JP2013116130A - Cell culture support, and method for producing the same - Google Patents
Cell culture support, and method for producing the same Download PDFInfo
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- JP2013116130A JP2013116130A JP2013055770A JP2013055770A JP2013116130A JP 2013116130 A JP2013116130 A JP 2013116130A JP 2013055770 A JP2013055770 A JP 2013055770A JP 2013055770 A JP2013055770 A JP 2013055770A JP 2013116130 A JP2013116130 A JP 2013116130A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Clinical Laboratory Science (AREA)
- Molecular Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
本発明は細胞シートを安定的に量産作製するための細胞培養支持体に関する。 The present invention relates to a cell culture support for stably mass-producing production of cell sheets.
細胞シートとは、細胞間結合で細胞同士が少なくとも単層で連結されたシート状の細胞集合体である。細胞シートは再生医療などで用いられる。細胞シートはシャーレなどの支持体上で細胞培養を行うことにより得られるが、支持体上で形成された細胞シートは接着分子などを介して支持体表面と強固に結合しているため、細胞−細胞間の結合を壊さずに培養支持体から細胞シートを迅速に剥離することは容易ではない。 The cell sheet is a sheet-like cell aggregate in which cells are connected to each other in at least a single layer by intercellular bonding. Cell sheets are used in regenerative medicine. The cell sheet can be obtained by culturing cells on a support such as a petri dish, but the cell sheet formed on the support is firmly bonded to the support surface via an adhesion molecule or the like. It is not easy to quickly peel the cell sheet from the culture support without breaking the bonds between the cells.
そこで、細胞培養支持体から細胞シートを効率的に剥離する方法がこれまで検討されてきた。剥離方法は２つに大別できる。第一の方法は、酵素反応を用いて支持体と細胞間の結合を弱める方法である。第二の方法は、細胞接着力の弱い支持体や細胞接着力の変化する支持体を用いる方法である。 Thus, methods for efficiently peeling a cell sheet from a cell culture support have been studied so far. The peeling method can be roughly divided into two. The first method is a method of weakening the bond between the support and the cells using an enzyme reaction. The second method is a method using a support with weak cell adhesion or a support with varying cell adhesion.
第一の方法は、具体的には、酵素を用いて、プロテアーゼ（タンパク質分解酵素）やコラーゲナーゼ（コラーゲン分解酵素）といった細胞間接着分子（密着結合、接着結合、デスモゾーム結合、ギャップ結合、ヘミデスモゾーム結合）を構成するタンパク質や、培養物の周囲を取り巻くコラーゲン結合織や、細胞と支持体間に形成される細胞外マトリクス（Extracellular Matrix: ECM）を分解する方法である。この方法では細胞−支持体表面の結合だけでなく、細胞−細胞間の結合も弱まる。この方法は細胞培養の分野で古くから使われている。この方法で分解される結合物質は、培養される細胞、組織、器官において作られる物質であるから、剥離後においても一定の条件と期間で分解された結合物質を再生することができる。 In the first method, specifically, an enzyme is used to intercellular adhesion molecules such as protease (proteolytic enzyme) and collagenase (collagen degrading enzyme) (tight bond, adhesive bond, desmosome bond, gap bond, hemidesmosome). It is a method of degrading the protein constituting the binding), the collagen-binding tissue surrounding the periphery of the culture, and the extracellular matrix (Extracellular Matrix: ECM) formed between the cells and the support. This method weakens not only cell-support surface binding but also cell-cell binding. This method has long been used in the field of cell culture. Since the binding substance decomposed by this method is a substance produced in cells, tissues, and organs to be cultured, the binding substance decomposed under a certain condition and period can be regenerated even after detachment.
しかしながら、結合物質の再生には時間がかかるという問題がある。また、この方法では、細胞シートが少なからず損傷を受けるため、再生医療に用いられる細胞シートの作出方法としては望ましくないといえる。 However, there is a problem that it takes time to regenerate the binding substance. In addition, this method is not desirable as a method for producing a cell sheet used for regenerative medicine because the cell sheet is damaged in many ways.
そこで、細胞接着力の弱い支持体や細胞接着力の変化する支持体を用いる第二の方法が新たに開発されている。 Then, the 2nd method using the support body with weak cell adhesive force and the support body with which cell adhesive force changes is newly developed.
細胞接着力の弱い支持体としては特許文献１や特許文献２に開示されているものが挙げられる。これらの文献には、支持体表面にナノピラーと呼ばれる極微小の柱を立て、その上で培養を行う技術が開示されている。この技術では支持体と培養材料は非常に小さい面積でしか接着されず、回収剥離が容易でダメージも少ないとされる。 Examples of the support having a weak cell adhesion include those disclosed in Patent Document 1 and Patent Document 2. These documents disclose a technique in which ultra-fine pillars called nanopillars are set on the surface of a support and culture is performed thereon. According to this technique, the support and the culture material are adhered to each other only in a very small area, and the recovery and peeling are easy and the damage is small.
しかしながら非特許文献１及び２に記載されているように、細胞接着や接着した細胞の挙動は平面に接着する場合と凹凸表面に接着する場合とでは異なり、ナノピラー上では細胞の接着、伸展が遅くなったり、細胞表面から仮足が発生するという問題がある。また凹部が２０μｍ以上の幅を有する場合には細胞が潜入してしまうという問題もある。 However, as described in Non-Patent Documents 1 and 2, the cell adhesion and the behavior of the adhered cells are different between the case of adhering to a flat surface and the case of adhering to an uneven surface, and cell adhesion and extension are slow on the nanopillar. And there is a problem that a temporary foot is generated from the cell surface. In addition, when the concave portion has a width of 20 μm or more, there is a problem that cells infiltrate.
細胞接着力の変化する支持体として、細胞増殖表面を温度応答性ポリマーで被覆した支持体がある（特許文献３）。細胞接着力を変化させる目的には、温度応答性ポリマーを用いることが最も好ましいが、それ以外でもｐＨ応答性ポリマーや、イオン応答性ポリマーを用いることもできる。特許文献１及び２では温度応答性ポリマーをナノピラーを用いた培養に組合せることが言及されている。細胞培養において温度応答性ポリマーを用いることについては特許文献４にも言及がある。 As a support that changes cell adhesion, there is a support in which a cell growth surface is coated with a temperature-responsive polymer (Patent Document 3). For the purpose of changing the cell adhesive force, it is most preferable to use a temperature-responsive polymer, but other than that, a pH-responsive polymer or an ion-responsive polymer can also be used. Patent Documents 1 and 2 mention that a temperature-responsive polymer is combined with culture using a nanopillar. Patent Document 4 also mentions the use of a temperature-responsive polymer in cell culture.
特許文献３には、温度応答性ポリマー層の製造方法として、電子線照射により、モノマーの重合反応と、温度応答性ポリマーの少なくとも一端を基材を構成する分子に共有結合させて基材表面に温度応答性ポリマーを固定化する（グラフト化する）反応とを行うグラフト重合法が記載されている。しかし、特許文献４にも記載されるように、グラフト重合法により形成された温度応答性ポリマー層は電子線照射時の原材料の比率や電子線の照射条件が一定でないと温度応答性を示さないという問題があった。 In Patent Document 3, as a method for producing a temperature-responsive polymer layer, a monomer polymerization reaction and at least one end of the temperature-responsive polymer are covalently bonded to a molecule constituting the substrate by electron beam irradiation to form a substrate surface. A graft polymerization method in which a temperature responsive polymer is immobilized (grafted) is described. However, as described in Patent Document 4, the temperature-responsive polymer layer formed by the graft polymerization method does not exhibit temperature responsiveness unless the ratio of the raw materials at the time of electron beam irradiation and the electron beam irradiation conditions are constant. There was a problem.
特許文献４では、電子線照射前に基材表面に塗布する原料モノマー／溶媒混合物における溶媒残留量が低くなるよう溶媒の量を規定することが開示されている。しかしながら、特許文献４の実施例および比較例の方法では残留溶剤の量に再現性を確保するために恒温恒湿槽内における放置乾燥、窒素気流下での乾燥、真空乾燥、残存モノマーの重合に影響しない範囲内での加熱乾燥等の煩雑な工程管理が必要であった。また、特許文献４の実施例および比較例では、電子線エネルギーによる重合効率を良くする目的で被覆原料を溶解するための溶媒として沸点120℃以下のものを用いている。このような溶媒中にモノマー原料を溶解した組成物をディシュ上に被覆した場合、モノマーが結晶化しやすいという問題があった。肉眼で結晶が観察されなくても顕微鏡観察で微小結晶が存在することも確認されている。 Patent Document 4 discloses that the amount of solvent is regulated so that the residual amount of solvent in the raw material monomer / solvent mixture applied to the surface of the substrate before electron beam irradiation is low. However, in the methods of Examples and Comparative Examples of Patent Document 4, in order to ensure reproducibility of the amount of residual solvent, it is used for standing drying in a constant temperature and humidity chamber, drying under a nitrogen stream, vacuum drying, and polymerization of residual monomers. Complicated process management such as heating and drying within the range not affected was necessary. In Examples and Comparative Examples of Patent Document 4, a solvent having a boiling point of 120 ° C. or lower is used as a solvent for dissolving the coating raw material for the purpose of improving the polymerization efficiency by electron beam energy. When a composition in which a monomer raw material is dissolved in such a solvent is coated on a dish, there is a problem that the monomer is easily crystallized. Even if crystals are not observed with the naked eye, it is confirmed by microscopic observation that microcrystals are present.
本発明者らによる検討で明らかとなったことであるが、モノマー結晶が存在する塗膜に電子線を照射すると、結晶部分で重合が阻害されるため、十分な量の温度応答性ポリマーが被覆されないという問題があった。温度応答性ポリマーは放射線によるグラフト重合後、余分な遊離のポリマーを洗浄することでグラフト化されたポリマーのみを露出させ利用するが、被覆面の凹凸や洗浄時間を短くするために原料の塗布量を減らすことは、結晶化を促進させるので問題があった。 As clarified by the study by the present inventors, when a coating film containing monomer crystals is irradiated with an electron beam, polymerization is inhibited at the crystal portion, so that a sufficient amount of temperature-responsive polymer is coated. There was a problem of not being. For temperature-responsive polymers, after graft polymerization by radiation, the excess free polymer is washed to expose only the grafted polymer, but the coating amount of the raw material is used to shorten the unevenness of the coated surface and cleaning time. Reducing the value is problematic because it promotes crystallization.
