JP4507845B2 - Cell culture container and cultured cell - Google Patents

Cell culture container and cultured cell Download PDF

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JP4507845B2
JP4507845B2 JP2004331324A JP2004331324A JP4507845B2 JP 4507845 B2 JP4507845 B2 JP 4507845B2 JP 2004331324 A JP2004331324 A JP 2004331324A JP 2004331324 A JP2004331324 A JP 2004331324A JP 4507845 B2 JP4507845 B2 JP 4507845B2
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cell culture
culture container
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cells
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JP2005168494A (en
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孝介 桑原
昭浩 宮内
拓司 安藤
雅彦 荻野
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Hitachi Ltd
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    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings

Description

本発明は細胞培養容器とその容器で培養した培養細胞に関する。   The present invention relates to a cell culture container and cultured cells cultured in the container.

近年、医療目的に使われる細胞培養の技術が進歩し、実際に皮膚の移植などに用いられている。また、少量の細胞から皮膚などの組織にとどまらず角膜，歯，骨，臓器など複雑な器官へと自家移植や他家移植に向けた培養技術の進歩が見られる。   In recent years, cell culture technology used for medical purposes has advanced and is actually used for skin transplantation and the like. In addition, culture techniques for autotransplantation and allogeneic transplantation are seen from small amounts of cells to complex organs such as the cornea, teeth, bones, and organs as well as tissues such as skin.

細胞の培養にはガラス製や樹脂製のペトリシャーレなどの容器が用いられる。例えば、以下のようなペトリシャーレが開示されている。所定の濃度のコラーゲン溶液を、培養容器に、ピペットで表面が一様になるよう分注して塗布した後、１５分〜７２時間乾燥する。あるいは、所定の濃度のコラーゲン溶液を、シリコン膜等の伸縮性を有する培養基材上に塗布し、１５〜４２℃のインキュベーター内で２０〜１２０分間重合させた後、このシリコーン膜等の伸縮性を有する培養基材をＵＶ灯下で１５分〜７２時間放置し、コラーゲンが乾燥した後、リン酸緩衝液で再び湿らせ、その後、１０〜４０％伸展させて固定するなどの手法により細胞培養用シャーレを作成する方法がある（特許文献１参照）。   Containers such as glass or resin petri dishes are used for cell culture. For example, the following petri dishes are disclosed. A collagen solution having a predetermined concentration is dispensed and applied to a culture vessel with a pipette so that the surface is uniform, and then dried for 15 minutes to 72 hours. Alternatively, a collagen solution having a predetermined concentration is applied on a culture substrate having elasticity such as a silicon film, polymerized in an incubator at 15 to 42 ° C. for 20 to 120 minutes, and then the elasticity of this silicone film or the like. The cell culture medium is allowed to stand under a UV lamp for 15 minutes to 72 hours, and after collagen has dried, it is again moistened with a phosphate buffer, and then stretched and fixed by 10 to 40%. There is a method of creating a petri dish (see Patent Document 1).

また、別の細胞培養容器として以下のような培養容器が開示されている。培養容器の底面に温度によって親水性，疎水性の入れ替わるポリマーを塗布し、紫外線照射などで定着させることにより培養後の細胞の剥離に適した培養容器を作ることが出来る（特許文献２参照）。   Moreover, the following culture containers are disclosed as another cell culture container. A culture vessel suitable for detachment of cells after culturing can be prepared by applying a polymer whose hydrophilicity and hydrophobicity are switched depending on the temperature to the bottom surface of the culture vessel and fixing it by ultraviolet irradiation or the like (see Patent Document 2).

特開２００２−１４２７５１号公報（実施例Ａ）JP 2002-142751 A (Example A) 特開平５−２４４９３８号公報（実施例１）Japanese Patent Laid-Open No. 5-244938 (Example 1)

上記第一の方法によれば、細胞をその細胞と親和性のあるコラーゲンの上において培養することが出来るが、反面、細胞と培養容器の密着力が強い為に細胞の剥離が困難であった。この密着力に対して、機械的に細胞を剥がすと細胞に物理的な損傷を与え、トリプシンなどの酵素によって化学的な処理を行うと細胞表面の膜タンパク質が破壊されて移植後の細胞の組織への定着率が低下するという問題があった。   According to the first method, cells can be cultured on collagen having affinity for the cells, but on the other hand, the adhesion between the cells and the culture vessel is strong, so that it was difficult to detach the cells. . When the cells are mechanically peeled against this adhesion, the cells are physically damaged, and when chemically treated with an enzyme such as trypsin, the membrane proteins on the cell surface are destroyed and the tissue of the cells after transplantation. There was a problem that the fixing rate to the lowering.

上記第２の方法は剥離の問題を培養容器表面の親水性を調整することにより細胞表面の密着力を減少させることで解決することが出来る。しかし、細胞培養容器表面の状態が特定の物質に限定され、そのほかに、例えば様々な細胞に対応した任意の表面状態を作る、培養容器を作るのにかかる時間の短縮，シート状の細胞の中央部への栄養物の運搬と老廃物排出の促進、等の課題が残っていた。   The second method can solve the peeling problem by reducing the adhesion of the cell surface by adjusting the hydrophilicity of the culture vessel surface. However, the condition of the surface of the cell culture vessel is limited to a specific substance. In addition, for example, an arbitrary surface state corresponding to various cells can be created, the time taken to make the culture vessel can be reduced, the center of the sheet-like cell Issues such as transportation of nutrients to the department and promotion of waste discharge were still left.

本発明の目的は簡便に簡単な構造で剥離時の細胞への損傷を防ぎ、栄養物の運搬，老廃物排出を促進する細胞培養容器を提供することである。   An object of the present invention is to provide a cell culture container that prevents damage to cells during detachment with a simple and simple structure, and promotes transport of nutrients and discharge of waste products.

