JPH037577A - Orientation controlling tool for cell, production thereof and method for controlling orientation of cell - Google Patents
Orientation controlling tool for cell, production thereof and method for controlling orientation of cellInfo
- Publication number
- JPH037577A JPH037577A JP1141965A JP14196589A JPH037577A JP H037577 A JPH037577 A JP H037577A JP 1141965 A JP1141965 A JP 1141965A JP 14196589 A JP14196589 A JP 14196589A JP H037577 A JPH037577 A JP H037577A
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- JP
- Japan
- Prior art keywords
- cell
- pattern
- mask
- adhesive
- radiation
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Links
- 238000000034 method Methods 0.000 title claims description 23
- 238000004519 manufacturing process Methods 0.000 title claims description 16
- 239000000463 material Substances 0.000 claims abstract description 20
- 239000000178 monomer Substances 0.000 claims abstract description 20
- 230000004956 cell adhesive effect Effects 0.000 claims abstract description 19
- 230000005855 radiation Effects 0.000 claims abstract description 18
- 125000000524 functional group Chemical group 0.000 claims abstract description 14
- 230000001070 adhesive effect Effects 0.000 claims description 14
- 239000000853 adhesive Substances 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 6
- 230000000977 initiatory effect Effects 0.000 claims description 5
- 238000006116 polymerization reaction Methods 0.000 claims description 5
- 230000000379 polymerizing effect Effects 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 11
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 abstract description 6
- 125000003277 amino group Chemical group 0.000 abstract description 6
- 238000010559 graft polymerization reaction Methods 0.000 abstract description 4
- 230000001939 inductive effect Effects 0.000 abstract description 3
- 239000004033 plastic Substances 0.000 abstract description 2
- 229920003023 plastic Polymers 0.000 abstract description 2
- 238000004381 surface treatment Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 67
- 208000028659 discharge Diseases 0.000 description 8
- 229920000642 polymer Polymers 0.000 description 7
- -1 polytetrafluoroethylene Polymers 0.000 description 7
- 241000283690 Bos taurus Species 0.000 description 5
- 210000002889 endothelial cell Anatomy 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 5
- 210000003556 vascular endothelial cell Anatomy 0.000 description 5
- 150000001408 amides Chemical class 0.000 description 4
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 4
- 239000004810 polytetrafluoroethylene Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- 230000021164 cell adhesion Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- BAPJBEWLBFYGME-UHFFFAOYSA-N Methyl acrylate Chemical compound COC(=O)C=C BAPJBEWLBFYGME-UHFFFAOYSA-N 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229920001477 hydrophilic polymer Polymers 0.000 description 2
- 230000005660 hydrophilic surface Effects 0.000 description 2
- 230000005661 hydrophobic surface Effects 0.000 description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 229920002939 poly(N,N-dimethylacrylamides) Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229920002818 (Hydroxyethyl)methacrylate Polymers 0.000 description 1
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010894 electron beam technology Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 230000002747 voluntary effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Exposure Of Semiconductors, Excluding Electron Or Ion Beam Exposure (AREA)
Abstract
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、その製法および細胞の
配列制御法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a cell arrangement control tool, a method for producing the same, and a cell arrangement control method.
［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、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, along with rapid advances in cell engineering, LSI technology, medical engineering, etc., ultra-small biosensors using cells, switching elements, bioreactors, hybrid artificial organs, and even neurocomputers are attracting attention. gather,
These developments are actively underway.
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。It is difficult to arrange cells as desired and maintain their functions, which is one of the obstacles to realizing devices using cells. Cell arrangement control technology, which allows cells to be arranged in a desired manner to form a circuit network, could be a major key technology for realizing these devices.
また、紫外線や放射線による表面親水化や親水性モノマ
ーのグラフト重合は、材料表面の接着性や生体適合性を
向上させる手法として応用されているが、これを細胞の
配列制御へ応用しようという試みはなされていない。In addition, surface hydrophilization using ultraviolet rays or radiation and graft polymerization of hydrophilic monomers have been applied as methods to improve the adhesiveness and biocompatibility of material surfaces, but there have been no attempts to apply these to control cell arrangement. Not done.
［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。[Problem to be solved by the invention] As an attempt to control the arrangement of cells, there is an example in which a pattern is formed by applying fibronectin, a cell adhesion protein, using an inkjet printer, and cells are cultured on this pattern. However, the resolution is poor and non-uniform, making it unsuitable for microfabrication.
