JP2017042094A - Dish-type cell culture vessel - Google Patents
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Abstract
Description
本発明は、シャーレ型細胞培養容器に関する。 The present invention relates to a petri dish type cell culture container.
培養基板上の指定した位置に細胞を付着させて培養する細胞パターニングを行う技術は、生体組織を模倣した組織化細胞培養が可能である。そのため、細胞パターニングは、基礎研究のみならず、実験動物に代わる薬剤スクリーニング系や細胞シート等の移植用組織・臓器の開発等、様々な研究・開発に有用である。 A technique for performing cell patterning in which cells are attached and cultured at a specified position on a culture substrate enables organized cell culture that mimics a living tissue. Therefore, cell patterning is useful not only for basic research but also for various research and development such as drug screening systems that replace laboratory animals and the development of transplanted tissues and organs such as cell sheets.
この細胞パターニングに利用可能な細胞容器が近年開発されており、例えば、非特許文献１に記載された、フィルム製のシャーレ型細胞培養容器も、細胞パターニングに利用可能である。 Cell containers that can be used for this cell patterning have been developed in recent years. For example, a petri dish-type cell culture container made of film described in Non-Patent Document 1 can also be used for cell patterning.
しかしながら、非特許文献１に記載されたシャーレ型細胞培養容器は、エタノール等の有機溶媒により殺菌することにより、接着剤の部分が溶出して接着力が弱くなるばかりでなく、その接着剤が細胞培養に悪影響を及ぼし、細胞の接着性を制御できずに所望の細胞パターニングを得られない場合がある。 However, the petri dish-type cell culture container described in Non-Patent Document 1 is not only sterilized with an organic solvent such as ethanol, but the adhesive part is eluted and the adhesive force is weakened. The culture may be adversely affected, and cell adhesion may not be controlled, resulting in failure to obtain desired cell patterning.
他方、細胞パターニングの中には、光等の外部刺激に応じて細胞付着性が変化する、いわゆる「動的パターニング」というものが知られている。この動的パターニングは、刺激位置を変えながら随時細胞を添加することで、複数種の細胞を位置選択的に配置できるものであり、つまり細胞付着を任意のタイミングで操作可能であるため、例えば、細胞の運動性の評価等、通常の細胞パターニングとは異なる研究・開発に利用可能である。動的パターニングのうち、光により培養基板の細胞付着性を変化させるためには、光分解性基を細胞基板の表面に結合させることが必要となる。 On the other hand, as cell patterning, so-called “dynamic patterning” is known in which cell adhesion changes in response to an external stimulus such as light. In this dynamic patterning, by adding cells at any time while changing the stimulation position, a plurality of types of cells can be placed selectively, that is, cell attachment can be manipulated at an arbitrary timing. It can be used for research and development different from normal cell patterning, such as evaluation of cell motility. In dynamic patterning, in order to change the cell adhesion of the culture substrate by light, it is necessary to bond a photodegradable group to the surface of the cell substrate.
しかしながら、非特許文献１に記載されたシャーレ型細胞培養容器は、培養基板の外周が容器の底面に光硬化性接着剤により直接接着されている。このように、光硬化性接着剤を用いて培養基板を接着した場合、光硬化のための光照射によって上述の光分解性基が分解してしまうことから、非特許文献１に記載されたような光硬化性接着剤を利用したシャーレ型細胞培養容器は、光応答性の動的パターニングには利用できない。 However, in the petri dish type cell culture container described in Non-Patent Document 1, the outer periphery of the culture substrate is directly bonded to the bottom surface of the container with a photocurable adhesive. Thus, when the culture substrate is adhered using a photocurable adhesive, the above-described photodegradable group is decomposed by light irradiation for photocuring, so that it is described in Non-Patent Document 1. A petri dish type cell culture container using a photocurable adhesive cannot be used for photoresponsive dynamic patterning.
本発明は以上の実情に鑑みてなされたものであり、培養基板の底面への接着に接着剤を利用する必要がなく、有機溶媒により殺菌を行っても培養基板の培養容器への接着力が損なわれにくく、更に、有機溶媒による殺菌による細胞培養への影響が低減された、光応答性の動的パターニングに適したシャーレ型細胞培養容器を提供することを目的とする。 The present invention has been made in view of the above circumstances, and it is not necessary to use an adhesive for adhesion to the bottom surface of the culture substrate. Even if sterilization is performed with an organic solvent, the adhesion force of the culture substrate to the culture vessel is improved. It is an object of the present invention to provide a petri dish-type cell culture vessel suitable for photoresponsive dynamic patterning, which is less likely to be damaged and further has a reduced influence on cell culture by sterilization with an organic solvent.