上記課題を解決するために、本発明は、細胞培養容器の底面に相当直径が１０ｎｍ以上１０μｍ以下であり、高さが１０ｎｍ以上１mm以下である突起群を形成し、この突起群の相当直径が、細胞培養容器内で培養する細胞の直径の５分の１以下であり、これら突起群を構成する隣り合う突起物の間隔が細胞培養容器内で培養する細胞の直径よりも小さくする細胞培養容器を特徴とする。これにより、培養液を細胞の下部に行き渡らせ、細胞の必要とする栄養物の供給と細胞の放出する老廃物の排出を促進するとともに細胞と容器の接触を点接触にすることにより細胞の剥離時に生じる細胞の損傷を防ぐことのできる細胞培養容器を提供するものである。なお、相当直径という語を用いたのは、突起の断面が必ずしも円形ではなく、楕円，多角形，非対称形などの場合があるためで、本発明ではこれらを全て包含するために相当直径を用いている。本発明で相当直径は、突起物底面の断面の直径とする。

In order to solve the above problems, the present invention forms a projection group having an equivalent diameter of 10 nm to 10 μm and a height of 10 nm to 1 mm on the bottom surface of a cell culture container, and the equivalent diameter of the projection group is A cell culture container that is not more than one-fifth of the diameter of cells cultured in the cell culture container, and the interval between adjacent protrusions constituting these protrusion groups is smaller than the diameter of cells cultured in the cell culture container It is characterized by. This spreads the culture solution to the lower part of the cell, promotes the supply of nutrients required by the cell and the discharge of waste products released by the cell, and makes the cell-container contact a point contact. It is an object of the present invention to provide a cell culture container that can prevent damage to cells that sometimes occurs. The term equivalent diameter is used because the cross section of the protrusion is not necessarily circular but may be an ellipse, a polygon, an asymmetric shape, etc. In the present invention, the equivalent diameter is used to encompass all of these. ing. In the present invention, the equivalent diameter is the diameter of the cross section of the bottom surface of the protrusion.

細胞培養容器の表面は培養液を行き渡らせるために親水性に処理する必要がある。しかし、培養する細胞の種類によっては容器表面を疎水性に処理をすることや、容器表面を金属やタンパク質などで覆うことが必要となることがある。このため、本発明は細胞培養容器に形成された突起群が細胞と接する先端部のみに細胞培養に必要な疎水化処理，金属によるコート，タンパク質によるコートなど細胞培養に必要な処理を施した細胞培養容器を提供するものである。   It is necessary to treat the surface of the cell culture vessel to be hydrophilic in order to spread the culture solution. However, depending on the type of cells to be cultured, it may be necessary to treat the surface of the container to be hydrophobic, or to cover the surface of the container with metal or protein. For this reason, the present invention provides a cell that has been subjected to treatments necessary for cell culture, such as a hydrophobic treatment necessary for cell culture, a metal coating, a protein coating, etc., only at the tip portion where the projection group formed in the cell culture container contacts the cell A culture vessel is provided.

また、培養する細胞に配向性など、特定の形状を求める場合には培養する細胞の形状に応じて疎水処理などの特定の処理を細胞培養容器の底面に部分的に処理することがある。本発明は細胞培養容器表面の突起群の先端部に対して施す疎水性，金属によるコート，タンパク質によるコートなど細胞培養に必要な処理を容器表面に選択的に施した細胞培養容器を提供するものである。   In addition, when a specific shape such as orientation is required for the cells to be cultured, a specific treatment such as a hydrophobic treatment may be partially processed on the bottom surface of the cell culture container according to the shape of the cells to be cultured. The present invention provides a cell culture container in which treatment necessary for cell culture, such as hydrophobicity, metal coating, and protein coating, is applied to the tip of the projection group on the surface of the cell culture container. It is.

本発明を適用することにより、簡便に簡単な構造で剥離時の細胞への損傷を防ぎ、栄養物の運搬，老廃物排出を促進する細胞培養容器を提供できる。突起群を用いた形状効果で上記の効果を出しているため、形成プロセスを簡便にすることができ、従来に比べて新たな薬品や処理装置の導入も無いことから新しく廃棄方法を考慮する必要がないという効果を得ることもできた。   By applying the present invention, it is possible to provide a cell culture container that prevents damage to cells at the time of detachment with a simple and simple structure, and promotes transport of nutrients and waste discharge. Since the above effect is achieved by the shape effect using the projection group, the formation process can be simplified, and there is no introduction of new chemicals or processing equipment compared to the conventional method, so it is necessary to consider a new disposal method It was also possible to obtain the effect that there was no.

以下、図面を参照しながら本発明の細胞培養容器について詳細に説明する。   Hereinafter, the cell culture container of the present invention will be described in detail with reference to the drawings.

図１は本発明における細胞培養容器１００を示す鳥瞰図である。細胞、及びその培養液を入れる容器１００の底面に相当直径が１０ｎｍ以上１０μｍ以下であり、高さが１０
ｎｍ以上１mm以下である突起物集合体１０１が形成されている。また、突起物集合体101の中には培養液を流れやすくするための隙間１０３が形成されている。 FIG. 1 is a bird's-eye view showing a cell culture container 100 according to the present invention. The equivalent diameter is 10 nm or more and 10 μm or less on the bottom surface of the cell 100 and the container 100 in which the culture solution is placed, and the height is 10
A projection aggregate 101 having a thickness of not less than nm and not more than 1 mm is formed. Further, a gap 103 for facilitating the flow of the culture solution is formed in the projection aggregate 101.

図１の突起物集合体１０１の相当直径は、細胞との接触面積を減らすために細胞の直径よりも十分に小さく、例えば細胞の直径の５分の１以下に設定される。また、突起物の下部に十分に培養液を浸透させる必要があるためこれらの突起物の高さは十分に、例えば相当直径以上、より好ましくは相当直径の５倍以上であることが好まれる。しかし、構造強度的な観点からは１００倍以下が好まれる。   The equivalent diameter of the protrusion assembly 101 in FIG. 1 is sufficiently smaller than the diameter of the cell to reduce the contact area with the cell, and is set to, for example, one fifth or less of the diameter of the cell. Further, since it is necessary to sufficiently infiltrate the culture solution into the lower part of the protrusions, it is preferable that the height of these protrusions is sufficiently larger than the equivalent diameter, for example, more preferably five times the equivalent diameter. However, 100 times or less is preferable from the viewpoint of structural strength.

図２は本発明における細胞培養容器１００に対して突起群の先端への表面処理を行った後の状態を示す鳥瞰図である。細胞、及びその培養液を入れる容器１００の底面に相当直径が１０ｎｍ以上１０μｍ以下であり、高さが１０ｎｍ以上１mm以下である突起物集合体１０１が形成されており、その突起物の先端部のみに疎水化処理，金属によるコート，タンパク質によるコートなど細胞培養に必要な処理１０２が施されている。   FIG. 2 is a bird's-eye view showing a state after the surface treatment of the tip of the projection group is performed on the cell culture container 100 according to the present invention. A protrusion assembly 101 having an equivalent diameter of 10 nm or more and 10 μm or less and a height of 10 nm or more and 1 mm or less is formed on the bottom surface of the container 100 in which the cells and the culture solution are placed. Only the tip of the protrusion is formed. Further, a treatment 102 necessary for cell culture, such as a hydrophobic treatment, a metal coat, and a protein coat, is applied.

