JPH0284174A - Cell culture substrate and production thereof - Google Patents

Cell culture substrate and production thereof

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Publication number
JPH0284174A
JPH0284174A JP1015058A JP1505889A JPH0284174A JP H0284174 A JPH0284174 A JP H0284174A JP 1015058 A JP1015058 A JP 1015058A JP 1505889 A JP1505889 A JP 1505889A JP H0284174 A JPH0284174 A JP H0284174A
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Japan
Prior art keywords
pattern
cell culture
cells
culture substrate
substrate
Prior art date
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Granted
Application number
JP1015058A
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Japanese (ja)
Other versions
JP2777392B2 (en
Inventor
Yoichi Takahashi
洋一 高橋
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Dai Nippon Printing Co Ltd
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Dai Nippon Printing Co 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Electromagnetism (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

PURPOSE:To obtain a cell culture medium, excellent in adhesion and propagating properties of cells and capable of advantageously culturing the cells by forming a fine pattern having regularity on the surface of a substrate. CONSTITUTION:For example, a desired pattern is drawn on a substrate consisting of a polyacetal, polyamide or polystyrene, with ultraviolet rays, ionizing radiation, electron beams, ion beans or laser beams. Thereby, the substrate in the part irradiated with the beams is slightly deteriorated to form the pattern having surface energy different from that in the unirradiated part. Any pattern having the regularity can be formed as the pattern in this case and a pattern consisting of lines and spaces is especially preferred. The size of the pattern is especially preferably about 50Angstrom to 500mum. The depth thereof is normally suitably about 0.01-100mum. Cells can be stably adhered to the surface of the substrate and propagated for a long period by forming the above- mentioned pattern.

Description

【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、細胞を培養するための細胞培養基板およびそ
の製法に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a cell culture substrate for culturing cells and a method for producing the same.

〔従来の技術〕[Conventional technology]

近年、動植物の細胞を種々の条件下において培養する研
究、あるいは特定の培養細胞の代謝活動による産生物の
研究が活発に行なわれており、特に人工的には合成が不
可能であったり、あるいは、合成が極めて困難な物質を
、特定の細胞活動を利用して製造することが多方面にお
いて検討されている。In recent years, research has been actively conducted on culturing animal and plant cells under various conditions, and on products produced by the metabolic activities of specific cultured cells. The production of substances that are extremely difficult to synthesize by utilizing specific cellular activities is being studied in many fields.

このような細胞の培養は、通常、細胞を多Ｉ！類、タン
白質、ポリスチレンなどの高分子物質（特開昭５８−８
９１７９．特開昭５９−１６４０１５．特開昭６０−２
５７７４５゜特開昭６ｌ−５２２８１）あるいはそれら
を表面処理した基体などの培養床に植込みあるいは接種
したものを、例えば培地中に置き、当該細胞株に適応し
た環境条件下でインキユベーシゴンすることによって行
なわれている。Cultivation of such cells usually involves culturing the cells at multiple levels. Polymer substances such as polystyrene, proteins, and polystyrene (Japanese Patent Application Laid-open No. 58-8
9179. Japanese Patent Publication No. 59-164015. JP-A-60-2
57745° JP-A-6L-52281) or those implanted or inoculated onto a culture bed such as a surface-treated substrate, placed in a medium, for example, and incubated under environmental conditions suitable for the cell line concerned. It is carried out by

〔発明が解決しようとする課題〕[Problem to be solved by the invention]

上述したように、細胞の培養は、通常、培養床に細胞を
播種したものを培地中に存在させ、当該細胞をその培養
に適した環境下におくことによって行なわれているが、
このような細胞の培養においては、培養床の特性が大き
な影響を及ぼし、例えば正常２倍体細胞などの接着依存
性細胞は培養床に接着し単層で増殖するものであるので
、培養効率は培養床の特性、特にその接着性、増殖性の
良否に大きく左右される。As mentioned above, cell culture is usually carried out by seeding cells on a culture bed and placing them in a culture medium, and placing the cells in an environment suitable for culturing.
In culturing such cells, the characteristics of the culture bed have a great influence; for example, adhesion-dependent cells such as normal diploid cells adhere to the culture bed and proliferate in a monolayer, so the culture efficiency is It greatly depends on the characteristics of the culture bed, especially its adhesion and proliferation.

この様な細胞培養床としては、酸素プラズマあるいは空
気プラズマ中で表面を酸化し、親水化処理したポリスチ
レンが最も一般的に用いられているが、プラズマ処理に
よって表面を親水化しても、親水性が不安定で、表面の
均一性が悪く、かつ経時変化を起こし時間とともに親水
性が劣化するなどの欠点があり、このような細胞培養床
による培養においては、培養効率が必ずしも十分とはい
えず、新規で有用な細胞培養床の出現が強く望まれてい
る。The most commonly used cell culture bed is polystyrene whose surface has been oxidized and made hydrophilic in oxygen plasma or air plasma, but even if the surface is made hydrophilic by plasma treatment, the hydrophilicity remains There are drawbacks such as instability, poor surface uniformity, and deterioration of hydrophilicity over time due to changes over time.Culture efficiency cannot always be said to be sufficient when culturing with such cell culture beds. The emergence of new and useful cell culture beds is strongly desired.

更に、親水化処理した培養床上では播種した細胞は自由
に増殖するため所望の形態に沿った細胞培養床あるいは
細胞の培養範囲を制御できる細胞培養床の出現が強く望
まれていた。細胞の培養床への接着は、細胞表面の膜流
動・溶存タンパク質・基体表面構造に大きく関与してい
ることを科学的に解析している片開−則、岡野光夫、桜
井端久らが、材料表面のミクロ相分離構造が細胞の粘着
性と形態変化を左右する重要な因子であることを、明ら
かにしたことから〔例えば、′バイオメディカルポリマ
ー　（化学増刊８２）化学同人、　１９８０）いくらか
の研究者がソフトセグメントとハードセグメントあるい
は親水性と疎水性のセグメントを有するブロックあるい
はグラフト共重合体を合成し、種々のミクロ相分離構造
を有する材料を培養床へ応用しようとしている。Furthermore, since the seeded cells proliferate freely on a hydrophilized culture bed, there has been a strong desire for a cell culture bed that can conform to a desired morphology or that can control the range of cell culture. Noriyuki Katakai, Mitsuo Okano, Hisashi Sakurai, and others have scientifically analyzed that cell adhesion to the culture bed is greatly involved in membrane flow, dissolved proteins, and substrate surface structure on the cell surface. It has been clarified that the microphase-separated structure on the material surface is an important factor that influences cell adhesion and morphological changes [for example, 'Biomedical Polymers (Kagaku Special Edition 82) Kagaku Dojin, 1980]. Researchers are trying to synthesize block or graft copolymers with soft and hard segments, or hydrophilic and hydrophobic segments, and to apply materials with various microphase-separated structures to culture beds.

