート材が剥離し、それが原因で細胞の分化が起こらなかった。また、コート材中の分子間の架橋度が不十分であったため、水−エタノール混合溶液で溶解した。
In the biochemical container of the example, since the coating material is sufficiently adhered to the container, bovine serum albumin does not adhere even if pure water treatment is performed, and the degree of cross-linking between molecules in the coating material is sufficiently high. Therefore, even the water-ethanol mixed solution did not dissolve. On the other hand, in the biochemical container of the comparative example, the coating material was not sufficiently adhered to the container, and the coating material was peeled off by performing pure water treatment, which caused bovine serum albumin adhesion. Further, since the degree of cross-linking between molecules in the coating material was insufficient, it was dissolved in a water-ethanol mixed solution.
In addition, since the coating material of the cell culture container of Example is sufficiently adhered to the container, cell differentiation occurs even when pure water treatment is performed, and the degree of cross-linking between molecules in the coating material is sufficiently high. Therefore, even the water-ethanol mixed solution did not dissolve. On the other hand, in the cell culture container of the comparative example, the coating material was not sufficiently adhered to the container, and the coating material was peeled off by performing the pure water treatment, and thus cell differentiation did not occur. Further, since the degree of cross-linking between molecules in the coating material was insufficient, it was dissolved in a water-ethanol mixed solution.
本発明に開示された高分子化合物を使用することにより、容器と共有結合でき、かつ分子内でも架橋するため強固なコーティング材を得ることが可能となり、さらに上記特徴を有する細胞培養容器を得ることが可能となった。
By using the polymer compound disclosed in the present invention, it is possible to obtain a strong coating material because it can be covalently bonded to the container and also crosslinks within the molecule, and further, a cell culture container having the above characteristics can be obtained. Became possible.
Claims (10)
（式中、Ｒ１、Ｒ３及びＲ６は互いに独立して、共重合して得られた状態の重合性原子団を表わし、下記（３）又は（４）であり、；Ｒ２、Ｒ４及びＲ７は互いに独立して、置換基を有しても良いフェニル基又は−Ｃ（Ｏ）−、−Ｃ（Ｏ）Ｏ−、−Ｏ−、もしくは−Ｓ−で示される基を表わし；Ｒ５は置換基を有しても良いフェニル基又は−ＯＣ（Ｏ）−、−Ｃ（Ｏ）−、−Ｏ−で示される基を表わし；ｌ及びｍは互いに独立して、２以上の整数を表わし；ｎは１以上の整数を表わし；ａ及びｂは互いに独立して２以上の整数を表わし；ｃは０もしくは１以上の整数を表わす。；Ｒ１を含む構造単位とＲ３を含む構造単位とＲ６を含む構造単位はランダムな順序で結合するか、またはブロック状態で結合している。）
(In the formula, R1, R3 and R6 independently represent a polymerizable atomic group obtained by copolymerization, and are the following (3) or (4) ; R2, R4 and R7 are Independently, it represents a phenyl group which may have a substituent or a group represented by -C (O)-, -C (O) O-, -O- or -S-; R5 represents a substituent. An optionally substituted phenyl group or a group represented by —OC (O) —, —C (O) —, —O—; and l and m each independently represent an integer of 2 or more; A and b each independently represent an integer of 2 or more; c represents an integer of 0 or 1; a structural unit containing R1, a structural unit containing R3, and a structure containing R6 Units are combined in a random order or in a block state.)
（式中、Ｒ１、Ｒ３及びＲ６は互いに独立して、共重合して得られた状態の重合性原子団を表わし、下記（３）又は（４）であり、；ａ、ｂは２以上の整数であり、ｃは０もしくは１以上の整数であり；ｎは１ないし１０の整数であり；ホスホリルコリン基を含む側鎖を有する構造単位とアジドシンナモイル基を含む構造単位とアルキレート基を含む構造単位とはランダムな順序またはブロックな順序で結合している。）
(Wherein R1, R3 and R6 each independently represent a polymerizable atomic group in a state obtained by copolymerization, and are the following (3) or (4) ; a and b are 2 or more C is an integer of 0 or 1 or more; n is an integer of 1 to 10; includes a structural unit having a side chain containing a phosphorylcholine group, a structural unit containing an azidocinnamoyl group, and an alkylate group (The structural units are connected in a random or block order.)
１項に記載の高分子化合物。 In the polymer compound having the structure represented by the general formulas (1) and (2), the value of a / (a + b + c) is 0.30 or more and 0.90 or less, and the value of b / (a + b + c) is 0.10 or more. The polymer compound according to any one of claims 1 to 3, wherein the polymer compound is 0.60 or less and a value of c / (a + b + c) is 0 or more and 0.50 or less.
The method for producing a molded body according to claim 9, wherein, in the second step according to claim 9, the light irradiation amount is 10 mJ / cm 2 or more and 1000 mJ / cm 2 or less.
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| JP7047321B2 (en) * | 2017-10-17 | 2022-04-05 | 東洋製罐グループホールディングス株式会社 | Laminated structure and method for manufacturing the laminated structure |
| WO2019021748A1 (en) * | 2017-07-22 | 2019-01-31 | 東洋製罐グループホールディングス株式会社 | Culture vessel, method for manufacturing culture vessel, laminated structure, and method for producing laminated structure |
| JP2019026825A (en) * | 2017-08-03 | 2019-02-21 | 住友ベークライト株式会社 | Copolymer, coating composition, and article |
| JP7352146B2 (en) * | 2019-07-23 | 2023-09-28 | 住友ベークライト株式会社 | Separator and culture system |
| WO2021157480A1 (en) * | 2020-02-06 | 2021-08-12 | 富士フイルム株式会社 | Compound, liquid crystal composition, and liquid crystal film |
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