JP2004321040A - Cell culture bed chip, method for producing the same, and cell culture method - Google Patents

Abstract

A cell culture bed chip having a plurality of partitioned cell culture regions of an intended size, each having a suitable cell-adhesive surface formed thereon, and which can be industrially advantageously produced, and a method for producing the same. And a cell culture method thereby.
A cell culture bed chip includes a substrate having a cell adhesive surface, and a cell culture region partition membrane covering the surface of the substrate and made of a material exhibiting an adhesion-inhibiting property to adhesion-dependent cells. Become. The cell culture region partitioning film is formed by exposing and developing the coating of the photosensitive resin composition, and partitions a plurality of minute cell culture regions where the surface of the substrate is exposed. The cell culture method includes a step of seeding the adhesion-dependent cells on the entire outer surface of the cell culture bed chip on which the cell culture region partition film is formed and fixing the cells in the cell culture region. In the biological reaction method, a cell culture bed chip on which adhesion-dependent cells are cultured by this cell culture method is used.
[Selection] Figure 2

Description

〔式（Ａ）中、ｎは０〜８の数を示し、Ｒ^１ は水素原子または炭素数１〜９のアルキル基を示す。〕
【００３７】
上記式（Ａ）で表わされる化合物の具体例としては、市販品のアロニックスＭ−１０１（ｎ：約２、Ｒ^１ ：Ｈ）、同Ｍ−１０２（ｎ：約４、Ｒ^１ ：Ｈ）、同Ｍ−１１１（ｎ：約２、Ｒ^１ ：ｎ−Ｃ_８ Ｈ_１９）、同Ｍ−１１３（ｎ：約４、Ｒ^１ ：ｎ−Ｃ_８ Ｈ_１９）、同Ｍ−１１４（ｎ：約１、Ｒ^１ ：ｎ−Ｃ_８ Ｈ_１９）、同Ｍ−１１７（ｎ：約２、Ｒ^１ ：ｎ−Ｃ_８ Ｈ_１９）〔以上、東亜合成化学工業（株）製〕、ＫＡＹＡＲＡＤ Ｒ−５６４（ｎ：約２． ３、Ｒ^１ ：Ｈ）〔日本化薬（株）製〕などを挙げることができる。
【００３８】
上記式（Ａ）で表わされる化合物以外の１個のエチレン性不飽和二重結合を有する化合物として、市販品のＫＡＹＡＲＡＤ ＴＣ−１１０Ｓ、同ＴＣ−１２０Ｓ〔以上、日本化薬（株）製〕、Ｖ−１５８、Ｖ−２３１１〔以上、大阪有機化学工業（株）製〕などを挙げることができる。また、マレイン酸ジメチル、マレイン酸ジエチルなどの不飽和カルボン酸ジエステルを用いることもできる。
【００３９】
２個のエチレン性不飽和二重結合を有する化合物としては、２価のアルコールの（メタ）アクリル酸ジエステルを挙げることができる。この（メタ）アクリル酸ジエステルのうち、下記式（Ｂ）で表わされる化合物を好適に使用することができる。
【００４０】
【化２】

〔式（Ｂ）中、ｎおよびｍはそれぞれ０〜８の数を示し、Ｒ^２ は水素原子またはメチル基を示す。〕
【００４１】
上記式（Ｂ）で表わされる化合物の具体例としては、市販品のアロニックスＭ−２１０（ｎ：約２、ｍ：約２、Ｒ^２ ：ＣＨ_３ ）〔東亜合成化学工業（株）製〕、ＫＡＹＡＲＡＤ Ｒ５５１（ｎ＋ｍ：約４、Ｒ^２ ：ＣＨ_３ ）、同Ｒ−７１２（ｎ＋ｍ：約４、Ｒ^２ ：Ｈ）、〔日本化薬（株）製〕を挙げることができる。
【００４２】
また、下記式（Ｃ）で表わされる化合物も好適に使用することができる。
【化３】
式（Ｃ） ＣＨ_２ ＝ＣＨＣＯＯ−Ｒ^３ −ＣＯＣＨ＝ＣＨ_２
〔式（Ｃ）中、Ｒ^３ はエチレングリコール残基、プロピレングリコール残基が１〜１０繰り返した基を示す。〕
【００４３】
上記式（Ｃ）で表わされる化合物の具体例としては、市販品のアロニックスＭ−２４０〔Ｒ^３ ： −（ＣＨ_２ ＣＨ_２ Ｏ）ｎ −、ｎ：約４〕、同Ｍ−２４５〔Ｒ^３ ： −（ＣＨ_２ ＣＨ_２ Ｏ）ｎ −、ｎ：約９〕〔以上、東亜合成化学工業（株）製〕、同ＰＥＧ４００ＤＡ〔Ｒ^３ ： −（ＣＨ_２ ＣＨ_２ Ｏ）ｎ −、ｎ：約８〕〔以上、日本化薬（株）製〕などを挙げることができる。
【００４４】
〔光重合開始剤〕