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, there have been recent attempts to control the direction of neuronal synapse growth using artificially uneven surfaces, but this has not led to the formation of the desired arrangement.
［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 the above-mentioned circumstances, the present inventors have conducted extensive research on methods for easily controlling cell arrangement, and have found that The arrangement of cells can be easily controlled by culturing cells on a material surface having an arrangement pattern of They discovered that it can be easily manufactured through a process and 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
ターンを有することを特徴とする細胞の配列制御用具、
細胞の配列制御用具の細胞非接着性表面上にパターンマ
スクを設置したのち、紫外線または放射線よって表面処
理し、非マスク部のみに細胞接着性官能基を誘導して配
列パターンを形成することを特徴とする前記細胞の配列
制御用具の製法、
細胞の配列制御用具の材料表面上にパターンマスクを設
置したのち、紫外線または放射線によって表面処理し、
非マスク部のみに重合開始種を誘導し、これにより、細
胞接着性モノマーまたは細胞非接着性モノマーを非マス
ク部のみにグラフト重合させて配列パターンを形成する
ことを特徴とする前記細胞の配列制御用具の製法、およ
び
前記細胞の配列制御用具を用いて細胞を培養することを
特徴とする細胞の配列制御法に関する。That is, the present invention provides a cell array control device characterized by having an array pattern consisting of a cell adhesive surface and a cell non-adhesive surface, and a pattern mask is provided on the cell non-adhesive surface of the cell array control device. A method for manufacturing the device for controlling cell array, which is characterized in that the surface is treated with ultraviolet rays or radiation to form an array pattern by inducing cell-adhesive functional groups only in the non-mask portion, and materials for the device for controlling cell array. After placing a pattern mask on the surface, the surface is treated with ultraviolet rays or radiation,
Cell arrangement control characterized in that a polymerization initiating species is induced only in the non-masked region, thereby graft-polymerizing a cell adhesive monomer or a cell non-adhesive monomer only in the non-masked region to form an array pattern. The present invention relates to a method for manufacturing a device, and a cell array control method characterized by culturing cells using the cell array control device.
［実施例］
本発明の細胞の配列制御用具は、パターン化した細胞接
着性表面と細、胞非接着性表面とが本発明の細胞の配列
制御用具となる材料の表面に形成されたものである。[Example] The cell array control device of the present invention is one in which a patterned cell adhesive surface and a cell/cell non-adhesive surface are formed on the surface of a material serving as the cell array control device of the present invention. be.
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） Ｒ，ＧＤ
Ｓ（Ａｒｇ−Ｇｌｙ−Ａｓｐ−８ｅｒ）のような細胞接
着性ペプチドを有する表面をいう。これらの官能基やペ
プチドは、材料表面に直接導入されていてもよく、これ
らの官能基やペプチドを有する高分子が材料表面に固定
されているものでもよい。The cell-adhesive surface refers to charged functional groups such as carboxyl groups and amino groups, and (also) R, GD.
A surface having a cell adhesive peptide such as S (Arg-Gly-Asp-8er). These functional groups and peptides may be directly introduced onto the material surface, or polymers having these functional groups and peptides may be fixed to the material surface.
これらの高分子を材料表面に固定する方法としては、た
とえば材料表面の官能基と該高分子の官能基とを化学反
応により結合させる方法、カルボキシル基などの電荷を
有する官能基含有モノマーを材料表面にグラフト重合す
る方法、材料表面にグラフト重合されたポリマーにカル
ボキシル基やアミノ基を誘導する方法などがある。たと
えばアクリルアミドを材料表面にグラフト重合したのち
、アルカリ処理によりアミドを加水分解してカルボキシ
ル基とする方法、または次亜塩素酸ソーダのアルカリ溶
液で処理（ホフマン分解）することによりアミドをアミ
ノ基に変換する方法などがある。Methods for fixing these polymers to the material surface include, for example, a method of bonding a functional group on the material surface with a functional group of the polymer through a chemical reaction, and a method of attaching a monomer containing a charged functional group such as a carboxyl group to the material surface. There are methods such as graft polymerization on the surface of the material, and methods of inducing carboxyl groups or amino groups on the polymer grafted onto the surface of the material. For example, after graft polymerizing acrylamide onto the material surface, the amide is hydrolyzed into a carboxyl group by alkali treatment, or the amide is converted to an amino group by treatment with an alkaline solution of sodium hypochlorite (Hoffmann degradation). There are ways to do this.
