JPH06153905A - Cell culture base and method for cell culture - Google Patents
Cell culture base and method for cell cultureInfo
- Publication number
- JPH06153905A JPH06153905A JP4338163A JP33816392A JPH06153905A JP H06153905 A JPH06153905 A JP H06153905A JP 4338163 A JP4338163 A JP 4338163A JP 33816392 A JP33816392 A JP 33816392A JP H06153905 A JPH06153905 A JP H06153905A
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- Japan
- Prior art keywords
- cell
- cells
- cell culture
- culture
- substrate
- Prior art date
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は細胞培養方法および細胞
培養基材に関するものである。TECHNICAL FIELD The present invention relates to a cell culture method and a cell culture substrate.
【0002】[0002]
【従来の技術】人工臓器などバイオテクノロジー分野の
急速な発展に伴い、生体内の細胞が有する機能を生体外
で培養した細胞に発現させ長期にわたり維持することが
緊急な要件となっている。細胞培養基材および細胞培養
方法には、従来、表面がコラーゲンなどの接着性蛋白質
からなる細胞培養基材等を用い、増殖とともに単層を形
成させる単層培養方法などが良く知られている。2. Description of the Related Art With the rapid development of biotechnology fields such as artificial organs, it is an urgent requirement to express the function of cells in vivo in cells cultured in vitro and maintain it for a long period of time. Conventionally well-known cell culture substrates and cell culture methods include a monolayer culture method in which a cell culture substrate having a surface made of an adhesive protein such as collagen is used to form a monolayer with proliferation.
【0003】また、近年、組織を生体外で再構築するこ
とにより、細胞の機能発現を試みようという考え方のも
と、陰性荷電を持たないプラッチック製の培養容器中
で、細胞接着のための基質及び細胞外間質物質を添加せ
ずに、無血清培地を用いて培養し、球状に集合して浮遊
した細胞凝集塊塊を形成させることを特徴とする肝細胞
の球状培養方法(特開平1−277486号公報)やポ
リ−N−p−ビニルベンジル−D−ラクトンアミドを合
成後、この水溶液をコーティングして、無血清培養液を
用いて肝実質細胞を培養することで、高機能発現性と長
期生存性を有する細胞凝集塊を形成し培養する方法(小
林一清ら、人工臓器第19巻,1156−1160頁,
1990年)などの三次元培養方法が提案されている。Further, in recent years, under the concept of attempting to express the function of cells by reconstructing tissues in vitro, a substrate for cell adhesion is provided in a culture container made of plastic which does not have negative charge. And a method for culturing hepatocytes in a spherical shape, which comprises culturing in a serum-free medium without adding an extracellular stromal substance to form spherical aggregates of floating cells that are aggregated (Japanese Patent Application Laid-Open No. HEI-1) No. 277486) or poly-N-p-vinylbenzyl-D-lactonamide, and then coated with this aqueous solution and culturing hepatocytes using a serum-free culture medium to obtain high functional expression. And a method for forming and culturing cell aggregates having long-term viability (Kazubayashi Kobayashi et al., Artificial Organ Vol. 19, pp. 1156-1160,
Three-dimensional culture methods such as 1990) have been proposed.
【0004】[0004]
【発明が解決しようとする課題】しかしながら、接着性
蛋白質からなる表面での単層培養法においては、生体内
で有していた細胞機能を維持することは困難であり、細
胞は生存、増殖はするものの、急速に脱分化して機能を
失ってしまう。また接着性蛋白質は高価で、かつその安
定性にも課題を有している。However, in the monolayer culture method on the surface composed of the adhesive protein, it is difficult to maintain the cell function possessed in the living body, and the cells survive and proliferate. However, it rapidly dedifferentiates and loses its function. Further, the adhesive protein is expensive and has a problem in its stability.
【0005】特開平1−277486号公報の培養方法
においては、浮遊した細胞凝集塊は、生体内での細胞の
機能を長期にわたって維持する可能性を有しているにも
かかわらず、浮遊細胞凝集塊同士が凝集して大きな塊と
なり、短期間の内に内部細胞が壊死するという課題を有
していた。またポリ−N−p−ビニルベンジル−D−ラ
クトンアミド水溶液をコーティングした表面で肝実質細
胞を培養する方法においても、細胞凝集塊は細胞培養基
材表面から剥離しやすく、前記と同様の課題を有してい
る。またポリ−N−p−ビニルベンジル−D−ラクトン
アミドの合成は非常に煩雑であるという課題も有してい
る。In the culturing method of Japanese Patent Application Laid-Open No. 1-277486, suspended cell aggregates have the potential to maintain the function of cells in vivo for a long period of time, even though the suspended cell aggregates have a possibility of being suspended. There was a problem that the clumps aggregated into a large clump and the inner cells were necrotic within a short period of time. Also in the method of culturing hepatocytes on the surface coated with an aqueous poly-N-p-vinylbenzyl-D-lactonamide solution, the cell aggregates are easily separated from the surface of the cell culture substrate, and the same problems as described above are encountered. Have There is also a problem that the synthesis of poly-Np-vinylbenzyl-D-lactone amide is very complicated.