光重合開始剤は、光の照射を受けてラジカルを生成する光ラジカル生成化合物であって、例えば、ベンジル、ジアセチルなどのα−ジケトン類；ベンゾインなどのアシロイン類；ベンゾインメチルエーテル、ベンゾインエチルエーテル、ベンゾインイソプロピルエーテルなどのアシロインエーテル類；チオキサントン、２，４−ジエチルチオキサントン、チオキサントン−４−スルホン酸、ベンゾフェノン、４，４’ −ビス（ジメチルアミノ）ベンゾフェノン、４，４’ −ビス（ジエチルアミノ）ベンゾフェノンなどのベンゾフェノン類；アセトフェノン、ｐ−ジメチルアミノアセトフェノン、α，α’ −ジメトキシアセトキシベンゾフェノン、２，２’ −ジメトキシ−２−フェニルアセトフェノン、ｐ−メトキシアセトフェノン、２−メチル〔４−（メチルチオ）フェニル〕−２−モルフォリノ−１−プロパノン、２−ベンジル−２−ジメチルアミノ−１−（４−モルフォリノフェニル）−ブタン−１−オンなどのアセトフェノン類；アントラキノン、１，４−ナフトキノンなどのキノン類；フェナシルクロライド、トリブロモメチルフェニルスルホン、トリス（トリクロロメチル）−ｓ−トリアジンなどのハロゲン化合物；ジ−ｔ −ブチルパーオキサイドなどの過酸化物；２，４，６−トリメチルベンゾイルジフェニルフォスフィンオキサイドなどのアシルフォスフィンオキサイド類などが挙げられ、また、市販品としては、イルガキュア１８４、６５１、５００、９０７、ＣＧＩ３６９、ＣＧ２４−６１（チバガイギー（株）製）、ルシリンＬＲ８７２８、ＴＰＯ（ＢＡＳＦ（株）製）、ダロキュア１１１６、１１７３（メルク（株）製）、ユベクリルｐ３６（ＵＣＢ（株）製）などを挙げることができる。
【００４５】
上述した種々の光重合開始剤の中で好ましい化合物としては、２−メチル〔４−（メチルチオ）フェニル〕−２−モルフォリノ−１−プロパノン、２−ベンジル−２−ジメチルアミノ−１−（４−モルフォリノフェニル）−ブタン−１−オンなどのアセトフェノン類またはフェナシルクロライド、トリブロモメチルフェニルスルホン、２，４，６−トリメチルベンゾイルジフェニルフォスフインオキサイドなどを挙げることができる。これらの化合物は単独でまたは併用することができる。これらの光重合開始剤は、放射線増感剤と共に使用することも可能である。
【００４６】
光重合開始剤は、その全部または一部が、光増感作用を有する炭素クラスターおよび／またはその誘導体、光ラジカル重合開始剤、光カチオン重合開始剤などであってもよい。
ここに、炭素クラスターおよび／またはその誘導体としては、例えばフラーレンおよびその誘導体を挙げることができ、また光カチオン重合開始剤としては、例えば、ジアゾニウム塩であるアデカウルトラセットＰＰ−３３（旭電化工業（株）製）、スルホニウム塩であるオプトマーＳＰ−１５０、１７０（旭電化工業（株）製）、メタロセン化合物であるイルガキュア２６１（チバ・スペシャルティ・ケミカルズ（株）製）などの市販品を挙げることができる。
【００４７】
〔共重合性化合物〕
感光性樹脂組成物に含有させることのできる共重合性化合物は、少なくとも１個の不飽和二重結合を有し、ポリエチンレングリコール構造を有しないラジカル重合性化合物であって、その具体例としては、例えば、メチルメタクリレート、メチルアクリレート、などの（メタ）アクリル酸エステル、アクリル酸、メタクリル酸、クロトン酸などのモノカルボン酸；マレイン酸、フマル酸、シトラコン酸、メサコン酸、イタコン酸などのジカルボン酸、２−サクシノロイルエチルメタクリレート、２−マレイノロイルエチルメタクリレート、２−ヘキサヒドロフタロイルエチルメタクリレートなどのカルボキシル基およびエステル結合を有するメタアクリル酸誘導体、並びに、シクロヘキシル（メタ）アクリレート、２−メチルシクロヘキシル（メタ）アクリレート、ジシクロペンタニルオキシエチル（メタ）アクリレート、イソボルニル（メタ）アクリレート、ジシクロペンタニル（メタ）アクリレート、スチレン、α−メチルスチレン、α−メチルスチレン、ｐ −クロロスチレン、ジビニルベンゼン、ビニルピリジンなどの芳香族ビニル化合物、その他を挙げることができる。
【００４８】
感光性樹脂組成物を構成する各成分の割合は、例えば、アルカリ可溶性重合体成分が２０〜９０質量％、ポリエチレングリコール変性ラジカル重合性化合物成分が１０〜５０質量％、共重合性化合物が０〜５０質量％であることが好ましい。
【００４９】
また、光重合開始剤の使用量は、アルカリ可溶性重合体成分１００重量部に対して１〜５０重量部とされることが好ましく、特に１０〜４０重量部とされることが好ましい。この光重合開始剤の割合が１重量部未満の場合には、酸素によるラジカルの失活の影響を受けやすくて感度が低下したものとなり、一方、５０重量部を超えると、他の成分との相溶性が悪くなったり、感光性樹脂組成物の保存安定性が低下する傾向がある。
【００５０】
〔感光性樹脂組成物溶液の調製〕
上記の感光性樹脂組成物を適宜の溶剤に溶解して組成物溶液を調製し、この組成物溶液を、基板１０の表面上に塗布し、加熱により溶媒を除去することによって所望の感光性被膜１２が形成される。
【００５１】
〔有機溶剤〕
感光性樹脂組成物溶液を調製するための溶剤としては、各組成成分の種類に応じて、適宜の有機溶剤を用いることができる。
この有機溶剤は、感光性樹脂組成物の各成分を溶解するもの、または均一に分散するものであればよい。有機溶剤の具体例としては、例えば、メチルエチルケトン、メチルイソブチルケトン、２−ヘプタノン、シクロヘキサノン、エチレングリコールモノメチルエーテル、プロピレングリコールモノメチルエーテル、プロピレングリコールモノメチルエーテルアセテート、３−メトキシプロピオン酸メチル、３−エトキシプロピオン酸エチル、乳酸エチル、γ−ブチロラクトンなどを挙げることができ、これらから選ばれる少なくとも１ 種を用いることができる。
【００５２】
これらの有機溶剤うち、塗膜に均一性が得られる特性および安全性の点から、エチレングリコールモノメチルエーテル、プロピレングリコールモノメチルエーテル、プロピレングリコールモノメチルエーテルアセテート、３−メトキシプロピオン酸メチル、シクロヘキサノン、３−エトキシプロピオン酸エチル、乳酸エチル、γ−ブチロラクトンなどを好ましく用いることができる。
【００５３】
有機溶剤の使用量は、感光性樹脂組成物の溶液または分散液が、所望の粘度あるいは濃度となる量とすればよい。
【００５４】
〔感光性被膜の形成〕
感光性樹脂組成物溶液を基板１０の表面に塗布することにより、感光性被膜が形成される。
感光性樹脂組成物溶液を基板１０の表面に塗布する方法としては、例えば、スピンコート法、ロールコート法、スクリーン印刷法、アプリケーター法などの方法を用いることができる。