ところが、これらの共重合体は合成した重合体の塊の断
面を観れば、ミクロ相分離構造を形成している部分はあ
るものの、細胞の粘着機構にとって最も重要であるミク
ロドメインの均一性が悪く、また制御が極めて困難であ
るとの問題がある。However, when looking at the cross-section of a synthesized polymer block, these copolymers show that although some parts form a microphase-separated structure, the uniformity of the microdomains, which are most important for the cell adhesion mechanism, is poor. There is also the problem that control is extremely difficult.

また、細胞培養用の容器等を成形あるいは、従来容器に
コーティング等をする場合、実際の細胞が接する表面は
共重合体の一方のポリマーが表面を覆って安定化するた
め、ミクロ相分離構造が形成されないという致命的な問
題がある。In addition, when molding containers for cell culture or coating conventional containers, one polymer of the copolymer covers and stabilizes the surface in contact with the actual cells, resulting in a microphase separation structure. There is a fatal problem that it is not formed.

本発明は、以上の如き事情に基づいてなされたものであ
って、細胞の接着性及び増殖性に優れていて細胞を有利
に培養することができる細胞培養床或いは細胞の培養範
囲を任意に制御できる細胞培養床を提供するものである
。The present invention has been made based on the above-mentioned circumstances, and provides a cell culture bed that has excellent cell adhesion and proliferation properties and can advantageously culture cells, or a cell culture bed that can arbitrarily control the cell culture range. This provides a cell culture bed that can be used for cell culture.

〔課題を解決するための手段〕[Means to solve the problem]

本発明者らは、上記の如き従来の合成樹脂等を用いた細
胞培養床の問題点を解決すべく鋭意研究を重ねた結果、
基板表面に規則性を有する微細なパターンを形成するこ
とによって基板表面に細胞を安定的に粘着し、長期にわ
たって細胞の生育・増殖に関して安定したデータが得ら
れるという事実を見い出し、また、基板表面に規則性を
有する微細なパターンの集合体からなる所望のパターン
を形成することで該パターンに所望の細胞を該パターン
の形状に沿って選択的に吸着し、培養し得るという事実
を見出し本発明に到達したものである。The present inventors have conducted intensive research to solve the problems of conventional cell culture beds using synthetic resins as described above.
We discovered that by forming a regular fine pattern on the substrate surface, we could stably adhere cells to the substrate surface and obtain stable data on cell growth and proliferation over a long period of time. The present invention was based on the discovery that by forming a desired pattern consisting of a collection of regular fine patterns, desired cells can be selectively adsorbed and cultured along the shape of the pattern. It has been reached.

すなわち、本発明の要旨は、基板表面に規則性を有する
微細なパターンを形成することによって基板表面に細胞
を安定的に粘着し、培養し得るようにした細胞培養基板
であり、前記パターンは凹凸状のパターン、あるいは、
細胞の粘着性を付与する各種官能基を有するパターン、
あるいは、細胞の粘着性を付与する所定の電荷を有する
パターンであることを特徴とする細胞培養基板または、
基板表面に所望のパターンを形成し、該パターン上に所
望の細胞を該パターンの形状に沿って選択的に吸着し、
培養し得るようにした選択的細胞培養基板、前記パター
ンが凹凸状の微細パターン、前記パターンが微細なパタ
ーンの集合体、前記パターンか十文字パターンから成る
もの、あるいは、細胞の吸着性を付与する各種官能基を
有する微細パターン、あるいは細胞の吸着性を付与する
所定の電荷を有する微細パターンの集合体であることを
特徴とする細胞培養基板およびそれらの製法である。That is, the gist of the present invention is a cell culture substrate in which cells can be stably adhered to and cultured by forming a regular fine pattern on the substrate surface, and the pattern has irregularities. pattern, or
Patterns with various functional groups that give cells adhesiveness,
Alternatively, a cell culture substrate characterized by a pattern having a predetermined charge that imparts adhesion to cells;
Forming a desired pattern on the surface of the substrate, selectively adsorbing desired cells onto the pattern along the shape of the pattern,
A selective cell culture substrate capable of culturing, a fine pattern in which the pattern is uneven, an aggregate of fine patterns, a substrate consisting of the pattern or a cross pattern, or various types that impart adsorption properties to cells. A cell culture substrate characterized by being a fine pattern having a functional group or an aggregate of fine patterns having a predetermined charge that imparts cell adsorption properties, and a method for producing the same.

なお、基板表面に形成される微細なパターンとしては培
養する細胞及び培地の種類にもよるが、規則性を有する
パターンなら何でも可能であり、特にラインとスペース
から成るパターン・水玉模様状のパターン・格子パター
ンなどが好ましく、またパターンのサイズは５０人〜５
（１０μｍ位が特に好ましく用いられ得る。パターンの
凹凸としては、これも培養する細胞及び培地の種類にも
よるが、その深さは一般に０．０１〜１１（１０ａ位が
適している。The fine pattern formed on the substrate surface depends on the cells to be cultured and the type of culture medium, but any regular pattern is possible, especially patterns consisting of lines and spaces, polka dot patterns, etc. A grid pattern is preferable, and the size of the pattern is 50 to 5 people.
(About 10 μm may be particularly preferably used. The depth of the unevenness of the pattern also depends on the cells to be cultured and the type of medium, but the depth is generally 0.01 to 11 (about 10 a is suitable).

また、細胞の粘着性を付与する官能基としては、細胞に
もよるが、カルボニル基、カルボキシル基。Furthermore, functional groups that impart adhesiveness to cells include carbonyl groups and carboxyl groups, depending on the cell.

エポキシ基、アミノ基、アミド基、イミド基、ニトロ基
、スルホン基、水酸基、ハロゲン基などが用いられ得る
。Epoxy groups, amino groups, amide groups, imido groups, nitro groups, sulfone groups, hydroxyl groups, halogen groups, etc. can be used.

さらに、細胞の粘着性を付与する電荷は、細胞にもよる
が、カルボキシル基あるいはその塩・アンモニウム塩・
スルホン酸基あるいはその塩などとして官能基として電
荷を有する場合と基板自体を導電性材料として外部から
直接電荷を持たせる場合のいずれでも良い。Furthermore, the charge that gives cells adhesiveness depends on the cell, but carboxyl groups or their salts, ammonium salts,
The substrate may be charged as a functional group such as a sulfonic acid group or a salt thereof, or it may be directly charged externally by using the substrate itself as a conductive material.