ところで本出願人は電子線材料による印刷塗膜技術の分野で防曇フィルムや化粧合板の多くの技術を有する。本出願人らは、電子線照射による印刷、コーティング、接着硬化に重合性オリゴマーやプレポリマーを添加した塗膜が優れた化粧塗膜特性を持っていることを見出している。（特許文献５） By the way, the present applicant has many technologies for anti-fogging films and decorative plywood in the field of printed coating technology using electron beam materials. The present applicants have found that a coating film obtained by adding a polymerizable oligomer or prepolymer to printing, coating, and adhesion curing by electron beam irradiation has excellent cosmetic coating film properties. (Patent Document 5)
放射線を用いて温度応答性ポリマーを基材表面にグラフト化させる場合の各種照射条件は、グラフト化されるポリマーが基材上に均一に存在するように決定される。しかしながら首尾よく基材表面上にポリマーが固定化され該ポリマーによる親疎水変化が起きても、基材を構成する材料によっては細胞シート剥離が進みにくいことがあった。 Various irradiation conditions when the temperature-responsive polymer is grafted onto the substrate surface using radiation are determined so that the polymer to be grafted exists uniformly on the substrate. However, even when the polymer is successfully immobilized on the surface of the base material and the hydrophilicity / hydrophobicity change due to the polymer occurs, the cell sheet peeling may be difficult to proceed depending on the material constituting the base material.
上記の通り、細胞シートを作製するための従来の細胞培養支持体の製造方法では、基材表面に均一な温度応答性グラフトポリマー層を形成するためには、残留溶媒量の少ないモノマー／溶媒混合物を基材表面に塗布し、放射線照射を行う必要があった。しかしながら、溶媒量が少ない原料モノマー含有組成物を基材上に塗布して形成された塗膜中ではモノマーの微小結晶が形成されやすい。その結果、塗膜が、モノマー微小結晶とモノマー溶解液とからなる海島状となる。この塗膜に電子線を照射して重合反応とポリマーのグラフト化反応とを行うと、島状の結晶部分はグラフト化が進みにくいため細胞剥離機能が弱い部分となり、海状のモノマー溶解液部分はグラフト化が進みやすいため細胞剥離機能が強い部分となる。こうして得られるポリマー被覆細胞培養支持体上では、細胞が均一に接着又は増殖できないという問題があった。そして、細胞培養支持体上で細胞が均一にコンフルエントになりにくいという問題があった。そして、コンフレントな細胞を細胞シートとして得ようとしても、シートが剥離しない、シート剥離が遅い、シートが破損する、破損せず剥離してもシートに歪みができる、細胞にダメージが生ずる等問題があった。 As described above, in the conventional method for producing a cell culture support for producing a cell sheet, in order to form a uniform temperature-responsive graft polymer layer on the substrate surface, a monomer / solvent mixture with a small amount of residual solvent is used. It was necessary to apply to the surface of the substrate and to perform radiation irradiation. However, monomer microcrystals are easily formed in a coating film formed by applying a raw material monomer-containing composition having a small amount of solvent on a substrate. As a result, the coating film has a sea-island shape composed of monomer microcrystals and a monomer solution. When this coating film is irradiated with an electron beam to carry out a polymerization reaction and a grafting reaction of the polymer, the island-like crystal part is difficult to proceed with grafting, so the cell peeling function is weak, and the sea-like monomer solution part Is a part with a strong cell detachment function because of easy grafting. On the polymer-coated cell culture support thus obtained, there is a problem that the cells cannot adhere or grow uniformly. And there existed a problem that a cell did not become confluent uniformly on a cell culture support body. And even if trying to obtain confluent cells as a cell sheet, the sheet does not peel off, the sheet peeling is slow, the sheet breaks, the sheet can be distorted even if it is peeled without breakage, and the cells are damaged. there were.
また、モノマーの結晶化を避けるために、高沸点溶媒を用いて調製された原料モノマー／溶媒混合物を基材上に塗布すると、放射線効率が悪くなり、必要なグラフトポリマー量が得られないという問題があった。また原料モノマー／溶媒混合物の塗布量を増やすと長時間の洗浄が必要になるという問題に加えて、均一な乾燥や放射線照射が難しく、残留溶剤量の多い時同様に表面凹凸が発生し、培養基材としては平滑な表面が得られないという問題があった。温度変化による親疎水変化、そして細胞シート剥離という現象も、部分的に変化し難い島部分のポリマーを有するため、剥離の場所依存や全体としてのダイナミックな剥離を妨げるという問題があった。 In addition, when a raw material monomer / solvent mixture prepared using a high boiling point solvent is applied on a substrate to avoid crystallization of the monomer, the radiation efficiency is deteriorated and the necessary amount of graft polymer cannot be obtained. was there. In addition to the problem that increasing the coating amount of the raw material monomer / solvent mixture requires long-term cleaning, uniform drying and radiation irradiation are difficult, and surface unevenness occurs as in the case of a large amount of residual solvent. There was a problem that a smooth surface could not be obtained as a substrate. The phenomenon of hydrophilicity / hydrophobicity change due to temperature change and cell sheet exfoliation also has a problem in that it has an island portion polymer that is not easily changed in part, thereby hindering the location dependence of exfoliation and dynamic exfoliation as a whole.
また、特許文献３や４には、被覆が施される支持体の材質は通常細胞培養に用いられるガラス、改質ガラス、ポリスチレン、ポリメチルメタクリレート等の物質のみならず、一般に形態付与が可能である物質、例えば上記以外の高分子化合物、セラミックス、金属類など全て用いることができ、その形状はペトリディッシュに限定されることは無く、プレート、ファイバー、（多孔質）粒子、また、一般に細胞培養等に用いられる容器（フラスコ等）の形状を付与されていても構わない、と記載されている。しなしながら、実際には、汎用基材としてシャーレに用いたポリスチレン以外では、スライドガラス表面やガラス表面にシランカップリング剤処理をした基材等での研究報告・学会発表があるだけである。ガラス表面では、放射線による温度応答性グラフトポリマー表面が形成できることが解っているが、こうして形成された温度応答性グラフトポリマー表面はポリスチレンシャーレ表面のそれと比べ顕著な細胞シート剥離を示さないという問題があった。 In Patent Documents 3 and 4, the material of the support to be coated is not only a substance usually used for cell culture, such as glass, modified glass, polystyrene, polymethylmethacrylate, but generally can be given a form. A certain substance, for example, polymer compounds other than those mentioned above, ceramics, metals, etc. can be used. The shape is not limited to petri dishes, but plates, fibers, (porous) particles, and generally cell culture. It is described that the shape of a container (flask or the like) used in the above may be given. However, in fact, there are only research reports and conference presentations on slide glass surfaces and substrates treated with a silane coupling agent other than polystyrene used for petri dishes as general purpose substrates. Although it has been found that a temperature-responsive graft polymer surface can be formed by radiation on the glass surface, the temperature-responsive graft polymer surface formed in this way has a problem that it does not show significant cell sheet detachment compared to that of a polystyrene petri dish surface. It was.
本発明の予備実験でも汎用プラスチックであるポリエチレンテレフタレート基材上に形成された温度応答性グラフトポリマー表面は細胞シート剥離機能を有していないことが確認されている。また、ポリカーボネートを基材した場合においても、ポリスチレンを基材とした場合に比べ親疎水変化も細胞シート剥離も弱いものであった。 Also in the preliminary experiment of the present invention, it has been confirmed that the temperature-responsive graft polymer surface formed on the polyethylene terephthalate base material, which is a general-purpose plastic, does not have a cell sheet peeling function. Further, even when polycarbonate was used as a base material, both the hydrophilicity / hydrophobicity change and the cell sheet peeling were weaker than when polystyrene was used as the base material.
本発明は上記問題点を解消した細胞培養支持体を提供することを目的とする。 It is an object of the present invention to provide a cell culture support in which the above problems are eliminated.
本出願は以下の発明を包含する。
（１）温度応答性ポリマー、ｐＨ応答性ポリマー及びイオン応答性ポリマーからなる群から選択される少なくとも１種のポリマーが共有結合により表面に固定化された細胞培養支持体の製造方法であって、放射線照射により重合して前記ポリマーを形成し得るモノマーと、前記モノマーが重合してなるオリゴマー又はプレポリマーと、有機溶媒とを含む組成物を、前記ポリマーと放射線照射により共有結合し得る材料を含む表面を備えた基材に塗布して、前記基材の表面上に塗膜を形成する塗布工程と、前記塗膜に放射線を照射して、重合反応及び基材表面と前記ポリマーとの結合反応を進行させる放射線照射工程と、前記塗膜を乾燥させる乾燥工程とを含む前記方法。
（２）前記ポリマーが温度応答性ポリマーである、（１）記載の方法。
（３）温度応答性ポリマーがアクリル系ポリマー又はメタクリル系ポリマーである、（２）記載の方法。
（４）アクリル系ポリマーがポリ−Ｎ−イソプロピルアクリルアミドである、（３）記載の方法。
（５）前記組成物が５×１０−３Ｐａ・ｓ〜１０Ｐａ・ｓの粘度を有する組成物である、（１）〜（４）のいずれかに記載の方法。
（６）前記オリゴマー又はプレポリマーの分子量が３，０００以上である、（１）〜（５）のいずれかに記載の方法。
（７）前記モノマーと、前記オリゴマー又はプレポリマーとの重量比が５００：１〜１：２０である、（１）〜（６）のいずれかに記載の方法。
（８）放射線照射工程が１回の放射線照射により行われる、（１）〜（７）のいずれかに記載の方法。
（９）放射線がγ線又は電子線である、（１）〜（８）のいずれかに記載の方法。
（１０）放射線が５Ｍｒａｄ〜５０Ｍｒａｄの線量の電子線であるか、あるいは０．５Ｍｒａｄ〜５Ｍｒａｄの線量のγ線である、（１）〜（９）のいずれかに記載の方法。
（１１）乾燥工程が放射線照射工程の前に行われる、（１）〜（１０）のいずれかに記載の方法。
（１２）基材の形状がフィルム形状である、（１）〜（１１）のいずれかに記載の方法。
（１３）基材が、ポリスチレン、低密度ポリエチレン、中密度ポリエチレン、高密度ポリエチレン、ポリウレタン、アクリル系樹脂、ポリアミド、ポリカーボネート、共役結合を持つ天然ゴム、共役結合を持つ合成ゴム、及びポリシリコンを含有するシリコンゴムからなる群から選択される少なくとも１種の材料からなる、（１）〜（１２）のいずれかに記載の方法。
（１４）基材が、表面が易接着処理されたポリエチレンテレフタレート、表面がコロナ処理またはプラズマ処理された合成樹脂、及び表面がアクリル系樹脂により被覆された合成樹脂からなる群から選択される少なくとも１種の材料からなる、（１）〜（１２）のいずれかに記載の方法。
（１５）合成樹脂がナイロン、低密度ポリエチレン、中密度ポリエチレン、ポリプロピレン、ポリエチレンテレフタレート、ポリカーボネート、及びポリスチレンからなる群から選択される少なくとも１種の材料である、（１４）記載の方法。
（１６）（１）〜（１５）のいずれかに記載の方法により製造された、温度応答性ポリマー、ｐＨ応答性ポリマー及びイオン応答性ポリマーからなる群から選択される少なくとも１種のポリマーが共有結合により表面に固定化された細胞培養支持体。
（１７）細胞培養支持体の表面に固定化された前記ポリマーの層の乾燥時の厚さが０．００１〜１０μｍである、（１６）記載の細胞培養支持体。
（１８）（１６）又は（１７）記載の細胞培養支持体上で細胞培養する工程を含む、細胞シートの作製方法。
（１９）（１８）記載の方法により作製された細胞シート。
This application includes the following inventions.