組成物溶液による塗膜の乾燥条件は、当該組成物における各成分の種類、配合割合、塗布膜厚などによって異なるが、通常は７０〜１２０℃で５〜２０分間程度であり、好ましくは８０〜１００℃である。乾燥時間が短すぎると、現像時の密着状態が悪くなり、一方、長すぎると熱かぶりによる解像度の低下を招くようになる。
【００５５】
〔露光処理〕
感光性被膜１２にパターンのマスク１４を介して照射される光は、例えば、波長が３００〜５００ｎｍの紫外線または可視光線である。このような光の光源として、低圧水銀灯、高圧水銀灯、超高圧水銀灯、メタルハライドランプ、アルゴンガスレーザーなどを用いることができる。また、紫外線または可視光線の代わりに、遠紫外線、Ｘ線、電子線などを用いることもでき、これらも露光処理のための光として有効である。
露光処理における光の照射量は、感光性樹脂組成物における各成分の種類、配合量、塗膜の膜厚などによって異なるが、例えば超高圧水銀灯を使用する場合には、１００〜５００ｍＪ／ｃｍ^２ である。
【００５６】
〔現像処理〕
露光処理の後の感光性被膜１２に対して現像処理が行われる。現像処理は、アルカリ性水溶液を現像液として用いてこれに可溶な部分、すなわち露光処理において光が照射されなかった領域の膜部分を溶解除去し、光照射領域（露光領域）のみを残存させる処理であり、これにより、残存する感光性被膜によって細胞培養領域区画膜１６が形成され、同時に、目的とする区画された細胞培養領域Ｓが形成される。
【００５７】
現像液としては、例えば、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム、ケイ酸ナトリウム、メタケイ酸ナトリウム、アンモニア水、エチルアミン、ｎ−プロピルアミン、ジエチルアミン、ジ−ｎ−プロピルアミン、トリエチルアミン、メチルジエチルアミン、ジメチルエタノールアミン、トリエタノールアミン、テトラメチルアンモニウムヒドロキシド、テトラエチルアンモニウムヒドロキシド、ピロール、ピペリジン、１，８−ジアザビシクロ［５，４，０］−７−ウンデセン、１，５−ジアザビシクロ［４，３，０］−５−ノナンなどのアルカリ類の水溶液を使用することができる。また上記アルカリ類の水溶液にメタノール、エタノールなどの水溶性有機港媒や界面活性剤を適当量添加した水溶液を現像液として使用することもできる。
【００５８】
現像時間、すなわち現像液に接触する時間は、組成物における各成分の種類、配合割合、組成物の乾燥膜厚によって異なるが、通常３０〜３６０秒間であり、また現像の方法は液盛り法、ディッピング法、パドル法、スプレー現像法などのいずれでもよい。現像後は、流水洗浄を例えば３０〜９０秒間行い、エアーガンなどを用いて風乾させたり、オーブン中で乾燥させる。
【００５９】
〔後処理〕
上記の感光性樹脂組成物は、上記の露光処理のみによっても十分に硬化させることができるが、用途に応じて、追加の露光処理（以下、「後露光」という。）や加熱によってさらに硬化させることができる。後露光は、上記の露光処理と同様の方法で行なうことができ、光照射量は特に限定されるものではないが、高圧水銀灯を使用する場合には、１００〜１０００ｍＪ／ｃｍ^２ の条件で行われることが好ましい。また、加熱の方法は、ホットプレート、オーブンなどの加熱装置を用いて、所定の温度、例えば１００〜２００℃で所定の時間、例えばホットプレートによる場合には５〜６０分間、オーブン中では３０〜９０分間加熱処理をすればよい。
この後処理を施すことによって、さらに良好な特性を有するパターン状の硬化物を得ることができる。
【００６０】
〔その他の成分〕
感光性樹脂組成物には、その効果を損なわない範囲において、必要に応じて上記以外の添加成分を含有させることができる。このような添加成分としては、例えば、従来公知のネガ型フォトレジスト組成物の添加剤として使用可能なものが挙げられる。
【００６１】
本発明によれば、細胞培養領域Ｓが、フォトリソグラフィの技法によって形成される細胞培養領域区画膜１６によって形成されるので、当該細胞培養領域Ｓを所期の面積および形状を有する微小領域としてパターン状に形成することができる。従って、当該細胞培養領域Ｓの面積および形状を、目的とする接着依存性細胞の種類に応じて適正なものとすることが容易であり、所期の接着依存性細胞の接着およびその増殖を確実に行うことができる細胞培養床チップを確実に得ることができる。
【００６２】
また、上記の理由により、当該細胞培養床チップを用いることにより、細胞培養領域Ｓにおいては所期の接着依存性細胞の接着をきわめて有利に行うことができる。すなわち、細胞培養領域Ｓが当該接着依存性細胞に対して好適な条件を備えているのみでなく、細胞培養領域区画膜１６の表面は、当該接着依存性細胞に対して接着阻害性を有するものであるから、当該細胞培養床チップの全面に接着依存性細胞を播種すれば、いわば自動的に細胞培養領域Ｓにのみ当該接着依存性細胞を接着させることができる。これは、細胞培養領域区画膜１６の特性によって、細胞培養領域Ｓ以外の表面における当該接着依存性細胞の接着が阻害されるからである。
【００６３】
更に、そのような細胞培養領域区画膜により区画された細胞を有する細胞培養床チップを用いることにより、当該接着依存性細胞に特異的な生体反応を行わせることができ、これを利用して、種々の検査や診断を有利に行うことができる。
【００６４】
【実施例】
以下、本発明の具体的な実施例について説明するが、本発明は以下の実施例に限定されるものではない。
【００６５】
実施例１
図１〜図３の例に従い、下記の条件によって細胞培養床チップを作製した。
（１）感光性樹脂組成物溶液の調製
下記の種類および量の各成分、並びに有機溶剤を用いて感光性樹脂組成物溶液を調製した。
・アルカリ可溶性重合体成分
ポリエチレングリコール（分子量３，０００）、７０質量部
・ポリエチレングリコール変性ラジカル重合性化合物
ナノエチレングリコールジアクリレート、１３質量部
・共重合性化合物
メタクリル酸、１７質量部
・光重合開始剤
２−メチル〔４−（メチルチオ）フェニル〕−２−モルフォリノ−１−プロパノン、１２質量部
・有機溶剤
乳酸エチル、２５０質量部
【００６６】
（２）感光性被膜の形成
縦７５ｍｍ、横２５ｍｍ、厚さ１ｍｍのガラス板よりなる基板の表面に、感光性樹脂組成物溶液をスピンコート法によって塗布し、温度９０℃で５分間乾燥して乾燥後の膜厚が２μｍの感光性被膜を形成した。
