JPH0751061B2 - Manufacturing method of cell array control tool - Google Patents
Manufacturing method of cell array control toolInfo
- Publication number
- JPH0751061B2 JPH0751061B2 JP1141964A JP14196489A JPH0751061B2 JP H0751061 B2 JPH0751061 B2 JP H0751061B2 JP 1141964 A JP1141964 A JP 1141964A JP 14196489 A JP14196489 A JP 14196489A JP H0751061 B2 JPH0751061 B2 JP H0751061B2
- Authority
- JP
- Japan
- Prior art keywords
- cell
- adhesive
- adhesive surface
- pattern
- hydrophilic polymer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 12
- 230000004956 cell adhesive effect Effects 0.000 claims description 32
- 229920001477 hydrophilic polymer Polymers 0.000 claims description 16
- 239000000853 adhesive Substances 0.000 claims description 14
- 230000001070 adhesive effect Effects 0.000 claims description 14
- 206010034972 Photosensitivity reaction Diseases 0.000 claims description 7
- 230000036211 photosensitivity Effects 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 4
- 210000004027 cell Anatomy 0.000 description 43
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 229920000642 polymer Polymers 0.000 description 16
- 238000000034 method Methods 0.000 description 13
- 150000001875 compounds Chemical class 0.000 description 11
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 7
- 229910052753 mercury Inorganic materials 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- -1 dextran sulfate Polymers 0.000 description 5
- 102000016359 Fibronectins Human genes 0.000 description 4
- 108010067306 Fibronectins Proteins 0.000 description 4
- 230000021164 cell adhesion Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 229920001577 copolymer Polymers 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 229920002939 poly(N,N-dimethylacrylamides) Polymers 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 230000005660 hydrophilic surface Effects 0.000 description 2
- 230000005661 hydrophobic surface Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 229940088644 n,n-dimethylacrylamide Drugs 0.000 description 2
- YLGYACDQVQQZSW-UHFFFAOYSA-N n,n-dimethylprop-2-enamide Chemical compound CN(C)C(=O)C=C YLGYACDQVQQZSW-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- HSVFKFNNMLUVEY-UHFFFAOYSA-N sulfuryl diazide Chemical compound [N-]=[N+]=NS(=O)(=O)N=[N+]=[N-] HSVFKFNNMLUVEY-UHFFFAOYSA-N 0.000 description 2
- 229920001059 synthetic polymer Polymers 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- UFHSKEHEWFGEFH-UHFFFAOYSA-N (2,5-dioxopyrrolidin-3-yl) prop-2-enoate Chemical compound C=CC(=O)OC1CC(=O)NC1=O UFHSKEHEWFGEFH-UHFFFAOYSA-N 0.000 description 1
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- NNRFRJQMBSBXGO-CIUDSAMLSA-N (3s)-3-[[2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-4-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-oxobutanoic acid Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NNRFRJQMBSBXGO-CIUDSAMLSA-N 0.000 description 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- 239000004342 Benzoyl peroxide Substances 0.000 description 1
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 229920000045 Dermatan sulfate Polymers 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 101000829980 Homo sapiens Ral guanine nucleotide dissociation stimulator Proteins 0.000 description 1
- 229920000288 Keratan sulfate Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 102100023320 Ral guanine nucleotide dissociation stimulator Human genes 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 235000019400 benzoyl peroxide Nutrition 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- VQXINLNPICQTLR-UHFFFAOYSA-N carbonyl diazide Chemical compound [N-]=[N+]=NC(=O)N=[N+]=[N-] VQXINLNPICQTLR-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 1
- 229940051593 dermatan sulfate Drugs 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- KXCLCNHUUKTANI-RBIYJLQWSA-N keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 description 1
- 238000001459 lithography Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- GYVGXEWAOAAJEU-UHFFFAOYSA-N n,n,4-trimethylaniline Chemical compound CN(C)C1=CC=C(C)C=C1 GYVGXEWAOAAJEU-UHFFFAOYSA-N 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000083 poly(allylamine) Polymers 0.000 description 1
- 229920000172 poly(styrenesulfonic acid) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000004584 polyacrylic acid Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229940005642 polystyrene sulfonic acid Drugs 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000012966 redox initiator Substances 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Landscapes
- Photosensitive Polymer And Photoresist Processing (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Exposure Of Semiconductors, Excluding Electron Or Ion Beam Exposure (AREA)
Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明は、細胞の配列制御用具の製法に関する。TECHNICAL FIELD The present invention relates to a method for producing a cell array control tool.