(1) A method for producing a cell culture support in which at least one polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer is immobilized on a surface by a covalent bond, A material comprising a monomer that can be polymerized by irradiation with radiation, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent, and a material that can be covalently bonded to the polymer by irradiation. A coating process for forming a coating film on the surface of the substrate by coating the substrate with a surface, and irradiating the coating film with radiation to cause a polymerization reaction and a bonding reaction between the substrate surface and the polymer. The said method including the radiation irradiation process to advance, and the drying process which dries the said coating film.
(2) The method according to (1), wherein the polymer is a temperature-responsive polymer.
(3) The method according to (2), wherein the temperature-responsive polymer is an acrylic polymer or a methacrylic polymer.
(4) The method according to (3), wherein the acrylic polymer is poly-N-isopropylacrylamide.
(5) The method according to any one of (1) to (4), wherein the composition is a composition having a viscosity of 5 × 10 −3 Pa · s to 10 Pa · s.
(6) The method according to any one of (1) to (5), wherein the oligomer or prepolymer has a molecular weight of 3,000 or more.
(7) The method according to any one of (1) to (6), wherein a weight ratio of the monomer to the oligomer or prepolymer is 500: 1 to 1:20.
(8) The method according to any one of (1) to (7), wherein the radiation irradiation step is performed by one radiation irradiation.
(9) The method according to any one of (1) to (8), wherein the radiation is γ rays or electron beams.
(10) The method according to any one of (1) to (9), wherein the radiation is an electron beam with a dose of 5 Mrad to 50 Mrad, or a gamma ray with a dose of 0.5 Mrad to 5 Mrad.
(11) The method according to any one of (1) to (10), wherein the drying step is performed before the radiation irradiation step.
(12) The method according to any one of (1) to (11), wherein the substrate has a film shape.
(13) The base material contains polystyrene, low density polyethylene, medium density polyethylene, high density polyethylene, polyurethane, acrylic resin, polyamide, polycarbonate, natural rubber having conjugated bond, synthetic rubber having conjugated bond, and polysilicon. The method according to any one of (1) to (12), comprising at least one material selected from the group consisting of silicone rubbers.
(14) At least one selected from the group consisting of a polyethylene terephthalate whose surface is easily adhered, a synthetic resin whose surface is corona-treated or plasma-treated, and a synthetic resin whose surface is coated with an acrylic resin. The method according to any one of (1) to (12), comprising a seed material.
(15) The method according to (14), wherein the synthetic resin is at least one material selected from the group consisting of nylon, low density polyethylene, medium density polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, and polystyrene.
(16) At least one polymer selected from the group consisting of a temperature-responsive polymer, a pH-responsive polymer, and an ion-responsive polymer produced by the method according to any one of (1) to (15) is shared A cell culture support immobilized on a surface by binding.
(17) The cell culture support according to (16), wherein the thickness of the polymer layer immobilized on the surface of the cell culture support is 0.001 to 10 μm.
(18) A method for producing a cell sheet, comprising a step of culturing cells on the cell culture support according to (16) or (17).
(19) A cell sheet produced by the method according to (18).
本発明に係る細胞培養支持体は、放射線照射前に残留溶剤量を下げた場合でもモノマーの析出、結晶化が発生しないため、基材全面に各種ポリマーをグラフト化できる。 Since the cell culture support according to the present invention does not cause monomer precipitation or crystallization even when the residual solvent amount is lowered before irradiation, various polymers can be grafted on the entire surface of the substrate.
本発明に係る細胞培養支持体は、放射線照射前に残留溶剤量を下げた場合でもモノマーの析出、結晶化が発生しないため、薄膜コートが可能で、放射線照射後の洗浄時間を短縮できる。 Since the cell culture support according to the present invention does not cause monomer precipitation or crystallization even when the residual solvent amount is lowered before irradiation, thin film coating is possible, and the cleaning time after irradiation can be shortened.
本発明に係る細胞培養支持体を用いて細胞シートを作製した場合、不要なポリマーを含まないため、細胞接着、細胞増殖が早く、より短時間で基材表面全体にコンフレントな状態（隣接する細胞同士が隙間なく接着した、集合、群集した状態）またはそれに近い状態をつくれ、細胞シート剥離前の培養時間を短くできる。 When a cell sheet is produced using the cell culture support according to the present invention, since it does not contain unnecessary polymers, cell adhesion and cell growth are fast, and the entire surface of the substrate can be confined in a shorter time (adjacent cells). A state in which the members adhere together without gaps, or a state in which they are gathered or gathered together, or a state close thereto, and the culture time before cell sheet peeling can be shortened.
本発明に係る細胞培養支持体を用いて細胞シートを作製した場合、部分的に培養の遅れる現象がなくコンフレント（コンフレントに近く）になるため、剥離した細胞シートに切れ目や損傷が少なく、均質な細胞シートが得られる。 When a cell sheet is prepared using the cell culture support according to the present invention, the cell sheet is partially confused and becomes confluent (close to confluence). A cell sheet is obtained.
本発明に係る細胞培養支持体がその表面に温度応答性グラフトポリマーの層を含むものである場合、温度に応じて表面の濡れ性が変化するとともに、可逆的に良好な細胞接着・剥離性を示す。 When the cell culture support according to the present invention includes a layer of a temperature-responsive graft polymer on its surface, the wettability of the surface changes according to the temperature and reversibly shows good cell adhesion / peelability.
本発明に係る細胞培養支持体を用いて作製された細胞シートは表面の接着因子が損なわれていないため再生医療などへの利用に適する。 The cell sheet produced using the cell culture support according to the present invention is suitable for use in regenerative medicine and the like because the surface adhesion factor is not impaired.
（応答性ポリマー）
本発明は、温度応答性ポリマー、ｐＨ応答性ポリマー及びイオン応答性ポリマーからなる群から選択される少なくとも１種のポリマーが共有結合により表面に固定化（すなわちグラフト化）された細胞培養支持体の製造方法に関する。
(Responsive polymer)
The present invention relates to a cell culture support in which at least one polymer selected from the group consisting of a temperature responsive polymer, a pH responsive polymer, and an ion responsive polymer is covalently immobilized (ie, grafted) to a surface. It relates to a manufacturing method.
応答性ポリマーとしては特に温度応答性ポリマーが好ましいがこれには限定されない。 The responsive polymer is particularly preferably a temperature responsive polymer, but is not limited thereto.
本発明に好適に使用できる温度応答性ポリマーは細胞培養温度下（通常、３７℃程度）において疎水性を示し、培養した細胞シートの回収時の温度下において親水性を示すものである。なお、温度応答性ポリマーが、疎水性から親水性に変化する温度（水に対する臨界溶解温度（Ｔ））としては、特に限定されないが、培養後の細胞シートの回収の容易さの観点からは、細胞培養温度よりも低い温度であることが好ましい。このような温度応答性ポリマー成分を含むことで、細胞培養時においては、細胞の足場（細胞接着面）が充分に確保されるため細胞培養を効率よく行うことができる。その一方、培養後の細胞シートの回収時においては、疎水性部分を親水性に変化させ、培養された細胞シートを細胞培養基材から分離させることで、細胞シートの回収をより一層容易にすることができる。特に所定の臨界溶解温度未満の温度で親水性を示し、同温度以上の温度で疎水性を示す温度応答性ポリマーが好ましい。このような温度応答性ポリマーにおける臨界溶解温度を特に下限臨界溶解温度と呼ぶ。 The temperature-responsive polymer that can be suitably used in the present invention is hydrophobic at the cell culture temperature (usually about 37 ° C.) and hydrophilic at the temperature at the time of recovering the cultured cell sheet. The temperature at which the temperature-responsive polymer changes from hydrophobic to hydrophilic (critical solution temperature in water (T)) is not particularly limited, but from the viewpoint of ease of recovery of the cell sheet after culture, The temperature is preferably lower than the cell culture temperature. By including such a temperature-responsive polymer component, since cell scaffolds (cell adhesion surfaces) are sufficiently secured during cell culture, cell culture can be performed efficiently. On the other hand, at the time of collecting the cell sheet after culturing, the hydrophobic part is changed to hydrophilic, and the cultured cell sheet is separated from the cell culture substrate, thereby making it easier to collect the cell sheet. be able to. In particular, a temperature-responsive polymer that exhibits hydrophilicity at a temperature lower than a predetermined critical dissolution temperature and exhibits hydrophobicity at a temperature equal to or higher than the same temperature is preferable. The critical solution temperature in such a temperature-responsive polymer is particularly called the lower critical solution temperature.