【0006】[0006]
【課題を解決するための手段】本発明者は、長期にわた
って脱分化を起こさない細胞凝集塊を形成し、浮遊細胞
凝集塊同士の凝集による内部細胞の壊死をなくし、細胞
が有する機能を生体外で培養した細胞に発現させ長期に
わたり維持することを可能とする、作成が容易で安価で
あり、安定性の高い細胞培養基材;ならびに細胞培養方
法について鋭意検討した結果、基材表面への細胞の接着
力を、安価なビニル系共重合体を用い表面状態を変化さ
せて調節することで、特定組成のビニル系共重合体表面
のみが、細胞凝集塊を形成し、かつ、その細胞凝集塊の
剥離がないことを見いだし、本発明に到達した。Means for Solving the Problems The present inventor has formed a cell aggregate that does not undergo dedifferentiation for a long period of time, eliminates necrosis of internal cells due to aggregation of floating cell aggregates, and has the function of cells in vitro. A cell culture substrate that is easy to make, inexpensive, and highly stable, and that can be expressed in cells cultured in E. coli and maintained for a long period of time; By controlling the adhesive force of the vinyl copolymer by using an inexpensive vinyl copolymer and changing the surface condition, only the vinyl copolymer surface with a specific composition forms cell aggregates and the cell aggregates are formed. It was found that there was no peeling of the above, and the present invention was reached.
【0007】すなわち本発明は、三次元培養方法におい
て、基材表面が、下記一般式(1)で表される重合性モ
ノマー(a)を共重合させたビニル系共重合体(A)か
らなる、細胞凝集塊を形成し、かつ、該表面に細胞凝集
塊が接着維持することを特徴とする動物細胞培養基材
(I);並びに該動物細胞培養基材表面(I)に細胞凝
集塊を接着維持して培養することを特徴とする動物細胞
培養方法である。 (但し、R1は水素原子またはメチル基、R2は炭素数1
〜4の直鎖あるいは分岐のアルキレン基)That is, according to the present invention, in the three-dimensional culture method, the surface of the substrate is composed of a vinyl-based copolymer (A) obtained by copolymerizing a polymerizable monomer (a) represented by the following general formula (1). An animal cell culture substrate (I), characterized by forming a cell aggregate and maintaining the cell aggregate adherent to the surface; and a cell aggregate on the animal cell culture substrate surface (I) An animal cell culture method characterized by culturing while maintaining adhesion. (However, R 1 is a hydrogen atom or a methyl group, R 2 is a carbon atom 1
4 straight or branched alkylene groups)
【0008】本発明の三次元培養方法とは、充填型バイ
オリアクターで多用される、多孔質基材の内部に細胞を
保持して立体的に高密度に培養する方法ではなく、細胞
同士が密接に接着した細胞凝集塊を形成して培養を行な
う方法である。以下、本発明の細胞培養基材について詳
細に説明する。重合性モノマー(a)としては、2−ヒ
ドロキシメチルメタクリレート、2−ヒドロキシエチル
メタクリレート、2−ヒドロキシエチルアクリレート、
2−ヒドロキシイソプロピルメタクリレート等が挙げら
れる。特に好ましくは、2−ヒドロキシメチルメタクリ
レート、2−ヒドロキシエチルメタクリレートである。The three-dimensional culturing method of the present invention is not a method which is frequently used in a packed bioreactor, in which cells are held inside a porous substrate and cultivated in a three-dimensional high density. It is a method of culturing by forming cell aggregates adhered to the. Hereinafter, the cell culture substrate of the present invention will be described in detail. As the polymerizable monomer (a), 2-hydroxymethyl methacrylate, 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate,
2-hydroxy isopropyl methacrylate etc. are mentioned. Particularly preferred are 2-hydroxymethyl methacrylate and 2-hydroxyethyl methacrylate.
【0009】重合性モノマー(a)を共重合させたビニ
ル系共重合体(A)としては、特に限定されないが、例
えば、メチルメタクリレート、エチルメタクリレート、
ジメチルアミノエチルメタクリレート、ジエチルアミノ
エチルメタクリレート、アクリル酸、アクリル酸メチ
ル、メタクリル酸、メタクリル酸メチル、スチレン等の
モノマーと共重合して得られるビニル系共重合体(A)
等が挙げられる。上記モノマー1種類との共重合体でも
良いが、1種類以上のモノマーと共重合して得られるビ
ニル系共重合体(A)でも良い。またランダムあるいは
ブロック共重合どちらの重合様式でもかまわない。The vinyl copolymer (A) obtained by copolymerizing the polymerizable monomer (a) is not particularly limited, but for example, methyl methacrylate, ethyl methacrylate,
Vinyl-based copolymer (A) obtained by copolymerizing with monomers such as dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, acrylic acid, methyl acrylate, methacrylic acid, methyl methacrylate and styrene.
Etc. It may be a copolymer with one kind of the above-mentioned monomer, or may be a vinyl-based copolymer (A) obtained by copolymerizing with one or more kinds of monomers. Further, either random or block copolymerization may be used.