［従来の技術］ 近年、細胞工学、LSI技術、医工学などの急激な進歩と
ともに、細胞を用いた超小型バイオセンサー、スイッチ
ング素子、バイオリアクター、ハイブリッド型人工臓
器、さらにはニューロコンピューターなどが注目を集
め、これらの開発が活発に行なわれている。[Conventional technology] In recent years, with the rapid progress of cell engineering, LSI technology, medical engineering, etc., ultra-compact biosensors using cells, switching elements, bioreactors, hybrid artificial organs, and neurocomputers are attracting attention. Collectively, these developments are actively carried out.
細胞を望むように配列させ、しかもその機能を維持させ
ておくことは難しく、細胞を用いたデバイス実現の一つ
の障壁となっている。細胞を望むように配列させて回路
網を形成させるというような細胞の配列制御技術は、こ
れらのデバイス実現のための大きなキーテクノロジーと
なりうる。Arranging cells in a desired manner and maintaining their functions is difficult, which is one of the obstacles to realizing devices using cells. Cell array control technology such as arranging cells as desired to form a circuit network can be a key technology for realizing these devices.
［発明が解決しようとする課題］ 細胞の配列を制御する試みとしては、インクジェットプ
リンターを用いて細胞接着性蛋白質であるフィブロネク
チンを塗布してパターンを形成し、この上で細胞を培養
させた例があるが、解像度がわるく不均一であり、微細
加工には適していない。[Problems to be Solved by the Invention] As an attempt to control the cell arrangement, an example in which an inkjet printer is used to apply fibronectin, which is a cell adhesive protein, to form a pattern, and then the cells are cultured However, the resolution is poor and uneven, and it is not suitable for fine processing.
また、最近、人工的な凹凸面を用いて神経細胞シナプス
成長の方向制御を試みた例があるが、望むような配列を
形成させるまでには至っていない。In addition, recently, there has been an example of trying to control the direction of nerve cell synapse growth using an artificial uneven surface, but it has not been possible to form a desired array.
［課題を解決するための手段］ 本発明者らは、このような実状に鑑み、細胞の配列を容
易に制御する方法について鋭意研究を重ねた結果、細胞
接着性表面および細胞非接着性表面よりなる配列パター
ンを有する材料表面上で細胞を培養することにより、細
胞の配列が容易に制御できること、細胞接着性表面およ
び細胞非接着性表面よりなる配列パターンを有する細胞
の配列制御用具が、特定の工程を経て容易に製造できる
ことを見出し、本発明を完成するに至った。[Means for Solving the Problems] In view of such circumstances, the present inventors have earnestly conducted research on a method for easily controlling the cell arrangement, and as a result, from the cell adhesive surface and the cell non-adhesive surface, By culturing the cells on the surface of the material having the following arrangement pattern, the arrangement of the cells can be easily controlled, and the device for controlling the arrangement of the cells having the arrangement pattern of the cell adhesive surface and the cell non-adhesive surface has The inventors have found that they can be easily manufactured through steps, and have completed the present invention.
すなわち、本発明は 細胞接着性表面および細胞非接着性表面よりなる配列パ
ターンが、 （１）感光性を有する細胞非接着性親水性高分子を細胞
接着性表面に塗布もしくは吸着させて存在させる工程、 （２）（１）でえられた表面上に望む配列パターンを有
するフォトマスクを設置してパターン露光する工程およ
び （３）洗浄により現像し、細胞非接着性親水性高分子よ
りなる像を細胞接着性表面に形成させる工程 または （１）感光性を有する細胞接着性親水性高分子を細胞非
接着性表面に塗布もしくは吸着させて存在させる工程、 （２）（１）でえられた表面上に望む配列パターンを有
するフォトマスクを設置してパターン露光する工程およ
び （３）洗浄により現像し、細胞接着性親水性高分子より
なる像を細胞非接着性表面に形成させる工程 を経て形成されることを特徴とする細胞の配列制御用具
の製法に関する。That is, according to the present invention, an arrangement pattern comprising a cell-adhesive surface and a cell-non-adhesive surface is (1) a step of coating or adsorbing a cell-adhesive hydrophilic polymer having photosensitivity on the cell-adhesive surface (2) A step of setting a photomask having a desired arrangement pattern on the surface obtained in (1) and performing pattern exposure, and (3) developing by washing to form an image of a cell-nonadhesive hydrophilic polymer. Or (1) a step of applying or adsorbing a cell-adhesive hydrophilic polymer having photosensitivity to a cell-non-adhesive surface to allow it to exist, (2) the surface obtained in (1) A step of placing a photomask having a desired arrangement pattern on the surface and performing pattern exposure and (3) developing by washing to form an image of the cell-adhesive hydrophilic polymer on the cell-nonadhesive surface. That process relates to a process for the production of sequence control device of cells, characterized by being formed through.
［実施例］ 本発明により製造される細胞の配列制御用具は、パター
ン化した細胞接着表面と細胞非接着性表面とが本発明に
より製造される細胞の配列制御用具となる材料の表面に
形成されたものである。[Example] In the cell alignment control device produced by the present invention, a patterned cell adhesion surface and a cell non-adhesive surface are formed on the surface of a material which is the cell alignment control device produced by the present invention. It is a thing.