【００６７】
（３）露光処理
形成された感光性被膜に対し、パターンマスクを介して、超高圧水銀灯より得られる波長３５０ｎｍの紫外線を１００ｍＪ／ｃｍ^２ の照射量で照射した。
【００６８】
（４）現像処理
露光処理の後の感光性被膜に対して、濃度２．４質量％のテトラメチルアンモニウムヒドロキシド（ＴＭＡＨ）水溶液よりなる現像液を用いてディッピング法により６０秒間接触させて現像を行い、流水洗浄を６０秒間行い、エアーガンを用いて風乾させて未露光領域の膜部分を溶解除去することにより、各々直径１００μｍの円形の細胞培養領域（Ｓ）が縦２２５個、横１１０個で縦横に並んだパターンによる細胞培養領域区画膜（１６）によって区画された細胞培養床チップを作製した。
【００６９】
（５）後処理
乾燥後の細胞培養床チップに対し、その硬化を完全なものとするために、高圧水銀灯よりの光を１００ｍＪ／ｃｍ^２ の照射量で２０分間照射した。
【００７０】
（６）培養テスト
上記のようにして得られた細胞培養床チップをエチレンオキシドガスにより滅菌した後、クリーンベンチ内において、ラット肝臓をコラゲナーゼ処理して得られた肝実質細胞を、シャーレ内の１０％牛胎児血清含有ＤＭＥＭ培地中、チップ当たり５×１０^６ 個の数で播種し、これを温度３７℃、５％二酸化炭素ガス雰囲気のインキュベータにより培養した。
そして、２４時間経過後、当該細胞培養床チップをインキュベータより取り出して顕微鏡で観察したところ、細胞培養領域区画膜上には細胞の存在はほとんど認められないが、一方、播種した肝実質細胞のほとんど全部が、区画された細胞培養領域に、直径約８０μｍの二次元的に一様な広がりをもった細胞塊として綺麗に並んだ状態となっていることが認められた。
【００７１】
以上のように、当該細胞培養床チップによれば、所期のパターンに従って区画されて配列された細胞培養領域の各々においては肝実質細胞が当該細胞培養領域に一様に広がった状態で接着しているが、それ以外の領域では肝実質細胞が存在しておらず、従って、当該肝実質細胞の接着領域が、細胞培養領域のパターンに従ってパターン状に配列された状態を実現することができる。
【００７２】
【発明の効果】
本発明の細胞培養床チップは、細胞接着性表面を有する基板の表面に、接着依存性細胞に対して接着阻害性の材質よりなる細胞培養領域区画膜が形成され、この細胞培養領域区画膜によって細胞培養領域が区画されて形成されたものであるところ、当該細胞培養領域は、細胞培養領域区画膜の一部がいわゆるフォトリソグラフィーの技法によって除去されることにより形成されることにより、確実に好適な大きさのものとされることができ、しかも各細胞培養領域では基板の表面による細胞接着性が発揮されるので十分な細胞接着性を有するものであり、従って工業的に有利に製造することができる。
【００７３】
本発明の細胞培養床チップの製造方法によれば、細胞培養領域は、細胞培養領域区画膜の一部がいわゆるフォトリソグラフィーの技法によって除去することにより形成されるので、確実に好適な大きさのものとすることができ、しかも各細胞培養領域では基板の表面による細胞接着性が発揮されるので十分な細胞接着性を有するものとすることができ、従って工業的に有利に所期の細胞培養床チップを製造することができる。
【００７４】
本発明の細胞培養方法によれば、上記の細胞培養床チップにおいて細胞培養領域が所期の微小な面領域とされており、他の領域は接着阻害性領域とされているので、当該細胞培養床チップを用いることにより、所期の接着依存性細胞の培養を有利に行うことができる。
【００７５】
そして、上記のようにして接着依存性細胞が培養された細胞培養床チップを用いることにより、当該接着依存性細胞に特異的な生体反応を生じさせることができるので、目的とする診断や検査などを有利に実行することができる。
【図面の簡単な説明】
【図１】本発明による細胞培養床チップの製造に用いられる基板の一例を示す説明用断面図である。
【図２】図１に示す基板に形成された感光性被膜に対する露光処理についての説明用断面図である。
【図３】本発明の細胞培養床チップの構成の一例を示す説明用断面図である。
【符号の説明】
１０ 基板
１２ 感光性被膜
１４ マスク
１６ 細胞培養領域区画膜
Ｓ 細胞培養領域[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention particularly relates to a cell culture bed chip for adhesion-dependent cells, a method for producing the same, and a cell culture method.
[0002]
[Prior art]
With recent advances in cell engineering, attempts to utilize biological reactions of cells for diagnosis and testing have become active in various fields. The type of cells used for such a diagnosis or test is appropriately selected depending on the purpose, but generally, adhesion-dependent vesicles such as vascular endothelial cells, parenchymal cells and nerve cells have high utility. Things. Adhesion-dependent cells live, proliferate, differentiate, and maintain their functions as a living body while adhered to the surface of a substrate having a cell-adhesive surface. Will die. Under such circumstances, it is presently required to provide a substrate having a cell adhesive surface for culturing adhesion-dependent cells.