【0010】細胞凝集塊を形成し、かつ、その細胞凝集
塊を接着維持させることで、生体内の細胞が有する機能
を生体外で培養した細胞に発現させ長期にわたり維持す
る培養を行なうためには、ビニル系共重合体(A)に対
して重合性モノマー(a)の含有率が10〜90モル%
の範囲内でなければならない。重合性モノマー(a)の
含有率が10モル%未満であるなら、細胞は共重合体表
面に接着して単層を形成し、細胞凝集塊を形成すること
なく、生体内と全く異なる偏平な細胞形態となる。この
偏平な細胞形態では、生存、増殖はするものの急速に脱
分化して、生体内で本来細胞が持つ機能を生体外で長期
間にわたって発現し維持することができない。ビニル系
共重合体(A)に対して重合性モノマー(a)の含有率
が90モル%より高い場合は、細胞凝集塊は形成される
が、細胞凝集塊はビニル系共重合体表面に接着維持する
ことができず、浮遊し、細胞凝集塊同士が凝集して大き
な塊を形成し、内部細胞の壊死で短期間のうちに細胞が
死滅してしまう。To form a cell aggregate and to maintain the adhesion of the cell aggregate by adhering and maintaining the cell aggregate, the function of the cell in the living body can be expressed in the cell cultured in vitro and the culture can be performed for a long period of time. , The content of the polymerizable monomer (a) with respect to the vinyl-based copolymer (A) is 10 to 90 mol%
Must be within the range. When the content of the polymerizable monomer (a) is less than 10 mol%, the cells adhere to the surface of the copolymer to form a monolayer, and form a cell aggregate without forming a flat surface completely different from that in the living body. It becomes a cell morphology. In this flat cell morphology, although it survives and proliferates, it is rapidly dedifferentiated, and the function originally possessed by cells in vivo cannot be expressed and maintained in vitro for a long period of time. When the content of the polymerizable monomer (a) is higher than 90 mol% based on the vinyl copolymer (A), cell aggregates are formed, but the cell aggregates adhere to the vinyl copolymer surface. It cannot be maintained and floats, cell aggregates aggregate to form a large aggregate, and the cells die in a short period due to necrosis of the inner cells.
【0011】ビニル系共重合体(A)の重合方法は、有
機希釈剤、重合開始剤の存在下、溶液重合する方法より
実施できる。有機希釈剤としては、水酸基などの官能基
に対して不活性であれば特に限定されないが、通常はト
ルエンなどが用いられる。有機希釈剤のモノマーに対す
る添加量は、全モノマー成分に対して0.5〜20重量
倍であることが好ましい。また、重合開始剤としては、
過酸化ベンゾイル、過酸化ラウロイル、アゾビスイソブ
チロニトリル、アゾビスイソバレロニトリルなどが用い
られ、通常その使用量は、全モノマーに対して0.01
〜5.0重量%の範囲である。好ましくは0.01〜
1.0重量%の濃度範囲から選ばれる。The vinyl copolymer (A) can be polymerized by solution polymerization in the presence of an organic diluent and a polymerization initiator. The organic diluent is not particularly limited as long as it is inactive to a functional group such as a hydroxyl group, but toluene or the like is usually used. The amount of the organic diluent added to the monomer is preferably 0.5 to 20 times by weight the total amount of the monomer components. Further, as the polymerization initiator,
Benzoyl peroxide, lauroyl peroxide, azobisisobutyronitrile, azobisisovaleronitrile and the like are used, and the amount thereof is usually 0.01
Is in the range of 5.0% by weight. Preferably 0.01-
It is selected from the concentration range of 1.0% by weight.
【0012】本発明における細胞培養基材(I)は、該
ビニル系共重合体(A)を種々の形状の材料にコーティ
ングしたり、あるいは該ビニル系共重合体(A)を種々
の材料に成形することで作成できる。コーティングする
材料としては、例えば、合成高分子材料、天然高分子材
料および無機材料があげられる。The cell culture substrate (I) in the present invention is obtained by coating the vinyl-based copolymer (A) on various shaped materials or by coating the vinyl-based copolymer (A) on various materials. It can be created by molding. Examples of the material to be coated include synthetic polymer materials, natural polymer materials and inorganic materials.
【0013】合成高分子材料としては、例えば、ポリエ
チレン、ポリプロピレン、塩素化ポリエチレン、アイオ
ノマーなどのオレフィン系重合体,ポリテトラフルオロ
エチレン、ポリフッ化ビニリデンなどのフッ素系樹脂,
ポリスチレンなどのスチレン系樹脂、ポリメチルメタク
リレートなどのアクリル系樹脂,ポリビニルアルコー
ル、ポリ酢酸ビニル、ポリビニルアセタール、ポリアク
リロニトリル、ポリ塩化ビニル、ポリ塩化ビニリデン、
ポリカーボネイト、ポリアクリレート、ポリフェニレン
オキサイド、ポリエチレンテレフタレート、ポリブチレ
ンテレフタレートなどのポリエステル樹脂,エポキシ樹
脂,ポリアミド,ポリイミド,ポリスルホン,シリコー
ン樹脂,ポリウレタンなどの種々の重合体もしくは共重
合体またはそれらのブレンドが例示できる。Examples of the synthetic polymer material include olefin polymers such as polyethylene, polypropylene, chlorinated polyethylene and ionomer, fluorine resins such as polytetrafluoroethylene and polyvinylidene fluoride,
Styrene resins such as polystyrene, acrylic resins such as polymethylmethacrylate, polyvinyl alcohol, polyvinyl acetate, polyvinyl acetal, polyacrylonitrile, polyvinyl chloride, polyvinylidene chloride,
Examples include polyester resins such as polycarbonate, polyacrylate, polyphenylene oxide, polyethylene terephthalate and polybutylene terephthalate, various polymers or copolymers such as epoxy resin, polyamide, polyimide, polysulfone, silicone resin and polyurethane, or blends thereof.