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、または電荷を有さす接触角が５０度以
下の親水性表面をいう。Further, the cell non-adhesive surface refers to a hydrophobic surface with a contact angle of 100 degrees or more, or a hydrophilic surface with a 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 made of polytetrafluoroethylene, silicone, and the like.
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。Specific examples of the hydrophilic surface having a contact angle of 50 degrees or less include surfaces made of uncharged hydrophilic polymers, such as polyvinyl alcohol, polyethylene glycol, polyacrylamide, polydimethylacrylamide, polyhydroxyethyl methacrylate, and Examples include copolymers of monomers constituting these, cellulose, etc., but are not limited thereto.
また、これらの電荷を持たない親水性モノマーを材料表
面にグラフト重合することにより材料表面を細胞非接着
性表面に改質することもできる。Furthermore, by graft polymerizing these uncharged hydrophilic monomers onto the surface of the material, the surface of the material can be modified into a non-cell-adhesive surface.
さらに、本発明の細胞の配列制御用具となる材料として
は、たとえば各種プラスチック製の培養用器、フィルム
、チューブなどがあげられる。Furthermore, examples of materials that can be used as the cell arrangement control device of the present invention include various plastic culture vessels, films, tubes, and the like.
つぎに前記細胞の配列制御用具の製法について説明する
。Next, a method for manufacturing the cell arrangement control device will be described.
まず第１の製法として、細胞の配列制御用具の細胞非接
着性表面上にパターンマスクを設置したのち、紫外線ま
たは放射線によって表面処理し、非マスク部のみに細胞
接着性官能基を誘導して配列パターンを形成する方法を
説明する。First, as the first manufacturing method, a pattern mask is placed on the cell non-adhesive surface of a cell arrangement control device, and then the surface is treated with ultraviolet rays or radiation, and cell adhesion functional groups are induced and arranged only in the non-mask area. Explain how to form a pattern.
前記細胞非接着性表面としては、たとえばポリジメチル
アクリルアミドなどの電荷を持たない親水性高分子など
よりなる表面があげられる。Examples of the cell non-adhesive surface include surfaces made of uncharged hydrophilic polymers such as polydimethylacrylamide.
本発明において誘導される細胞接着性官能基は、カルボ
キシル基やアミノ基であり、空気中紫外線照射またはプ
ラズマのような放射線処理により周知のごとく誘導され
る。The cell-adhesive functional groups induced in the present invention are carboxyl groups and amino groups, and are induced by radiation treatment such as ultraviolet irradiation in the air or plasma, as is well known.
前記放射線としては、たとえばコロナ放電やグロー放電
などのプラズマ、電子線、γ線などがあげられるが、こ
れらに限定されるものではない。なかでも適用対象の多
様性、効果の有用性の点でグロー放電のようなプラズマ
を用いるのが好ましく、さらには官能基を効率よく誘導
させうるという点から Ｎ２プラズマや０２プラズマが
好ましい。Examples of the radiation include, but are not limited to, plasma such as corona discharge and glow discharge, electron beams, and gamma rays. Among these, it is preferable to use plasma such as glow discharge in terms of the diversity of applications and the usefulness of the effect, and N2 plasma and 02 plasma are more preferable in terms of their ability to efficiently induce functional groups.
また、高解像度の処理をしうるという点で紫外線が好ま
しい。Furthermore, ultraviolet light is preferable because it allows high-resolution processing.
前記パターンマスクとは、描きたいパターンの孔を有し
、それに対応する部分を、照射する紫外線または放射線
に暴露させつる構造を有するもので、一般のフォトマス
クが利用できる。The pattern mask has a hole in the pattern to be drawn and has a structure that exposes the corresponding portion to ultraviolet rays or radiation, and a general photomask can be used.
とくにプラズマのような放射線を利用するばあいは、金
属製のマスクではマスクからの放電で解像度が低下する
可能性があるため、非金属性のマスクが望ましい。Particularly when using radiation such as plasma, a non-metallic mask is preferable, since a metallic mask may reduce resolution due to discharge from the mask.
一方、細胞の配列制御用具の第２の製法は、細胞の配列
制御用具の材料表面上にパターンマスクを設置したのち
、紫外線または放射線によっテ表面処理し、非マスク部
のみに重合開始種を誘導し、これにより、細胞接着性モ
ノマーまたは細胞非接着性モノマーを非マスク部のみに
グラフト重合させて配列パターンを形成する方法である
。On the other hand, the second method for manufacturing a cell array control device is to place a pattern mask on the material surface of the cell array control device, and then treat the surface with ultraviolet rays or radiation to inject polymerization initiating species only in the non-mask area. In this method, a cell-adhesive monomer or a cell-non-adhesive monomer is graft-polymerized only on the non-mask area to form an array pattern.