以上のような規則性を有するパターンを形成する基板と
しては、ガラス、セラミックス、各種金属板はもちろん
透明性のため、細胞の観察を容易にする点では、ポリア
セタール、ポリアミド、ポリカーボネート、ポリブチレ
ンテレフタレート。Substrates that form the above-mentioned regular patterns include glass, ceramics, and various metal plates, as well as polyacetal, polyamide, polycarbonate, and polybutylene terephthalate, which are transparent and allow for easy observation of cells.

ポリエチレンテレフタレートＰＲ−ＡＳ樹脂、　ＦＲ−
ＡＢＳ樹脂、　ＡＳ樹脂、　　ＡＢＳ樹脂、ポリフェニ
レンオキサイド、ポリフェニレンサルファイド、ポリス
ルホン、ポリエーテルスルホン、ポリエーテルエーテル
ケトン、ボリアリレート、ポリオキシベンゼン、フッ素
系樹脂、ポリイミド、ポリアミドイミド、ポリアミドビ
スマレイミド、ポリエーテルイミド、シリコーン樹脂、
　ＢＴ樹脂、　ＧＬ樹脂、ポリメチルペンテン、超高分
子量ポリエチレン、　　ＦＲ−ポリプロピレン、ポリス
チレン及びこれらの誘導体等の高分子材料が特に好まし
く用いられ得る。Polyethylene terephthalate PR-AS resin, FR-
ABS resin, AS resin, ABS resin, polyphenylene oxide, polyphenylene sulfide, polysulfone, polyether sulfone, polyether ether ketone, polyarylate, polyoxybenzene, fluorine resin, polyimide, polyamide imide, polyamide bismaleimide, polyether imide, Silicone resin,
Polymer materials such as BT resin, GL resin, polymethylpentene, ultra-high molecular weight polyethylene, FR-polypropylene, polystyrene, and derivatives thereof can be particularly preferably used.

次に基板表面に規則性の微細パターンを形成させる細胞
培養基板の製法について説明する。Next, a method for manufacturing a cell culture substrate in which a regular fine pattern is formed on the surface of the substrate will be described.

１、例えばポリアセクール、ポリアミド、ポリスチレン
、ポリメチルメタクリレートなどの高分子から成る基板
に紫外光・レーザー・電離放射線・電子ビーム・イオン
ビーム・レーザービーム等で所望のパターンを描画する
。そうするとビームの照射部分の基材が若干変質し、未
照射部分との間で表面エネルギーの異なる規則的なパタ
ーンが形成される（第１図）。1. A desired pattern is drawn on a substrate made of a polymer such as polyacecool, polyamide, polystyrene, polymethyl methacrylate, etc. using ultraviolet light, laser, ionizing radiation, electron beam, ion beam, laser beam, etc. As a result, the quality of the base material in the beam irradiated area changes slightly, and a regular pattern with different surface energy is formed between the beam irradiated area and the unirradiated area (FIG. 1).

２．１と同様な基板に、高融点金属の微細パターンから
成るマスクを介して、Ｘ線、γ線、シンクロトロン放射
光を照射する、あるいは遮光材の微細パターンから成る
マスクを介してレーザ、紫外光等を照射する。そうする
と１と同じ様にビームの照射部分の基材が変質し、表面
エネルギーの異なる規則的なパターンを有する基板が形
成される（第２図）。A substrate similar to 2.1 is irradiated with X-rays, γ-rays, or synchrotron radiation through a mask made of a fine pattern of a high-melting point metal, or laser, Irradiate with ultraviolet light, etc. Then, as in 1, the base material in the beam irradiated area changes in quality, and a substrate having a regular pattern with different surface energies is formed (FIG. 2).

３８１および２の光照射の際及び光照射後基板表面が活
性化しているうちに、基板周辺をハロゲン系ガス雰囲気
、シリコン系ガス雰囲気、アミン系ガス雲囲気などにす
ることでパターンに各種官能基を容易に導入することが
できる（第３図）。During the light irradiation of 381 and 2, and while the substrate surface is activated after light irradiation, various functional groups can be added to the pattern by creating a halogen gas atmosphere, silicon gas atmosphere, amine gas cloud atmosphere, etc. around the substrate. can be easily introduced (Figure 3).

４、ガラス、セラミックス、金属、高分子などの基板上
に電子ビーム、Ｘ線など電離放射線または、紫外光に反
応して溶媒への溶解性の差の生じるいわゆるレジスト性
のポリマーをスピンコーティング等により所望の厚さに
塗布する。これに当該ビームで微細パターンを描画し、
現像して、ガラス、セラミックス、金属、高分子などの
基板上に所望の厚さ及び大きさを有するパターンが形成
できる。4. On a substrate such as glass, ceramics, metal, or polymer, a so-called resist polymer that reacts with ionizing radiation such as electron beams, Apply to desired thickness. A fine pattern is drawn on this using the beam,
By development, a pattern having a desired thickness and size can be formed on a substrate such as glass, ceramics, metal, or polymer.

このようなパターンを形成可能なレジスト性のポリマー
としては、はとんどのポリマーが可能であるが、二重結
合エポキシ基、・スルホン基。As a resistive polymer that can form such a pattern, most polymers are possible, including double-bonded epoxy groups and sulfone groups.

アミノ基、アミド基、イミド基、ニトロ基、フェニル基
等を含有しているポリマーが特に好ましい。Particularly preferred are polymers containing amino groups, amide groups, imido groups, nitro groups, phenyl groups, and the like.

以上から分るように、レジスト性ポリマーとして官能基
あるいは電荷を有する官能基を有するポリマーを用いる
ことにより、容易に官能基や電荷のパターンを有する基
板が形成できる（第４図）。As can be seen from the above, by using a polymer having a functional group or a charged functional group as a resistive polymer, a substrate having a pattern of functional groups or charges can be easily formed (FIG. 4).

５、細胞の粘着性が特に培養床の電荷に依存する細胞の
場合は、上記基板に導電性の金属、　　ＩＴＯ１高分子
等を用いてその上に規則性のパターンを形成することに
より、スイッチのＯＮ　−ＯＦＦにて容易に粘着細胞の
粘着−剥離を行うことができるという利点を有している
。5. In the case of cells whose adhesion is particularly dependent on the charge of the culture bed, the switch can be made by forming a regular pattern on the substrate using conductive metal, ITO1 polymer, etc. It has the advantage that adhesion and detachment of adherent cells can be easily performed by ON-OFF.

以上１〜５を組み合わせることにより、より複雑なパタ
ーンを有する基板が形成できる。By combining 1 to 5 above, a substrate having a more complicated pattern can be formed.