【0014】天然高分子材料としては、例えば、セルロ
ース,セルロース誘導体,キトサン,キチンなどが例示
できる。Examples of natural polymer materials include cellulose, cellulose derivatives, chitosan and chitin.
【0015】無機材料としては、例えば、ガラス,セラ
ミックなどが例示できる。Examples of inorganic materials include glass and ceramics.
【0016】コーティングするものの形状あるいは成形
する形状としては、例えば、シャーレ、フラスコ、フィ
ルム、チューブ、繊維、微粒子、不織布、多孔質フォー
ム、多孔質ビーズ状、中空糸などが例示できる。これら
の形状のうち、細胞の機能を高度に維持して高密度で大
量に培養するためには、高密度大量培養装置への適用が
容易な中空糸、多孔質ビーズがなどの多孔質材料が好ま
しい。また小スケールの場合は、その扱い易さの観点か
ら、シャーレ、ボトルなどの形状が好ましい。コーティ
ングする場合、その膜厚は、表面特性が共重合体の性質
に変化し、かつ、耐久性を有していれば特に限定されな
い。Examples of the shape of the material to be coated or the shape to be molded include petri dishes, flasks, films, tubes, fibers, fine particles, non-woven fabrics, porous foams, porous beads, hollow fibers and the like. Among these shapes, porous materials such as hollow fibers and porous beads that can be easily applied to high-density mass-culturing devices are used in order to maintain high cell function and perform high-density mass-culturing. preferable. In the case of a small scale, the shape of a petri dish, a bottle, etc. is preferable from the viewpoint of easy handling. In the case of coating, the film thickness is not particularly limited as long as the surface characteristics are changed to the properties of the copolymer and the coating has durability.
【0017】次に、本発明の細胞培養方法について詳細
に説明する。本発明の細胞培養方法は、細胞を、本発明
の細胞培養基材上に播種することで行える。細胞播種
後、細胞は立体的に高密度に凝集した細胞凝集塊を形成
し、細胞培養基材表面に接着維持する。Next, the cell culture method of the present invention will be described in detail. The cell culture method of the present invention can be performed by seeding cells on the cell culture substrate of the present invention. After the cell seeding, the cells form a three-dimensionally densely aggregated cell aggregate, and adhere and maintain the cell culture substrate surface.
【0018】培養する細胞としては動物細胞のうち、生
体内で細胞が有する機能を生体外で高度に発現、維持す
ることが困難な初代培養系の細胞や、培養は困難ではな
いが、生体内で細胞が本来持つ機能を生体外で高度に発
現させることが望まれる株化細胞などがあげられる。The cells to be cultured include, among animal cells, cells of a primary culture system in which it is difficult to highly express and maintain the functions of the cells in vivo in vitro, and cells in vivo that are not difficult to culture. The cell line and the like in which it is desired to highly express the function originally possessed by the cell in vitro are mentioned.
【0019】初代細胞系の細胞としては、例えば、肝実
質細胞、乳腺上皮細胞、膵島細胞、骨芽細胞、破骨細
胞、軟骨細胞、尿細管上皮細胞、糸球体細胞、血管内皮
細胞、ランゲルハンス細胞、胆管上皮細胞、皮膚線維芽
細胞などの各種線維芽細胞などがあげられる。これらが
癌化した細胞であってもかまわない。株化細胞として
は、例えば、肝癌由来の細胞株(HepG2、Huh−
3など)、膵臓癌由来の細胞株などの各種癌細胞由来の
株化細胞、正常皮膚由来の線維芽細胞株、正常血管内皮
由来の正常血管内皮細胞株などの各種正常細胞由来の株
化細胞などが例示できる。これら細胞を1種類で培養し
ても良いが、1種類以上の細胞を同時に培養しても良
い。この場合の混合比率は適宜変えることができるが、
組合せは初代細胞系の細胞だけに限られず、株化細胞も
自由に選択し利用できる。Examples of the cells of the primary cell line include hepatocytes, mammary epithelial cells, pancreatic islet cells, osteoblasts, osteoclasts, chondrocytes, renal tubular epithelial cells, glomerular cells, vascular endothelial cells, Langerhans cells. , Various types of fibroblasts such as bile duct epithelial cells and skin fibroblasts. These may be cancerous cells. Examples of the established cell lines include cell lines derived from liver cancer (HepG2, Huh-).
3), cell lines derived from various cancer cells such as cell lines derived from pancreatic cancer, fibroblast cell lines derived from normal skin, cell lines derived from various normal cells such as normal vascular endothelial cell lines derived from normal vascular endothelium Can be exemplified. These cells may be cultivated by one kind, but one or more kinds of cells may be cultivated at the same time. The mixing ratio in this case can be appropriately changed,
The combination is not limited to cells of the primary cell line, and cell lines can be freely selected and used.