本発明において誘導される重合開始種とは、材料自体に
由来するポリマーラジカルであり、紫外線処理またはプ
ラズマのような放射線処理により周知のごとく誘導され
る。紫外線処理では一般に増感剤を必要とするため、こ
れを必要としない放射線処理が好ましい。The polymerization initiating species induced in the present invention are polymer radicals originating from the material itself and induced by radiation treatment, such as ultraviolet or plasma, as is well known. Since ultraviolet light treatment generally requires a sensitizer, radiation treatment that does not require this is preferred.
前記放射線としては、前述のごとく種々のものがあげら
れるが、なかでもグロー放電のようなプラズマが好まし
い。As the radiation, there are various types as mentioned above, and among them, plasma such as glow discharge is preferable.
このようにして誘導したラジカルによる細胞接着性モノ
マーまたは細胞非接着性モノマーのグラフト重合は、七
ツマ−を直接ラジカルを生成させた系内に導入して重合
させてもよく、−旦ラジカルを空気と接触させてパーオ
キサイドとしたのちモノマーを添加し、パーオキサイド
を分解して再びラジカルとして重合させてもよいＯ
前記細胞接着性モノマーとしては、たとえばアクリル酸
、アクリル酸メチルなどの電荷を有するモノマーなどが
あげられる。また細胞非接着性モノマーとしては、たと
えばアクリルアミド、エチレングリコール、ヒドロキシ
エチルメタクリレートなどの電荷を有さない親水性のモ
ノマーなどがあげられる。Graft polymerization of a cell-adhesive monomer or a cell-nonadhesive monomer using radicals induced in this way may be carried out by directly introducing the hexamer into the system in which the radicals have been generated, and then the radicals are first removed by air. The cell adhesion monomer may be a monomer having a charge such as acrylic acid or methyl acrylate. etc. Examples of the cell non-adhesive monomer include uncharged hydrophilic monomers such as acrylamide, ethylene glycol, and hydroxyethyl methacrylate.
なお、パターンマスクは、前記第１の製法のばあいと同
様のものを用いることができる。Note that the same pattern mask as in the first manufacturing method can be used.
前記のごとく形成された配列パターンを冑する細胞の配
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、高解像度の微細パター
ンを形成することができる。By culturing cells in a conventional manner using a cell array control tool that covers the array pattern formed as described above, the cell array can be easily controlled and a fine pattern with high resolution 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
器などの製造や、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 resulting fine patterns are useful for manufacturing ultra-small biosensors, switching devices, bioreactor hybrid artificial organs, and even developing neurocomputers.
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
、本発明はこれらに限定されるものではない。Next, the present invention will be explained in more detail using Examples, but the present invention is not limited thereto.
実施例１
５％（重量％、以下同様）のビスアジド化合物（４，４
−ジアジドスチルベン−２，２°−ジスルホン酸ソーダ
）を混合したポリ（Ｎ、Ｎ−ジメチルアクリルアミド）
のメタノール溶液を、ポリスチレンシャーレにキャスト
製膜したのち、高圧水銀灯を用いて紫外線を照射し、ポ
リ（Ｎ、Ｎ−ジメチルアクリルアミド）を固定したシャ
ーレ（以下、ＰＤＭＡＡ−シャーレという）をえた。Example 1 5% (wt%, same applies hereinafter) of bisazide compound (4,4
-Poly(N,N-dimethylacrylamide) mixed with diazidostilbene-2,2°-sodium disulfonate)
The methanol solution was cast into a film on a polystyrene petri dish, and then irradiated with ultraviolet rays using a high-pressure mercury lamp to obtain a petri dish on which poly(N,N-dimethylacrylamide) was fixed (hereinafter referred to as PDMAA-petri dish).
ＰＤＭＡＡ−シャーレ上に第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
トを有するパターンマスクをセットし、イオンコーター
（エイコーエンジニアリンク社製、ＩＢ−３型）を用い
てグロー放電処理したのち、空気と接触させて放置し、
非マスク部にカルボキシル基を誘導した。なお、第３図
はフォトマスクの写真のスケッチ図である。A pattern mask having a pair of slits with a width of 250 μm, consisting of an aperture part and a non-aperture part, as shown in Fig. 3, was set on a PDMAA petri dish, and an ion coater (manufactured by Eiko Engineering Co., Ltd., IB- After glow discharge treatment using 3 type), leave it in contact with air,
Carboxyl groups were induced in the non-mask area. Note that FIG. 3 is a sketch diagram of a photo of the photomask.