本発明者らは、シャーレ型細胞培養容器の底面部に形成された穴部に細胞基板を嵌合し、底面部の裏側から固定することで、有機溶媒により殺菌を行っても培養基板の培養容器への接着力が損なわれにくく、有機溶媒での殺菌による細胞培養への影響が低減されることを見出し、本発明を完成するに至った。より具体的には、本発明は以下のようなものを提供する。 The present inventors can cultivate a culture substrate even when sterilized with an organic solvent by fitting the cell substrate into a hole formed in the bottom portion of the petri dish type cell culture container and fixing it from the back side of the bottom portion. It has been found that the adhesive force to the container is not easily impaired, and the influence on cell culture by sterilization with an organic solvent is reduced, and the present invention has been completed. More specifically, the present invention provides the following.
（１） 底面部と側壁部とを備えるシャーレ型細胞培養容器であって、
前記底面部に、該底面部の厚さ方向に貫通した穴部が形成され、
光分解性基が表面に結合された培養基板が、前記穴部に前記表面が容器の内側方向を向くように配置された状態で嵌合された、シャーレ型細胞培養容器。
(1) A petri dish type cell culture container comprising a bottom surface portion and a side wall portion,
In the bottom surface portion, a hole portion penetrating in the thickness direction of the bottom surface portion is formed,
A petri dish-type cell culture container in which a culture substrate having a photodegradable group bonded to a surface is fitted in the hole portion so that the surface faces the inner side of the container.
（２） 前記培養基板が容器の外側方向からフィルムにより前記底面部に固定された、（１）に記載の細胞培養容器。 (2) The cell culture container according to (1), wherein the culture substrate is fixed to the bottom surface portion with a film from the outside of the container.
（３） 前記光分解性基が、シランカップリング剤である、（１）又は（２）に記載の細胞培養容器。 (3) The cell culture container according to (1) or (2), wherein the photodegradable group is a silane coupling agent.
本発明によれば、培養基板の底面への接着に接着剤を利用する必要がなく、有機溶媒により殺菌を行っても培養基板の培養容器への接着力が損なわれにくく、更に、有機溶媒での殺菌による細胞培養への影響が低減された、光応答性の動的パターニングに適したシャーレ型細胞培養容器を提供することができる。 According to the present invention, it is not necessary to use an adhesive for adhesion to the bottom surface of the culture substrate, and even when sterilized with an organic solvent, the adhesive force of the culture substrate to the culture vessel is not easily impaired. It is possible to provide a petri dish-type cell culture container suitable for photoresponsive dynamic patterning, in which the influence on cell culture due to the sterilization is reduced.
以下、本発明の実施形態について説明するが、本発明はこれに限定されない。 Hereinafter, although embodiment of this invention is described, this invention is not limited to this.
＜シャーレ型細胞培養容器＞
本発明のシャーレ型細胞培養容器は、底面部と側壁部とを備えるシャーレ型細胞培養容器であって、底面部に、該底面部の厚さ方向に貫通した穴部が形成され、光分解性基が表面に結合された培養基板が、穴部に表面が容器の内側方向を向くように配置された状態で嵌合されたものである。以下、図１を参照しながら、本発明の好ましい一実施形態であるシャーレ型細胞培養容器１について説明する。
<Petri dish type cell culture vessel>
The petri dish-type cell culture container of the present invention is a petri dish-type cell culture container having a bottom surface portion and a side wall portion, and a hole portion penetrating in the thickness direction of the bottom surface portion is formed in the bottom surface portion. The culture substrate having the base bonded to the surface is fitted into the hole in a state where the surface is arranged so as to face the inner side of the container. Hereinafter, with reference to FIG. 1, a petri dish type cell culture vessel 1 which is a preferred embodiment of the present invention will be described.
本発明のシャーレ型細胞培養容器１は、図１に示すように、底面部１１と側壁部１２とを備え、底面部１１に形成された穴部１１１に、培養基板１３が嵌合される。該培養基板１３は、フィルム１４により底面部１１に固定される。また、図１には示さないが、蓋部を更に備える。 As shown in FIG. 1, the petri dish-type cell culture container 1 of the present invention includes a bottom surface portion 11 and a side wall portion 12, and a culture substrate 13 is fitted into a hole portion 111 formed in the bottom surface portion 11. The culture substrate 13 is fixed to the bottom surface portion 11 by a film 14. Moreover, although not shown in FIG. 1, a cover part is further provided.