【0020】培養液としては、培養する細胞の種類に応
じて種々の培養液が用いられる。具体的には、ダルベッ
コ改変イーグル培養液にインシュリン(10μg/m
l)、上皮細胞成長因子(10ng/ml)、アスコル
ビン酸(20μg/ml)を添加し、水素イオン濃度を
10-7mol/lに調整した無血清培養液などが例示で
きる。また、血清を添加しない培養液で培養を開始した
後、血清を添加した培養液に切り替え培養を行なうとい
う培養液の使い方なども例示できる。As the culture solution, various culture solutions are used depending on the type of cells to be cultured. Specifically, insulin (10 μg / m 2) was added to Dulbecco's modified Eagle medium.
l), epidermal growth factor (10 ng / ml), and ascorbic acid (20 μg / ml) are added to adjust the hydrogen ion concentration to 10 −7 mol / l. In addition, a method of using the culture medium may be exemplified, in which after the culture is started in the culture medium to which the serum is not added, the culture medium is switched to the culture medium to which the serum is added to perform the culture.
【0021】培養環境は細胞の機能維持に適した温度、
炭酸ガス濃度などの条件で培養が行える。例えば、温度
37℃、湿度100%、炭酸ガス濃度5%の雰囲気中で
の培養などが例示できる。The culture environment is a temperature suitable for maintaining the function of cells,
Culture can be performed under conditions such as carbon dioxide concentration. For example, culturing in an atmosphere having a temperature of 37 ° C., a humidity of 100% and a carbon dioxide gas concentration of 5% can be exemplified.
【0022】この細胞培養方法および細胞培養用基材の
用途としては、生体内の細胞が有する機能を生体外で培
養した細胞に発現させ、長期間にわたり維持することが
できるため、例えば、動物実験の代替として化学物質の
毒性判断、有用物質の探索、細胞機能解明および病理組
織から得た細胞を培養し、生体外で各種薬剤の効果をス
クリーニングし治療に役立てる等の使用方法を目的とし
た細胞培養キット,細胞が産生する有用物質の大量生
産,ハイブリット人工臓器の構築などに利用できる。The cell culture method and the cell culture substrate can be used, for example, in animal experiments because the functions of cells in vivo can be expressed in cells cultured in vitro and maintained for a long period of time. As a substitute for cells, cells for the purpose of usage such as judgment of toxicity of chemical substances, search for useful substances, elucidation of cell function, culturing cells obtained from pathological tissues, screening effects of various drugs in vitro and utilizing for treatment It can be used for culture kits, mass production of useful substances produced by cells, construction of hybrid artificial organs, etc.
【0023】[0023]
【実施例】以下本発明を実施例により具体的に示すが、
本発明はこれらの実施例に限定されるものではない。EXAMPLES The present invention will be specifically described below with reference to Examples.
The invention is not limited to these examples.
【0024】基材作成例1 2−ヒドロキシエチルメタクリレート10.4部、ジメ
チルアミノエチルメタタクリレート3.1部、トルエン
121.5部、アゾビスイソブチロニトリル0.07部
を仕込み、65℃、8時間重合を行なった。析出した共
重合体をトルエンで洗浄後、乾燥した。NMRによる分
析の結果、2−ヒドロキシエチルメタクリレートを80
モル%を含有するビニル系共重合体を得た。Example 1 of base material preparation 10.4 parts of 2-hydroxyethyl methacrylate, 3.1 parts of dimethylaminoethyl methacrylate, 121.5 parts of toluene and 0.07 parts of azobisisobutyronitrile were charged and the temperature was 65 ° C. Polymerization was carried out for 8 hours. The precipitated copolymer was washed with toluene and then dried. As a result of NMR analysis, 80% of 2-hydroxyethyl methacrylate was obtained.
A vinyl-based copolymer containing mol% was obtained.
【0025】次に、ビニル系共重合体をジメチルホルム
アミドに2.0重量%に溶解して直径50mmの円形の
ガラス板にスピンコーター(スピンコーター1H−DX
ミカサ(株))でスピンコーティング(2000rp
m,80秒)し細胞培養基材1を得た。Next, the vinyl-based copolymer was dissolved in dimethylformamide at 2.0% by weight, and a spin coater (spin coater 1H-DX was used on a circular glass plate having a diameter of 50 mm).
Spin coating (2000rp) by Mikasa Co., Ltd.
m, 80 seconds) to obtain a cell culture substrate 1.
【0026】基材作成例2 2−ヒドロキシメチルメタクリレート2.3部、メチル
メタクリレート8.0部をトルエン92.7部、アゾビ
スイソブチロニトリル0.05部を仕込むこと以外は基
材作成例1と同様に行なった。NMRによる分析の結
果、2−ヒドロキシメチルメタクリレートを20モル%
含有するビニル系共重合体を得た。次に、基材作成例1
と同様にスピンコートを行い細胞培養基材2を得た。Substrate preparation example 2 Substrate preparation example except that 2.3 parts of 2-hydroxymethyl methacrylate, 8.0 parts of methyl methacrylate and 92.7 parts of toluene and 0.05 parts of azobisisobutyronitrile were charged. The same procedure as in 1 was performed. As a result of analysis by NMR, 20 mol% of 2-hydroxymethyl methacrylate was obtained.
The contained vinyl-based copolymer was obtained. Next, base material preparation example 1
Spin coating was performed in the same manner as above to obtain a cell culture substrate 2.