前記細胞接着性表面とは、カルボキシル基やアミノ基な
どの電荷を有する官能基および（または）RGDS（Arg−G
ly−Asp−Ser）のような細胞接着性ペプチドを導入した
表面、または細胞接着性を有する高分子を固定した表面
をいう。The cell adhesive surface means a functional group having a charge such as a carboxyl group or an amino group and / or RGDS (Arg-G
ly-Asp-Ser) or a surface on which a cell adhesive peptide such as ly-Asp-Ser) has been introduced or a polymer having cell adhesive properties is immobilized.
前記カルボキシル基やアミノ基などの官能基は、本発明
の配列制御用具となる材料表面をプラズマなどの放射線
で処理することにより導入することができる。この際の
前記材料としてはプラスチック製の培養用皿、フィル
ム、チューブなどを利用しうる。The functional groups such as the carboxyl group and the amino group can be introduced by treating the surface of the material to be the arrangement control tool of the present invention with radiation such as plasma. In this case, as the material, a plastic culture dish, film, tube or the like can be used.
前記細胞接着性を有する高分子の具体例としては、たと
えばポリアクリル酸、ポリビニル硫酸、ポリスチレンス
ルホン酸、ポリアリルアミンなどの電荷を有する合成高
分子、コンドロイチン硫酸、デルマタン硫酸、デキスト
ラン硫酸、ケラタン硫酸、ヘパラン硫酸、ヒアルロン
酸、キチンなどの電荷を有する多糖類、コラーゲン、ゼ
ラチン、フイブロネクチン、ハイドロネクチンなどの細
胞接着性蛋白質、さらには細胞接着性蛋白質や細胞接着
性ペプチドを固定した合成高分子などがあげられるが、
これらに限定されるものではない。これらは単独で用い
てもよく、２種以上を併用してもよい。Specific examples of the polymer having cell adhesiveness include, for example, polyacrylic acid, polyvinyl sulfate, polystyrene sulfonic acid, polyallylamine, and other charged synthetic polymers, chondroitin sulfate, dermatan sulfate, dextran sulfate, keratan sulfate, heparan. Charged polysaccharides such as sulfuric acid, hyaluronic acid and chitin, cell adhesion proteins such as collagen, gelatin, fibronectin and hydronectin, as well as cell adhesion proteins and synthetic polymers with cell adhesion peptides immobilized. But,
It is not limited to these. These may be used alone or in combination of two or more.
また、前記細胞非接着性表面とは、接触角が100度以上
の疎水性表面、または電荷を有さず接触角が50度以下の
親水性表面をいう。The cell non-adhesive surface means a hydrophobic surface having a contact angle of 100 degrees or more, or a hydrophilic surface having no charge and a contact angle of 50 degrees or less.
前記疎水性表面の具体例としては、たとえばポリテトラ
フルオロエチレン、シリコーンなどから形成された表面
があげられるが、これらに限定されるものではない。Specific examples of the hydrophobic surface include, but are not limited to, surfaces formed of polytetrafluoroethylene, silicone, and the like.
また、前記接触角が50度以下の親水性表面の具体例とし
ては、電荷を持たない親水性高分子よりなる表面、たと
えばポリビニルアルコール、ポリエチレングリコール、
ポリアクリルアミド、ポリジメチルアクリルアミド、ポ
リヒドロキシエチルメタクリレート、さらにはこれらを
構成する単量体の共重合体、セルロースなどがあげられ
るが、これらに限定されるものではない。Further, as a specific example of the hydrophilic surface having a contact angle of 50 degrees or less, a surface made of a hydrophilic polymer having no electric charge, for example, polyvinyl alcohol, polyethylene glycol,
Examples thereof include, but are not limited to, polyacrylamide, polydimethylacrylamide, polyhydroxyethyl methacrylate, and copolymers of the monomers constituting these, cellulose and the like.
さらに、本発明により製造される細胞の配列制御用具を
形成しうる素材としては、たとえば各種プラスチック、
ガラス、金属などがあげられ、すでにデバイスとして用
いられているたとえば培養用皿、半導体基盤などの材料
も利用できる。Further, as a material capable of forming the cell array control tool produced by the present invention, for example, various plastics,
Examples thereof include glass and metal, and materials such as culture dishes and semiconductor substrates that have already been used as devices can also be used.
つぎに前記細胞の配列制用具の製法について説明する。Next, a method for producing the cell arraying device will be described.