本発明は、表面性状を高精度に制御した基板を形成する
ものである。The present invention forms a substrate whose surface properties are controlled with high precision.

本発明の適用においては、培養されるべき接着依存性細
胞は、何ら制限されるべきものではなく、例えばヒト胎
児繊維芽細胞、ヒト血管内皮細胞。In the application of the present invention, the adhesion-dependent cells to be cultured are not limited in any way, and include, for example, human fetal fibroblasts and human vascular endothelial cells.

ヒト子宮ガン細胞、チャイニーズ−ハムスター肺細胞、
サル腎繊維芽細胞、ヒト***細胞、ヒナ繊維芽細胞、ウ
サギ胎児の腎細胞、サル肺細胞、ブタ血管内皮細胞、ヒ
ト膵臓Ｂ細胞、ヒト膵臓Ｔ細胞などを挙げることができ
、更に従来の培養床では全く培養できなかった細胞をも
培養することが可能である。Human uterine cancer cells, Chinese hamster lung cells,
Monkey kidney fibroblasts, human foreskin cells, chick fibroblasts, fetal rabbit kidney cells, monkey lung cells, porcine vascular endothelial cells, human pancreatic B cells, human pancreatic T cells, etc. can be mentioned, as well as conventional culture. It is possible to culture cells that could not be cultured at all on the bed.

また、この基板によれば、任意の形態の細胞増殖体（組
＄６）の培養が可能であるので、例えば神経細胞を細長
い状態に培養したり、皮膚細胞を損傷形態に合わせて任
意の形に培養したりすることが可能となる。Furthermore, according to this substrate, it is possible to culture cell proliferation bodies (set $6) in any shape, so for example, nerve cells can be cultured in an elongated state, and skin cells can be cultured in any shape according to the type of damage. This makes it possible to cultivate the cells.

また、この方法を利用して単位面積当りの培養細胞の定
性分析や定量分析に有効に利用できる。Furthermore, this method can be effectively used for qualitative and quantitative analysis of cultured cells per unit area.

〔作　用〕[For production]

本発明は、基板表面に性状の異なる規則的な微細パター
ンを形成することによって種々の細胞を効率的に培養し
ようとするものであるり、更に、基板表面に性状の異な
る規則的な微細パターンの集合体を任意の形態に形成す
ることによって種々の細胞を任意の形態に培養しようと
するものである。The present invention aims to efficiently culture various cells by forming regular micropatterns with different properties on the surface of a substrate, and furthermore, by forming regular micropatterns with different properties on the surface of a substrate. The aim is to culture various cells in arbitrary shapes by forming aggregates in arbitrary shapes.

本発明の規則的な微細パターンの上で細胞が増殖しやす
いという機構の詳細は不明であるが、発明者らは次の如
く考えている。Although the details of the mechanism by which cells tend to proliferate on the regular fine pattern of the present invention are unknown, the inventors believe as follows.

細胞の粘着機構は細胞の表面の粘着因子（レセプター）
及びその膜流動と粘着に関与する培地中のタンパク質と
、培養基板の表面高次構造に深い関係があると考えられ
る。従って制御された表面構造を示す基板に、まず粘着
に関与するタンパク質が選択的に吸着する。この際、吸
着タンパク質も基板の表面高次構造の影響を受けて吸着
パターンに制御性が生じている。このタンパク層の上に
当該細胞が、膜表面のレセプターを介して粘着する。こ
の時、基板表面がタンパク質や細胞の大きさ、レセプタ
ーの種類９間隔、厚さなどに最適な条件になっているた
めに、細胞が安定的に生育するものと考えている。The cell adhesion mechanism is an adhesion factor (receptor) on the cell surface.
It is thought that there is a deep relationship between the proteins in the medium that are involved in membrane flow and adhesion, and the surface conformation of the culture substrate. Therefore, proteins involved in adhesion are first selectively adsorbed to a substrate that exhibits a controlled surface structure. At this time, the adsorbed protein is also influenced by the surface higher-order structure of the substrate, resulting in controllability of the adsorption pattern. The cells adhere to this protein layer via receptors on the membrane surface. At this time, we believe that the cells will grow stably because the substrate surface has optimal conditions for the size of the proteins and cells, the spacing of the receptor types, and the thickness.

以下、実施例を用いて本発明を更に詳しく説明する。Hereinafter, the present invention will be explained in more detail using Examples.

実施例１ （基板の作成） ポリスチレン（分子量ｉｏｏ、ｏｏｏ、分散度１．０１
以下）の３％キシレン溶液を０．２μｍのメンブランフ
ィルタ−で濾過したものを滅菌・洗浄したガラス基板上
にスピンコーティング法により塗布し、１１０°Ｃにて
３０分間、加熱処理して厚さ０．１μｍの均一なポリス
チレン膜を得た。次にこの基板に加速電圧１０にνの電
子線描画装置にて５０μＣ／　ｃｉの照射量で露光して
幅０．２μｍのラインとスペースからなる繰り返しパタ
ーンの描画を行った。露光後、この基板を酢酸イソアミ
ルを主成分とする溶剤中に６０秒間浸漬して現像し、続
いてエチルアルコールで１２０秒間すすいで０．２μｍ
のラインとスペースから成るポリスチレンのパターンを
有するガラス基板を得た。Example 1 (Creation of substrate) Polystyrene (molecular weight ioo, ooo, dispersity 1.01
A 3% xylene solution (below) was filtered through a 0.2 μm membrane filter and applied onto a sterilized and washed glass substrate by spin coating, and heated at 110°C for 30 minutes to achieve a thickness of 0. A uniform polystyrene film of .1 μm was obtained. Next, this substrate was exposed to light at a dose of 50 μC/ci using an electron beam drawing device with an acceleration voltage of 10 and ν to draw a repetitive pattern consisting of lines and spaces each having a width of 0.2 μm. After exposure, the substrate was developed by immersing it in a solvent mainly composed of isoamyl acetate for 60 seconds, and then rinsed with ethyl alcohol for 120 seconds to obtain a 0.2 μm film.
A glass substrate with a polystyrene pattern consisting of lines and spaces was obtained.

（細胞の培養） 細胞：ヒト血管内皮細胞 培地：ダルベツコ変法イーグル培地（ＧＩＢＣＯ製）９
０ｄに牛胎児血清１０ｄ、内皮細胞増殖因子２．０■、
ペパリン９．０■を混合し、全量１（１０＋ＪＩを濾過
滅菌したもの。(Culture of cells) Cells: Human vascular endothelial cells Medium: Dulbecco's modified Eagle medium (manufactured by GIBCO) 9
0d, fetal bovine serum 10d, endothelial cell growth factor 2.0■,
A total of 1 (10+JI) was mixed with 9.0 μ of pepperin and sterilized by filtration.