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（Ｆｅ２）を含むＤＭＥＭ（Ｄｕｌ
ｂｄｃｃｏ’ｓ Ｍｏｄｉｒｌｅｄ 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、カルボキシル基が導
入された部分（非マスク部）のみに、内皮細胞が選択的
に伸展・増殖し、細胞の配列パターンが形成された。In the petri dish thus obtained, bovine vascular endothelial cells were seeded, and DMEM (Dul.
bdcco's Modirled Eagle's
When the cells were cultured in a CO2 incubator at 37°C using a medium (Medium) as a medium, endothelial cells selectively expanded and proliferated only in the areas where carboxyl groups were introduced (non-masked areas), forming an array pattern of cells. It was done.
実施例２
実施例１と同様にしてえたＰＤＭＡＡ−シャーレ上に第
３図に示すパターンマスクをセットし、低圧水銀灯を用
いて空気中で紫外線を１０分間照射し、非マスク部にカ
ルボキシル基を誘導した。Example 2 The pattern mask shown in Figure 3 was set on a PDMAA petri dish prepared in the same manner as in Example 1, and ultraviolet rays were irradiated in the air using a low-pressure mercury lamp for 10 minutes to induce carboxyl groups in the non-mask areas. did.
このようにしてえたシャーレ上で実施例１と同様にして
牛血管内皮細胞を培養したところ、カルホキシル基が導
入された部分（非マスク部）のみに、内皮細胞が選択的
に伸展−増殖し、細胞の配列パターンが形成、された。When bovine vascular endothelial cells were cultured on the petri dish thus obtained in the same manner as in Example 1, the endothelial cells selectively spread and proliferated only in the area where carboxyl groups were introduced (non-masked area). A cell arrangement pattern was formed.
実施例３
組織培養用ポリスチレンシャーレ（コーニング（ＣＯＲ
ＮＩＮＧ）社製）上に第３図に示すパターンマスクをセ
ットし、実施例１と同様にしてグロー放電処理し、非マ
スク部（こパーオキサイドを導入した。Example 3 Polystyrene petri dish for tissue culture (Corning (COR)
A pattern mask shown in FIG. 3 was set on the mask (manufactured by NING), and glow discharge treatment was performed in the same manner as in Example 1, and peroxide was introduced into the non-mask area.
続いて、このシャーレを５％アクリルアミド水溶液中に
浸し、充分脱気したのち密封し、２時間６０℃に保ち、
非マスク部のみにアクリルアミドをグラフト重合した。Next, this petri dish was immersed in a 5% acrylamide aqueous solution, thoroughly degassed, sealed, and kept at 60°C for 2 hours.
Acrylamide was graft-polymerized only on the non-mask area.
反応終了後、シャーレを充分水洗し、非マスク部のみに
ポリアクリルアミドがグラフトされた組織培養用ポリス
チレンシャーレをえた。After the reaction was completed, the petri dish was thoroughly washed with water to obtain a polystyrene petri dish for tissue culture in which polyacrylamide was grafted only on the non-mask area.
このようにしてえたシャーレ上で実施例１と同様にして
牛血管内皮細胞を培養したところ、ポリアクリルアミド
がグラフトされていない部分（マスク部）のみに、内皮
細胞が選択的に伸展・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形成された。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。When bovine vascular endothelial cells were cultured on the petri dish thus obtained in the same manner as in Example 1, endothelial cells selectively spread and proliferated only in the area where polyacrylamide was not grafted (mask area). The cell arrangement pattern shown in FIGS. 1 and 2 was formed. Figure 1 is a sketch of a photograph of the array pattern of stained cells (the magnification is the same as the photograph on which Figure 3 is based), and Figure 2 is a more enlarged photograph than the photograph on which Figure 1 is based. This is a sketch of the photograph taken.
実施例４
ポリテトラフルオロエチレンフィルム上に第３図に示す
パターンマスクをセットし、実施例１と同様にしてグロ
ー放電処理し、非マスク部にパーオキサイドを導入した
。Example 4 A pattern mask shown in FIG. 3 was set on a polytetrafluoroethylene film, and glow discharge treatment was performed in the same manner as in Example 1 to introduce peroxide into non-mask areas.