まず第１の製法として、 細胞接着性表面および細胞非接着性表面よりなる配列パ
ターンを （１）感光性を有する細胞非接着性親水性高分子を細胞
接着性表面に塗布もしくは吸着させて存在させる工程、 （２）（１）でえられた表面上に望む配列パターンを有
するフォトマスクを設置してパターン露光する工程およ
び （３）洗浄により現像し、細胞非接着性親水性高分子よ
りなる像を細胞接着性表面に形成させる工程 を経て形成する方法を説明する。First, as a first production method, an array pattern consisting of a cell-adhesive surface and a cell-non-adhesive surface is present (1) by coating or adsorbing a cell-adhesive hydrophilic polymer having photosensitivity on the cell-adhesive surface Step, (2) a step of placing a photomask having a desired arrangement pattern on the surface obtained in (1) and performing pattern exposure, and (3) an image composed of a cell-nonadhesive hydrophilic polymer developed by washing. A method for forming the cells on the cell adhesive surface will be described.
第１の製法においては、たとえば細胞非接着性親水性高
分子、好ましくは前記電荷を持たない親水性高分子が、
該高分子と２個以上のアジド基を有する化合物とからな
る組成物を本発明の細胞の配列制御用具となる細胞接着
性表面に存在させたのち、光照射することにより、細胞
接着性表面に容易に固定される。また、前記高分子に直
接アジド基を導入したものを用いることもできるが、ア
ジド基を導入する特別な操作が不要な点および現像時の
未反応物の除去が容易な点で、該高分子と２個以上のア
ジド基を有する化合物よりなる組成物を用いるのが好ま
しい。In the first production method, for example, a cell-non-adhesive hydrophilic polymer, preferably the non-charged hydrophilic polymer is
A composition comprising the polymer and a compound having two or more azido groups is allowed to exist on the cell-adhesive surface, which serves as a device for controlling the arrangement of cells of the present invention, and then irradiated with light to form a cell-adhesive surface. Easily fixed. Further, it is also possible to use a polymer in which an azido group is directly introduced, but the polymer does not require a special operation for introducing an azide group and can easily remove unreacted substances during development. And a compound having two or more azido groups is preferably used.
前記２個以上のアジド基を有する化合物としては、たと
えば第１表に示すような一般のビスアジド化合物、１分
子中に２個以上のアジド基を導入したアジド化ポリマー
などが利用できるが、これらに限定されるものではな
い、上記アジド基には、たとえばカルボニルアジド（Ｒ
−CON3）、スルホニルアジド（Ｒ−SO2N3）、芳香族ア
ジド などがあるが、安定性のよい芳香族アジドまたはスルホ
ニルアジドが好ましい。また、より長波長域の光でナイ
トレンに転化できる点で、ニトロ基のような電子吸引性
置換基を有する芳香族アジド、ｉ線またはｇ線感光性の
ビスアジド化合物がさらに好ましい。As the compound having two or more azido groups, for example, a general bisazide compound as shown in Table 1 and an azide polymer in which two or more azido groups are introduced in a molecule can be used. Non-limiting examples of the azido group include carbonyl azide (R
-CON 3 ), sulfonyl azide (R-SO 2 N 3 ), aromatic azide However, an aromatic azide or a sulfonyl azide having good stability is preferable. Further, an aromatic azide having an electron-withdrawing substituent such as a nitro group and an i-ray or g-ray-sensitive bisazide compound are more preferable because they can be converted to nitrene by light having a longer wavelength region.
該高分子は、アジド基が光照射により転化したナイトレ
ン基が、細胞接着性表面および該高分子に対して、たと
えば次式に示すような化学反応、すなわち、水素引抜き
反応（１）、二重結合への付加やＣ−Ｈ結合への挿入
（２）、およびカップリング反応（３）を行なうことに
より固定される。 In the polymer, a nitrene group obtained by converting an azide group by light irradiation is reacted with the cell-adhesive surface and the polymer by, for example, a chemical reaction represented by the following formula, that is, a hydrogen abstraction reaction (1), a double reaction. It is fixed by addition to a bond, insertion into a C—H bond (2), and a coupling reaction (3).
なお、ナイトレン基はきわめて反応性が高いため、上記
の反応以外の反応による結合が生じるばあいもある。ま
た、上記反応が該高分子間に生じ、架橋が生じるばあい
があるが、これにより該高分子がより安定的に細胞接着
性表面に固定されるばあいがある。さらに、該高分子が
前述のごとく細胞接着性表面に結合していなくても、皮
膜として付着し固定されていてもよい。 Since the nitrene group has extremely high reactivity, a bond other than the above reaction may occur. In addition, the above reaction may occur between the polymers to cause cross-linking, which may cause the polymers to be more stably fixed on the cell-adhesive surface. Further, the polymer may not be bound to the cell adhesive surface as described above, but may be attached and fixed as a film.