（底面部１１）
底面部１１は、図１に示すように、該底面部１１の厚さ方向に貫通した穴部１１１が形成されている。該穴部１１１は、後述する培養基板１３が嵌合可能に構成され、円形状に形成される。また、底面部１１は、円形平面状に構成される。
(Bottom part 11)
As shown in FIG. 1, the bottom surface portion 11 is formed with a hole portion 111 penetrating in the thickness direction of the bottom surface portion 11. The hole 111 is configured to be able to fit a culture substrate 13 described later, and is formed in a circular shape. Moreover, the bottom face part 11 is comprised by circular planar shape.
底面部１１の直径は、特に限定されず、例えば、φ１０〜１５０ｍｍのものを用いることができる。穴部１１１の直径は、培養基板１３が嵌合可能に構成されれば、特に限定されないが、例えば、φ５〜５０ｍｍのものを用いることができる。また、底面部１１の厚さは特に限定されず、例えば、０．１〜２．０ｍｍであってもよい。 The diameter of the bottom face part 11 is not specifically limited, For example, the thing of (phi) 10-150mm can be used. Although the diameter of the hole 111 will not be specifically limited if the culture substrate 13 is comprised so that fitting is possible, For example, a (phi) 5-50 mm thing can be used. Moreover, the thickness of the bottom face part 11 is not specifically limited, For example, 0.1-2.0 mm may be sufficient.
底面部１１の材質は、特に限定されず、ガラスにより構成してもよく、樹脂（例えば、ポリスチレン、ポリプロピレン、ポリエステル、ポリカーボネート）により構成してもよい。 The material of the bottom face part 11 is not particularly limited, and may be made of glass, or may be made of resin (for example, polystyrene, polypropylene, polyester, polycarbonate).
（側壁部１２）
側壁部１２は、底面部１１の外周に設けられる。側壁部１２の材質は、特に限定されず、底面部１１と同様の材質を用いることができる。側壁部１２は、蓋部により覆うことが可能となるように構成される。また、側壁部１２の厚さは、特に限定されず、例えば、０．１〜２．０ｍｍであってもよい。また、側壁部１２の高さは、例えば、５．０〜３０ｍｍであってもよい。
(Sidewall 12)
The side wall portion 12 is provided on the outer periphery of the bottom surface portion 11. The material of the side wall portion 12 is not particularly limited, and the same material as that of the bottom surface portion 11 can be used. The side wall part 12 is comprised so that it can cover with a cover part. Moreover, the thickness of the side wall part 12 is not specifically limited, For example, 0.1-2.0 mm may be sufficient. Moreover, 5.0-30 mm may be sufficient as the height of the side wall part 12, for example.
（培養基板１３）
培養基板１３は、表面に光分解性基が結合される。また、培養基板１３は、穴部１１１に培養基板１３の表面が容器の内側方向を向くように配置された状態で嵌合される。培養基板１３は、細胞を培養する場となる部分である。
(Culture substrate 13)
The culture substrate 13 has a photodegradable group bonded to its surface. In addition, the culture substrate 13 is fitted in the hole 111 in a state where the surface of the culture substrate 13 faces the inner side of the container. The culture substrate 13 is a part that serves as a place for culturing cells.
培養基板１３は穴部１１１に嵌合可能に構成されるものであり、本実施形態においては、穴部１１１の円形形状に合わせて円形平面状に構成される。 The culture substrate 13 is configured to be able to fit into the hole 111, and in the present embodiment, is configured in a circular flat shape in accordance with the circular shape of the hole 111.
培養基板１３は、表面に光分解性基が結合可能な基を有する素材であり、通常、光透過性の素材が用いられるが、例えば、ガラス、石英ガラス、シリコン、ダイアモンド、金等が挙げられる。 The culture substrate 13 is a material having a group to which a photodegradable group can be bonded on the surface. Usually, a light transmissive material is used, and examples thereof include glass, quartz glass, silicon, diamond, and gold. .
「光分解性基」とは、紫外線等の高エネルギーの光の照射により、分解する基を意味し、従来の公知の光分解性基を使用可能であるが、光分解性のシランカップリング剤であることが好ましい。 “Photodegradable group” means a group that decomposes when irradiated with high-energy light such as ultraviolet rays. Conventionally known photodegradable groups can be used, but photodegradable silane coupling agents It is preferable that
光分解性基は、特に限定されず、例えば、以下の一般式（Ｉ）〜（ＩＶ）で表される基が挙げられる。 The photodegradable group is not particularly limited, and examples thereof include groups represented by the following general formulas (I) to (IV).
Ｒ１は、単結合又は炭素数１〜２０の直鎖又は分鎖のアルキル基もしくはアルコキシ基（酸素原子は、培養基板側又はその逆側のいずれに位置してもよい。）であり、Ｒ２は、細胞付着抑制基である。 R 1 is a single bond or a linear or branched alkyl group or alkoxy group having 1 to 20 carbon atoms (the oxygen atom may be located on the culture substrate side or the opposite side thereof). 2 is a cell adhesion inhibitory group.