続いて、実施例３と同様にして非マスク部のみにアクリ
ルアミドをグラフト重合したポリテトラフルオロエチレ
ンフィルム（以下、ＰＡＡｍ−ＰＴＰＥという）をえた
。Subsequently, in the same manner as in Example 3, a polytetrafluoroethylene film (hereinafter referred to as PAAm-PTPE) in which acrylamide was graft-polymerized only in the non-mask area was obtained.
えられたＰＡＡｍ−ＰＴＦＥをＬＮ−ＮａＯＨ水溶液中
に浸漬して１時間５０℃に保ち、アミド部分を加水分解
してカルボキシル基に変換した。The obtained PAAm-PTFE was immersed in an aqueous LN-NaOH solution and kept at 50°C for 1 hour to hydrolyze the amide moiety and convert it into a carboxyl group.
このようにしてえたフィルム上で実施例１と同様にして
牛血管内皮細胞を培養したところ、ポリマーがグラフト
されている部分（非マスク部）のみに、内皮細胞が選択
的に伸展・増殖し、細胞の配列パターンが形成された。When bovine vascular endothelial cells were cultured on the film thus obtained in the same manner as in Example 1, endothelial cells selectively spread and proliferated only in the areas where the polymer was grafted (non-masked areas). An array pattern of cells was formed.
実施例５
実施例４と同様にしてえたＰＡＡｍ−ＰＴＰＥを、次亜
塩素酸ソーダのＮ ａ ＯＨアルカリ水溶液中に浸漬し
て１時間０ ’Ｃに保ってホフマン分解し、アミド部分
をアミノ基に変換した。Example 5 PAAm-PTPE obtained in the same manner as in Example 4 was immersed in an aqueous NaOH alkaline solution of sodium hypochlorite and kept at 0'C for 1 hour for Hofmann decomposition, converting the amide moiety into an amino group. Converted.
このようにしてえたフィルム上で実施例１と同様にして
牛血管内皮細胞を培養したところ、ポリマーがグラフト
されている部分（非マスク部）のみに内皮細胞が選択的
に伸展・増殖し、細胞の配列パターンが形成された。When bovine vascular endothelial cells were cultured on the film thus obtained in the same manner as in Example 1, the endothelial cells selectively spread and proliferated only in the areas where the polymer was grafted (non-masked areas). An array pattern was formed.
［発明の効果］
本発明の細胞の配列制御用具は、細胞の付着の有無を選
択的に制御でき、これを用いて従来の細胞培養と同様に
して培養を行なうことにより、容易に細胞の配列を制御
することができ、精度の高い細胞配列パターンを形成す
ることができる。[Effects of the Invention] The cell arrangement control device of the present invention can selectively control the presence or absence of cell attachment, and can be used to easily control cell arrangement by culturing in the same manner as conventional cell culture. can be controlled and a highly accurate cell arrangement pattern can be formed.
また、本発明の製法により、望むような配列パターンを
容易に解像度よく形成させた細胞の配列制御用具をうろ
ことができる。Furthermore, the manufacturing method of the present invention allows for the production of a cell array control tool that easily forms a desired array pattern with high resolution.
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、ハイブリッド型人工臓器、バイ
オリアクター、ニューロコシビ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。The present invention will greatly contribute to the development of ultra-small biosensors, switching elements, hybrid artificial organs, bioreactors, neurocoscibutors, etc. that utilize various cellular functions. It can also be applied to 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図はパターンマスクの
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。
第
図
第
３
図
手続補正書
（自発）
５補正の対象
（１）明細書の「特許請求の範囲」の欄（２）明細書の
「発明の詳細な説明」の欄１事件の表示
平成１年特許願第１４１９６５号
２発明の名称
細胞の配列制御用具、
その製法および細胞の配列制御法
３補正をする者
事件との関係 特許出願人
住 所 大阪市北区中之島三丁目２番４号名 称
（０９４）鐘淵化学工業株式会社代表者 舘 糾
６補正の内容
（１）明細書の「特許請求の範囲」を別紙「補正された
特許請求の範囲」のとおり補正する。
（２） 明細書４頁末行の「よって」を「によって」
と補正する。
（３）同１３頁２行のｒ ＤｕｌｂｄｃｃｏｏＳ」をｒ
Ｄｕｌｂｅｃｃｏ’ｓＪと補正する・（４）同１３頁
１１行の「１０分間」を「１時間」と補正する。
７添付書類の目録
（１）補正された特許請求の範囲 １通４代理人
住所
〒５４０
大阪市中央区谷町２丁目２番２２号
補正された特許請求の範囲
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
ターンを冑することを特徴とする細胞の配列制御用具。
２ 細胞の配列制御用具の細胞非接着性表面上にパター
ンマスクを設置したのち、紫外線または放射線によって
表面処理し、非マスク部のみに細胞接着性官能基を誘導
して配列パターンを形成することを特徴とする請求項１
記載の細胞の配列制御用具の製法。
３ 細胞の配列制御用具の材料表面上にパターンマスク
を設置したのち、紫外線または放射線によって表面処理
し、非マスク部のみに重合開始種を誘導し、これにより
、細胞接着性モノマーまたは細胞非接着性モノマーを非
マスク部のみにグラフト重合させて配列パターンを形成
することを特徴とする請求項１記載の細胞の配列制御用
具の製法。
４ 請求項１記載の細胞の配列制御用具を用い「 １
て細胞を培養することを特徴とする細胞の配列制御法。
」