内径６０ｍｍのガラス製フラットシャレー内で、前記培
養基板上に、上記液体培地を満たし、これに２、３　Ｘ
ＩＯ’個の上記細胞を播種し、インキュベーター中で炭
酸ガス下温度３７°Ｃの環境下で培養を行った。４８時
間経過後において、細胞培養床の表面層に接着していた
細胞数は７．２　ＸＩＯ’個であり、７２時間後の細胞
数は９．７　ＸＩＯ’個であり、良好な増殖能を示した
。In a glass flat chalet with an inner diameter of 60 mm, the culture substrate was filled with the liquid medium, and 2 to 3×
IO' of the above cells were seeded and cultured in an incubator under carbon dioxide gas at a temperature of 37°C. After 48 hours, the number of cells adhering to the surface layer of the cell culture bed was 7.2 XIO', and after 72 hours, the number of cells was 9.7 XIO', indicating good proliferation ability. Indicated.

実施例２ （基板の作成） 表面に厚さ４（１０人のシリコン窒化膜を設けた石英ガ
ラス基板にポリアミノスチレンの４％キシレン溶液をス
ピンコーティング法により塗布し、１（１０°Ｃにて３
０分間加熱処理して厚さ０．２μｍの均一な膜を得た。Example 2 (Preparation of substrate) A 4% xylene solution of polyaminostyrene was applied by spin coating to a quartz glass substrate having a silicon nitride film with a thickness of 4 (10%) on the surface,
A uniform film with a thickness of 0.2 μm was obtained by heat treatment for 0 minutes.

次にこの基板に長さ１μｍ、幅０．１μｍのタンタルか
ら成る繰り返しパターンを有するＸ線マスクを用いて３
０ｍＪ／ｃ＋ｆｌのエネルギーにてＸ線−括露光を行っ
た。Next, an X-ray mask having a repeating pattern made of tantalum with a length of 1 μm and a width of 0.1 μm was used to
X-ray exposure was performed at an energy of 0 mJ/c+fl.

続いて、この基板をメチルエチルケトンを主成分とする
溶剤中で６０秒間現像し、つづいてエチルアルコールで
１２０秒間すすいで、Ｘ線マスクと同一ノハターンを有
する基板を得た。この基板のパターンは表面にアミノ基
を有するものであった。Subsequently, this substrate was developed in a solvent containing methyl ethyl ketone as a main component for 60 seconds, and then rinsed with ethyl alcohol for 120 seconds to obtain a substrate having the same pattern as the X-ray mask. The pattern of this substrate had amino groups on the surface.

尚、この方法は、他の実施例に比較して量産性が極めて
高い方法であった。Note that this method had extremely high mass productivity compared to other examples.

（細胞の培養） 細胞：　ＷＫＡラット胎児肺由来のＲＦＬ細胞培地：　
ＲＰＭＩ　１６４０（Ｇｉｂｃｏ社製）の培養液に牛胎
児血清（Ｇｉｂｃｏ社製）を１０％加えたもの。(Culture of cells) Cells: RFL cell culture medium derived from WKA rat fetal lung:
10% fetal bovine serum (manufactured by Gibco) was added to the culture solution of RPMI 1640 (manufactured by Gibco).

前記基板上に上記液体培地中にて、上記細胞を播種し、
５％炭炭酸ガスゼインキュベーター内３７°Ｃで培養し
た。Seeding the cells on the substrate in the liquid medium,
The cells were cultured at 37°C in a 5% carbon dioxide incubator.

細胞播種後、９６時間後の細胞増殖率は、同じ大きさの
ポリスチレンシャレーで培養したのに比べて２．１倍で
あった。The cell proliferation rate 96 hours after cell seeding was 2.1 times that of culture in a polystyrene chalet of the same size.

実施例３ （基板の作成） 内径６０鵬のガラス製フラットシャレーの容器内面にポ
リメチルメタクリレートのエチルセロソルブアセテート
溶液を塗布した後、乾燥及び２（１０°Ｃ３０分間の加
熱処理を施して厚さ約３μｍのポリメチルメタクリレー
ト層を有してなるシャレーを得た０次に、これに加速電
圧２０ＫＶの電子線描画装置にて１（１０μＣ／ｃｔｆ
ｌの照射量で露光して幅０．ＩＩＩｍの繰り返しパター
ンの描画を行った。この電子線描画によってポリメチル
メタクリレート層の表面は露光部と未露光部とで表面エ
ネルギーの異なる規則的なパターンを有する表面が形成
できた。Example 3 (Preparation of substrate) After applying an ethyl cellosolve acetate solution of polymethyl methacrylate to the inner surface of a glass flat chalet container with an inner diameter of 60 mm, it was dried and subjected to heat treatment at 10°C for 30 minutes to a thickness of approximately After obtaining a chalet having a polymethyl methacrylate layer of 3 μm, this was subjected to 1 (10 μC/ctf) using an electron beam lithography device with an accelerating voltage of 20 KV.
Exposure with a radiation dose of 0.1 and a width of 0. A repeating pattern of IIIm was drawn. By this electron beam drawing, the surface of the polymethyl methacrylate layer had a regular pattern in which the exposed and unexposed areas had different surface energies.

（細胞の培養） 細胞：Ｈｅ１ａ（ヒト子宮頚部癌） 培地２イ一グルＭＥ？１培地Ｉｏｖｌに牛血清２ｍｌ、
？、５％ＮａＨＣＯｓ　２　ｔｌを加えたもの。(Culture of cells) Cell: He1a (human cervical cancer) Medium 2 single ME? 2 ml of bovine serum in 1 medium Iovl,
? , plus 2 tl of 5% NaHCOs.

作成したシャレー内に上記液体培地を満たし、これに１
×１０ｓ個の上記細胞を播種し、３７゛Ｃの保温下、５
％炭酸ガス気流中でインキュベーターンした。４８時間
後、０．２５％トリプシン溶液にてシャーレ−壁面に生
育した細胞を離脱させ、この細胞浮遊液中の細胞数を測
定した。その結果、培地中の細胞数は５．２〜ｌ０ＸＩ
Ｏ’個であり、良好な増殖能を示した。Fill the created chalet with the above liquid medium, and add 1
x 10 s of the above cells were seeded, and kept at 37°C for 5 s.
% carbon dioxide gas stream. After 48 hours, the cells grown on the wall of the petri dish were detached using a 0.25% trypsin solution, and the number of cells in this cell suspension was measured. As a result, the number of cells in the medium was 5.2 to 10XI
The number of cells was O', indicating good proliferation ability.