光分解性基は、上記一般式（Ｉ）〜（ＩＶ）のように、細胞培養の動的パターニングを行うために、細胞付着抑制基を置換基として有するものであることが好ましい。細胞付着抑制基とは、細胞の付着（又は接着）を抑制（又は阻害）する官能基を意味し、代表的には、ポリエチレングリコール（ＰＥＧ）、ポリ（２−メタクリロイルオキシエチルホスホリルコリン）（ＰＭＰＣ）、ポリ（２−ヒドロキシエチルメタクリレート）（ＰＨＥＭＡ）を置換基として有する炭化水素基又はアミノ基が挙げられる。細胞付着抑制基は、好ましくは、以下の一般式（Ｖ）で表されるポリエチレングリコール（ＰＥＧ）を置換基として有する炭化水素基又はアミノ基である。炭化水素基としては、炭素数１〜２０の直鎖又は分鎖のアルキル基が挙げられる。アミノ基は、第１級アミノ基、第２級アミノ基が挙げられる。 The photodegradable group preferably has a cell adhesion inhibitory group as a substituent in order to perform dynamic patterning of cell culture as in the general formulas (I) to (IV). The cell adhesion inhibitory group means a functional group that inhibits (or inhibits) cell adhesion (or adhesion), and is typically polyethylene glycol (PEG), poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC). And a hydrocarbon group or amino group having poly (2-hydroxyethyl methacrylate) (PHEMA) as a substituent. The cell adhesion-inhibiting group is preferably a hydrocarbon group or an amino group having a polyethylene glycol (PEG) represented by the following general formula (V) as a substituent. Examples of the hydrocarbon group include linear or branched alkyl groups having 1 to 20 carbon atoms. Examples of the amino group include a primary amino group and a secondary amino group.
（式中、ｍは３〜１２００の整数であり、Ｒ３は、水素原子又はメチル基である。）
(In the formula, m is an integer of 3 to 1200, and R 3 is a hydrogen atom or a methyl group.)
培養基板１３の表面を構成する基としては、培養基板１３の素材がガラスの場合は、以下の一般式（ＶＩ）で表される結合ができるシランカップリング剤が好ましく用いられる。また、培養基板１３の素材が金の場合は、以下の一般式（ＶＩＩ）で表される結合ができるチオールまたはジスルフィドが好ましく用いられる。 As the group constituting the surface of the culture substrate 13, when the material of the culture substrate 13 is glass, a silane coupling agent capable of bonding represented by the following general formula (VI) is preferably used. Moreover, when the raw material of the culture substrate 13 is gold, a thiol or disulfide capable of binding represented by the following general formula (VII) is preferably used.
培養基板１３の表面への光分解性基の結合は、従来の公知の方法に行うことができ、例えば、特許５１６７７３８号に記載された方法を用いることができる。 Bonding of the photodegradable group to the surface of the culture substrate 13 can be performed by a conventionally known method, for example, the method described in Japanese Patent No. 5166738 can be used.
（フィルム１４）
フィルム１４は、培養基板１３を容器の外側方向から底面部１１に固定させる。フィルム１４の形状は、上記固定を可能に構成されるよう、従来の公知のフィルムを用いることができる。本実施形態においては、図１に示すように、フィルム１４は、底面部１１における穴部１１１と培養基板１３との両方に接することで貼着され、培養基板１３の中央部と重ならないように貫通した環状（リング状）の形状で構成されている。このようにフィルム１４を環状の形状とすることで、細胞培養時に、培養基板１３に付着した細胞を観察しやすくなる。フィルム１４の大きさは、穴部１１１の大きさに合わせて適宜選択されるが、例えば、外径φ１０〜６０ｍｍ、内径φ１〜４５ｍｍであってもよい。
(Film 14)
The film 14 fixes the culture substrate 13 to the bottom surface part 11 from the outside direction of the container. As the shape of the film 14, a conventionally known film can be used so as to enable the fixing. In the present embodiment, as shown in FIG. 1, the film 14 is attached by being in contact with both the hole portion 111 and the culture substrate 13 in the bottom surface portion 11 so as not to overlap the central portion of the culture substrate 13. It has a circular (ring-shaped) shape that penetrates. Thus, by making the film 14 into an annular shape, it becomes easy to observe cells attached to the culture substrate 13 during cell culture. Although the magnitude | size of the film 14 is suitably selected according to the magnitude | size of the hole part 111, outer diameter (phi) 10-60mm and inner diameter (phi) 1-45mm may be sufficient, for example.