図３は本発明における細胞培養容器１００に対して突起物の先端部の一部への表面処理を行った後の状態を示す鳥瞰図である。細胞、及びその培養液を入れる容器１００の底面に相当直径が１０ｎｍ以上１０μｍ以下であり、高さが１０ｎｍ以上１mm以下である突起物集合体１０１が形成されており、その突起物の一部の先端部に細胞培養に必要な疎水化処理，金属によるコート，タンパク質によるコートなど細胞培養に必要な処理１０２を施されている。   FIG. 3 is a bird's eye view showing a state after subjecting the cell culture container 100 according to the present invention to surface treatment on a part of the tip of the protrusion. A protrusion assembly 101 having an equivalent diameter of 10 nm or more and 10 μm or less and a height of 10 nm or more and 1 mm or less is formed on the bottom surface of the container 100 in which the cells and the culture solution are placed. The tip portion is subjected to a treatment 102 necessary for cell culture, such as a hydrophobic treatment necessary for cell culture, a metal coat, and a protein coat.

本発明における細胞培養容器１００の材質は特に限定されないが、所望する加工精度，表面特性，光学特性，強度などに応じて、選択される。具体的には、ポリエチレン，ポリプロピレン，ポリビニルアルコール，ポリ塩化ビニリデン，ポリエチレンテレフタレート，ポリ塩化ビニール，ポリスチレン，ＡＢＳ樹脂，ＡＳ樹脂，アクリル樹脂，ポリアミド，ポリアセタール，ポリブチレンテレフタレート，ガラス強化ポリエチレンテレフタレート，ポリカーボネート，変性ポリフェニレンエーテル，ポリフェニレンスルフィド，ポリエーテルエーテルケトン，液晶性ポリマー，フッ素樹脂，ポリアレート，ポリスルホン，ポリエーテルスルホン，ポリアミドイミド，ポリエーテルイミド，熱可塑性ポリイミド等の熱可塑性樹脂や、フェノール樹脂，メラミン樹脂，ユリア樹脂，エポキシ樹脂，不飽和ポリエステル樹脂，アルキド樹脂，シリコーン樹脂，ジアリルフタレート樹脂，ポリアミドビスマレイミド，ポリビスアミドトリアゾール等の熱硬化性樹脂、及びこれらを２種以上ブレンドした材料を用いることが可能である。また、この他に石英，ガラス類などの無機物も使用することが可能である。   The material of the cell culture container 100 in the present invention is not particularly limited, but is selected according to desired processing accuracy, surface characteristics, optical characteristics, strength, and the like. Specifically, polyethylene, polypropylene, polyvinyl alcohol, polyvinylidene chloride, polyethylene terephthalate, polyvinyl chloride, polystyrene, ABS resin, AS resin, acrylic resin, polyamide, polyacetal, polybutylene terephthalate, glass reinforced polyethylene terephthalate, polycarbonate, modified Thermoplastic resins such as polyphenylene ether, polyphenylene sulfide, polyether ether ketone, liquid crystalline polymer, fluororesin, polyarate, polysulfone, polyethersulfone, polyamideimide, polyetherimide, thermoplastic polyimide, phenol resin, melamine resin, urea Resin, epoxy resin, unsaturated polyester resin, alkyd resin, silicone resin, diallyl phthalate resin, poly Bromide bismaleimide, poly bisamide thermosetting resin triazole and the like, and it is possible to use two or more kinds of these blended material. In addition, inorganic materials such as quartz and glass can also be used.

本発明における突起物集合体１０１の材質は特に限定されないが、やはり所望する加工精度，表面特性，光学特性，強度などに応じて前述の樹脂組成物、若しくは石英，ガラス類など無機物よりなる。突起群の強度が問題となる場合はこれらの突起群が細胞培養容器と同じ材料でできており、一体化されていることが好まれる。   The material of the projection aggregate 101 in the present invention is not particularly limited, but it is also made of the above-described resin composition or an inorganic substance such as quartz or glass depending on desired processing accuracy, surface characteristics, optical characteristics, strength, and the like. When the strength of the protrusion group becomes a problem, it is preferable that these protrusion groups are made of the same material as the cell culture container and are integrated.

本発明における細胞培養容器の底面に突起群を形成する手法を図４に示す。容器４０１を加熱して軟化し、その容器４０１に微細な凹凸パターンが形成された金型４０２を押し付けることにより、金型４０２の微細形状４０３を容器４０１に転写し、突起群を形成した細胞培養容器１００を得ることができる。   FIG. 4 shows a method for forming a protrusion group on the bottom surface of the cell culture container in the present invention. A cell culture in which a container 401 is heated and softened, and a mold 402 having a fine uneven pattern formed thereon is pressed against the container 401 to transfer the fine shape 403 of the mold 402 to the container 401, thereby forming a projection group. A container 100 can be obtained.

金型４０２上の微細形状４０３は細胞培養容器４００上の突起群を前述の効果を与えるのに必要な大きさ，相当直径が１０ｎｍ以上１０μｍ以下であり、高さが１０ｎｍ以上１mm以下であることが求められる。このため、金型は金属，カーボンやシリコンなどの無機物、及び樹脂組成物の少なくとも１つを含み、その表面形状は光リソグラフィ法，電子線直接描画法，粒子線ビーム加工法，走査プローブ加工法などの微細加工法や微粒子の自己組織化、又はこれらの手法により形成されたマスタよりナノプリント法，射出成型法，無電解めっき法などにより形状転写することにより形成される。また、図４では突起群を形成する手法として軟化した容器に金型を押し付けるいわゆるナノプリント法を用いたが、これ以外の射出成型法を代表とする樹脂の成型法や、金型を用いずレーザ加工法などで直接加工することも可能であるのは明らかである。   The fine shape 403 on the mold 402 has a size necessary for providing the above-mentioned effects on the projection group on the cell culture vessel 400, an equivalent diameter of 10 nm to 10 μm, and a height of 10 nm to 1 mm. Is required. Therefore, the mold includes at least one of a metal, an inorganic material such as carbon and silicon, and a resin composition, and the surface shape thereof is an optical lithography method, an electron beam direct writing method, a particle beam beam processing method, a scanning probe processing method. It is formed by transferring a shape by a nano-printing method, an injection molding method, an electroless plating method or the like from a fine processing method such as self-organization of fine particles, or a master formed by these methods. In FIG. 4, the so-called nano-printing method in which the mold is pressed against the softened container is used as a method for forming the projection group. However, other resin molding methods such as injection molding methods and molds are not used. Obviously, it is also possible to directly process by a laser processing method or the like.