実施例４ （基板の作成） 内径６０ｍｍのポリスチレン製のフラットシャレーの容
器内面にポリ　（セパシルピペラジン）の５％メタノー
ル溶液を塗布した後、乾燥及び８０“Ｃ，３０分の加熱
処理を施して厚さ０．１μｍのポリ　（セパシルピペラ
ジン）層を有して成るシャレーを得た。Example 4 (Preparation of substrate) A 5% methanol solution of poly (sepacyl piperazine) was applied to the inner surface of a polystyrene flat chalet container with an inner diameter of 60 mm, and then dried and heat-treated at 80"C for 30 minutes. A chalet was obtained having a layer of poly(sepacylpiperazine) with a thickness of 0.1 μm.

次にこれに加速電圧１０ＫＶの電子線描画装置にて１０
μＣ／ｄの照射量で露光して０．３μｍφのドツトパタ
ーンの描画を行った。露光後、このシャレーをメタノー
ルに６０秒間浸漬して現像し、水で１２０秒間すすいで
０．３μｍφのドツトパターンを有するシャレーを得た
。Next, this was applied to an electron beam lithography system with an acceleration voltage of 10 KV for 10
A dot pattern of 0.3 μmφ was drawn by exposure at a dose of μC/d. After exposure, the chalet was immersed in methanol for 60 seconds for development, and rinsed with water for 120 seconds to obtain a chalet having a dot pattern of 0.3 μmφ.

（細胞の培養） 細胞：ヒト血管内皮細胞 培地：ダルベツコ変法イーグル培地（Ｇ　ｉ　ｂｃｏ社
製）９０ｃｉａに牛胎児血清１０ｃｄ、内皮細胞増殖因
子２．０■、ヘパリン９．０■を混合し、全量１（１０
ｃｄを濾過滅菌したもの。(Culture of cells) Cells: Human vascular endothelial cell medium: 90 cia of Dulbecco's modified Eagle medium (manufactured by Gibco) mixed with 10 cd of fetal bovine serum, 2.0 ■ endothelial cell growth factor, and 9.0 ■ heparin. Total amount 1 (10
Filter-sterilized CD.

作成したシャレー内に前記液体培地を満たし、上記細胞
を播種し、５％炭酸ガス中、インキュベーター内にて３
７℃で培養した。Fill the prepared chalet with the liquid medium, seed the cells, and incubate in an incubator in 5% carbon dioxide gas for 3
Cultured at 7°C.

細胞播種後、３時間後にはほとんどの細胞が付着し、９
６時間後の細胞増殖率は、同じ大きさのポリスチレン製
シャレーで培養したものに比べて２〜５倍であった。Three hours after cell seeding, most of the cells had attached, and 9
The cell proliferation rate after 6 hours was 2 to 5 times that of cells cultured in polystyrene chalets of the same size.

実施例５ （基板の作成） ポリ　（スチンスルホン酸アンモニウム）の０．５％水
溶液を０．２μｍのメンブランフィルタ−で濾過したも
のを滅菌・洗浄したポリスチレン製のシャレーに注ぎ、
加熱真空乾燥して、シャレー内にポリ（スチレンスルホ
ン酸アンモニウム）から成る厚さ約２（１０人の腫を得
た。Example 5 (Preparation of substrate) A 0.5% aqueous solution of poly(ammonium stinsulfonate) was filtered through a 0.2 μm membrane filter and poured into a sterilized and washed polystyrene chalet.
By heating and vacuum drying, a tumor of about 2 (10) thickness made of poly(ammonium styrene sulfonate) was obtained in a chalet.

次に、この基板に加速電圧５０ｋＶの電子線描画装置に
て４３０ｕＣ／ｃＴ１の照射量で露光して幅約２５０人
ラインの繰り返しパターンから成る幅ＩＣＩＩの十文字
パターンの描画を行った。露光後、このシャレーを水に
て現像して幅２５０人ラインの繰り返し微細パターンの
集合体から成る十文字パターンを有するシャレーを得た
。Next, this substrate was exposed to light at a dose of 430 uC/cT1 using an electron beam drawing device with an accelerating voltage of 50 kV to draw a cross pattern with a width of ICII consisting of a repeating pattern of about 250 lines in width. After exposure, this chalet was developed with water to obtain a chalet having a criss-cross pattern consisting of an aggregate of repeated fine patterns with a width of 250 lines.

（細胞の培養） 細胞：ヒト血管内皮細胞 培地：ダルベツコ変法イーグル培地に牛胎児血清、内皮
細胞増殖因子、ヘパリンを加え濾過滅菌したもの。(Culture of cells) Cells: Human vascular endothelial cell medium: Dulbecco's modified Eagle's medium added with fetal bovine serum, endothelial cell growth factor, and heparin and sterilized by filtration.

前記培養シャレーに、上記液体培地を満たし、これにヒ
ト胎児請帯より離脱した血管内皮細胞を播種し、炭酸ガ
ス下、３７°Ｃにてインキュベートした。７２時間経過
後において観察したところ十文字のパターンの範囲に血
管内皮細胞が効率良く増殖しており、培養細胞の形態を
制御することができた。The culture chalet was filled with the liquid medium, inoculated with vascular endothelial cells detached from the human fetal cord, and incubated at 37°C under carbon dioxide gas. Observation after 72 hours showed that vascular endothelial cells were efficiently proliferating within the cross-shaped pattern, and the morphology of the cultured cells could be controlled.

実施例６ （基板の作成） ポリ（セパシル−３−アミノ−ペルハイドロアセヒレ）
の０．５％水溶液を０．２μｍのメンブランフィルタ−
で濾過したものを滅菌・洗浄したガラス製のシャレーに
注ぎ、加熱真空乾燥したシャレー内にポリ（セパシル−
３−アミノーペルハイドロアゼピレ）から成る厚さ約３
（１０人の膜を得た。Example 6 (Creation of substrate) Poly(sepacyl-3-amino-perhydroacefin)
A 0.5% aqueous solution of
The filtered material was poured into a sterilized and washed glass chalet, and poly(sepasil) was poured into the chalet which was heated and vacuum dried.
3-aminoperhydroazepyre) with a thickness of approx.
(10 people's membranes were obtained.

次に、この基板に長さ０．１μｍ、幅０．（１５μｍの
タンタルの繰り返しパターンの集合体から成る直径１ｃ
１１の水玉模様パターンを有するＸ線マスクを用いて５
（１０■／ｃ４のエネルギーにてＸ線−括露光を行った
。Next, this substrate has a length of 0.1 μm and a width of 0.1 μm. (Diameter 1c consisting of an aggregate of 15 μm tantalum repeating patterns)
5 using an X-ray mask with 11 polka dot patterns.
(X-ray exposure was carried out at an energy of 10 .mu./c4.