フィルム１４は、従来の公知の貼着可能なものであれば、特に限定されず、例えば、理化学実験用のマイクロプレート用シール等を用いることができる。また、フィルム１４の貼着に用いられる粘着剤層を構成する粘着剤は、従来の公知の粘着剤を用いることができ、例えば、ゴム系粘着剤、アクリル系粘着剤、シリコーン系粘着剤、エチレン−酢酸ビニル共重合体系粘着剤、ポリビニルエーテル系粘着剤、ポリウレタン系粘着剤等を１種又は２種以上用いることができる。また、細胞培養時に、培養基板１３に付着した細胞を観察しやすくなることから、透明な素材を用いることが好ましい。 The film 14 is not particularly limited as long as it can be pasted and can be used, for example, a microplate seal for physics and chemistry experiments. Moreover, the adhesive which comprises the adhesive layer used for sticking of the film 14 can use the conventionally well-known adhesive, for example, a rubber adhesive, an acrylic adhesive, a silicone adhesive, ethylene -1 type (s) or 2 or more types can be used for a vinyl acetate copolymer adhesive, a polyvinyl ether adhesive, a polyurethane adhesive, etc. FIG. In addition, it is preferable to use a transparent material because cells attached to the culture substrate 13 can be easily observed during cell culture.
（蓋部）
蓋部は、図１には示さないが、側壁部１２の外側を覆うように構成され、従来の公知のシャーレに用いられる蓋を、底面部１１の直径や側壁部１２の高さ等に合わせて適宜選択できる。また、蓋部の素材は、底面部１１や側壁部１２と同様の素材を用いることができる。
(Cover)
Although the lid portion is not shown in FIG. 1, the lid portion is configured to cover the outside of the side wall portion 12, and the lid used for a conventional well-known petri dish is matched with the diameter of the bottom surface portion 11, the height of the side wall portion 12, and the like. Can be selected as appropriate. Moreover, the material similar to the bottom face part 11 and the side wall part 12 can be used for the raw material of a cover part.
（シャーレ型細胞培養容器１の作製方法）
本発明のシャーレ型細胞培養容器１は、例えば、底面部１１に、切削にて穴部１１１を設け、環状にくりぬいたフィルム１４に培養基板１３を接着し、次いで、穴部１１１に培養基板１３が嵌合するように底面部１１を被せ、フィルム１４を抑えて固定することで、作製することができる。
(Production method of petri dish type cell culture container 1)
In the petri dish type cell culture container 1 of the present invention, for example, a hole portion 111 is provided in the bottom surface portion 11 by cutting, the culture substrate 13 is adhered to the annularly cut film 14, and then the culture substrate 13 is attached to the hole portion 111. Can be manufactured by covering the bottom surface portion 11 so that the film 14 is fitted and holding the film 14 in place.
（効果）
ここで、従来の接着剤を用いた型細胞培養容器１Ａを、図２に示す。図２に示すように、接着剤１５が、培養基板１３上にあるため、有機溶媒により殺菌を行った場合、接着剤１５が溶出しやすい。そのため、接着力が弱くなり、更に、溶出した有機溶媒が細胞培養を阻害するおそれがある。
(effect)
Here, FIG. 2 shows a type cell culture vessel 1A using a conventional adhesive. As shown in FIG. 2, since the adhesive 15 is on the culture substrate 13, the adhesive 15 is likely to be eluted when sterilized with an organic solvent. For this reason, the adhesive force is weakened, and the eluted organic solvent may inhibit cell culture.