該高分子と２個以上のアジド基を有する化合物よりなる
組成物を細胞接着性表面に存在させる方法としては、該
組成物をメタノールのような揮発性有機溶媒に溶解また
は懸濁し、この液を細胞接着性表面に、塗布または噴霧
したのち乾燥し、該組成物の薄層を細胞接着性表面に形
成させる方法、該組成物の水溶液またはコロイド溶液と
細胞接着性表面とを接触させ、細胞接着性表面に吸着さ
せる方法などがある。これらのなかでも均質な薄層がえ
られる点で、揮発性有機溶媒の溶液を用いてキャスト製
膜する方法が好ましい。As a method for allowing a composition comprising the polymer and a compound having two or more azido groups to be present on the cell adhesive surface, the composition is dissolved or suspended in a volatile organic solvent such as methanol, and the solution is A method of forming a thin layer of the composition on the cell-adhesive surface by applying or spraying on the cell-adhesive surface, or by contacting an aqueous solution or colloidal solution of the composition with the cell-adhesive surface, There is a method of adsorbing to the surface. Among these, the method of forming a cast film using a solution of a volatile organic solvent is preferable because a uniform thin layer can be obtained.
また、２個以上のアジド基を有する化合物を細胞接着性
表面に存在させたのち、その上に該高分子を存在させて
もよい。Alternatively, a compound having two or more azido groups may be allowed to exist on the cell adhesive surface, and then the polymer may be allowed to exist thereon.
前記光照射に用いる光源としては、高圧水銀灯、低圧水
銀灯、超高圧水銀灯など各種水銀灯、エキシマレーザー
などがあるが、長波長域で感光可能なアジド化合物を用
いるばあいは、フィルターにより短波長域をカットする
ことにより、短波長紫外線による該高分子や材料表面へ
の影響を軽減することができる。これは蛋白質などの親
水性高分子を用いるばあいとくに好ましい。As the light source used for the light irradiation, there are various mercury lamps such as high-pressure mercury lamp, low-pressure mercury lamp, and ultra-high-pressure mercury lamp, excimer laser, and the like. By cutting, it is possible to reduce the influence of short-wavelength ultraviolet light on the surface of the polymer or the material. This is particularly preferable when a hydrophilic polymer such as protein is used.
また、ナイトレン基の反応は極めて短時間で完了するた
め、露光時間は５分以内でよい。Moreover, since the reaction of the nitrene group is completed in an extremely short time, the exposure time may be within 5 minutes.
パターン露光の方法は、パターンを有するフォトマスク
を設置した上より光照射する方法、エキシマレーザーに
よるリソグラフィーを利用する方法などがある。Examples of the pattern exposure method include a method in which a photomask having a pattern is placed and then light irradiation, a method in which lithography with an excimer laser is used, and the like.
一方、細胞の配列制御用具の第２の製法は、前記第１の
製法の（１）の工程において、感光性を有する細胞非接
着性親水性高分子を細胞接着性表面に塗布もしくは吸着
させるかわりに、感光性を有する前記細胞接着性親水性
高分子を細胞非接着性表面に塗布もしくは吸着させるほ
かは、第１の製法と同様にして製造する方法である。On the other hand, in the second method of manufacturing a device for controlling cell arrangement, in the step (1) of the first manufacturing method, instead of coating or adsorbing a cell non-adhesive hydrophilic polymer having photosensitivity on the cell adhesive surface. In addition, the method is the same as the first method except that the cell-adhesive hydrophilic polymer having photosensitivity is applied to or adsorbed on the cell-non-adhesive surface.
第２の製法によれば、たとえば前記細胞接着性を有する
高分子が、該高分子と２個以上のアジド基を有する化合
物よりなる組成物を本発明により製造される細胞の配列
制御用具となる細胞非接着性表面に存在させたのち、光
照射することにより、細胞非接着性表面に容易に固定さ
れる。According to the second production method, for example, a composition in which the polymer having cell adhesiveness is composed of the polymer and a compound having two or more azido groups is used as a cell arrangement control tool produced by the present invention. After being allowed to exist on the cell non-adhesive surface, it is easily fixed to the cell non-adhesive surface by irradiation with light.
前記のごとく製造される細胞の配列制御用具を用い、常
法により細胞を培養することにより、細胞配列を容易に
制御でき、μｍオーダーまでの高解像度の微細パターン
を形成することができる。By culturing the cells by a conventional method using the cell array control tool manufactured as described above, the cell array can be easily controlled and a high-resolution fine pattern up to the μm order can be formed.
えられた微細パターンは、超小型バイオセンサー、スイ
ッチング素子、バイオリアクター、ハイブリッド型人工
臓器などの製造、さらにはニューロコンピューターなど
の開発に有用である。The obtained fine pattern is useful for manufacturing microminiature biosensors, switching elements, bioreactors, hybrid artificial organs, etc., and also for developing neurocomputers, etc.