以 上Figure 1 is a sketch of a photograph of the array pattern of stained cells, Figure 2 is a sketch of a photograph that is further enlarged than the photograph on which Figure 1 is based, and Figure 3 is a photograph of the pattern mask ( This is a sketch with the same magnification as the photograph on which Figure 1 is based. Figure 3 Written amendment to the figure procedure (voluntary) 5. Subject of amendment (1) "Claims" column of the specification (2) "Detailed description of the invention" column 1 of the description 1 Case Representation 1999 Patent Application No. 141965 2 Name of the invention Cell array control device, its manufacturing method and relationship with the cell array control method 3 Amendment case Patent applicant address 3-2-4 Nakanoshima, Kita-ku, Osaka City name
(094) Kanekabuchi Chemical Industry Co., Ltd. Representative Takashi Tate Contents of the 6th amendment (1) The “Claims” of the specification will be amended as shown in the attached “Amended Scope of Claims”. (2) Change “by” to “by” on the last line of page 4 of the specification.
and correct it. (3) "rDulbdccooS" on page 13, line 2
Correct with Dulbecco's J. (4) Correct "10 minutes" in line 11 of page 13 to "1 hour". 7 List of attached documents (1) Amended claims 1 copy 4 Agent address 2-2-22 Tanimachi, Chuo-ku, Osaka 540 Amended claims Cell adhesive surface and cell non-adhesive property A cell arrangement control tool characterized by forming an arrangement pattern on the surface. 2. After installing a pattern mask on the cell non-adhesive surface of the cell array control device, the surface is treated with ultraviolet rays or radiation to induce cell adhesive functional groups only in the non-mask areas to form an array pattern. Claim 1
A method for manufacturing the cell arrangement control device described above. 3. After installing a pattern mask on the material surface of the cell arrangement control device, the surface is treated with ultraviolet rays or radiation to induce polymerization initiation species only in the non-mask area, thereby creating a cell adhesive monomer or cell non-adhesive monomer. 2. The method for manufacturing a cell array control device according to claim 1, wherein the monomer is graft-polymerized only on the non-mask portion to form an array pattern. 4. A method for controlling cell arrangement, which comprises culturing cells using the device for controlling cell arrangement according to claim 1.
Claims (1)
パターンを有することを特徴とする細胞の配列制御用具
。 ２細胞の配列制御用具の細胞非接着性表面上にパターン
マスクを設置したのち、紫外線または放射線よって表面
処理し、非マスク部のみに細胞接着性官能基を誘導して
配列パターンを形成することを特徴とする請求項１記載
の細胞の配列制御用具の製法。 ３細胞の配列制御用具の材料表面上にパターンマスクを
設置したのち、紫外線または放射線によって表面処理し
、非マスク部のみに重合開始種を誘導し、これにより、
細胞接着性モノマーまたは細胞非接着性モノマーを非マ
スク部のみにグラフト重合させて配列パターンを形成す
ることを特徴とする請求項１記載の細胞の配列制御用具
の製法。 ４請求項１記載の細胞の配列制御用具を用いて細胞を培
養することを特徴とする細胞の配列制御法。[Scope of Claim] A cell arrangement control device characterized by having an arrangement pattern consisting of a cell-adhesive surface and a cell-nonadhesive surface. 2. After installing a pattern mask on the cell non-adhesive surface of the cell array control device, the surface is treated with ultraviolet rays or radiation to induce cell adhesive functional groups only in the non-mask areas to form an array pattern. A method for producing a cell arrangement control device according to claim 1. 3. After placing a pattern mask on the material surface of the cell arrangement control tool, the surface is treated with ultraviolet rays or radiation to induce polymerization initiating species only in the non-mask areas, thereby
2. The method for manufacturing a cell array control device according to claim 1, wherein the array pattern is formed by graft polymerizing a cell adhesive monomer or a cell non-adhesive monomer only on the non-mask portion. 4. A method for controlling cell arrangement, which comprises culturing cells using the device for controlling cell arrangement according to claim 1.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1141965A JPH037577A (en) | 1989-06-03 | 1989-06-03 | Orientation controlling tool for cell, production thereof and method for controlling orientation of cell |