[0003]
In order to form a cell culture bed for adhesion-dependent cells, a substrate having a cell-adhesive surface may be used, and such a cell-adhesive surface is, for example, one having a cell-adhesive factor on its surface. Also, materials having moderate hydrophilicity and forming such a cell-adhesive surface are being energetically developed at present.
[0004]
However, in recent years, the number of diagnoses, tests, powerful drug screenings, and toxicology tests that have been performed has been dramatically increasing, and the amount of specimens subjected to the tests has been required to be very small. In response to the request, it is advantageous that a large number of cell culture regions are formed on one substrate, and each of them can adhere and fix adhesion-dependent cells.
[0005]
On the other hand, in the adhesion and proliferation of certain types of adhesion-dependent cells, the surface area actually provided for adhesion in the culture bed may be an appropriately sized area that differs depending on the type of the cell. is important. For example, in the case of hepatic spheroid cells, it is actually necessary that a cell culture area having a diameter of about 50 to 500 μm is formed as a unit culture area in a culture bed. This is because if the area of the unit culture area is large, the area of adhesion of the cells becomes larger than that of the seeded cells, and as a result, the number of cells adhered to each area does not become uniform. This is because the utility value is low.
[0006]
From the above situation, a cell culture bed chip formed by forming a large number of microscopic cell culture regions each having an expected size area or shape on the surface of a substrate in a pattern-divided state. Attention has been paid.
[0007]
At present, as a method for obtaining such a cell culture bed chip, the surface of the substrate is patterned into a hydrophilic region and a hydrophobic region using a microlithography technique, and the partitioned hydrophilic region is formed. A method for forming a cell culture region has been proposed.For example, a method has been proposed in which gold is vapor-deposited on a surface of a substrate material through a pattern mask, and the vapor-deposited film of gold is patterned with a compound having a thiol group. (See Non-Patent Document 1).
In addition, there has been proposed a method of forming a patterned cell culture region by applying a polymer substance such as polylactic acid to a substrate, and then removing a part thereof by plasma etching.
[0008]
[Non-patent document 1]
Presentation paper of the 31st Medical Polymer Symposium (Fujii et al.)