【0027】比較基材作成例1 2−ヒドロキシエチルメタクリレート10部、トルエン
90部、アゾビスイソブチロニトリル0.05部を仕込
むこと以外は基材作成例1と同様に行った。得られたポ
リヒドロキシエチルメタクリレートを基材作成例1と同
様にスピンコートを行い細胞培養基材3を得た。Comparative Substrate Preparation Example 1 The same procedure as in Substrate Preparation Example 1 was repeated except that 10 parts of 2-hydroxyethyl methacrylate, 90 parts of toluene and 0.05 part of azobisisobutyronitrile were charged. The obtained polyhydroxyethyl methacrylate was spin-coated in the same manner as in the substrate preparation example 1 to obtain a cell culture substrate 3.
【0028】比較基材作成例2 メチルメタクリレート10部をトルエン90部、アゾビ
スイソブチロニトリル0.05部を仕込むこと以外は基
材作成例1と同様に重合を行った後、メタノールで再沈
精製した。得られたポリメチルメタクリレートを基材作
成例1と同様にスピンコートし、細胞培養基材4を得
た。Comparative Base Material Preparation Example 2 Polymerization was carried out in the same manner as in Base Material Preparation Example 1 except that 10 parts of methyl methacrylate was charged with 90 parts of toluene and 0.05 part of azobisisobutyronitrile, and then re-used with methanol. It was purified by precipitation. The obtained polymethylmethacrylate was spin-coated in the same manner as in Substrate preparation example 1 to obtain a cell culture substrate 4.
【0029】比較基材作成例3 メチルメタクリレート10部、スチレン10部、トルエ
ン100部、アゾビスイソブチロニトリル0.1部を仕
込み、基材作成例1と同様に重合を行なった後、メタノ
ールで再沈精製し、メチルメタクリレートとスチレンの
共重合体を得た。次に、基材作成例1と同様にスピンコ
ートを行い細胞培養基材5を得た。Comparative Base Material Preparation Example 3 Methyl methacrylate (10 parts), styrene (10 parts), toluene (100 parts) and azobisisobutyronitrile (0.1 parts) were charged, polymerization was carried out in the same manner as in the base material preparation example 1, and then methanol was prepared. Was purified by reprecipitation, and a copolymer of methyl methacrylate and styrene was obtained. Then, spin coating was performed in the same manner as in Example 1 for preparing a substrate to obtain a cell culture substrate 5.
【0030】培養例1 細胞培養基材1を滅菌後、無菌的に直径60mmのポリ
スチレンシャーレに移し培養に供した。評価はラット肝
実質細胞の初代培養系を用いて行なった。肝実質細胞は
ウイスターラットから常法によりIn situコラゲ
ナーゼ灌流法により得た(初代培養肝細胞実験法、6〜
10頁、中村敏一著 学会出版センター)。肝実質細胞
は生存率98%以上のものを使用した。培養液は10%
血清含有培養液を用いた(基礎培養液はダルベッコ改変
イーグル培養液:インシュリン10μg/ml、上皮細
胞成長因子10ng/ml、アスコルビン酸20μg/
ml、プロリン20μg/ml、デキサメサゾン10-6
M添加)。Culture Example 1 After sterilizing the cell culture substrate 1, it was aseptically transferred to a polystyrene dish having a diameter of 60 mm and subjected to culture. The evaluation was performed using a primary culture system of rat liver parenchymal cells. Liver parenchymal cells were obtained from Wistar rats by a conventional in situ collagenase perfusion method (primary culture hepatocyte test method, 6-).
Page 10, Toshikazu Nakamura, Academic Publishing Center). The liver parenchymal cells used had a survival rate of 98% or more. Culture solution is 10%
Serum-containing medium was used (basal medium was Dulbecco's modified Eagle medium: insulin 10 μg / ml, epidermal growth factor 10 ng / ml, ascorbic acid 20 μg / ml)
ml, proline 20 μg / ml, dexamethasone 10 -6
M addition).
【0031】肝実質細胞は培養液1ml当り、5x10
5 個に細胞密度を調整した後、細胞培養基材1を移した
シャーレに細胞懸濁液を4ml播種して肝細胞培養を開
始した。培養開始24時間後、無血清培養液に切り替え
培養を継続した。培養液は24時間毎に4mlずつ全交
換した。Liver parenchymal cells were 5 × 10 5 per 1 ml of the culture medium.
After adjusting the cell density to 5 cells, 4 ml of the cell suspension was seeded on a petri dish to which the cell culture substrate 1 was transferred, and hepatocyte culture was started. Twenty-four hours after the start of culture, the culture was switched to a serum-free culture medium and continued to culture. The culture solution was completely replaced by 4 ml every 24 hours.
【0032】評価は、経日的に肝特異的機能であるアル
ブミン産生量をELISAで定量し、肝実質細胞 1x
106個が、24時間に産生するアルブミンの量で肝細
胞の生きの良さを評価した。また培養開始3日目に細胞
塊形成率[(細胞凝集塊形成に関与する細胞数÷播種し
た細胞)×100]を評価し、培養3日目及び20日目
に細胞凝集塊接着維持率[((形成された細胞凝集塊数
−剥離した総細胞凝集塊数)÷形成された細胞凝集塊
数)×100]を評価した。For the evaluation, the amount of albumin production, which is a liver-specific function, was quantified daily by ELISA, and 1x liver parenchymal cells were evaluated.