本発明において、細胞培養容器は必要に応じて過酸化水素やオゾンなどの酸化剤を含む溶媒を用いた溶媒への浸漬や紫外線照射，プラズマ処理などの気相処理などによる親水化処理や、溶液への浸漬などによるタンパクによるコート、若しくはめっき，気相蒸着法による金属コート，光や電子線，粒子線などによる表面改質が行われ、細胞培養に必要な表面処理が施される。   In the present invention, the cell culture vessel may be hydrophilized by immersion in a solvent using a solvent containing an oxidizing agent such as hydrogen peroxide or ozone, or vapor phase treatment such as ultraviolet irradiation or plasma treatment, or a solution as necessary. Surface modification necessary for cell culture is performed by coating with protein by dipping in a metal or by surface modification by plating, metal coating by vapor deposition, light, electron beam, particle beam or the like.

続いて本発明における突起群の先端のみへの表面修飾法を図５を用いて説明する。突起群を形成し、全面に必要な表面処理が施された細胞培養容器１００に対し、処理剤５０３を表面に付着したスタンプ５０２を密着させ、必要なら加熱，光照射などを施すことにより、スタンプ５０２と接触した突起物集合体１０１の先端部のみに所望の表面処理５０１を施すことができる。   Next, the surface modification method for only the tips of the projection groups in the present invention will be described with reference to FIG. A stamp 502 having a treatment agent 503 attached to the surface thereof is brought into close contact with the cell culture vessel 100 in which the protrusions are formed and the necessary surface treatment is applied to the entire surface, and if necessary, the stamp is applied by heating, light irradiation, or the like. A desired surface treatment 501 can be applied only to the tip of the projection assembly 101 in contact with 502.

スタンプ５０２は細胞培養容器１００表面の多くの突起物集合体１０１に均等に密着する必要がある。そのため、スタンプ５０２は必要な弾性を有するシリコーンゴム，樹脂フィルム，金属薄膜などの弾性体よりなることが望ましい。また、ここで図６に示すようにスタンプ５０２に凹凸６０１を形成しておくことにより、突起群先端部への処理の分布を形成することができる。   The stamp 502 needs to be in close contact with many protrusion assemblies 101 on the surface of the cell culture container 100. Therefore, the stamp 502 is preferably made of an elastic body such as a silicone rubber, a resin film, or a metal thin film having necessary elasticity. Further, as shown in FIG. 6, by forming the unevenness 601 on the stamp 502, it is possible to form the distribution of processing to the tip end of the projection group.

処理剤５０３としては樹脂ペースト，シリコーングリス，フッ素系コート剤などの疎水化剤やタンパクを含有する溶媒が挙げられる。処理剤５０３をスタンプ５０２に塗布する際に処理剤の有無，濃度などで面内分布をつけることによっても突起群先端部への処理の分布を形成することができる。また、加熱などにより直接突起物集合体１０１の先端部を変化させる場合は処理剤５０３を省くことができる。   Examples of the treatment agent 503 include a hydrophobic agent such as resin paste, silicone grease, and fluorine-based coating agent, and a solvent containing protein. By applying an in-plane distribution based on the presence / absence, concentration, etc. of the processing agent when the processing agent 503 is applied to the stamp 502, the distribution of processing to the tip of the projection group can be formed. Further, the treatment agent 503 can be omitted when the tip of the projection aggregate 101 is directly changed by heating or the like.

図７に本発明における細胞培養容器１００の使用法を示す。細胞培養容器１００内部を培養液７０１で満たし、突起物集合体１０１の上に細胞７０２を配置する。この後、常法により細胞７０２を培養する。本発明の細胞培養容器１００では、細胞７０２と容器との接触が点接触となるため、細胞７０２の剥離時に生じる損傷を防ぐことができる。また、突起物集合体１０１の形状や突起物集合体１０１の表面処理を、培養する細胞７０２によって変化させることにより、品質の良い培養細胞を剥離時の損傷なしに形成できる。なお、培養細胞とは、培養組織，器官に代表される組織化された培養細胞をも含む概念である。   FIG. 7 shows how to use the cell culture container 100 in the present invention. The inside of the cell culture vessel 100 is filled with the culture solution 701, and the cell 702 is placed on the protrusion assembly 101. Thereafter, the cells 702 are cultured by a conventional method. In the cell culture container 100 of the present invention, since the contact between the cell 702 and the container is a point contact, damage caused when the cell 702 is detached can be prevented. Further, by changing the shape of the projection aggregate 101 and the surface treatment of the projection aggregate 101 according to the cells 702 to be cultured, high-quality cultured cells can be formed without damage during peeling. The cultured cell is a concept including organized cultured cells represented by cultured tissues and organs.

以下、実施例により本発明をさらに詳述する。   Hereinafter, the present invention will be described in more detail by way of examples.

以下、本発明の一実施例を説明する。図８は本実施例で作製した突起物集合体１０１の走査型電子顕微鏡写真の模式図である。突起物集合体１０１は複数の柱状微小突起物801から成る。柱状微小突起物８０１の材質はポリスチレンで分子量は２０００から６０万であり、更に分子量の上限は６００万まで拡張することができる。   An embodiment of the present invention will be described below. FIG. 8 is a schematic diagram of a scanning electron micrograph of the protrusion assembly 101 produced in this example. The projection aggregate 101 is composed of a plurality of columnar microprojections 801. The material of the columnar microprojections 801 is polystyrene, the molecular weight is 2000 to 600,000, and the upper limit of the molecular weight can be extended to 6 million.

図９は突起物集合体１０１を拡大した走査型電子顕微鏡写真の模式図である。柱状微小突起物８０１の高さは３μｍ、一辺の長さは根元で３００ｎｍである。柱状微小突起物
８０１は上部約１μｍの部分は平滑な表面状態であり、根元から約２μｍの部分の表面は縞模様である。 FIG. 9 is a schematic diagram of a scanning electron micrograph in which the projection aggregate 101 is enlarged. The height of the columnar microprojection 801 is 3 μm, and the length of one side is 300 nm at the root. The columnar microprojection 801 has a smooth surface state at the upper portion of about 1 μm, and the surface of the portion of about 2 μm from the root has a striped pattern.