[0009]
[Problems to be solved by the invention]
However, the conventional method as described above requires forming a reduced-pressure atmosphere or a vacuum atmosphere in the vapor deposition step or the plasma etching step, and requires a large number of processing steps, and is not an industrially advantageous method. Moreover, these methods have a problem that the formed cell culture region does not necessarily have a sufficiently high cell adhesive surface.
[0010]
The present invention has been made in view of the above circumstances, and an object of the present invention is to have a plurality of partitioned cell culture regions of a desired size, and to have a suitable cell adhesion in each cell culture region. It is an object of the present invention to provide a cell culture bed chip which has an acidic surface and can be produced by an industrially advantageous method.
Another object of the present invention is to provide a cell culture bed chip having a plurality of cell culture regions partitioned into a desired size and having a suitable cell adhesive surface in each cell culture region, which is industrially advantageous. It is to provide a method that can be manufactured.
Still another object of the present invention is to provide a cell culture method that can advantageously culture adhesion-dependent cells.
[0011]
[Means for Solving the Problems]
The cell culture bed chip of the present invention is a substrate having a cell-adhesive surface, and a cell culture region partition membrane made of a material having an adhesion-inhibiting property to adhesion-dependent cells, provided so as to cover the surface of the substrate. Having
The cell culture region partition film is formed by exposing a photosensitive film made of a photosensitive resin composition and exposing the photosensitive film to remove a part of the photosensitive film, and a plurality of minute cells exposing the surface of the substrate. It is characterized by partitioning a culture area.
[0012]
In the above, the cell culture region partition membrane is characterized in that the adhesion-dependent cell adhesion rate on the surface of the cell culture region partition membrane is 10% or less.
[0013]
Further, the substrate may be a non-treated substrate made of a cell-adhesive substance, a surface-treated substrate whose surface has been made cell-adhesive by being subjected to surface treatment, or a surface having a coating made of a cell-adhesive substance formed on the surface. It is a coated substrate.
[0014]
The method for producing a cell culture bed chip according to the present invention comprises a photosensitive resin which is provided so as to cover the surface of a substrate having a cell-adhesive surface, and which is cured and exhibits adhesion-inhibiting properties to adhesion-dependent cells. The photosensitive film of the composition is selectively exposed to light and then developed to remove a part thereof, thereby forming a cell culture region partition film for partitioning a plurality of minute cell culture regions exposing the surface of the substrate. A step of producing the above-mentioned cell culture bed chip.
[0015]
In the above, the photosensitive resin composition for forming the photosensitive film for the cell culture region partition film comprises an alkali-soluble polymer component, a photopolymerization initiator for generating radicals by light, and the alkali-soluble polymer by radical polymerization. A cross-linkable monomer component that forms a cross-linked copolymer insoluble in a developer composed of an aqueous alkali solution by copolymerization with a coalescing component,
The crosslinkable monomer component preferably comprises a polyethylene glycol-modified monomer having a plurality of unsaturated double bonds.
[0016]
The cell culture method of the present invention uses the above-described cell culture bed chip, seeds the adhesion-dependent cells on the entire outer surface of the cell culture bed chip where the cell culture region partition membrane is formed, and in the cell culture region. A step of fixing, wherein the adhesion-dependent cells are cultured in the cell culture region.
[0017]
The biological reaction method of the present invention is characterized in that a specific biological reaction is caused to the adhesion-dependent cells using a cell culture bed chip on which the adhesion-dependent cells are cultured by the above-described cell culture method.
[0018]
[Action]
In the cell culture bed chip of the present invention, a cell culture region partition membrane made of a material having an adhesion-inhibiting property to adhesion-dependent cells is formed on a surface of a substrate having a cell adhesive surface, and the cell culture region partition membrane Where the cell culture region is formed by partitioning, the cell culture region is preferably formed by removing a part of the cell culture region partition film by a so-called photolithography technique, so that it is preferably used. In each cell culture area, the cell surface exhibits sufficient cell adhesiveness due to the surface of the substrate, and therefore has sufficient cell adhesiveness. Can be.
[0019]
According to the method for producing a cell culture bed chip of the present invention, the cell culture region is formed by removing a part of the cell culture region partition membrane by a so-called photolithography technique, so that the cell culture region has a suitable size. In each cell culture region, cell adhesion is exhibited by the surface of the substrate, so that sufficient cell adhesion can be obtained. Floor chips can be manufactured.
[0020]
According to the cell culture method of the present invention, in the cell culture bed chip, the cell culture region is a desired minute surface region, and the other region is an adhesion-inhibiting region. By using a floor chip, the desired culture of adhesion-dependent cells can be advantageously performed.
[0021]
Then, by using a cell culture bed chip on which the adhesion-dependent cells are cultured as described above, a specific biological reaction can be caused to the adhesion-dependent cells, so that a target diagnosis or test can be performed. Can be performed advantageously.
[0022]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
1 to 3 are sectional views for explaining a specific method for producing a cell culture bed chip according to the present invention in the order of steps.
[0023]
In the figure, reference numeral 10 denotes a substrate, and the surface of the substrate 10 has a cell-adhesive property. The substrate 10 may be an untreated substrate made of a cell-adhesive substance, a surface-treated substrate made cell-adhesive by surface treatment, or a surface-coated substrate having a surface made of a film made of a cell-adhesive substance formed thereon. It is.