続いて、このシャレーをエタノールと水との混合液にて
６０秒間現像し、Ｘ線マスクと同一のパターンを有する
基板を得た。Subsequently, this chalet was developed with a mixture of ethanol and water for 60 seconds to obtain a substrate having the same pattern as the X-ray mask.

（細胞の培養） 細胞：ミニブタ大動脈血管内皮細胞 培地：Ｆ−１０培地＋１０％ウシ胎児血清前記培養シャ
レーに、上記液体培地を満たし、これにミニブタ大動脈
よりトリプシンやコラゲナーゼなどの酵素を用いて採取
した血管内皮細胞を播種し、炭酸ガス下、３７°Ｃにて
インキュベートした。４８時間経過後において、培養シ
ャレーを観察したところ、細胞は水玉模様状に増殖して
いた。(Culture of cells) Cells: Minipig aorta vascular endothelial cell culture medium: F-10 medium + 10% fetal bovine serum The culture chalet was filled with the above liquid medium, and cells were collected from the minipig aorta using enzymes such as trypsin and collagenase. Vascular endothelial cells were seeded and incubated at 37°C under carbon dioxide gas. After 48 hours, the culture chalet was observed and the cells were found to have proliferated in a polka dot pattern.

〔発明の効果〕〔Effect of the invention〕

本発明は、基板表面に規則性を有する凹凸パターン、細
胞の粘着を制御する官能基のパターン、荷電分布の異な
るパターン、表面エネルギーの異なるパターン等を高精
度に形成することにより、基板表面に細胞を安定的に粘
着し、長期にわたって細胞を増殖できるものである。本
発明によれば、肝細胞や肝細胞のような今まで培養が不
可能、あるいは培養が極めて難しいとされていた細胞も
培養することが可能である。更に、本発明は培養細胞の
形態を任意に制御することが可能であり細胞工学に寄与
するばかりでなく生化学的に極めて有用である。The present invention enables cells to be formed on the substrate surface by forming with high precision a regular uneven pattern, a pattern of functional groups that control cell adhesion, a pattern with different charge distribution, a pattern with different surface energy, etc. on the substrate surface. It is capable of stably adhering to cells and allowing cells to proliferate over a long period of time. According to the present invention, it is possible to culture cells such as hepatocytes and hepatocytes, which have hitherto been considered impossible or extremely difficult to culture. Furthermore, the present invention makes it possible to arbitrarily control the morphology of cultured cells, which not only contributes to cell engineering but also is extremely useful biochemically.

【図面の簡単な説明】[Brief explanation of drawings]

第１図は基板表面に紫外光、レーザー、電離放射線等で
所望のパターンを描画する方法を示す図、第２図はＸ線
マスクを通して、放射光を照射する方法を示す図、第３
図は紫外光、レーザー、電離放射線の照射をガス雰囲気
中で行う方法を示す図、第４図は基板表面にレジスト性
ポリマーを塗布した後に紫外光、レーザー光、電離放射
線を照射する方法をそれぞれ示す図、第５図は十文字パ
ターンの斜視図である。Figure 1 shows how to draw a desired pattern on the substrate surface using ultraviolet light, laser, ionizing radiation, etc. Figure 2 shows how to irradiate synchrotron radiation through an X-ray mask, Figure 3
The figure shows a method of irradiating ultraviolet light, laser light, and ionizing radiation in a gas atmosphere, and Figure 4 shows a method of irradiating ultraviolet light, laser light, and ionizing radiation after coating a resistive polymer on the substrate surface. The figure shown in FIG. 5 is a perspective view of a cross pattern.

Claims (15)