また、柱状微小突起物８０１は底面の一辺が３００ｎｍで高さが３μｍなので高さと一辺の比は１０となり、１より大きいことが分かる。   In addition, since the columnar microprojection 801 has one side of the bottom surface of 300 nm and a height of 3 μm, the ratio of the height to one side is 10 and is larger than 1.

また、柱状微小突起物８０１は先端部が底面部より小さくなっており、末広がり状であることが分かる。本実施例では、柱状微小突起物８０１の形状は根本から先端にかけて細くなっていく形状であるが、例えば、根本から先端にかけて細くなり先端部に太い部分を有するきのこのような形状でもよい。本発明の柱状微小突起物８０１は、根本から先端部にかけて細くなる部分を有することを特徴の一つとしている。   Further, it can be seen that the columnar microprojection 801 has a tip end portion smaller than the bottom surface portion, and has a divergent shape. In this embodiment, the columnar microprojection 801 has a shape that narrows from the root to the tip, but may be a mushroom shape that narrows from the root to the tip and has a thick portion at the tip. One feature of the columnar microprojection 801 of the present invention is that it has a portion that narrows from the root to the tip.

また、柱状微小突起物８０１は下地８０２と同じポリスチレンでできている。   The columnar microprojection 801 is made of the same polystyrene as the base 802.

また、柱状微小突起物８０１は下地８０２に接続されており、一体化していることが分かる。   Further, it can be seen that the columnar microprojections 801 are connected to the base 802 and are integrated.

また、突起物の先端部が突起物の底面部より小さく末広がり状であるため、突起物が基板から取れにくい効果を得られる。また、突起物が下地の材料と同じであるため突起物が下地から取れにくい効果を得られる。また、突起物が基板と一体化しているため基板から取れにくい効果を得られる。   In addition, since the tip of the protrusion is smaller than the bottom surface of the protrusion and has a divergent shape, an effect that the protrusion is difficult to remove from the substrate can be obtained. Further, since the protrusion is the same as the base material, it is possible to obtain an effect that the protrusion is difficult to remove from the base. Moreover, since the protrusion is integrated with the substrate, it is difficult to remove the protrusion from the substrate.

なお、本実施例では、柱状微小突起物８０１と底面８０２の材料はポリスチレンを用いたが、細胞培養容器の材質として先に例示した材料を用いてもよい。   In this embodiment, polystyrene is used as the material for the columnar microprojections 801 and the bottom surface 802, but the materials exemplified above may be used as the material for the cell culture container.

上述の柱状微小突起物８０１は以下に述べる方法で作製した。図１０は柱状微小突起物８０１の製造工程である。容器４０１を１００℃に加熱し、表面に直径０.５μｍ ，深さ１.０μｍの微細孔が１.０μｍピッチで全面に形成された金型４０２をプレス圧力４MPaで３００ｓ間プレスした。プレス装置内にて７０℃に冷却後に金型４０２と容器４０１をプレス装置より取り出し、金型４０２を容器４０１から垂直に引き上げることで剥離し、柱状微小突起物８０１を有する細胞培養容器１００を作製した。金型４０２は結晶方位
(１００)，直径２５mmのシリコンウエハであり、成型時における容器４０１との接着を防止する為にフッ素系の離型剤により離型処理を施した柱状微小突起物８０１のアスペクト比は金型４０２の凹凸のアスペクト比の約３倍である。このような高アスペクト比の構造はアスペクト比の大きい凹凸を金型４０２に形成することによっても細胞培養容器１００上に形成できるが、本実施例の手法を用いれば高いアスペクト比の柱状微小突起物８０１を比較的作成が容易である低アスペクト比の金型４０２から形成できる効果を得られる。 The columnar microprojections 801 described above were produced by the method described below. FIG. 10 shows a manufacturing process of the columnar microprojection 801. The container 401 was heated to 100 ° C., and a die 402 having micropores with a diameter of 0.5 μm and a depth of 1.0 μm formed on the entire surface at a pitch of 1.0 μm was pressed at a pressing pressure of 4 MPa for 300 s. After cooling to 70 ° C. in the press apparatus, the mold 402 and the container 401 are taken out of the press apparatus and peeled off by pulling the mold 402 vertically from the container 401 to produce the cell culture container 100 having the columnar microprojections 801. did. Mold 402 has crystal orientation
(100), a silicon wafer having a diameter of 25 mm, and the columnar microprojection 801 having a mold release treatment with a fluorine-based mold release agent to prevent adhesion to the container 401 during molding has an aspect ratio of the mold 402 The aspect ratio of the unevenness is about 3 times. Such a high aspect ratio structure can also be formed on the cell culture vessel 100 by forming irregularities having a large aspect ratio on the mold 402. However, by using the method of this embodiment, a columnar microprojection having a high aspect ratio. The effect of being able to form 801 from the low aspect ratio mold 402 that is relatively easy to produce can be obtained.

なお、本実施例では、ポリスチレンを容器４０１に用いたが、容器４０１はポリスチレンに限らず例えばポリカーボネートなどの有機物，ガラスなどの無機物、あるいはこれらの積層構造体でもよい。   In this embodiment, polystyrene is used for the container 401. However, the container 401 is not limited to polystyrene, but may be an organic substance such as polycarbonate, an inorganic substance such as glass, or a laminated structure thereof.

また、本実施例では、金型４０２に結晶方位（１００），直径２５ミリメートルのシリコンウエハを用いたが、特に結晶方位が（１００）であったり、材質が単結晶シリコンである必要はなく、ニッケルなどの金属薄膜やＰＤＭＳ（polydimethylsiloxane）などの有機物でもよい。また、加熱した容器４０１より金型４０２を離型する際の容器４０１や柱状微小突起物８０１への損傷を防ぐため金型４０２にはフッ素系、若しくはシリコーン系などの離型剤によりコートされていることが望ましい。   In this embodiment, a silicon wafer having a crystal orientation (100) and a diameter of 25 millimeters was used for the mold 402, but it is not particularly necessary that the crystal orientation is (100) or the material is single crystal silicon. It may be a metal thin film such as nickel or an organic material such as PDMS (polydimethylsiloxane). Further, in order to prevent damage to the container 401 and the columnar microprojections 801 when the mold 402 is released from the heated container 401, the mold 402 is coated with a release agent such as fluorine or silicone. It is desirable.

また、金型４０２の凹部の深さや容器４０１の材質を調整することで柱状微小突起物
８０１の直径や高さを制御できる。 Further, the diameter and height of the columnar microprojection 801 can be controlled by adjusting the depth of the concave portion of the mold 402 and the material of the container 401.