これに対し、シャーレ型細胞培養容器１は、底面部１１に、該底面部１１の厚さ方向に貫通した穴部１１１が形成され、光分解性基が表面に結合された培養基板１３が、穴部１１１に培養基板１３の表面が容器の内側方向を向くように配置された状態で嵌合されるように構成した。これにより、シャーレ型細胞培養容器１は、培養基板１３の底面部１１への接着に接着剤を利用する必要がなく、培養基板１３を固定できるため、有機溶媒により殺菌を行っても培養基板１３の培養容器への接着力が損ないにくい。また、有機溶媒による殺菌を行っても、接着剤を使用してないことから、溶出した接着剤による細胞培養への影響を低減することができる。また、仮に、接着剤を使用したとしても、培養基板１３の表面に接着剤を塗布せずに固定が可能であり（例えば、穴部１１１と培養基板１３との間に接着剤を塗布すればよいため）、接着剤が培養した細胞に接しにくいため、接着剤による細胞培養への影響を低減することができる。以上で述べた理由により、シャーレ型細胞培養容器１は、光照射による動的パターニングに好適に使用することができる。 On the other hand, the petri dish-type cell culture container 1 has a culture substrate 13 in which a hole portion 111 penetrating in the thickness direction of the bottom surface portion 11 is formed in the bottom surface portion 11 and a photodegradable group is bonded to the surface. The hole 111 was configured to be fitted in a state where the surface of the culture substrate 13 was arranged so as to face the inner side of the container. Thereby, the petri dish type cell culture container 1 does not need to use an adhesive for adhesion to the bottom surface portion 11 of the culture substrate 13 and can fix the culture substrate 13, so that the culture substrate 13 can be sterilized with an organic solvent. It is difficult to lose the adhesive strength to the culture container. Moreover, even if sterilization with an organic solvent is performed, since no adhesive is used, the influence of the eluted adhesive on cell culture can be reduced. Even if an adhesive is used, it can be fixed to the surface of the culture substrate 13 without applying the adhesive (for example, if an adhesive is applied between the hole 111 and the culture substrate 13). Because it is difficult for the adhesive to come into contact with the cultured cells, the influence of the adhesive on the cell culture can be reduced. For the reasons described above, the petri dish type cell culture container 1 can be suitably used for dynamic patterning by light irradiation.
また、シャーレ型細胞培養容器１において、培養基板１３が容器の外側方向からフィルムにより底面部１１に固定されるように構成した。これにより、接着剤を用いずとも、より強固に培養基板１３を底面部１１に固定可能となる。 Moreover, in the petri dish type cell culture container 1, the culture substrate 13 was configured to be fixed to the bottom surface part 11 with a film from the outside of the container. Thereby, the culture substrate 13 can be more firmly fixed to the bottom surface portion 11 without using an adhesive.
（変形例）
シャーレ型細胞培養容器は、上述したシャーレ型細胞培養容器１に限定されず、必要に応じて適宜変更することができる。
(Modification)
The petri dish type cell culture container is not limited to the petri dish type cell culture container 1 described above, and can be appropriately changed as necessary.
シャーレ型細胞培養容器１においては、底面部１１、培養基板１３は円形平面状に構成したが、これに限定されず、適宜形状を変更でき、例えば、凸状や凹状であってもよく、球状であってもよい。また、培養基板１３は、穴部１１１の形状に合わせればどのような形状であってもよく、例えば、穴部１１１が四角形状であった場合、培養基板１３も四角形状に構成できる。 In the petri dish-type cell culture vessel 1, the bottom surface portion 11 and the culture substrate 13 are configured in a circular plane shape, but the shape is not limited to this, and the shape can be changed as appropriate, for example, a convex shape or a concave shape, or a spherical shape. It may be. Further, the culture substrate 13 may have any shape as long as it matches the shape of the hole 111. For example, when the hole 111 has a quadrangular shape, the culture substrate 13 can also be configured to have a quadrangular shape.
シャーレ型細胞培養容器１においては、フィルム１４を環状に構成したが、環状でなくてもよく、貫通部のない円形状、四角形状等であってもよい。 In the petri dish type cell culture container 1, the film 14 is formed in an annular shape, but may not be in an annular shape, and may be a circular shape without a penetrating portion, a rectangular shape, or the like.
＜培養基板への光分解性基の結合＞
（表面修飾）
まず、培養基板として用いるカバーガラス（松浪硝子工業株式会社製、Ｎｏ．１、丸型φ１５ｍｍ）に、ＵＶ−Ｏ３クリーナーにてＵＶを１時間ずつ両面にそれぞれ照射し、前処理を行った。次いで、５００ｍＬセパラブルフラスコに以下の式（１）で表されるシランカップリング剤の０．３ｍＭ乾燥トルエン溶液１４０ｍＬを調製し、これに酢酸０．４ｍＬ（５０ｍＭ）を添加し、磁製染色器（池本理化工業株式会社製）に立掛けた上述の前処理済みのカバーガラスを入れ、窒素雰囲気下で、アルミ箔で遮光して室温で２４時間静置して、表面修飾を行った。その後、カバーガラスをエタノールで洗浄し、窒素気流で乾燥した。この際のカバーガラスの水の接触角は６８°であった。この表面修飾の反応スキームを以下に示す。
<Binding of photodegradable group to culture substrate>
(Surface modification)
First, a cover glass (manufactured by Matsunami Glass Industry Co., Ltd., No. 1, round φ15 mm) used as a culture substrate was pretreated by irradiating UV on both sides with a UV-O 3 cleaner for 1 hour. Next, 140 mL of a 0.3 mM dry toluene solution of a silane coupling agent represented by the following formula (1) is prepared in a 500 mL separable flask, and 0.4 mL (50 mM) of acetic acid is added thereto, and a magnetic stainer The above-mentioned pre-treated cover glass leaned on (Ikemoto Rika Kogyo Co., Ltd.) was put in, light-shielded with aluminum foil in a nitrogen atmosphere, and allowed to stand at room temperature for 24 hours for surface modification. Thereafter, the cover glass was washed with ethanol and dried with a nitrogen stream. At this time, the water contact angle of the cover glass was 68 °. The reaction scheme for this surface modification is shown below.