[0024]
On the surface of the substrate 10, a photosensitive film 12 made of a photosensitive resin composition that is cured to exhibit adhesion inhibition properties to adhesion-dependent cells is provided so as to cover the surface of the substrate 10.
[0025]
As shown in FIG. 2, the photosensitive film 12 is selectively exposed by irradiating the photosensitive film 12 with light, for example, ultraviolet rays, through a pattern mask 14 so as to expose the copolymer in the irradiated area. Then, as shown in FIG. 3, by developing and removing the coating portion in the unexposed area, the surface of the substrate 10 is exposed in the removal area. The cell culture region partitioning film 16 that partitions the cell culture region S is formed. Here, each cell culture region S is formed by the exposed surface of the substrate 10, and the periphery thereof is partitioned by the cell culture region partition film 16.
[0026]
〔substrate〕
In the above, the substrate 10 is, for example, a non-processed substrate formed of a cell-adhesive substance exhibiting suitable adhesion dependence for specific adhesion-dependent cells to be cultured. Examples of the substance for forming the non-processed substrate include glass, styrene- (meth) acrylate copolymer, and collagen sheet.
[0027]
The substrate 10 can also be constituted by a surface-treated substrate whose surface is made cell-adhesive by treating the surface of an appropriate substrate material. In this case, the degree of freedom in selecting a material that can be used as the substrate material is increased.
Specific examples of the surface treatment of the substrate material include, for example, sulfonation-hydrophilization treatment, corona discharge treatment, plasma oxidation treatment, and immobilization treatment of the adhesion factor vitronectin or fibronectin.
[0028]
The substrate 10 may be a surface-coated substrate formed by coating the surface of an appropriate substrate material with a coating of a cell-adhesive substance exhibiting suitable adhesion dependency on the adhesion-dependent cells. Also in this case, the degree of freedom in selecting a material that can be used as the substrate material is increased.
Examples of the cell-adhesive substance for forming the coating include those selected from the above-mentioned cell-adhesive substances listed for the substrate material. The method for forming the coating is not particularly limited, and a known method can be used.
[0029]
(Photosensitive resin composition)
The photosensitive resin composition for forming the photosensitive film 12 includes, for example, an alkali-soluble polymer component, a photopolymerization initiator that generates radicals by light, and a crosslinkable monomer component. It is.
Here, the alkali-soluble polymer component is a polymer component itself soluble in a developer composed of an alkali aqueous solution, and the crosslinkable monomer component is copolymerized with the alkali-soluble polymer component by radical polymerization. And a crosslinked copolymer insoluble in a developing solution composed of an aqueous alkali solution. Further, the crosslinkable monomer component is preferably made of, for example, a polyethylene glycol-modified monomer having a plurality of unsaturated double bonds.
[0030]
In the photosensitive resin composition, although the cured film finally obtained has an adhesion inhibitory property to specific adhesion-dependent cells to be cultured, the survival of the adhesion-dependent cells in a cell culture region is reduced. It is necessary to have biocompatibility to the extent that it does not inhibit. However, the cause of the adhesion inhibition property of the cured film is not particularly limited. For example, the adhesion inhibition property may be expressed by the properties of the formed crosslinked copolymer itself. Alternatively, an adhesive inhibitor may be exhibited for other reasons.
[0031]
It is preferable that the coating constituting the cell culture region partition membrane 16 has an adhesion-dependent cell adhesion rate of 10% or less on the surface thereof. Here, the method of obtaining the adhesion ratio is as follows.
[Method for determining adhesion rate]
A bovine vascular endothelial cells a / cm on the sample surface²(However, a = 1 × 10⁴And ), And cultured for 12 hours in a MEM medium containing 10% fetal bovine serum, the number of unadhered cells b is counted, and the adhesion rate α is determined by the following equation (1).
(Equation 1)
Formula (1) Adhesion rate α = [(ab) / a] × 100 (unit:%)
[0032]
The photosensitive resin composition may contain, as a copolymerizable component, a radical polymerizable compound (hereinafter, referred to as a “copolymerizable compound”) other than a monomer that is a crosslinkable monomer component. The properties according to the type of the copolymerizable compound are imparted to the photosensitive film finally obtained, so that the photosensitive film can be made suitable.
[0033]
(Alkali-soluble polymer component)
In the photosensitive resin composition, as a polymer constituting the alkali-soluble polymer component, for example, polyvinyl alcohol, novolak resin, polyethylene glycol, polypropylene glycol, polyacrylamide, polyvinylpyrrolidone, polyacrylic acid, polymethacrylic acid, lactone Examples thereof include a ring polymer and a copolymer thereof.
[0034]
[Polyethylene glycol-modified radically polymerizable compound component having at least one ethylenically unsaturated double bond]
The polyethylene glycol-modified radical polymerizable compound having at least one ethylenically unsaturated double bond is, for example, a (meth) acrylic acid monoester or diester of polyethylene glycol. Is exemplified.
[0035]
As the (meth) acrylic acid monoester, there is a compound represented by the following formula (A).