| EP90110455A EP0402718B1 (en) | 1989-06-03 | 1990-06-01 | Control of cell arrangement |
| DE69013764T DE69013764T2 (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 |
| 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 |
|---|---|---|---|
| JP1141965A JPH037577A (en) | 1989-06-03 | 1989-06-03 | Orientation controlling tool for cell, production thereof and method for controlling orientation of cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH037577A true JPH037577A (en) | 1991-01-14 |
Family
ID=15304245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1141965A Pending JPH037577A (en) | 1989-06-03 | 1989-06-03 | Orientation controlling tool for cell, production thereof and method for controlling orientation of cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH037577A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0775547A (en) * | 1993-09-07 | 1995-03-20 | Bio Material Kenkyusho:Kk | Culture substrate |
| US5573942A (en) * | 1993-10-06 | 1996-11-12 | Nec Corporation | Method for arranging cells in a culture medium |
| US5602029A (en) * | 1994-06-27 | 1997-02-11 | Nec Corporation | Method for fabricating substrate for cell culture and method for cell arrangements |
| JP2005102612A (en) * | 2003-09-30 | 2005-04-21 | Toyota Central Res & Dev Lab Inc | Method for implanting cell and method for preparing cellular tissue |
| JP2006006214A (en) * | 2004-06-25 | 2006-01-12 | Institute Of Physical & Chemical Research | Method for producing substrate having immobilized cell and substrate |
| JP2008306977A (en) * | 2007-06-14 | 2008-12-25 | Nitto Denko Corp | Cell culture substrate |
| JP2009502243A (en) | 2005-07-20 | 2009-01-29 | サーモディクス，インコーポレイティド | Polymer coating and cell adhesion method |
| JP2010004755A (en) * | 2008-06-24 | 2010-01-14 | Tokai Univ | Endothelial cell-proliferative material |
| JP2011050295A (en) * | 2009-09-01 | 2011-03-17 | Scivax Kk | Cell-culturing structure, cell-culturing container and method for producing them |
| JP2013099272A (en) * | 2011-11-08 | 2013-05-23 | Dainippon Printing Co Ltd | Method for producing cell culture substrate, cell culture substrate, and method for producing cell sheet therewith |
-
1989
- 1989-06-03 JP JP1141965A patent/JPH037577A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0775547A (en) * | 1993-09-07 | 1995-03-20 | Bio Material Kenkyusho:Kk | Culture substrate |
| US5573942A (en) * | 1993-10-06 | 1996-11-12 | Nec Corporation | Method for arranging cells in a culture medium |
| US5591627A (en) * | 1993-10-06 | 1997-01-07 | Nec Corporation | Apparatus for arranging cells in culture medium |
| US5602029A (en) * | 1994-06-27 | 1997-02-11 | Nec Corporation | Method for fabricating substrate for cell culture and method for cell arrangements |
| JP2005102612A (en) * | 2003-09-30 | 2005-04-21 | Toyota Central Res & Dev Lab Inc | Method for implanting cell and method for preparing cellular tissue |
| JP2006006214A (en) * | 2004-06-25 | 2006-01-12 | Institute Of Physical & Chemical Research | Method for producing substrate having immobilized cell and substrate |
| JP2009502243A (en) | 2005-07-20 | 2009-01-29 | サーモディクス，インコーポレイティド | Polymer coating and cell adhesion method |
| JP2008306977A (en) * | 2007-06-14 | 2008-12-25 | Nitto Denko Corp | Cell culture substrate |
| JP2010004755A (en) * | 2008-06-24 | 2010-01-14 | Tokai Univ | Endothelial cell-proliferative material |
| JP2011050295A (en) * | 2009-09-01 | 2011-03-17 | Scivax Kk | Cell-culturing structure, cell-culturing container and method for producing them |
| JP2013099272A (en) * | 2011-11-08 | 2013-05-23 | Dainippon Printing Co Ltd | Method for producing cell culture substrate, cell culture substrate, and method for producing cell sheet therewith |
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