また、金型４０２の凹部の開口面積を大きくすることで柱状微小突起物８０１の底部の大きさを制御できる。   Further, the size of the bottom of the columnar microprojection 801 can be controlled by increasing the opening area of the concave portion of the mold 402.

さらに、金型４０２の凹部の位置を制御することで柱状微小突起物８０１を形成する位置を制御できる。   Furthermore, the position where the columnar microprojections 801 are formed can be controlled by controlling the position of the concave portion of the mold 402.

また、柱状微小突起物８０１の材料を熱可塑性にすることで、柱状微小突起物８０１の形成時の温度を調整することで柱状微小突起物８０１の形状を容易に制御できる効果を得られることは明らかである。   Further, by making the material of the columnar microprojections 801 thermoplastic, it is possible to obtain an effect that the shape of the columnar microprojections 801 can be easily controlled by adjusting the temperature at the time of forming the columnar microprojections 801. it is obvious.

また、柱状微小突起物８０１の材料を光硬化性にすることで、柱状微小突起物８０１の形成時に光を入射させることで柱状微小突起物８０１の形状を容易に制御できる効果を得られることは明らかである。   In addition, by making the material of the columnar microprojections 801 photocurable, it is possible to obtain an effect that the shape of the columnar microprojections 801 can be easily controlled by making light incident when the columnar microprojections 801 are formed. it is obvious.

以下、本発明の他の実施例を説明する。図１は本実施例で作製した細胞培養容器１００を示した鳥瞰図である。細胞培養容器１００は、厚さ２mmのポリスチレンを主成分とした直径３５mmのシャーレ状の形状を有している。細胞培養容器１００の底面には、実施例１で示した手法を用いて直径３０mmの領域に突起物集合体１０１を形成している。また、突起物集合体１０１の形成後に親水化処理として酸素プラズマ処理（１００Ｗ，３０ｓ）を施した。この突起物集合体１０１の先端部に、５０μｇ／ｍＬのコラーゲンＩ溶液
（Cultrex Bovine Collagen I（登録商標）、溶媒：０.０２Ｍ酢酸水溶液）を突起物集合体１０１の高さよりも薄くコートさせた表面親水化ＰＤＭＳ製の平滑スタンプを密着させ、突起物集合体１０１の先端部のみコラーゲンＩ溶液を付着させた。平滑スタンプを除いた後に室温にて１時間保持してＰＢＳ（リン酸緩衝液）で洗浄することで、突起物集合体１０１の先端部のみにコラーゲンによる修飾を加え、細胞培養に適した表面処理を行った。 Other embodiments of the present invention will be described below. FIG. 1 is a bird's-eye view showing a cell culture vessel 100 produced in this example. The cell culture container 100 has a petri dish-shaped shape with a diameter of 35 mm mainly composed of polystyrene having a thickness of 2 mm. On the bottom surface of the cell culture vessel 100, the projection aggregate 101 is formed in a region having a diameter of 30 mm by using the method shown in the first embodiment. Further, after the formation of the projection aggregate 101, oxygen plasma treatment (100 W, 30 s) was performed as a hydrophilic treatment. A 50 μg / mL collagen I solution (Cultrex Bovine Collagen I (registered trademark), solvent: 0.02 M acetic acid aqueous solution) was coated on the tip of the projection assembly 101 to be thinner than the height of the projection assembly 101. A smooth stamp made of surface hydrophilized PDMS was brought into close contact, and the collagen I solution was adhered only to the tip of the projection assembly 101. After removing the smooth stamp, it is kept at room temperature for 1 hour and washed with PBS (phosphate buffer), so that only the tip of the projection assembly 101 is modified with collagen, and a surface treatment suitable for cell culture. Went.

本実施例では、ポリスチレンを細胞培養容器１００に用いたが、ポリスチレンに限らず、施される表面処理との親和性があれば例えばポリカーボネートなどの有機物，ガラスなどの無機物、あるいはこれらの積層構造体でもよい。また、細胞培養容器１００の大きさを直径３５mm，突起物集合体１０１の形成領域を直径３０mmとしたが、これは培養する細胞の大きさに応じて変えられる。また、突起物集合体１０１は隙間１０３を設けて配置することが好ましく、本実施例では図１に示したよう十字形状に隙間１０３を設けている。このように隙間１０３を形成することで培養液が流れやすくなり細胞に対して効率良く栄養素を供給することができる。また、細胞培養時の細胞の老廃物を効率良く排出することができる。   In this example, polystyrene was used for the cell culture vessel 100. However, not only polystyrene but also an organic substance such as polycarbonate, an inorganic substance such as glass, or a laminated structure thereof, as long as it has an affinity for the surface treatment to be applied. But you can. Moreover, although the size of the cell culture vessel 100 is 35 mm in diameter and the formation area of the projection aggregate 101 is 30 mm in diameter, this can be changed according to the size of the cells to be cultured. Further, the projection aggregate 101 is preferably arranged with a gap 103, and in this embodiment, the gap 103 is provided in a cross shape as shown in FIG. By forming the gap 103 in this manner, the culture solution can easily flow and nutrients can be efficiently supplied to the cells. Moreover, the waste product of the cell at the time of cell culture can be discharged | emitted efficiently.

また、本実施例では突起物集合体１０１の形成後にタンパクの一種であるコラーゲンによる修飾を行ったが、この手法に限られず、培養を行う細胞の種類、及び細胞培養容器
１００、及び突起物集合体１０１の材質に応じて酸素プラズマ処理（例えば１００Ｗ，
３０ｓ），紫外線照射，過酸化水素水，オゾン水への浸漬などによる親水化処理，アミノ基，カルボキシル基，メチル基，ＣＦ3 基などに代表される官能基の導入など適宜異なる表面処理を施すこともできる。また、この表面処理を図５，図６に示す方法で突起物集合体１０１の一部にのみ施すことによって培養する細胞の形状を制御することができる。 In this embodiment, the modification with the collagen, which is a kind of protein, is performed after the formation of the protrusion aggregate 101. However, the present invention is not limited to this method, and the type of cell to be cultured, the cell culture container 100, and the protrusion aggregate. Oxygen plasma treatment (for example, 100 W, depending on the material of the body 101
30s), surface treatment such as UV irradiation, hydrophilization treatment by immersion in hydrogen peroxide water or ozone water, introduction of functional groups represented by amino groups, carboxyl groups, methyl groups, CF 3 groups, etc. You can also Further, the shape of cells to be cultured can be controlled by applying this surface treatment to only a part of the projection aggregate 101 by the method shown in FIGS.