本発明に好適に使用できる温度応答性ポリマーは具体的には下限臨界溶解温度Ｔが０〜８０℃、好ましくは０〜５０℃であるポリマーが好ましい。Ｔが８０℃を越えると細胞が死滅する可能性があるので好ましくない。またＴが０℃より低いと、一般に細胞増殖速度が極度に低下するか、または細胞が死滅してしまうため好ましくない。そのような好適なポリマーとしてはアクリル系ポリマー又はメタクリル系ポリマーが挙げられる。好適なポリマーは例えば特許文献３にも記載されている。具体的に適当なポリマーとしては、例えばポリ−Ｎ−イソプロピルアクリルアミド(Ｔ＝３２℃)、ポリ−Ｎ−ｎ−プロピルアクリルアミド(Ｔ＝２１℃)、ポリ−Ｎ−ｎ−プロピルメタクリルアミド(Ｔ＝３２℃)、ポリ−Ｎ−エトキシエチルアクリルアミド(Ｔ＝約３５℃)、ポリ−Ｎ−テトラヒドロフルフリルアクリルアミド(Ｔ＝約２８℃)、ポリ−Ｎ−テトラヒドロフルフリルメタクリルアミド（Ｔ＝約３５℃）、及びポリ−Ｎ,Ｎ−ジエチルアクリルアミド(Ｔ＝３２℃)等が挙げられる。その他のポリマーとしては、例えばポリ−Ｎ−エチルアクリルアミド、ポリ−Ｎ−イソプロピルメタクリルアミド、ポリ−Ｎ−シクロプロピルアクリルアミド、ポリ−Ｎ−シクロプロピルメタクリルアミド、ポリ−Ｎ−アクリロイルピロリジン、ポリ−Ｎ−アクリロイルピペリジン、ポリメチルビニルエーテル、メチルセルロース、エチルセルロース、ヒドロキシプロピルセルロース等のアルキル置換セルロース誘導体や、ポリポリプロピレンオキサイドとポリエチレンオキサイドとのブロック共重合体等に代表されるポリアルキレンオキサイドブロック共重合体や、ポリアルキレンオキサイドブロック共重合体が挙げられる。 Specifically, the temperature-responsive polymer that can be suitably used in the present invention is preferably a polymer having a lower critical solution temperature T of 0 to 80 ° C, preferably 0 to 50 ° C. If T exceeds 80 ° C., the cells may die, which is not preferable. If T is lower than 0 ° C., the cell growth rate is generally extremely reduced, or the cells are killed, which is not preferable. Such suitable polymers include acrylic or methacrylic polymers. Suitable polymers are also described, for example, in US Pat. Specific suitable polymers include, for example, poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), poly-Nn-propyl methacrylamide (T = 32 ° C.), poly-N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.) ), And poly-N, N-diethylacrylamide (T = 32 ° C.). Examples of other polymers include poly-N-ethylacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropylacrylamide, poly-N-cyclopropylmethacrylamide, poly-N-acryloylpyrrolidine, poly-N- Polyalkylene oxide block copolymers represented by alkyl-substituted cellulose derivatives such as acryloyl piperidine, polymethyl vinyl ether, methyl cellulose, ethyl cellulose, and hydroxypropyl cellulose, block copolymers of polypolypropylene oxide and polyethylene oxide, and polyalkylene An oxide block copolymer is mentioned.
これらのポリマーを形成するためのモノマーとしては、例えばモノマーの単独重合体がＴ＝０〜８０℃を有するようなモノマーであって、放射線照射によって重合し得るモノマーが挙げられる。モノマーとしては例えば、(メタ)アクリルアミド化合物、Ｎ−(若しくはＮ,Ｎ−ジ)アルキル置換(メタ)アクリルアミド誘導体、環状基を有する(メタ)アクリルアミド誘導体、及びビニルエーテル誘導体等が挙げられ、これらの１種以上を使用してよい。モノマーが一種類単独で使用された場合、基材上に形成されるポリマーはホモポリマーとなり、モノマーが複数種一緒に使用された場合、基材上に形成されるポリマーはコポリマーとなるが、どちらの形態も本発明に包含される。また、増殖細胞の種類によってＴを調節する必要がある場合や、被覆物質と細胞培養支持体との相互作用を高める必要が生じた場合や、細胞支持体の親水・疎水性のバランスを調整する必要がある場合などには、上記以外の他のモノマー類を更に加えて共重合してよい。更に本発明に使用する上記ポリマーとその他のポリマーとのグラフトまたはブロック共重合体、あるいは本発明のポリマーと他のポリマーとの混合物を用いてもよい。また、ポリマー本来の性質が損なわれない範囲で架橋することも可能である。 Examples of the monomer for forming these polymers include monomers having a monomer homopolymer having T = 0 to 80 ° C. and capable of being polymerized by irradiation. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. More than seeds may be used. When a single monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple monomers are used together, the polymer formed on the substrate is a copolymer. These forms are also encompassed by the present invention. In addition, when it is necessary to adjust T depending on the type of proliferating cell, when it is necessary to enhance the interaction between the coating substance and the cell culture support, and the balance between the hydrophilicity and hydrophobicity of the cell support is adjusted. If necessary, other monomers other than those described above may be further added for copolymerization. Further, a graft or block copolymer of the above-mentioned polymer used in the present invention and another polymer, or a mixture of the polymer of the present invention and another polymer may be used. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.
ｐＨ応答性ポリマーおよびイオン応答性ポリマーは作製しようとする細胞シートに適したものを適宜選択することができる。 As the pH responsive polymer and the ion responsive polymer, those suitable for the cell sheet to be prepared can be appropriately selected.
（塗布用組成物）
本発明の方法には、放射線照射により重合して前記ポリマーを形成し得るモノマーと、前記モノマーが重合してなるオリゴマー又はプレポリマーと、有機溶媒とを含む塗布用組成物を用いる。この塗布用組成物はオリゴマー又はプレポリマーを含むことから、有機溶媒が少量の場合にも結晶化しにくい。このため、この塗布用組成物を基材表面に塗布し、放射線照射により重合を進行させると、基材表面の全面に亘り均一なポリマー層を形成することができる。
(Coating composition)
In the method of the present invention, a coating composition comprising a monomer that can be polymerized by irradiation with radiation to form the polymer, an oligomer or prepolymer obtained by polymerizing the monomer, and an organic solvent is used. Since this coating composition contains an oligomer or a prepolymer, it is difficult to crystallize even when the amount of the organic solvent is small. For this reason, when this composition for application | coating is apply | coated to the base-material surface and superposition | polymerization is advanced by radiation irradiation, a uniform polymer layer can be formed over the whole surface of a base-material surface.
放射線重合成のモノマーについては上記の通りである。塗布用組成物にはモノマーが単独又は複数種含まれる。 The radiation polysynthetic monomer is as described above. The coating composition contains one or more monomers.
塗布用組成物に含まれるオリゴマー又はプレポリマーの大きさはダイマー以上のものであれば特に限定されず、分子量約３，３００（典型的には２８分子ポリマー）より大きいものが好ましく、分子量５，７００以上のものがより好ましい。上限は特に限定されず、分子量１００万以上であってもよい。なお本発明において「プレポリマー」という用語は放射線照射前のポリマーを指す。 The size of the oligomer or prepolymer contained in the coating composition is not particularly limited as long as it is a dimer or larger, and preferably has a molecular weight of more than about 3,300 (typically 28 molecular polymers). More than 700 are more preferable. The upper limit is not particularly limited, and may be a molecular weight of 1 million or more. In the present invention, the term “prepolymer” refers to a polymer before irradiation.
有機溶媒としてはモノマー、オリゴマー又はプレポリマーを溶解しうるものであれば特に限定されないが、常圧下に於いて沸点１２０℃以下、特に６０〜１１０℃のものが好ましい。好ましい溶媒としては、具体的にはメタノール、エタノール、ｎ(若しくはｉ)−プロパノール、２(若しくはｎ)−ブタノール、及び水等が挙げられ、それらの１種以上使用してよい。その他の溶媒、例えば１−ペンタノール、２−エチル−１−ブタノール、２−ブトキシエタノール、及びエチレン(若しくはジエチレン)グリコール又はそのモノエチルエーテル、等も１種以上使用してよい。上記溶液にはその他添加剤として、硫酸等で代表される酸類、モール塩等を配合してよい。 The organic solvent is not particularly limited as long as it can dissolve the monomer, oligomer or prepolymer, but those having a boiling point of 120 ° C. or less, particularly 60 to 110 ° C. under normal pressure are preferred. Preferable examples of the solvent include methanol, ethanol, n (or i) -propanol, 2 (or n) -butanol, and water, and one or more of them may be used. Other solvents such as 1-pentanol, 2-ethyl-1-butanol, 2-butoxyethanol, and ethylene (or diethylene) glycol or monoethyl ether thereof may be used. As other additives, acids such as sulfuric acid, Mole salt and the like may be added to the above solution.
使用するモノマーと、オリゴマー又はプレポリマーとの重量比は５００：1〜１：２０であることが最も好ましい。 Most preferably, the weight ratio of the monomer used to the oligomer or prepolymer is from 500: 1 to 1:20.
塗布用組成物中のモノマーの含有量は５〜７０重量％であることが好ましい。 The content of the monomer in the coating composition is preferably 5 to 70% by weight.
塗布用組成物中のオリゴマー又はプレポリマーの含有量は０．１〜２０重量％であることが好ましい。 The content of the oligomer or prepolymer in the coating composition is preferably 0.1 to 20% by weight.
塗布用組成物の粘度は５×１０−３Ｐａ・ｓ〜１０Ｐａ・ｓであることが好ましい。 The viscosity of the coating composition is preferably 5 × 10 −3 Pa · s to 10 Pa · s.
（基材）
塗布用組成物が塗布される基材は、その表面が、前記応答性ポリマーと放射線照射により共有結合し得る材料を含むものである限り特に限定されない。表面のみが、前記応答性ポリマーと放射線照射により共有結合し得る材料を含むものであってもよいし、基材の全部がそのような材料を含むものであってもよい。このような基材の材料は、通常細胞培養に用いられるガラス類、プラスチック類、セラミックス、金属等が挙げられるが、細胞培養が可能な材料であれば特に限定されない。基材の表面または中間層に本発明の目的を妨げない限り任意の層を設けてもよいし、任意の処理を施してもよい。例えば、支持体表面にオゾン処理、プラズマ処理、スパッタリング等の処理技術を用いて親水化を施すことができる。
(Base material)
The base material to which the coating composition is applied is not particularly limited as long as the surface thereof contains a material that can be covalently bonded to the responsive polymer by irradiation. Only the surface may contain a material that can be covalently bonded to the responsive polymer by irradiation, or the entire substrate may contain such a material. Examples of the material for the base material include glasses, plastics, ceramics, metals, and the like that are usually used for cell culture, but are not particularly limited as long as the material can be used for cell culture. An arbitrary layer may be provided on the surface of the substrate or the intermediate layer as long as the object of the present invention is not hindered, and an arbitrary treatment may be performed. For example, the support surface can be hydrophilized using a treatment technique such as ozone treatment, plasma treatment, or sputtering.