The viability of hepatocytes was evaluated by the amount of albumin produced by 10 6 cells in 24 hours. The cell aggregate formation rate [(the number of cells involved in cell aggregate formation / seeded cells) x 100] was evaluated 3 days after the start of culture, and the cell aggregate adhesion retention rate [on the 3rd and 20th days of culture] ((Number of formed cell aggregates-total number of detached cell aggregates) / number of formed cell aggregates) x 100] was evaluated.
【0033】培養例2 細胞培養基材2を用いること以外は、培養例1と同様の
方法で行なった。Culture Example 2 The same method as in Culture Example 1 was used except that the cell culture substrate 2 was used.
【0034】比較培養例1 細胞培養基材3を用いること以外は、培養例1と同様の
方法で行なった。Comparative Culture Example 1 The procedure of Culture Example 1 was repeated except that the cell culture substrate 3 was used.
【0035】比較培養例2 細胞培養基材4を用いること以外は、培養例1と同様の
方法で行なった。Comparative Culture Example 2 The procedure of Culture Example 1 was repeated except that the cell culture substrate 4 was used.
【0036】比較培養例3 細胞培養基材5を用いること以外は、培養例1と同様の
方法で行なった。Comparative Culture Example 3 The procedure of Culture Example 1 was repeated except that the cell culture substrate 5 was used.
【0037】比較培養例4 細胞培養基材として、陰性荷電を持たない市販のプライ
マリアデッシュ3802(直径60mm、ベクトン&デ
ィッキントン社製)用いること以外は、培養例1と同様
の方法で行なった。Comparative Culture Example 4 The same method as in Culture Example 1 was used except that a commercially available primary adish 3802 (60 mm in diameter, manufactured by Becton & Dickington) having no negative charge was used as the cell culture substrate. .
【0038】以上の培養例1、2および比較培養例1〜
4の評価結果を下記表1、表2に示す。The above-mentioned culture examples 1 and 2 and comparative culture examples 1-
The evaluation results of No. 4 are shown in Tables 1 and 2 below.
【0039】[0039]
【表1】 [Table 1]
【0040】(1):最大機能発現能→播種肝細胞数1
X106個の細胞当りに換算し、培養期間中で1日当り
最大のアルブミン産生量があった日の産生量 (μg) (2):機能発現能の維持期間→1X106個の細胞当
りに換算し、1日当り10μg以上の アルブミン産生
量が持続する培養日数 (日)(1): Ability to develop maximum function → number of seeded hepatocytes 1
Converted to X10 6 cells per day during the culture period
Amount of albumin produced on the day when the maximum amount of albumin was produced (μg) (2): Maintenance period of function expression ability → converted to per 1 × 10 6 cells, and the number of days of culture in which albumin production of 10 μg or more per day continues ( Day)
【0041】本発明の細胞培養基材を用いた培養方法で
は、最大機能発現能が比較例に比べ、約1.5倍高く、
機能維持期間も大幅に延長した。In the culture method using the cell culture substrate of the present invention, the maximum function expressing ability is about 1.5 times higher than that of the comparative example.
The function maintenance period has also been significantly extended.
【0042】[0042]
【表2】 [Table 2]
【0043】(1):細胞塊形成率=(細胞凝集塊形成
に関与する細胞数÷播種した細胞)×100 (2):細胞凝集塊接着維持率=((形成された細胞凝
集塊数−剥離した総細胞凝集塊数)÷形成された細胞凝
集塊数)×100(1): Cell aggregate formation rate = (number of cells involved in cell aggregate formation / seeded cells) × 100 (2): Cell aggregate adhesion retention rate = ((number of formed cell aggregates− Total number of detached cell aggregates) / number of formed cell aggregates) x 100
【0044】本発明の細胞培養基材を用いた培養方法で
は、播種細胞に対して、細胞凝集塊をほぼ100%形成
し、かつ、培養20日が経過しても約80%の細胞が細
胞培養基材表面に接着維持している。一方、細胞培養基
材3では細胞凝集塊を形成するものの、培養3日目で約
10%の細胞凝集塊しか細胞培養基材表面に接着維持し
ていない。その他の比較例の細胞培養基材においては、
細胞凝集塊を形成することができず、単層に細胞が接着
した。In the culture method using the cell culture substrate of the present invention, almost 100% of cell aggregates are formed in the seeded cells, and about 80% of the cells are formed even after 20 days of culture. Adhesion is maintained on the surface of the culture substrate. On the other hand, although cell aggregates are formed on the cell culture substrate 3, only about 10% of the cell aggregates are adhered and maintained on the surface of the cell culture substrate on day 3 of culture. In other comparative cell culture substrates,
Cell aggregates could not be formed and cells adhered to the monolayer.
【0045】[0045]
【発明の効果】本肝細胞培養基材および細胞培養方法
は、生体内の細胞が有する機能を生体外で培養した細胞
に発現させ、長期間にわたり維持することができるた
め、用途としては、例えば、動物実験の代替として化学
物質の毒性判断、有用物質の探索、細胞機能解明および
病理組織から得た細胞を培養し、生体外で各種薬剤の効
果をスクリーニングし治療に役立てる等の使用方法を目
的とした細胞培養キット,細胞が産生する有用物質の大
量生産,ハイブリット人工臓器の構築などに利用でき
る。INDUSTRIAL APPLICABILITY The present hepatocyte culturing substrate and cell culturing method allow the function of cells in vivo to be expressed in cells cultured in vitro and maintained for a long period of time. , As an alternative to animal experiments, to determine the toxicity of chemical substances, search for useful substances, elucidate cell functions, cultivate cells obtained from pathological tissues, screen the effects of various drugs in vitro, and use them for therapeutic purposes. The cell culture kit described above can be used for mass production of useful substances produced by cells, construction of hybrid artificial organs, and the like.