以下、本発明の細胞培養容器１００を用いて細胞を培養した例を示す。図７は本実施例２で作製した細胞培養容器１００を用いて細胞を培養している様子を表す模式図である。   Hereinafter, the example which cultured the cell using the cell culture container 100 of this invention is shown. FIG. 7 is a schematic diagram showing a state in which cells are cultured using the cell culture container 100 produced in the second embodiment.

細胞培養容器１００に培養液を浸した状態で入れ、細胞培養容器１００内にて、常法により正常ヒト表皮角化細胞を培養した（使用培地：HuMedia−ＫＢ２（クラボウ(株) 製。３７℃，５％ＣＯ2 下）。その結果、細胞培養容器１００上において表皮角化細胞は正常に付着し、シート形状に増殖した。 The cell culture vessel 100 was immersed in a culture solution, and normal human epidermal keratinocytes were cultured in the cell culture vessel 100 by a conventional method (used medium: HuMedia-KB2 (Kurabo Co., Ltd., 37 ° C.). under 5% CO 2). as a result, epidermal keratinocytes in cell culture vessel 100 is normally attached, and grown in a sheet shape.

培養開始の１４日後、培養した細胞の上に直径２cmのポリビニリデンジフルオライド
（ＰＶＤＦ）膜をかぶせて、培地を吸引することによって、細胞培養容器１００上に成長したシート状の表皮角化細胞を、ＰＶＤＦ膜と共に細胞培養容器１００から剥離した。このシート状の表皮角化細胞はかぶせたＰＶＤＦ膜から容易に剥がすことができた。細胞培養容器１００からの剥離によるこのシート状の表皮角化細胞への損傷は通常のガラスシャーレなどを使用した場合に比べて大幅に軽減することができた。 Fourteen days after the start of culture, a 2 cm diameter polyvinylidene difluoride (PVDF) membrane was placed on the cultured cells, and the medium was aspirated, so that sheet-like epidermal keratinocytes grown on the cell culture vessel 100 were obtained. Was peeled from the cell culture vessel 100 together with the PVDF membrane. The sheet-like epidermal keratinocytes could be easily peeled off from the PVDF membrane. Damage to the sheet-shaped epidermal keratinocytes due to peeling from the cell culture vessel 100 could be significantly reduced as compared with the case of using a normal glass petri dish or the like.

以下、本発明の細胞培養容器１００を用いて細胞を培養し、細胞の剥離性が向上した例を示す。   Hereinafter, an example in which cells are cultured using the cell culture container 100 of the present invention and cell detachability is improved will be described.

図１１は実施例１の手法を用いて本実施例で作製した細胞培養容器１００の形状例である。図１２は細胞培養容器１００上の突起物集合体１０１を構成する柱状微小突起物801の拡大図である。本実施例では培養した細胞の剥離性と突起物集合体１０１を構成する柱状微小突起物８０１の形状の関係を調べるために、柱状微小突起物８０１の直径Ｒを180ｎｍ，２４０ｎｍ，５００ｎｍ，２μｍの４通りとした４つの細胞培養容器１００を形成した。形成した柱状微小突起物８０１の高さＨは４つの細胞培養容器１００において全て３μｍである。柱状微小突起物８０１の配列は図１１，図１２に示すように２次元正方格子状であり、隣り合う柱状微小突起物８０１の間隔Ｄは直径Ｒの２倍である。   FIG. 11 shows an example of the shape of the cell culture container 100 produced in this example using the method of Example 1. FIG. 12 is an enlarged view of the columnar microprojections 801 constituting the projection aggregate 101 on the cell culture container 100. In this embodiment, in order to investigate the relationship between the peelability of cultured cells and the shape of the columnar microprojections 801 constituting the projection aggregate 101, the diameter R of the columnar microprojections 801 is 180 nm, 240 nm, 500 nm, and 2 μm. Four types of four cell culture vessels 100 were formed. The heights H of the formed columnar microprojections 801 are all 3 μm in the four cell culture containers 100. The arrangement of the columnar microprojections 801 is a two-dimensional square lattice as shown in FIGS. 11 and 12, and the interval D between adjacent columnar microprojections 801 is twice the diameter R.

剥離性の評価のために、それぞれの細胞培養容器１００上でヒト間葉系幹細胞(CAMBREX社、Cyro hMSC No.2051) を培地（CAMBREX社、ブレットキットMSCGM（間葉系幹細胞増殖用培地））中に播種し、３７℃・５％ＣＯ2 下のインキュータ内で５日間培養した。比較のために一般的に用いられる動物細胞培養用ディッシュ（Corning社）においても同様の条件でヒト間葉系幹細胞を培養した。 For evaluation of exfoliation property, human mesenchymal stem cells (CAMBREX, Cyro hMSC No.2051) were cultured on each cell culture vessel 100 (CAMBREX, Brett kit MSCGM (medium for stem cell proliferation)) The seeds were sown and cultured in an incubator at 37 ° C. and 5% CO 2 for 5 days. Human mesenchymal stem cells were cultured under the same conditions in animal cell culture dishes (Corning) generally used for comparison.

剥離性は、５日間培養後の細胞培養容器１００上のヒト間葉系幹細胞を培地中に発生させた水流によって剥離することで評価した。水流は２５０μｌ用電動ピペット（EDP plusEP-250）に容量２００μｌのピペットチップを装着して、細胞培養容器１００の底面近傍から底面に対して７０度の角度で培養に用いている培地を吐出することによって発生させた。   The detachability was evaluated by detaching human mesenchymal stem cells on the cell culture vessel 100 after culturing for 5 days by a water flow generated in the medium. For the water flow, attach a pipette tip with a capacity of 200 μl to an electric pipette for 250 μl (EDP plusEP-250), and discharge the medium used for culture at an angle of 70 degrees from the bottom of the cell culture vessel 100 to the bottom. Generated by.

顕微鏡観察下で、培地を１回吐出して細胞培養容器１００上の細胞の剥がれを観察した結果を表１に示す。本発明で形成したいずれの細胞培養容器１００においても、比較例として用いた動物細胞培養用ディッシュと比較してヒト間葉系幹細胞の剥離性が増すことが確認された。細胞培養容器１００からのヒト間葉系幹細胞の剥離性は柱状微小突起物801の直径Ｒに依存し、直径が大きいＲ＝２μｍの時に剥離能は最も大きくなることが示された。このことから、接着性細胞であるヒト間葉系幹細胞の接着力は柱状微小突起物８０１上では小さくなり、剥離性に優れることが示された。   Table 1 shows the results of observing the detachment of the cells on the cell culture container 100 by discharging the medium once under the microscope observation. In any cell culture vessel 100 formed in the present invention, it was confirmed that the peelability of human mesenchymal stem cells was increased as compared to the animal cell culture dish used as a comparative example. It was shown that the peelability of human mesenchymal stem cells from the cell culture vessel 100 depends on the diameter R of the columnar microprojections 801, and that the peelability is greatest when the diameter is large R = 2 μm. From this, it was shown that the adhesive force of human mesenchymal stem cells, which are adhesive cells, is reduced on the columnar microprojections 801 and is excellent in peelability.