基材を構成する材料であって、それ自体が上記応答性ポリマーと共有結合を形成し得るものとしては、ポリスチレン、低密度ポリエチレン、中密度ポリエチレン、高密度ポリエチレン、ポリウレタン、ウレタンアクリレート、ポリメチルメタクリレート等のアクリル系樹脂、ポリアミド(ナイロン)、ポリカーボネート、共役結合を持つ天然ゴム、共役結合を持つ合成ゴム、ポリシリコンを含有するシリコンゴム等が挙げられる。基材はこれらの材料を２種以上含むブレンドポリマー又はポリマーアロイからなるものであってもよい。 Materials that constitute the substrate and can themselves form a covalent bond with the responsive polymer include polystyrene, low-density polyethylene, medium-density polyethylene, high-density polyethylene, polyurethane, urethane acrylate, and polymethyl methacrylate. Examples thereof include acrylic resins such as polyamide (nylon), polycarbonate, natural rubber having a conjugated bond, synthetic rubber having a conjugated bond, and silicon rubber containing polysilicon. The substrate may be composed of a blend polymer or polymer alloy containing two or more of these materials.
上記応答性ポリマーと共有結合するように表面処理された基材としては、表面が易接着処理されたポリエチレンテレフタレート、表面がコロナ処理またはプラズマ処理された合成樹脂、表面がウレタンアクリレート等のアクリル系樹脂により被覆された合成樹脂等が挙げられる。基材はこれらの材料を２種以上含むブレンドポリマー又はポリマーアロイからなるものであってもよい。合成樹脂としてはナイロン、低密度ポリエチレン、中密度ポリエチレン、ポリプロピレン又はポリエチレンテレフタレート、ポリカーボネート、ポリスチレン等が挙げられる。合成樹脂はこれらの材料を２種以上含むブレンドポリマー又はポリマーアロイからなるものであってもよい。 Examples of the base material surface-treated so as to be covalently bonded to the responsive polymer include polyethylene terephthalate whose surface is easily bonded, synthetic resin whose surface is corona-treated or plasma-treated, and acrylic resin such as urethane acrylate. And synthetic resins coated with the above. The substrate may be composed of a blend polymer or polymer alloy containing two or more of these materials. Examples of the synthetic resin include nylon, low density polyethylene, medium density polyethylene, polypropylene or polyethylene terephthalate, polycarbonate, and polystyrene. The synthetic resin may be composed of a blend polymer or polymer alloy containing two or more of these materials.
基材の形状としては、ディッシュ形状や、フィルム形状などが挙げられる。フィルム形状基材を用いる場合、フィルム形状基材表面にグラフトポリマー層を形成した後、細胞培養に適した形状（例えばディッシュ形状）に加工することができる。加工の際は、必要に応じて他の材料からなる部材を前記基材と組み合わせて使用することもできる。ディッシュ形状基材を用いる場合、少なくとも細胞接着面となるディッシュ内底面部分がグラフトポリマー層により被覆されればよい。 Examples of the shape of the substrate include a dish shape and a film shape. When using a film-shaped base material, after forming a graft polymer layer on the film-shaped base material surface, it can be processed into a shape suitable for cell culture (for example, a dish shape). In processing, a member made of another material can be used in combination with the base material as necessary. In the case of using a dish-shaped substrate, it is sufficient that at least the inner bottom portion of the dish serving as the cell adhesion surface is covered with the graft polymer layer.
（塗布工程）
本発明の方法は、前記塗布用組成物を、前記基材の表面に塗布してその表面上に塗膜を形成する塗布工程を含む。
(Coating process)
The method of this invention includes the application | coating process which apply | coats the said composition for application | coating to the surface of the said base material, and forms a coating film on the surface.
本工程で形成される塗膜の塗布量はグラフトポリマーが機能（例えば温度応答性）を発揮する必要な塗布量である５０ｍｇ／ｍ２以上あればよい。塗布量の上限は特にないが、４０ｇ／ｍ２未満が好ましく、１０ｇ／ｍ２以下がより好ましい。塗布量が４０ｇ／ｍ２以上である場合には、厚みが増して塗膜厚が安定しないこと、厚みが増して放射線の貫通・照射量が安定しないこと、並びに照射エネルギーに由来する膜内の対流によりグラフトポリマーの被覆量にムラが生じることが本実施例中で確認されている。また、グラフトされない遊離のポリマーを洗浄するための洗浄時間を短くするためには塗膜量は１０ｇ／ｍ２以下が望ましい。 The coating amount of the coating film formed in this step may be 50 mg / m 2 or more which is a necessary coating amount for the graft polymer to exhibit a function (for example, temperature responsiveness). The upper limit of the coating amount is not particularly limited, but is preferably less than 40 g / m 2 and more preferably 10 g / m 2 or less. When the coating amount is 40 g / m 2 or more, the thickness is increased and the coating thickness is not stable, the thickness is increased and the radiation penetration / irradiation amount is not stable, and in the film derived from the irradiation energy It has been confirmed in this example that unevenness occurs in the coating amount of the graft polymer by convection. In order to shorten the washing time for washing the ungrafted free polymer, the coating amount is desirably 10 g / m 2 or less.
塗布用組成物の基材への小面積への塗布方法としては公知のいずれの方法でもよく、例えばスピンコーター、バーコーター等による塗布法、噴霧塗布法等が挙げられる。
大面積への塗布方法としてはブレードコーティング法、グラビアコーティング法、ロッドコーティング法、ナイフコーディング法、リバースロールコーティング法、オフセットグラビアコーティング法等が使用できる。
As a method for applying the coating composition to a substrate on a small area, any known method may be used, and examples thereof include a coating method using a spin coater, a bar coater and the like, and a spray coating method.
As a coating method for a large area, a blade coating method, a gravure coating method, a rod coating method, a knife coding method, a reverse roll coating method, an offset gravure coating method and the like can be used.
ベタ形成においては、グラビアコート法、ロールコート法、スロットコート法、キスコ−ト法、スプレーコート法、ファウンテンコーティング法等公知のコーティング法を用いて形成することが出来る。又、絵柄層のパターン形成においては、グラビア印刷法、スクリーン印刷法、オフセット印刷法等公知の印刷法を用いることが出来る。塗布用組成物の基材への塗布方法としては連続のコート法又は印刷法を使用することもできる。連続のコート法又は印刷法としては、具体的にはホットメルトコート、ホットラッカーコート、グラビアダイレクトコート、グラビアリバースコート、ダイコート、マイクログラビアコート、スライドコート、スリットリバースコート、カーテンコート、ナイフコート、エアコート、ロールコート等の塗布方法が使用できるが、これらは例示に過ぎず、当業者であれば暫時適用可能なものを使用することができる。 In the solid formation, a known coating method such as a gravure coating method, a roll coating method, a slot coating method, a kiss coating method, a spray coating method, or a fountain coating method can be used. In the pattern formation of the pattern layer, a known printing method such as a gravure printing method, a screen printing method, or an offset printing method can be used. As a method of applying the coating composition to the substrate, a continuous coating method or printing method can also be used. Specific coating methods or printing methods include hot melt coating, hot lacquer coating, gravure direct coating, gravure reverse coating, die coating, micro gravure coating, slide coating, slit reverse coating, curtain coating, knife coating, and air coating. Application methods such as roll coating can be used, but these are merely examples, and those skilled in the art can use those that can be applied for a while.
（放射線照射工程）
本発明の方法は、前記塗膜に放射線を照射して、重合反応及び基材表面と前記ポリマーとの結合反応（すなわちグラフト化）を進行させる放射線照射工程を含む。ここでいう結合反応（グラフト化）は、放射線照射による重合によってモノマー又はオリゴマーもしくはプレポリマーからin situで形成された遊離のポリマーが基材表面に結合する現象だけでなく、遊離のモノマーが基材表面に結合した後に当該モノマーを基点としてポリマー鎖が伸張する現象や、塗布用組成物に由来する遊離のプレポリマー又はオリゴマーが基材表面に結合する現象や、基材表面に結合したポリマー又はオリゴマーを基点としてポリマー鎖が伸張する現象などを包含する。
(Radiation irradiation process)
The method of the present invention includes a radiation irradiation step of irradiating the coating film with radiation to advance a polymerization reaction and a binding reaction (that is, grafting) between the substrate surface and the polymer. The bonding reaction (grafting) here is not only a phenomenon in which a free polymer formed in situ from a monomer, oligomer or prepolymer by polymerization by radiation irradiation binds to the surface of the substrate, but also a free monomer is bonded to the substrate. A phenomenon in which the polymer chain extends from the monomer as a starting point after bonding to the surface, a phenomenon in which a free prepolymer or oligomer derived from the coating composition is bonded to the substrate surface, or a polymer or oligomer bonded to the substrate surface Including the phenomenon that the polymer chain extends from the base point.
使用する放射線としては、α線、β線、γ線、電子線、紫外線等がある。所望のグラフトポリマーを作製するための合成にはγ線と電子線がエネルギー効率が良く、特に生産性の面からも電子線が好ましい。紫外線に関しては適当な重合開始剤や基材とのアンカー剤を組合せることで使用できる。 Examples of the radiation used include α rays, β rays, γ rays, electron beams, ultraviolet rays, and the like. In the synthesis for producing a desired graft polymer, γ rays and electron beams are energy efficient, and electron beams are particularly preferable from the viewpoint of productivity. With respect to ultraviolet rays, it can be used by combining an appropriate polymerization initiator and an anchor agent with a substrate.
放射線の線量の範囲は、電子線であれば５Mｒａｄ〜５０Mｒａｄが好ましく、γ線であれば０．５Mｒａｄ〜５Mｒａｄが好ましい。 The range of radiation dose is preferably 5 Mrad to 50 Mrad for electron beams, and preferably 0.5 Mrad to 5 Mrad for γ rays.
本発明の方法では、ある程度重合が進んだオリゴマー又はプレポリマーを含有する塗布用組成物を用いることから、１回のみの放射線照射によって、重合反応によるポリマーの形成と、基材表面とポリマーと間のグラフト化反応とを完了させることが可能である。 In the method of the present invention, a coating composition containing an oligomer or prepolymer that has been polymerized to some extent is used. It is possible to complete the grafting reaction.
（乾燥工程）
本発明の方法は、前記塗膜を乾燥させて塗布用組成物に由来する有機溶媒を除去する乾燥工程を含む。
(Drying process)
The method of the present invention includes a drying step of drying the coating film to remove an organic solvent derived from the coating composition.