Claims (3)
下記一般式(1)で表される重合性モノマー(a)を共
重合させたビニル系共重合体(A)からなることを特徴
とする動物細胞培養基材(I)。 (但し、R1は水素原子またはメチル基、R2は炭素数1
〜4の直鎖あるいは分岐のアルキレン基。)1. A three-dimensional culture method, wherein the substrate surface is
An animal cell culture substrate (I) comprising a vinyl copolymer (A) obtained by copolymerizing a polymerizable monomer (a) represented by the following general formula (1). (However, R 1 is a hydrogen atom or a methyl group, R 2 is a carbon atom 1
4 straight or branched alkylene groups. )
合性モノマー(a)の含有率が10〜90モル%の範囲
内であることを特徴とする請求項1記載の動物細胞培養
基材(I)。2. The animal cell culture medium according to claim 1, wherein the content of the polymerizable monomer (a) with respect to the vinyl copolymer (A) is in the range of 10 to 90 mol%. Material (I).
載の該動物細胞培養基材(I)表面で細胞を培養するこ
とで、細胞凝集塊を形成させ、該動物細胞培養基材
(I)表面に細胞凝集塊を接着維持して培養することを
特徴とする動物細胞培養方法。3. A method for culturing an animal cell, wherein by culturing cells on the surface of the animal cell culture substrate (I) according to claim 1, cell aggregates are formed to form the animal cell culture substrate (I). A method for culturing an animal cell, which comprises culturing while keeping the cell aggregate adhered to the surface.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4338163A JPH06153905A (en) | 1992-11-24 | 1992-11-24 | Cell culture base and method for cell culture |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4338163A JPH06153905A (en) | 1992-11-24 | 1992-11-24 | Cell culture base and method for cell culture |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH06153905A true JPH06153905A (en) | 1994-06-03 |
Family
ID=18315511
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP4338163A Pending JPH06153905A (en) | 1992-11-24 | 1992-11-24 | Cell culture base and method for cell culture |
Country Status (1)
Country | Link |
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JP (1) | JPH06153905A (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6303375B1 (en) | 1998-06-23 | 2001-10-16 | Terumo Kabushiki Kaisha | Cell supporting matrix, cell culture device, and fluid treating device |
JP4719824B2 (en) * | 2009-06-15 | 2011-07-06 | 株式会社 資生堂 | Cell aggregate formation container and cell aggregate formation method |
WO2012081470A1 (en) * | 2010-12-13 | 2012-06-21 | 株式会社資生堂 | Method for forming cell aggregate |
JP2013106599A (en) * | 2011-11-24 | 2013-06-06 | Sanyo Chem Ind Ltd | Substrate material for culturing cell aggregate, and method for producing cell aggregate |
US20210071147A1 (en) * | 2017-12-27 | 2021-03-11 | Sekisui Chemical Co., Ltd. | Scaffolding material for stem cell cultures and stem cell culture method using same |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5942890A (en) * | 1982-09-01 | 1984-03-09 | Japan Atom Energy Res Inst | Preparation of immobilized propiferated microbial cell composition containing cellulosic porous material |
JPS5942889A (en) * | 1982-09-01 | 1984-03-09 | Japan Atom Energy Res Inst | Preparation of immobilized proliferated microbial cell composition containing fibrous porous material |
-
1992
- 1992-11-24 JP JP4338163A patent/JPH06153905A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5942890A (en) * | 1982-09-01 | 1984-03-09 | Japan Atom Energy Res Inst | Preparation of immobilized propiferated microbial cell composition containing cellulosic porous material |
JPS5942889A (en) * | 1982-09-01 | 1984-03-09 | Japan Atom Energy Res Inst | Preparation of immobilized proliferated microbial cell composition containing fibrous porous material |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6303375B1 (en) | 1998-06-23 | 2001-10-16 | Terumo Kabushiki Kaisha | Cell supporting matrix, cell culture device, and fluid treating device |
JP4719824B2 (en) * | 2009-06-15 | 2011-07-06 | 株式会社 資生堂 | Cell aggregate formation container and cell aggregate formation method |
US9347031B2 (en) | 2009-06-15 | 2016-05-24 | Shiseido Company, Ltd. | Container for forming a cell aggregate and a method for forming a cell aggregate |
WO2012081470A1 (en) * | 2010-12-13 | 2012-06-21 | 株式会社資生堂 | Method for forming cell aggregate |
JP2012187097A (en) * | 2010-12-13 | 2012-10-04 | Shiseido Co Ltd | Method for forming cell aggregate |
JP2013106599A (en) * | 2011-11-24 | 2013-06-06 | Sanyo Chem Ind Ltd | Substrate material for culturing cell aggregate, and method for producing cell aggregate |
US20210071147A1 (en) * | 2017-12-27 | 2021-03-11 | Sekisui Chemical Co., Ltd. | Scaffolding material for stem cell cultures and stem cell culture method using same |
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