Figure 0004507845
Figure 0004507845

本発明による細胞培養容器の一例を示す模式図。The schematic diagram which shows an example of the cell culture container by this invention. 本発明による細胞培養容器の別の一例を示す模式図。The schematic diagram which shows another example of the cell culture container by this invention. 本発明による細胞培養容器の第３の例を示す模式図。The schematic diagram which shows the 3rd example of the cell culture container by this invention. 細胞培養容器表面に突起群を形成する手法を示す模式図。The schematic diagram which shows the method of forming a processus | protrusion group on the cell culture container surface. 突起群の先端のみへの表面修飾法を示す模式図。The schematic diagram which shows the surface modification method only to the front-end | tip of a processus | protrusion group. 突起群先端部への処理の分布を形成する修飾法を示す模式図。The schematic diagram which shows the modification method which forms distribution of the process to a processus | protrusion group front-end | tip part. 本発明における細胞培養容器の使用法を示す模式図。The schematic diagram which shows the usage method of the cell culture container in this invention. 第１の実施例において作製した突起物集合体の走査型電子顕微鏡写真の模式図。The schematic diagram of the scanning electron micrograph of the protrusion assembly produced in the first example. 突起物集合体を拡大した走査型電子顕微鏡写真を示す模式図。The schematic diagram which shows the scanning electron micrograph which expanded the protrusion aggregate | assembly. 柱状微小突起物の製造工程を示す模式図。The schematic diagram which shows the manufacturing process of a columnar microprotrusion. 第４の実施例において作製した細胞培養容器の一例を示す模式図。The schematic diagram which shows an example of the cell culture container produced in the 4th Example. 第４の実施例において作製した細胞培養容器上の柱状微小突起物の拡大図を示す模式図。The schematic diagram which shows the enlarged view of the columnar microprotrusion on the cell culture container produced in the 4th Example.

符号の説明Explanation of symbols

１００…細胞培養容器、１０１…突起物集合体、１０２，５０１…表面処理、４０１…容器、４０２…金型、４０３…金型の形状、５０２…スタンプ、５０３…表面処理剤、
７０１…培養液、７０２…細胞、８０１…柱状微小突起物、８０２…下地。



DESCRIPTION OF SYMBOLS 100 ... Cell culture container, 101 ... Protrusion assembly, 102,501 ... Surface treatment, 401 ... Container, 402 ... Mold, 403 ... Mold shape, 502 ... Stamp, 503 ... Surface treatment agent,
701 ... Culture medium, 702 ... Cells, 801 ... Columnar microprojections, 802 ... Base.



Claims (7)

細胞培養容器において、該細胞培養容器の底面に形成された、相当直径が１０ｎｍ以上１０μｍ以下であり、高さが１０ｎｍ以上１mm以下である突起群を有し、
前記細胞培養容器の底面に形成された突起群の相当直径が、前記細胞培養容器内で培養する細胞の直径の５分の１以下であり、且つ、前記突起群を構成する隣り合う突起物の間隔が前記細胞培養容器内で培養する細胞の直径よりも小さいことを特徴とする細胞培養容器。 A cell culture container, having a projection group formed on the bottom surface of the cell culture container, having an equivalent diameter of 10 nm to 10 μm and a height of 10 nm to 1 mm;
Equivalent diameter projection group formed on the bottom surface of the cell culture vessel, wherein 1 or less in 5 minutes of the diameter of the cells to be cultured in a cell culture vessel, and, of the projection adjacent constituting the projection group cell culture vessels gap being less than the diameter of the cells to be cultured in the cell culture vessel. 請求項１に記載の細胞培養容器において、前記細胞培養容器と前記突起群が同一材料であり、一体化されていることを特徴とする細胞培養容器。   The cell culture container according to claim 1, wherein the cell culture container and the projection group are made of the same material and are integrated. 請求項１に記載の細胞培養容器において、前記細胞培養容器の全体、若しくはその一部に、親水処理，疎水化処理，金属層の形成、又は、有機物質の被覆のいずれか１つ以上の処理が施されていることを特徴とする細胞培養容器。   The cell culture container according to claim 1, wherein the whole or a part of the cell culture container is subjected to any one or more treatments of hydrophilic treatment, hydrophobization treatment, formation of a metal layer, or coating of an organic substance. A cell culture vessel characterized by that. 請求項１に記載の細胞培養容器において、特定の領域に選択的に親水処理，疎水化処理，金属層の形成、又は、有機物質の被覆のいずれかの１つ以上の処理が施されていることを特徴とする細胞培養容器。   The cell culture container according to claim 1, wherein one or more treatments of hydrophilic treatment, hydrophobization treatment, formation of a metal layer, or coating of an organic substance are selectively applied to a specific region. A cell culture container characterized by the above. 請求項１に記載の細胞培養容器において、前記突起群を構成する個々の突起物に対して選択的に親水処理，疎水化処理，金属層の形成、又は有機物質の被覆のいずれか１つ以上の処理が施されていることを特徴とする細胞培養容器。   2. The cell culture container according to claim 1, wherein one or more of hydrophilic treatment, hydrophobization treatment, formation of a metal layer, or coating of an organic substance is selectively applied to individual protrusions constituting the protrusion group. The cell culture container characterized by the above-mentioned process. 請求項１に記載の細胞培養容器において、前記突起群の先端部のみに親水処理，疎水化処理，金属層の形成、または有機物質の被覆が施されていることを特徴とする細胞培養容器。   2. The cell culture container according to claim 1, wherein only the tip of the projection group is subjected to a hydrophilic treatment, a hydrophobic treatment, a metal layer formation, or an organic substance coating. 3. 請求項１に記載の細胞培養容器において、前記突起群が細胞培養容器表面に液体を通すことの出来る列状に空いた領域を有することを特徴とする細胞培養容器。   2. The cell culture container according to claim 1, wherein the projection group has a row of vacant regions through which a liquid can pass through the surface of the cell culture container.
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