前記塗布工程で形成される塗膜は残留溶剤量の影響により結晶が形成されることがないため、乾燥前の塗膜に放射線を照射した後、乾燥を行ってもよいし、塗膜を乾燥した後に放射線を照射してもよい。ただし、乾燥前のウェットな状態の塗膜に放射線照射を行うと、環境変化や異物、塗膜厚変動等の影響を受ける可能性があることから、塗膜を乾燥した後に放射線を照射することが好ましい。 Since the coating film formed in the coating process does not form crystals due to the effect of the residual solvent amount, the coating film before drying may be irradiated with radiation and then dried. After that, radiation may be applied. However, if radiation is applied to a wet coating before drying, it may be affected by environmental changes, foreign matter, coating thickness fluctuations, etc., so radiation should be applied after the coating is dried. Is preferred.
乾燥方法としては特に限定されないが、典型的にはドライエア乾燥法、熱風（温風）乾燥法、（遠）赤外乾燥法などが挙げられる。 Although it does not specifically limit as a drying method, Typically, a dry air drying method, a hot air (warm air) drying method, a (far) infrared drying method etc. are mentioned.
（洗浄工程）
上述の各工程を経て形成された細胞培養支持体のポリマー層には、基材表面上に共有結合により固定化されたポリマー分子だけでなく、固定化されていない遊離のポリマー分子や、未反応のモノマー又はオリゴマー分子等が存在している。そこでこれらの遊離ポリマー或いは未反応物を除去するために洗浄を行う洗浄工程を更に含むことが好ましい。
(Washing process)
The polymer layer of the cell culture support formed through the above-mentioned steps includes not only polymer molecules immobilized by covalent bonds on the substrate surface, but also free polymer molecules that are not immobilized and unreacted. The monomer or oligomer molecule is present. Therefore, it is preferable to further include a washing step for washing in order to remove these free polymers or unreacted substances.
洗浄方法としては特に限定されないが、典型的には浸漬洗浄、遥動洗浄、シャワー洗浄、スプレー洗浄、超音波洗浄等が挙げられる。また洗浄液としては典型的には各種水系、アルコール系、炭化水素系、塩素系、酸・アルカリ洗浄液が挙げられる。洗浄方法と洗浄液の組み合わせは洗浄される細胞培養支持体に応じて適宜選択すればよい。 Although it does not specifically limit as a washing | cleaning method, Typically, immersion washing | cleaning, swing washing | cleaning, shower washing | cleaning, spray washing | cleaning, ultrasonic washing | cleaning, etc. are mentioned. The cleaning liquid typically includes various water-based, alcohol-based, hydrocarbon-based, chlorine-based, acid / alkali cleaning liquids. The combination of the washing method and the washing solution may be appropriately selected according to the cell culture support to be washed.
（本発明の方法で製造された細胞培養支持体）
本発明はまた、本発明の方法により製造された細胞培養支持体に関する。本発明の細胞培養支持体は、原料モノマー／溶媒混合物を塗布用組成物として用いる従来法により製造された細胞培養支持体と比較してより均一なグラフトポリマー層を有することを特徴とする。
(Cell culture support produced by the method of the present invention)
The present invention also relates to a cell culture support produced by the method of the present invention. The cell culture support of the present invention is characterized by having a more uniform graft polymer layer as compared with a cell culture support produced by a conventional method using a raw material monomer / solvent mixture as a coating composition.
本発明の細胞培養支持体は、その表面に固定化されたグラフトポリマー層の乾燥時の厚さが０．００１〜１０μｍであることが好ましい。 The cell culture support of the present invention preferably has a dry thickness of the graft polymer layer immobilized on its surface of 0.001 to 10 μm.
また細胞培養支持体表面における各種応答性ポリマーの被覆量は、５〜８０μｇ／ｃｍ２であることが好ましく、６〜４０μｇ／ｃｍ２であることがより好ましい。ポリマー被覆量が８０μｇ／ｃｍ２を超過すると細胞は細胞培養支持体表面上に付着せず、逆に被覆量が５μｇ／ｃｍ２未満だと細胞は単層の状態で培養され組織状とならず、また培養細胞を支持体から剥離回収するのも困難となる。このようなポリマー被覆量は、例えばフーリエ変換赤外分光計全反射法（ＦＴ−ＩＲ−ＡＴＲ法）、被覆部若しくは非被覆部の染色や蛍光物質の染色による分析、更に接触角測定等による表面分析を単独或は併用して求めることが出来る。 The coating amount of the various responsive polymers in cell culture support surface is preferably 5~80μg / cm 2, more preferably 6~40μg / cm 2. When the polymer coating amount exceeds 80 μg / cm 2 , the cells do not adhere to the surface of the cell culture support, and conversely, when the coating amount is less than 5 μg / cm 2 , the cells are cultured in a monolayer and do not form a tissue. In addition, it becomes difficult to peel and collect the cultured cells from the support. Such a polymer coating amount is, for example, the surface by Fourier transform infrared spectrometer total reflection method (FT-IR-ATR method), analysis by coating or non-coating dyeing or fluorescent substance dyeing, and contact angle measurement. Analysis can be determined alone or in combination.
（細胞培養シートの作成方法）
本発明の細胞培養支持体を用いて、種々の細胞、例えば生体内の各組織、臓器を構成する上皮細胞や内皮細胞、収縮性を示す骨格筋細胞、平滑筋細胞、心筋細胞、神経系を構成するニューロン、グリア細胞、繊維芽細胞、生体の代謝に関係する肝実質細胞、非肝実質細胞や脂肪細胞、分化能を有する細胞として、種々組織に存在する幹細胞、さらには骨髄細胞、ＥＳ細胞等から細胞シートを作製することができる。こうして作製された細胞シートは表面の接着因子が損なわれていないことに加えて、細胞培養面に接した部分が均一な品質を有することから、再生医療などへの利用に適したものである。また、細胞シートを利用することでバイオセンサー等の検出デバイスへの応用へも展開できる。
(Method for creating cell culture sheet)
Using the cell culture support of the present invention, various cells, such as epithelial cells and endothelial cells constituting each tissue and organ in the living body, skeletal muscle cells exhibiting contractility, smooth muscle cells, cardiomyocytes, nervous system Constituent neurons, glial cells, fibroblasts, liver parenchymal cells related to metabolism in the living body, non-hepatic parenchymal cells and fat cells, stem cells existing in various tissues as cells having differentiation potential, bone marrow cells, ES cells A cell sheet can be produced from the above. The cell sheet thus prepared is suitable for use in regenerative medicine and the like because the adhesion factor on the surface is not impaired and the portion in contact with the cell culture surface has a uniform quality. In addition, the cell sheet can be applied to detection devices such as biosensors.
各種分子量のポリイソプロピルアクリルアミドの調整
以下の分子量のポリイソプロピルアクリルアミドは市販のものを購入した。
Preparation of polyisopropylacrylamide of various molecular weights Polyisopropylacrylamides having molecular weights below the same were purchased commercially.
レドックス合成ポリイソプロピルアクリルアミドの調整
500ｍＬセパラブルフラスコの中にＮ-イソプロピルアクリルアミド17.8ｇと純水150ｍＬを投入し、攪拌下、溶解・分散した。窒素ガス気流下、室温で過硫酸アンモニューム0.24ｇ、N,N,N',N',-テトラメチルエチレンジアミン0.30ｍＬを加えて重合を開始させた。重合終了後、加温してゲルを取り出し、100℃の電気乾燥器中で乾燥した。乾燥したゲルを粉砕して、ＮＭＰ溶媒でＧＰＣ分析したところ、市販の1、2および4比較で分子量約35〜40万であった。
Preparation of redox synthetic polyisopropylacrylamide
Into a 500 mL separable flask, 17.8 g of N-isopropylacrylamide and 150 mL of pure water were added, and dissolved and dispersed with stirring. Under a nitrogen gas stream, 0.24 g of ammonium persulfate and 0.30 mL of N, N, N ′, N ′,-tetramethylethylenediamine were added at room temperature to initiate polymerization. After completion of the polymerization, the gel was taken out by heating and dried in an electric dryer at 100 ° C. The dried gel was pulverized and subjected to GPC analysis with an NMP solvent. As a result, the molecular weight was about 350,000 to 400,000 in comparison with commercially available 1, 2 and 4.
連鎖移動剤使用のポリイソプロピルアクリルアミド合成
ｎ-ブチルメルカプタンを連鎖移動剤とした低分子量のポリイソプロピルアクリルアミドおよびイソプロピルアクリルアミドオリゴマーは非特許文献３のトレースによって合成した。ポリマー合成：１Ｌセパラブルフラスコの中にＮ-イソプロピルアクリルアミド11.3ｇとベンゼン300ｍＬを投入し、攪拌下、溶解・分散した。窒素ガス気流下、(a)4.31mLのｎ-ブチルメルカプタンおよび(b)40mLの0.1Mｎ-ブチルメルカプタンベンゼン溶液を加えたものを各々作製し、総量が400ｍＬになるように1.2gの過酸化ベンゾイルをベンゼンで溶かしたものを加え重合開始した。反応終了後、冷水抽出したものをファルマシア社のセファデックスＧ−２５ゲルでろ過し、ｎ-ブチルメルカプタンを除去、ポリマー分画のみを凍結乾燥して合成ポリマーを得た。脱硫は非特許文献４にも記載のあるレニーニッケル触媒を用いて行った。（ａ）条件から得たポリマーを５分子（分子量650）、（ｂ）条件から得たポリマーを28分子（分子量3300）として以下の実験を実施した。
Synthesis of Polyisopropylacrylamide Using Chain Transfer Agent Low molecular weight polyisopropylacrylamide and isopropylacrylamide oligomer using n-butyl mercaptan as a chain transfer agent were synthesized by the trace of Non-Patent Document 3. Polymer synthesis: Into a 1 L separable flask, 11.3 g of N-isopropylacrylamide and 300 mL of benzene were charged, and dissolved and dispersed under stirring. Under a nitrogen gas stream, (a) 4.31 mL of n-butyl mercaptan and (b) 40 mL of 0.1M n-butyl mercaptan benzene solution were prepared respectively, and 1.2 g of benzoyl peroxide was added so that the total amount was 400 mL. Polymerization was started by adding benzene dissolved in benzene. After completion of the reaction, the product extracted with cold water was filtered through Sephadex G-25 gel manufactured by Pharmacia, n-butyl mercaptan was removed, and only the polymer fraction was lyophilized to obtain a synthetic polymer. Desulfurization was performed using a Reny nickel catalyst described in Non-Patent Document 4. The following experiment was conducted with 5 molecules (molecular weight 650) obtained from the conditions (a) and 28 molecules (molecular weight 3300) obtained from the conditions (b).