つぎに実施例を用いて本発明をさらに詳しく説明する
が、本発明はこれに限定されるものではない。Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
実施例１ N,N−ジメチルアクリルアミドモノマー（（株）興人
製）をアセトン中、過酸化ベンゾイルおよびN,N−ジメ
チル−ｐ−トルイジンをレドックス系開始剤として重合
し、ポリ（N,N−ジメチルアクリルアミド）（以下、PDM
AAという）をえた。Example 1 N, N-dimethylacrylamide monomer (manufactured by Kojin Co., Ltd.) was polymerized in acetone using benzoyl peroxide and N, N-dimethyl-p-toluidine as a redox initiator to give poly (N, N- Dimethyl acrylamide) (hereinafter PDM
AA).
えられたPDMAA95部（重量部、以下同様）に対して、ビ
スアジド化合物である4,4′−ジアジドスチルベン−2,
2′−ジスルホン酸ソーダ５部を混合したものをメタノ
ールに溶かし、0.1％（重量％、以下同様）溶液とし
た。With respect to the obtained PDMAA 95 parts (parts by weight, the same applies hereinafter), 4,4′-diazidostilbene-2, which is a bisazide compound,
A mixture of 5 parts of 2'-sodium disulfonate was dissolved in methanol to prepare a 0.1% (wt%, the same applies hereinafter) solution.
この溶液を、組織培養用ポリスチレンシャーレ（コーニ
ング（CORNING）社製）上に滴下し、キャスト製膜して
風乾し、厚さ数十nmの膜を形成したのち、この上に第３
図に示すような開孔部と非開孔部とからなる一対の幅が
250μｍであるスリットを有するフォトマスクをセット
し、高圧水銀灯を用いて30秒間パターン露光した。な
お、第３図はフォトマスクの写真のスケッチ図である。This solution was dropped on a polystyrene dish for tissue culture (manufactured by CORNING), cast film-formed and air-dried to form a film having a thickness of several tens of nm, and then a third film was formed thereon.
As shown in the figure, a pair of widths consisting of open and non-open areas
A photomask having a slit of 250 μm was set, and pattern exposure was performed for 30 seconds using a high pressure mercury lamp. Note that FIG. 3 is a sketch drawing of a photo of the photomask.
つぎにメタノール、水で充分洗浄して現像し、PDMAAお
よびシャーレ表面よりなる微細パターンを形成したシャ
ーレをえた。Then, the dish was thoroughly washed with methanol and water and developed to obtain a petri dish having a fine pattern composed of PDMAA and the surface of the petri dish.
このようにしてえたシャーレに、牛血管内皮細胞を播種
し、15％子牛血清（FCS）を含むDMEM（Dulbecco′s Mod
ified Eagle′s Medium）を培地として用い、37℃のCO2
インキュベーター内で培養したところ、内皮細胞はPDMA
A非固定部（非露光部）のみに選択的に伸展・増殖し、
第１図および第２図に示す細胞の配列パターンがえられ
た。第１図は染色された細胞の配列パターンの写真（倍
率は第３図のもとになる写真と同じ）のスケッチ図、第
２図は第１図のもとになる写真よりもさらに拡大された
写真のスケッチ図である。The petri dish thus obtained was seeded with bovine vascular endothelial cells, and DMEM (Dulbecco's Mod) containing 15% calf serum (FCS) was added.
ified Eagle's Medium) is used as the medium and CO 2 at 37 ° C
When cultured in an incubator, endothelial cells showed PDMA
A Expands / proliferates selectively only in the non-fixed area (non-exposed area)
The cell arrangement patterns shown in FIGS. 1 and 2 were obtained. Fig. 1 is a sketch of a photograph of the stained cell array pattern (magnification is the same as the one on which Fig. 3 is based), and Fig. 2 is a larger image than the one on which Fig. 1 is based. It is a sketch drawing of the photograph.
実施例２ 実施例１のばあいと同様にして調製したビスアジド化合
物を含むPDMAAの0.1％メタノール溶液を、ポリスチレン
シャーレ上に滴下し、キャスト製膜して風乾したのち、
高圧水銀灯を用いて紫外線を照射し、PDMAAを光固定し
たシャーレ（以下、PDMAAシャーレという）をえた。Example 2 A 0.1% methanol solution of PDMAA containing a bisazide compound prepared in the same manner as in Example 1 was dropped on a polystyrene petri dish, and after cast film formation and air drying,
Ultraviolet rays were irradiated using a high-pressure mercury lamp to obtain a Petri dish (hereinafter referred to as PDMAA Petri dish) on which PDMAA was optically fixed.