（化学修飾）
５００ｍＬセパラブルフラスコにＰＥＧ１２Ｋ−ＮＨ２（日油株式会社製、ＳＵＮＢＲＩＧＨＴ（登録商標）ＭＥＰＡ−１２Ｔ）の０．５ｍＭアセトニトリル溶液１４０ｍＬを調製し、トリエチルアミン０．０１ｍＬ（０．５ｍＭ）を添加し、磁製染色器（池本理化工業株式会社製）に立掛けてあった表面修飾済みカバーガラスを入れ、窒素雰囲気下、アルミ箔で遮光して室温で２０時間静置して、光分解性基に細胞付着抑制基による化学修飾を行った。その後、カバーガラスをエタノールで洗浄し、窒素気流で乾燥した。この際のカバーガラスの水の接触角は３６°であった。この化学修飾の反応スキームを以下に示す。
(Chemical modification)
140 mL of a 0.5 mM acetonitrile solution of PEG 12K -NH 2 (manufactured by NOF Corporation, SUNBRIGHT (registered trademark) MEPA-12T) is prepared in a 500 mL separable flask, and 0.01 mL (0.5 mM) of triethylamine is added. Put the surface-modified cover glass that stood on a porcelain dyeing machine (Ikemoto Rika Kogyo Co., Ltd.), shield it with aluminum foil in a nitrogen atmosphere and let it stand at room temperature for 20 hours. Chemical modification with cell adhesion inhibitory group was performed. Thereafter, the cover glass was washed with ethanol and dried with a nitrogen stream. At this time, the water contact angle of the cover glass was 36 °. The reaction scheme for this chemical modification is shown below.
＜シャーレ型細胞培養容器の作製＞
ポリスチレン製のシャーレ（φ３５ｍｍ）の底面部に、切削にてφ１５ｍｍの貫通穴を設けた。機械加工後ハンドナイフにて両端にＣ面加工を施し、エアーにて異物を除去した。他方、培養基板の固定のためのフィルムとして用いられるマイクロプレートシール（カジックス株式会社製、製品名：タイタースティック、型番：ＨＣ、材質：ＰＥＴ）を外径φ２２ｍｍ、内径φ１２ｍｍのドーナツ形状にくり抜いた。上記のマイクロプレートシールの剥離紙をはがし、粘着面を上にして置き、マイクロプレートシールの中央に上記の修飾済みのカバーガラス（φ１５ｍｍ）を接着した。次いで、シャーレの底面部に形成された穴部に、カバーガラスが嵌合するように上記シャーレをカバーガラスに被せ、マイクロプレートシールを抑えて固定した。
<Production of petri dish type cell culture container>
A through hole having a diameter of 15 mm was formed by cutting on the bottom surface of a petri dish made of polystyrene (φ35 mm). After machining, C-face processing was applied to both ends with a hand knife, and foreign matters were removed with air. On the other hand, a microplate seal (manufactured by Kajix Corporation, product name: titer stick, model number: HC, material: PET) used as a film for fixing the culture substrate was cut into a donut shape having an outer diameter of φ22 mm and an inner diameter of φ12 mm. The release paper of the microplate seal was peeled off, the adhesive surface was placed on top, and the modified cover glass (φ15 mm) was adhered to the center of the microplate seal. Next, the petri dish was covered with the cover glass so that the cover glass was fitted into the hole formed in the bottom part of the petri dish, and the microplate seal was suppressed and fixed.
＜細胞培養試験＞
作製したシャーレ型細胞培養容器を、滅菌蒸留水で洗浄後、ＰＢＳ２ｍＬを添加した。蛍光顕微鏡にてフォトマスクを介して光照射し（１０Ｊ）、パターンを形成した。光照射による反応スキームを以下に示す。
<Cell culture test>
The prepared petri dish type cell culture container was washed with sterilized distilled water, and then 2 mL of PBS was added. Light was irradiated through a photomask with a fluorescence microscope (10J) to form a pattern. The reaction scheme by light irradiation is shown below.