細胞培養支持体の作製
（試験１）
実施例１〜８として、ＢＤ社１００８ペトリディッシュに表１の市販ポリイソプロピルアクリルアミド１〜５（実施例１〜５）、レドックス合成ポリイソプロピルアクリルアミド（実施例６）、連鎖移動剤使用の合成ポリイソプロピルアクリルアミド（ａ）（実施例７）、あるいは同（ｂ）（実施例８）がそれぞれ１ｗｔ％、イソプロピルアクリルアミドモノマーが40ｗｔ％になるようイソプロピルアルコールに溶解した液を室温２５℃、湿度60％で０．１ｍＬづつ添加した。比較例１として実施例のポリマーを含まない40ｗｔ％イソプロピルアクリルアミドモノマーのイソプロピルアルコール溶液を同様に添加したディッシュを作製した。この溶液を一時間放置したところ、表２に示すように比較例１では結晶が析出し、実施例１〜６および８では結晶は析出しなかった。実施例7では結晶が析出する場合としない場合があり、顕微鏡で観察すると微小な結晶が観察された。次にこの溶液の添加量を0.03ｍＬとし、デッシュを水平に静置して添加すると５秒以内にディッシュ底面は溶液で被覆されるが、１０分経過で比較例１では表２のように結晶が析出した。実施例１〜６および８では同様に結晶は析出しなかった。実施例7でも同様に結晶が析出する場合としない場合があり、顕微鏡で観察すると微小な結晶が観察された。
Preparation of cell culture support (Test 1)
As Examples 1-8, commercially available polyisopropylacrylamide 1-5 in Table 1 (Examples 1-5), redox synthetic polyisopropylacrylamide (Example 6), synthetic polyisopropyl using a chain transfer agent in 1008 Petri dish of BD. A solution prepared by dissolving acrylamide (a) (Example 7) or (b) (Example 8) in isopropyl alcohol so that the concentration of each component is 1 wt% and the isopropyl acrylamide monomer is 40 wt% is 0 at 25 ° C. and 60% humidity. Added in 1 mL increments. As Comparative Example 1, a dish was prepared in the same manner by adding an isopropyl alcohol solution of 40 wt% isopropylacrylamide monomer not containing the polymer of the example. When this solution was allowed to stand for 1 hour, as shown in Table 2, crystals were precipitated in Comparative Example 1, and no crystals were precipitated in Examples 1 to 6 and 8. In Example 7, crystals may or may not precipitate, and minute crystals were observed when observed with a microscope. Next, when the addition amount of this solution was 0.03 mL and the dish was left to stand horizontally and added, the bottom surface of the dish was covered with the solution within 5 seconds. Precipitated. In Examples 1 to 6 and 8, no crystals were similarly deposited. In Example 7 as well, crystals may or may not precipitate in the same manner, and minute crystals were observed when observed with a microscope.
（試験２）
次に実施例１〜６について同様の溶液を作製し、130μｍ厚の二軸延伸ポリスチレンシート（ＯＰＳ／防曇品として普及しているため、事前にエタノールで防曇剤は除去・乾燥した）にワイヤーバー４番手で約５ｇ／ｍ２の塗工厚で塗工し、ドライヤー乾燥および40℃、1×10２Ｐａ、１分の減圧乾燥で乾燥面質を確認した。ドライヤー乾燥においては表３のように実施例１〜６の全ての塗工膜の面質は透明平滑で微結晶も析出しなかった。比較例１は結晶白化して一部平滑性も壊れていた。40℃、1×10２Ｐａ、１分の減圧乾燥においては、実施例１〜６の全てで塗工膜の面質は透明平滑であったが、実施例１のみ微結晶を観察した。比較例１では結晶白化してシートからほとんど浮いた状態であった。 実施例１のポリマー量を0.5％に下げた場合および２％、５％に上げた場合、微結晶の析出頻度はポリマー添加量に反比例した。
(Test 2)
Next, a similar solution was prepared for Examples 1 to 6, and a 130 μm-thick biaxially stretched polystyrene sheet (because it was widely used as an OPS / antifogging product, the antifogging agent was removed and dried with ethanol in advance). Coating was performed at a coating thickness of about 5 g / m 2 with a 4th wire bar, and the dry surface quality was confirmed by drying with a dryer and drying under reduced pressure at 40 ° C., 1 × 10 2 Pa for 1 minute. In dryer drying, as shown in Table 3, the surface quality of all coating films of Examples 1 to 6 was transparent and smooth, and no fine crystals were deposited. Comparative Example 1 was crystallized and partly smooth. In 40 degreeC, 1 * 10 < 2 > Pa, and 1-minute reduced pressure drying, although the surface quality of the coating film was transparent and smooth in all of Examples 1-6, only Example 1 observed the microcrystal. In Comparative Example 1, the crystal was whitened and almost floated from the sheet. When the polymer amount of Example 1 was lowered to 0.5%, and when increased to 2% and 5%, the precipitation frequency of the microcrystals was inversely proportional to the amount of polymer added.
（試験３）
次に実施例１〜８および比較例１の溶液0.1ｍＬおよび0.03ｍＬをペトリディッシュに被覆したものに電子線を２回に分けて照射した。照射量を初回５Ｍｒａｄ、２回目２５Ｍｒａｄとした。電子線照射後、５℃のイオン交換水を用いて、ペトリディッシュを洗浄し、残留モノマーおよびペトリディッシュ表面に結合していないポリマーを取り除いた。クリーンベンチ内で乾燥させ、さらにエチレンオキサイド（ＥＯ）ガス滅菌を行い、さらに十分に脱気を行うことにより、最終的な製品である細胞培養支持体を得た。このものの被覆面の平滑性を光学顕微鏡下で表面に凹凸の有無を調べることにより検討した。結果を表４に示す。ウシ大動脈血管内皮細胞の培養は、得られた細胞培養支持体材料上にて、ウシ胎児血清(ＦＣＳ)を１０％含むダルベッコー改変イーグル培地(ＤＭＥＭ)を培養として、５％二酸化炭素中、３７℃で行なった。十分に細胞が増殖したのを確認した後、培養液を支持体ごと２０℃、５％ＣＯ２下のチャンバー内に移し、３０分間放置して、付着／増殖細胞を剥離させ、増殖細胞剥離回収率を下式に従って求めた。結果を表４に示す。
増殖細胞剥離回収率(％)＝１００×剥離回収した細胞総数／増殖させた細胞総数
その際、剥離回収した細胞総数および、増殖させた細胞総数を計測するためには、細胞を個々の状態にしなければならない。従って、剥離回収した細胞総数の測定は、２０℃に冷却、放置した後、回収した細胞塊に対し、トリプシン−ＥＤＴＡ処理を行ない細胞を個々の状態にして行なった。また増殖させた細胞総数は、上記方法で剥離回収した細胞総数に、２０℃に冷却、放置しても剥離しなかった細胞をトリプシン−ＥＤＴＡ処理にて、個々の状態に剥離させた細胞総数を加え合わせることにより求めた。また、３７℃のチャンバー内で５日間培養し、ディッシュ周囲以外全体がコンフレントになった細胞シート状塊についても、メスでディッシュ周囲のサブコンフレント状態の細胞層に切れ目を入れて、培養液を支持体ごと２０℃、５％ＣＯ２下のチャンバー内に移し、細胞シートの剥離を観察した。
(Test 3)
Next, an electron beam was irradiated in two portions on the samples 1 to 8 and Comparative Example 1 in which 0.1 mL and 0.03 mL were coated on a petri dish. The irradiation amount was 5 Mrad for the first time and 25 Mrad for the second time. After the electron beam irradiation, the Petri dish was washed with ion exchange water at 5 ° C. to remove residual monomers and polymers not bound to the Petri dish surface. It was dried in a clean bench, further sterilized with ethylene oxide (EO) gas, and further sufficiently degassed to obtain a cell culture support as a final product. The smoothness of the coated surface of this product was examined by examining the surface for unevenness under an optical microscope. The results are shown in Table 4. The bovine aortic vascular endothelial cells were cultured on the obtained cell culture support material using Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS) at 37 ° C. in 5% carbon dioxide. It was done in. After confirming that the cells have grown sufficiently, the culture solution is transferred together with the support into a chamber at 20 ° C. and 5% CO 2 and left for 30 minutes to detach the attached / proliferated cells and recover the detached cells. The rate was determined according to the following formula. The results are shown in Table 4.
Proliferated cell exfoliation recovery rate (%) = 100 × total number of exfoliated cells / total number of proliferated cells
At that time, in order to count the total number of cells collected and collected and the total number of proliferated cells, the cells must be in individual states. Therefore, the total number of detached and collected cells was measured by cooling the cells to 20 ° C. and allowing them to stand, and then treating the collected cell mass with trypsin-EDTA so that the cells were in individual states. The total number of cells grown is the total number of cells peeled and collected by the above method, and the total number of cells that have been peeled into individual states by trypsin-EDTA treatment after cooling to 20 ° C. Obtained by adding together. In addition, a cell sheet-like lump that has been cultured in a chamber at 37 ° C. for 5 days and is entirely confined except for the surroundings of the dish is cut with a scalpel into the sub-confluent cell layer surrounding the dish, The whole support was transferred into a chamber at 20 ° C. and 5% CO 2 , and detachment of the cell sheet was observed.
（試験４）
延伸ポリエチレンＯＰＳ上に実施例１〜６および比較例１と同様に作製した溶液をワイヤーバー塗工し、ドライヤー乾燥した塗工面に電子線を照射したものをペトリディッシュに被覆したもの同様に洗浄・乾燥し、ペトリディッシュ底面に両面テープで貼付した後、引き続き同様にＥＯＧ滅菌をして細胞培養支持体を得た。
(Test 4)
A solution prepared in the same manner as in Examples 1 to 6 and Comparative Example 1 on the stretched polyethylene OPS was coated with a wire bar, and the coated surface dried with a dryer was irradiated with an electron beam. After drying and affixing to the bottom surface of the Petri dish with a double-sided tape, EOG sterilization was performed in the same manner to obtain a cell culture support.