N,N−ジメチルアクリルアミド80部とアクリロキシコハ
ク酸イミド（国産化学製）20部よりなる共重合体とフィ
ブロネクチンとを、リン酸緩衝液（pH8.5）中で反応さ
せ、フィブロネクチンを固定したN,N−ジメチルアクリ
ルアミド共重合体（以下、FN−PDMAAという）をえた。Fibronectin was reacted with a copolymer consisting of 80 parts of N, N-dimethylacrylamide and 20 parts of acryloxysuccinimide (manufactured by Kokusan Kagaku Co., Ltd.) in a phosphate buffer (pH 8.5) to fix fibronectin. , N-dimethylacrylamide copolymer (hereinafter referred to as FN-PDMAA) was obtained.
FN−PDMAA95部に対してビスアジド化合物５部を混合し
たものをメタノールに溶かし、0.1％溶液とした。A mixture of 95 parts of FN-PDMAA and 5 parts of a bisazide compound was dissolved in methanol to prepare a 0.1% solution.
えられた溶液をPDMAAシャーレ上にキャスト製膜して風
乾し、厚さ数十nmの膜を形成したのち、フォトマスクを
セットし、高圧水銀灯を用いて30秒間パターン露光し
た。The obtained solution was cast on a PDMAA petri dish and air-dried to form a film having a thickness of several tens of nm, a photomask was set, and pattern exposure was performed for 30 seconds using a high pressure mercury lamp.
つぎにメタノール、水で充分洗浄して現像し、FN−PDMA
AおよびPDMAAシャーレ表面よりなる微細パターンを形成
したシャーレをえた。Next, wash thoroughly with methanol and water and develop, then use FN-PDMA.
A petri dish having a fine pattern composed of A and PDMAA petri dish surfaces was obtained.
このようにしてえたシャーレを用いて実施例１のばあい
と同様にして、牛血管内皮細胞を培養したところ、内皮
細胞は、FN−PDMAA固定部（露光部）のみに選択的に伸
展・増殖し、細胞による配列パターンがえられた。When the bovine vascular endothelial cells were cultured using the dish thus obtained in the same manner as in Example 1, the endothelial cells were selectively spread and proliferated only in the FN-PDMAA fixed part (exposed part). Then, a sequence pattern by cells was obtained.
［発明の効果］ 本発明により製造される細胞の配列制御用具は、細胞の
付着の有無の選択性がよく、これを用いることにより、
従来の細胞培養と同様にして培養を行なって容易に精度
の高い細胞配列制御をすることができ、極めて微細かつ
高解像度の細胞配列パターンを容易に形成することがで
きる。[Effects of the Invention] The cell array control device produced by the present invention has good selectivity for the presence or absence of cell attachment.
By culturing in the same manner as the conventional cell culture, the cell array can be easily controlled with high precision, and an extremely fine and high-resolution cell array pattern can be easily formed.
また、本発明の製法により、前記配列制御用具を容易に
製造することができる。Further, the array control tool can be easily manufactured by the manufacturing method of the present invention.
本発明は、各種細胞機能を応用した超小型バイオセンサ
ー、スイッチング素子、バイブリッド型人工臓器、バイ
オリアクター、ニューロコンピューターなどの開発に大
きく貢献するものである。また、細胞間の情報伝達など
の細胞機能の研究においても応用できるものである。INDUSTRIAL APPLICABILITY The present invention greatly contributes to the development of microminiature biosensors, switching elements, hybrid artificial organs, bioreactors, neurocomputers, etc., which apply various cell functions. It can also be applied in research on cell functions such as information transmission between cells.
第１図は染色された細胞の配列パターンの写真のスケッ
チ図、第２図は第１図のもとになる写真よりもさらに拡
大された写真のスケッチ図、第３図はフォトマスクの写
真（倍率は第１図のもとになる写真と同じ）のスケッチ
図である。Fig. 1 is a sketch drawing of a photograph of a stained cell array pattern, Fig. 2 is a sketch drawing of a photograph enlarged from the original photograph of Fig. 1, and Fig. 3 is a photomask photograph ( Magnification is a sketch drawing of the same as the original photo of FIG. 1).