パターン形成後のシャーレ型細胞培養容器をＰＢＳで洗浄し、次いで血清培地で洗浄した。その後、シャーレ型細胞培養容器に血清培地２ｍＬを添加し、ＨｅＬａ細胞を４×１０５ｃｅｌｌｓ播種し、インキュベーションした。インキュベーションを開始してから数時間後、血清培地で軽く洗浄し、インキュベーションを続け、逐次観察した。 The petri dish-type cell culture container after pattern formation was washed with PBS, and then washed with serum medium. Thereafter, 2 mL of serum medium was added to the petri dish-type cell culture vessel, and HeLa cells were seeded at 4 × 10 5 cells and incubated. Several hours after the start of incubation, the plate was gently washed with serum medium, and the incubation was continued and observed sequentially.
また、上記の修飾と同様の修飾を行ったカバーガラスのみを用いて、同様の細胞培養試験を行った。 Moreover, the same cell culture test was done only using the cover glass which performed the modification similar to said modification.
その結果を、図３、図４に示す。図３は、カバーガラスのみを用いて細胞培養を行った際の細胞パターニングを観察した画像を示す図である。図４は、上述の作製したシャーレ型細胞培養容器を用いて細胞培養を行った際の細胞パターニングを観察した画像を示す図である。図３のうち、（ａ）は、培養開始から２時間経過後、（ｂ）は、培養開始から１日経過後、（ｃ）は、培養開始から４日経過後、（ｄ）は、培養開始から７日経過後において、それぞれ顕微鏡で観察した画像である。図４のうち、（ａ）は、培養開始から４時間経過後、（ｂ）は、培養開始から１日経過後、（ｃ）は、培養開始から４日経過後、（ｄ）は、培養開始から７日経過後において、それぞれ顕微鏡で観察した画像である。 The results are shown in FIGS. FIG. 3 is a diagram illustrating an image obtained by observing cell patterning when cell culture is performed using only a cover glass. FIG. 4 is a diagram showing an image obtained by observing cell patterning when cell culture is performed using the above-described petri dish type cell culture container. In FIG. 3, (a) is 2 hours after the start of culture, (b) is 1 day after the start of culture, (c) is 4 days after the start of culture, (d) is after the start of culture. Each image is observed with a microscope after 7 days. 4, (a) is 4 hours after the start of culture, (b) is 1 day after the start of culture, (c) is 4 days after the start of culture, and (d) is after the start of culture. Each image is observed with a microscope after 7 days.
図３に示すように、カバーガラスのみで細胞パターニングを行った際に、問題なく明確な細胞パターンが観測され、パターンは７日間維持された。他方、図４に示すように、シャーレ型細胞培養容器を用いて細胞パターニングを行った際も、カバーガラスのみの場合と同様に、明確な細胞パターンが観測され、パターンは７日間維持されたことがわかった。 As shown in FIG. 3, when cell patterning was performed using only the cover glass, a clear cell pattern was observed without any problem, and the pattern was maintained for 7 days. On the other hand, as shown in FIG. 4, when cell patterning was performed using a petri dish-type cell culture vessel, a clear cell pattern was observed as in the case of only the cover glass, and the pattern was maintained for 7 days. I understood.
１ シャーレ型細胞培養容器
１Ａ シャーレ型細胞培養容器
１１ 底面部
１１１ 穴部
１２ 側壁部
１３ 培養基板
１４ フィルム
１５ 接着剤
DESCRIPTION OF SYMBOLS 1 Petri dish type cell culture container 1A Petri dish type cell culture container 11 Bottom face part 111 Hole part 12 Side wall part 13 Culture substrate 14 Film 15 Adhesive
Claims (3)
前記底面部に、該底面部の厚さ方向に貫通した穴部が形成され、
光分解性基が表面に結合された培養基板が、前記穴部に前記表面が容器の内側方向を向くように配置された状態で嵌合された、シャーレ型細胞培養容器。 A petri dish-type cell culture container comprising a bottom surface portion and a side wall portion,
In the bottom surface portion, a hole portion penetrating in the thickness direction of the bottom surface portion is formed,
A petri dish-type cell culture container in which a culture substrate having a photodegradable group bonded to a surface is fitted in the hole portion so that the surface faces the inner side of the container.
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| JP2015167060A JP6712073B2 (en) | 2015-08-26 | 2015-08-26 | Petri dish cell culture vessel |
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| JP2015167060A JP6712073B2 (en) | 2015-08-26 | 2015-08-26 | Petri dish cell culture vessel |
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| JP6712073B2 JP6712073B2 (en) | 2020-06-17 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018183070A (en) * | 2017-04-24 | 2018-11-22 | 株式会社イントロテック | Observation device |
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| JP6712073B2 (en) | 2020-06-17 |
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