【特許請求の範囲】[Claims] （１）基板表面に規則性を有する微細なパターンを形成
することによって基板表面に細胞を安定的に吸着し、培
養し得るようにした細胞培養基板。(1) A cell culture substrate that allows cells to be stably adsorbed and cultured on the substrate surface by forming a regular fine pattern on the substrate surface. （２）前記パターンが、凹凸状のパターンであることを
特徴とする請求項１記載の細胞培養基板。(2) The cell culture substrate according to claim 1, wherein the pattern is an uneven pattern. （３）前記パターンを形成する素材が、細胞の吸着性を
付与する各種官能基を有することを特徴とする請求項１
又は２記載の細胞培養基板。(3) Claim 1 characterized in that the material forming the pattern has various functional groups that impart adsorption properties to cells.
Or the cell culture substrate according to 2. （４）前記パターンを形成する素材が、細胞の吸着性を
付与する所定の電荷を有することを特徴とする請求項１
乃至３項のいずれかの項記載の細胞培養基板。(4) Claim 1, wherein the material forming the pattern has a predetermined charge that imparts cell adsorption properties.
The cell culture substrate according to any one of items 3 to 3. （５）基板表面に所望のパターンを形成し、該パターン
上に所望の細胞を該パターンの形状に沿って選択的に吸
着し、培養し得るようにした選択的細胞培養基板。(5) A selective cell culture substrate in which a desired pattern is formed on the surface of the substrate, and desired cells can be selectively adsorbed onto the pattern along the shape of the pattern and cultured. （６）前記パターンが、凹凸状のパターンであることを
特徴とする請求項１記載の選択的細胞培養基板。(6) The selective cell culture substrate according to claim 1, wherein the pattern is an uneven pattern. （７）前記パターンが微細なパターンの集合体から成る
ものであることを特徴とする請求項５又は６記載の選択
的な細胞培養基板。(7) The selective cell culture substrate according to claim 5 or 6, wherein the pattern is composed of an aggregate of fine patterns. （８）前記パターンか十文字パターンからなることを特
徴とする請求項５乃至７のいずれかの項記載の選択的細
胞培養基板。(8) The selective cell culture substrate according to any one of claims 5 to 7, wherein the pattern is a cross pattern. （９）前記パターンを形成する素材が、細胞の吸着性を
付与する各種官能基を有することを特徴とする請求項５
又は８記載の選択的細胞培養基板。(9) Claim 5, characterized in that the material forming the pattern has various functional groups that impart adsorption properties to cells.
or selective cell culture substrate according to 8. （１０）前記パターンを形成する素材が、細胞の吸着性
を付与する所定の電荷を有することを特徴とする請求項
５乃至８のいずれかの項記載の選択的細胞培養基板。(10) The selective cell culture substrate according to any one of claims 5 to 8, wherein the material forming the pattern has a predetermined electric charge that imparts cell adsorption properties. （１１）基板表面に電子ビーム、イオンビーム、レーザ
ービーム等を照射して、規則性を有する微細なパターン
を形成することを特徴とする請求項１乃至１０のいずれ
かの項記載の細胞培養基板の製法。(11) The cell culture substrate according to any one of claims 1 to 10, characterized in that a regular fine pattern is formed by irradiating the substrate surface with an electron beam, ion beam, laser beam, etc. manufacturing method. （１２）基板表面に高融点金属の微細パターンから成る
マスクを介してＸ線、γ線、或いはシンクロトロン放射
光を照射し、又は遮光材の微細パターンから成るマスク
を介してレーザー、紫外光等を照射することを特徴とす
る請求項１乃至１０のいずれかの項記載の細胞培養基板
の製法。(12) Irradiate the substrate surface with X-rays, gamma rays, or synchrotron radiation through a mask made of a fine pattern of high-melting point metal, or irradiate the substrate surface with laser, ultraviolet light, etc. through a mask made of a fine pattern of light-shielding material. The method for producing a cell culture substrate according to any one of claims 1 to 10, characterized in that the cell culture substrate is irradiated with. （１３）照射をハロゲン系ガス雰囲気、シリコン系ガス
雰囲気、アミン系ガス雰囲気のもとで行うことを特徴と
する請求項１１又は１２記載の製法。(13) The method according to claim 11 or 12, wherein the irradiation is performed in a halogen-based gas atmosphere, a silicon-based gas atmosphere, or an amine-based gas atmosphere. （１４）基板表面にレジスト性ポリマーを塗布し、次い
で電子ビーム、Ｘ線などを照射することを特徴とする請
求項１乃至１０のいずれかの項記載の細胞培養基板の製
法。(14) The method for producing a cell culture substrate according to any one of claims 1 to 10, characterized in that a resist polymer is applied to the surface of the substrate, and then irradiated with an electron beam, an X-ray, or the like. （１５）基板表面に導電性の金属、ＩＴＯ、高分子等で
規則性を有する微細なパターンを形成することを特徴と
する請求項１乃至１０のいずれかの項記載の細胞培養基
板の製法。（１６）請求項１１、請求項１２、請求項１
３、請求項１４及び請求項１５記載の製法のうちの少な
くとも２つ以上の製法を組み合わせることを特徴とする
細胞培養基板の製法。(15) The method for manufacturing a cell culture substrate according to any one of claims 1 to 10, characterized in that a regular fine pattern is formed on the surface of the substrate using a conductive metal, ITO, polymer, or the like. (16) Claim 11, Claim 12, Claim 1
3. A method for manufacturing a cell culture substrate, characterized by combining at least two or more of the manufacturing methods according to claims 14 and 15.
JP1015058A 1988-06-22 1989-01-26 Cell culture substrate and method for producing the same Expired - Lifetime JP2777392B2 (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
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US5202227A (en) * 1989-06-03 1993-04-13 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Control of cell arrangement
US5328843A (en) * 1990-02-27 1994-07-12 Hitachi, Ltd. Method for allocating cells and cell allocation device
WO2005118013A1 (en) * 2004-06-01 2005-12-15 Dai Nippon Printing Co., Ltd. Artificial blood vessel and process for producing the same
WO2006028274A1 (en) * 2004-09-08 2006-03-16 National University Corporation Nagoya University Production of cell culture product and material for use in said production
JP2011527888A (en) * 2008-07-12 2011-11-10 ユーライブ エンタープライゼズ リミテッド Materials and methods for cell proliferation
WO2012029731A1 (en) * 2010-08-31 2012-03-08 大阪有機化学工業株式会社 Method for manufacturing cell culture substrate

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US8592139B2 (en) 2006-11-10 2013-11-26 Dai Nippon Printing Co., Ltd. Test method using cells and test kit therefor
JP5261920B2 (en) 2006-11-10 2013-08-14 大日本印刷株式会社 Test methods and test kits using cells

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JPS63119754A (en) * 1986-11-10 1988-05-24 東京大学長 Artificial element having cell growth specificity
JPS63196279A (en) * 1987-02-10 1988-08-15 Sumitomo Electric Ind Ltd Substrate for cell culture
JPS63198976A (en) * 1987-02-13 1988-08-17 Sumitomo Electric Ind Ltd Base material for cultivating cell
JPH01141588A (en) * 1987-11-26 1989-06-02 Nippon Telegr & Teleph Corp <Ntt> Substrate for growing biological cell

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JPS5645189A (en) * 1979-09-20 1981-04-24 Sekisui Chem Co Ltd Formed product for tissue culture
JPS63119754A (en) * 1986-11-10 1988-05-24 東京大学長 Artificial element having cell growth specificity
JPS63196279A (en) * 1987-02-10 1988-08-15 Sumitomo Electric Ind Ltd Substrate for cell culture
JPS63198976A (en) * 1987-02-13 1988-08-17 Sumitomo Electric Ind Ltd Base material for cultivating cell
JPH01141588A (en) * 1987-11-26 1989-06-02 Nippon Telegr & Teleph Corp <Ntt> Substrate for growing biological cell

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5202227A (en) * 1989-06-03 1993-04-13 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Control of cell arrangement
US5593814A (en) * 1989-06-03 1997-01-14 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Control of cell arrangement
US5328843A (en) * 1990-02-27 1994-07-12 Hitachi, Ltd. Method for allocating cells and cell allocation device
WO2005118013A1 (en) * 2004-06-01 2005-12-15 Dai Nippon Printing Co., Ltd. Artificial blood vessel and process for producing the same
US8367411B2 (en) 2004-06-01 2013-02-05 Dai Nippon Printing Co., Ltd. Artificial blood vessel and method of manufacturing thereof
US9359594B2 (en) 2004-06-01 2016-06-07 Dai Nippon Printing Co., Ltd. Artificial blood vessel and method of manufacturing thereof
WO2006028274A1 (en) * 2004-09-08 2006-03-16 National University Corporation Nagoya University Production of cell culture product and material for use in said production
US7883865B2 (en) 2004-09-08 2011-02-08 National University Corporation Nagoya University Production of cell culture product and material for use in said production
JP2011527888A (en) * 2008-07-12 2011-11-10 ユーライブ エンタープライゼズ リミテッド Materials and methods for cell proliferation
WO2012029731A1 (en) * 2010-08-31 2012-03-08 大阪有機化学工業株式会社 Method for manufacturing cell culture substrate
JP5887272B2 (en) * 2010-08-31 2016-03-16 大阪有機化学工業株式会社 Method for producing cell culture substrate
US9434927B2 (en) 2010-08-31 2016-09-06 Osaka Organic Chemical Industry Ltd. Method for manufacturing cell culture substrate

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