電子線照射量は１５Ｍｒａｄおよび２５Ｍｒａｄの２通りで１回照射のみで行った。比較例１のペトリディシュに０．１ｍｌおよび０．０３ｍＬ被覆したものでも上記の照射量の１回照射を行い比較した。ＯＰＳに塗工した実施例１〜６は電子線照射量は１５Ｍｒａｄ、２５Ｍｒａｄともに塗工面は透明なままであったが、２５Ｍｒａｄの照射では熱によるシートの歪み・収縮が観察された。３０Ｍｒａｄでは両面テープの貼付に支障がある歪が観察された。細胞培養した細胞の回収率と細胞シート剥離は表５のようになった。結晶白化した比較例１からはどちらの照射量でも細胞も細胞シートも全く剥離しなかった。ディッシュに被覆してウェットな状態で照射したものでは、ディッシュの肉眼レベルの変形はどちらの照射量でも観察されなかったが、０．１ｍＬの被覆量ではどちらの照射量でも細胞が回収されたが、回収率が低く、細胞シートとしては剥離されなかった。０．０３ｍＬの被覆量ではどちらの照射量でも微結晶が析出し、細胞は回収されず、細胞シートも剥離しなかった。実施例１〜６については実施例１が細胞シート剥離に関して時間がかかったが、他は全て良好であった。 The amount of electron beam irradiation was 15 Mrad and 25 Mrad. Even when the Petri dishes of Comparative Example 1 were coated with 0.1 ml and 0.03 ml, the above irradiation dose was irradiated once and compared. In Examples 1 to 6 coated on the OPS, the electron beam irradiation amount was 15 Mrad and 25 Mrad, and the coated surface remained transparent. However, the sheet distortion and shrinkage due to heat were observed at 25 Mrad irradiation. At 30 Mrad, distortion that hinders the application of double-sided tape was observed. Table 5 shows the cell culture cell recovery rate and cell sheet detachment. From Comparative Example 1, which was crystallized, neither cells nor cell sheets were peeled at any dose. When the dish was coated and irradiated in a wet state, no deformation of the naked eye level of the dish was observed at either dose, but at the dose of 0.1 mL, cells were collected at either dose. The recovery rate was low and the cell sheet was not peeled off. At a coating amount of 0.03 mL, microcrystals were precipitated at both doses, the cells were not collected, and the cell sheet was not detached. As for Examples 1 to 6, Example 1 took time for cell sheet peeling, but everything else was good.
（試験５）
実施例２および６について、ポリマー含有量を０．５ｗｔ％、２ｗｔ％、５ｗｔ％にして４０ｗｔ％のモノマーを含むイソプロピルアルコール組成物を各１Ｋｇ調整し、粘着剤が塗工されて剥離フィルムで保護された易接着ＰＥＴのロール形状の連続ベースフィルムにグラビアダイレクトロールのベタ48線で９ｍｇ／ｍ２の塗工量を毎分５ｍ塗工し、連続の１ｍ、４５℃の熱風乾燥機で乾燥し、連続の電子線照射ユニットで酸素濃度50ppm以下の環境で電子線照射した。電子線の電子流量を変化させて照射量を１０、１２、１６、２０、２４Ｍｒａｄにした。各条件で作製した塗工フィルムを洗浄・乾燥し、ディッシュサイズに切り抜いたものを粘着剤でディッシュに固定してＥＯＧ滅菌して細胞培養支持体を得た。
(Test 5)
For Examples 2 and 6, the polymer content was adjusted to 0.5 wt%, 2 wt%, 5 wt%, and 1 kg each of an isopropyl alcohol composition containing 40 wt% monomer was prepared, and an adhesive was applied to protect it with a release film. A continuous base film in the form of easy-adhesive PET rolls is coated with a gravure direct roll with a solid 48 line at a coating amount of 9 mg / m 2 at a rate of 5 m / min and dried in a continuous 1 m, 45 ° C hot air dryer. The electron beam was irradiated in an environment having an oxygen concentration of 50 ppm or less with a continuous electron beam irradiation unit. The irradiation amount was adjusted to 10, 12, 16, 20, 24 Mrad by changing the electron flow rate of the electron beam. The coated film produced under each condition was washed and dried, and the one cut into a dish size was fixed to the dish with an adhesive and sterilized with EOG to obtain a cell culture support.
各組成物で作製した細胞培養支持体は全て平滑透明な表面で細胞回収、細胞シート剥離は表５に示すように全てで良好であった。ポリマー含有量２ｗｔ％の組成物に関しては２００ｍの塗工を行い、４０分の塗工の最後で出来た塗工フィルムからも細胞培養支持体を作製したが、塗工初期のフィルムからの細胞培養支持体との細胞回収、細胞シート剥離性能に差異はなく、良好であった。 As shown in Table 5, all cell culture supports prepared with the respective compositions had a smooth and transparent surface and cell recovery and cell sheet peeling were all good. For the composition having a polymer content of 2 wt%, 200 m of coating was performed, and a cell culture support was produced from the coated film formed at the end of the 40 minute coating. There was no difference in cell recovery and cell sheet peeling performance with the support, which was good.
（試験６）
実施例９として上記実施例２の連続塗工・乾燥・電子線照射工程の連続塗工フィルム作製時に電子線を照射しない状態のサンプルを作製した。このフィルムにコバルト60からのγ線を室温、１．５Ｍｒａｄを２時間で照射した。これを上記同様に洗浄・乾燥して、切り抜き・貼付したディッシュをＥＯＧ滅菌して細胞培養支持体を得た。作製した細胞培養支持体は全て平滑透明な表面で細胞回収、細胞シート剥離は良好であった。
(Test 6)
As Example 9, a sample was prepared in a state in which no electron beam was irradiated when the continuous coating film was prepared in the continuous coating / drying / electron beam irradiation process of Example 2 above. This film was irradiated with gamma rays from cobalt 60 at room temperature and 1.5 Mrad for 2 hours. This was washed and dried as described above, and the cut and pasted dish was EOG sterilized to obtain a cell culture support. The prepared cell culture supports were all smooth and transparent on the surface, and the cell recovery and cell sheet peeling were good.
（試験７）
実施例１０〜１６として表６のベースフィルム上に実施例２の組成物をワイヤーバー（番手４）コートし、ドライヤー乾燥後、塗工面に電子線を照射したものをペトリディッシュに被覆したもの同様に洗浄・乾燥した。電子線照射量は１０Ｍｒａｄ、１５Ｍｒａｄおよび２５Ｍｒａｄの３通りで１回照射のみで行った。ポリスチレンフィルムは２５Ｍｒａｄの照射で基材が70℃になり、実施例１〜６の時同様、多少の歪を生じた。ＰＥＴとポリイミドも基材が70℃になったが、基材の耐熱性があり、歪は見られなかった。ナイロン、ポリエチレン、ポリプロピレンは基材温度が上がらず、歪は見られなかった。洗浄・乾燥したフィルムを円形に切り、塗工裏面とペトリディッシュの底を両面テープで貼り合せた。これをＥＯＧ滅菌をして細胞培養支持体を得た。実施例１２の自調製フィルムは、実施例１１のフィルムに表７の組成物をワイヤーバー（番手４）コートし、高圧水銀灯を用いて、１７０ｍＪ／ｃｍ2（３６５ｎｍ）の条件で紫外線照射してコート面を硬化させた。この硬化コート面を他フィルムと同様に用いた。比較例２のポリスチレンフィルムは、事前にエタノールで防曇剤を除去・乾燥して用いた。
(Test 7)
Examples 10 to 16 are the same as those obtained by coating the base film of Table 6 with the composition of Example 2 with a wire bar (counter 4), drying the dryer, and then irradiating the coated surface with an electron beam on a Petri dish. Washed and dried. The amount of electron beam irradiation was 10 Mrad, 15 Mrad, and 25 Mrad. As for the polystyrene film, the substrate became 70 ° C. by irradiation with 25 Mrad, and some distortion occurred as in Examples 1-6. PET and polyimide also had a substrate temperature of 70 ° C., but the substrate had heat resistance and no distortion was observed. Nylon, polyethylene, and polypropylene did not raise the substrate temperature, and no distortion was observed. The washed and dried film was cut into a circle, and the back side of the coating and the bottom of the Petri dish were bonded together with a double-sided tape. This was EOG sterilized to obtain a cell culture support. The self-prepared film of Example 12 was obtained by coating the film of Example 11 with the composition shown in Table 7 on a wire bar (number 4) and irradiating it with ultraviolet light using a high-pressure mercury lamp at 170 mJ / cm 2 (365 nm). The coated surface was cured. This cured coated surface was used in the same manner as other films. The polystyrene film of Comparative Example 2 was used after removing and drying the antifogging agent with ethanol in advance.
実施例１〜８と同様の方法で細胞の回収率の測定と細胞シート剥離の観察を行った。結果は、以下の表８の通りでポリイミドでは細胞の回収も細胞シート剥離も見られず、ポリエステルではポリスチレンのそれより回収率、細胞シート剥離ともに機能性支持体として弱いものであった。易接着ＰＥＴ、ウレタンアクリレート、ナイロン、低密度ポリエチレン（ＬＤＰＥ）に関してはポリスチレンより低放射線量で作製した支持体から機能を示した。 Measurement of the cell recovery rate and observation of cell sheet peeling were performed in the same manner as in Examples 1-8. The results are as shown in Table 8 below. In polyimide, neither cell recovery nor cell sheet peeling was observed, and in polyester, both the recovery rate and cell sheet peeling were weaker as functional supports than polystyrene. For easy-adhesion PET, urethane acrylate, nylon, and low density polyethylene (LDPE), the function was demonstrated from a support produced with a lower radiation dose than polystyrene.
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| US10174172B2 (en) | 2013-08-27 | 2019-01-08 | Osaka University | Joined body joined by chemical bonding at material interface, and joining method for joined body |
| US11149249B2 (en) | 2015-09-25 | 2021-10-19 | Mitsubishi Gas Chemical Company, Inc. | Base material for cell culture and cell culture method using same, cell culture container, and use as base material |
| CN115466708A (en) * | 2022-09-16 | 2022-12-13 | 四川大学 | Method for controllably adhering and releasing cells |
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| US10174172B2 (en) | 2013-08-27 | 2019-01-08 | Osaka University | Joined body joined by chemical bonding at material interface, and joining method for joined body |
| WO2015115568A1 (en) | 2014-01-29 | 2015-08-06 | ダイキン工業株式会社 | Temperature-responsive base material, method for producing same, and method for evaluating same |
| JP2017025335A (en) * | 2014-01-29 | 2017-02-02 | ダイキン工業株式会社 | Temperature responsive substrate, method of producing the same, and evaluation method therefor |
| US10689615B2 (en) | 2014-01-29 | 2020-06-23 | Daikin Industries, Ltd. | Temperature-responsive base material, method for producing same, and method for evaluating same |
| US11149249B2 (en) | 2015-09-25 | 2021-10-19 | Mitsubishi Gas Chemical Company, Inc. | Base material for cell culture and cell culture method using same, cell culture container, and use as base material |
| CN115466708A (en) * | 2022-09-16 | 2022-12-13 | 四川大学 | Method for controllably adhering and releasing cells |
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