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｈ０１Ｌ 21/027 ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI technical display location H01L 21/027
Claims (1)
りなる配列パターンが、 （１）感光性を有する細胞非接着性親水性高分子を細胞
接着性表面に塗布もしくは吸着させて存在させる工程、 （２）（１）でえられた表面上に望む配列パターンを有
するフォトマスクを設置してパターン露光する工程およ
び （３）洗浄により現像し、細胞非接着性親水性高分子よ
りなる像を細胞接着性表面に形成させる工程 または （１）感光性を有する細胞接着性親水性高分子を細胞非
接着性表面に塗布もしくは吸着させて存在させる工程、 （２）（１）でえられた表面上に望む配列パターンを有
するフォトマスクを設置してパターン露光する工程およ
び （３）洗浄により現像し、細胞接着性親水性高分子より
なる像を細胞非接着性表面に形成させる工程 を経て形成されることを特徴とする細胞の配列制御用具
の製法。1. An array pattern comprising a cell-adhesive surface and a cell-non-adhesive surface, wherein (1) a step of applying or adsorbing a cell-adhesive hydrophilic polymer having photosensitivity on the cell-adhesive surface (2) A step of setting a photomask having a desired arrangement pattern on the surface obtained in (1) and performing pattern exposure, and (3) developing by washing to form an image of a cell-nonadhesive hydrophilic polymer. Or (1) a step of applying or adsorbing a cell-adhesive hydrophilic polymer having photosensitivity to a cell-non-adhesive surface to allow it to exist, (2) the surface obtained in (1) A step of placing a photomask having a desired arrangement pattern on it and performing pattern exposure, and (3) a step of developing by washing to form an image of a cell-adhesive hydrophilic polymer on a cell-nonadhesive surface A method for producing a device for controlling cell arrangement, which is characterized in that it is formed through
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1141964A JPH0751061B2 (en) | 1989-06-03 | 1989-06-03 | Manufacturing method of cell array control tool |
| EP90110455A EP0402718B1 (en) | 1989-06-03 | 1990-06-01 | Control of cell arrangement |
| US07/531,596 US5202227A (en) | 1989-06-03 | 1990-06-01 | Control of cell arrangement |
| DE69013764T DE69013764T2 (en) | 1989-06-03 | 1990-06-01 | Control of cell arrangement. |
| US08/308,204 US5593814A (en) | 1989-06-03 | 1994-09-19 | Control of cell arrangement |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1141964A JPH0751061B2 (en) | 1989-06-03 | 1989-06-03 | Manufacturing method of cell array control tool |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6287588A Division JP2609073B2 (en) | 1994-10-26 | 1994-10-26 | Cell arrangement control tool and cell arrangement control method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH037576A JPH037576A (en) | 1991-01-14 |
| JPH0751061B2 true JPH0751061B2 (en) | 1995-06-05 |
Family
ID=15304223
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1141964A Expired - Fee Related JPH0751061B2 (en) | 1989-06-03 | 1989-06-03 | Manufacturing method of cell array control tool |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0751061B2 (en) |
Families Citing this family (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3126269B2 (en) * | 1993-09-07 | 2001-01-22 | 株式会社バイオマテリアル研究所 | Culture substrate |
| JP2500472B2 (en) * | 1993-10-06 | 1996-05-29 | 日本電気株式会社 | Cell array culture device and method thereof |
| JP2570621B2 (en) * | 1994-06-27 | 1997-01-08 | 日本電気株式会社 | Cell culture substrate, method for producing the same, and method for forming cell array |
| US6893850B2 (en) * | 2000-03-17 | 2005-05-17 | President And Fellows Of Harvard College | Method for cell patterning |
| DE60228594D1 (en) | 2001-07-26 | 2008-10-09 | Transparent Inc | CONSTRUCTION OF CULTIVATED CELLS WITH SPHEROIDS OF CULTIVATED ANIMAL CELLS AND USE THEREOF |
| JP4589012B2 (en) * | 2004-02-19 | 2010-12-01 | 大日本印刷株式会社 | Patterning substrate for cell culture |
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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 |
| JP6105223B2 (en) * | 2012-07-30 | 2017-03-29 | 東京応化工業株式会社 | Cell culture plate production method, cell culture plate produced by this production method, cell culture method, cell sheet production method, cell sheet, and photosensitive resin composition |
| TWI613206B (en) | 2012-10-11 | 2018-02-01 | 國立大學法人富山大學 | Photodecomposition material, substrate and patterning method thereof |
| JP2020029468A (en) | 2016-11-10 | 2020-02-27 | 国立大学法人富山大学 | Material for cell patterning |
| WO2020071332A1 (en) | 2018-10-01 | 2020-04-09 | 株式会社片岡製作所 | Cell culture tool and method for manufacturing cell culture tool |
| JP7343119B2 (en) | 2019-04-26 | 2023-09-12 | 株式会社片岡製作所 | Cell culture substrate, cell culture container, cell culture method, cell manufacturing method, cell culture substrate manufacturing method, and cell culture container manufacturing method |
| US20220372540A1 (en) * | 2019-06-17 | 2022-11-24 | Toyo Gosei Co., Ltd. | Cell culture substrate, method for manufacturing the same and screening method by using the same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63173575A (en) * | 1987-01-14 | 1988-07-18 | Hitachi Ltd | Component for cell manipulating apparatus |
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1989
- 1989-06-03 JP JP1141964A patent/JPH0751061B2/en not_active Expired - Fee Related
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| Publication number | Publication date |
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| JPH037576A (en) | 1991-01-14 |
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