[0036]
Embedded image

[In the formula (A), n represents a number of 0 to 8;¹Represents a hydrogen atom or an alkyl group having 1 to 9 carbon atoms. ]
[0037]
Specific examples of the compound represented by the above formula (A) include commercially available Aronix M-101 (n: about 2, R¹: H) and M-102 (n: about 4, R¹: H) and M-111 (n: about 2, R¹: NC₈H₁₉) And M-113 (n: about 4, R¹: NC₈H₁₉M-114 (n: about 1, R¹: NC₈H₁₉M-117 (n: about 2, R¹: NC₈H₁₉) [Made by Toa Gosei Chemical Industry Co., Ltd.], KAYARAD R-564 (n: about 2.3, R¹: H) [manufactured by Nippon Kayaku Co., Ltd.].
[0038]
As the compound having one ethylenically unsaturated double bond other than the compound represented by the above formula (A), commercially available KAYARAD TC-110S and TC-120S (all manufactured by Nippon Kayaku Co., Ltd.), V-158 and V-2311 (all manufactured by Osaka Organic Chemical Industry Co., Ltd.). Further, unsaturated carboxylic acid diesters such as dimethyl maleate and diethyl maleate can also be used.
[0039]
Examples of the compound having two ethylenically unsaturated double bonds include a dihydric alcohol (meth) acrylate diester. Among these (meth) acrylic acid diesters, a compound represented by the following formula (B) can be suitably used.
[0040]
Embedded image

[In the formula (B), n and m each represent a number of 0 to 8, and R²Represents a hydrogen atom or a methyl group. ]
[0041]
Specific examples of the compound represented by the above formula (B) include commercially available Aronix M-210 (n: about 2, m: about 2, R²: CH₃) [Toa Gosei Chemical Industry Co., Ltd.], KAYARAD R551 (n + m: about 4, R²: CH₃) And R-712 (n + m: about 4, R²: H) and [Nippon Kayaku Co., Ltd.].
[0042]
Further, a compound represented by the following formula (C) can also be suitably used.
Embedded image
Formula (C) CH₂= CHCOO-R³-COCH = CH₂
[In the formula (C), R³Represents a group in which an ethylene glycol residue and a propylene glycol residue are repeated 1 to 10. ]
[0043]
Specific examples of the compound represented by the formula (C) include commercially available Aronix M-240 [R³:-(CH₂CH₂O) n-, n: about 4], and M-245 [R³:-(CH₂CH₂O) n-, n: about 9] [or more, manufactured by Toa Gosei Chemical Industry Co., Ltd.], PEG400DA [R³:-(CH₂CH₂O) n-, n: about 8] [all manufactured by Nippon Kayaku Co., Ltd.].
[0044]
(Photopolymerization initiator)
The photopolymerization initiator is a photoradical-forming compound that generates a radical upon irradiation with light, for example, α-diketones such as benzyl and diacetyl; acyloins such as benzoin; benzoin methyl ether, benzoin ethyl ether, Acyloin ethers such as benzoin isopropyl ether; thioxanthone, 2,4-diethylthioxanthone, thioxanthone-4-sulfonic acid, benzophenone, 4,4'-bis (dimethylamino) benzophenone, 4,4'-bis (diethylamino) benzophenone Benzophenones such as acetophenone, p-dimethylaminoacetophenone, α, α′-dimethoxyacetoxybenzophenone, 2,2′-dimethoxy-2-phenylacetophenone, p-methoxyacetophenone, 2- Acetophenones such as tyl [4- (methylthio) phenyl] -2-morpholino-1-propanone and 2-benzyl-2-dimethylamino-1- (4-morpholinophenyl) -butan-1-one; anthraquinone, Quinones such as 2,4-naphthoquinone; halogen compounds such as phenacyl chloride, tribromomethylphenylsulfone and tris (trichloromethyl) -s-triazine; peroxides such as di-t-butyl peroxide; Examples include acylphosphine oxides such as 6-trimethylbenzoyldiphenylphosphine oxide, and commercially available products include Irgacure 184, 651, 500, 907, CGI369, CG24-61 (manufactured by Ciba Geigy), and lucilin. LR8728, TPO (BA F (Ltd.)), manufactured by Darocur 1116,1173 (Merck & Co.), made Ubecryl p36 (UCB (Ltd.)), and the like.
[0045]
Preferred compounds among the various photopolymerization initiators described above include 2-methyl [4- (methylthio) phenyl] -2-morpholino-1-propanone and 2-benzyl-2-dimethylamino-1- (4- Examples include acetophenones such as morpholinophenyl) -butan-1-one or phenacyl chloride, tribromomethylphenylsulfone, and 2,4,6-trimethylbenzoyldiphenylphosphine oxide. These compounds can be used alone or in combination. These photopolymerization initiators can be used together with a radiosensitizer.
[0046]
The photopolymerization initiator may be entirely or partially a carbon cluster having photosensitizing action and / or a derivative thereof, a photoradical polymerization initiator, a photocationic polymerization initiator, or the like.
Here, as the carbon cluster and / or its derivative, for example, fullerene and its derivative can be mentioned, and as the photocationic polymerization initiator, for example, a diazonium salt, ADEKA ULTRASET PP-33 (Asahi Denka Kogyo Co., Ltd.) Commercially available products such as Optomer SP-150 and 170 (manufactured by Asahi Denka Kogyo KK), which are sulfonium salts, and Irgacure 261 (manufactured by Ciba Specialty Chemicals) which is a metallocene compound. it can.
[0047]
(Copolymerizable compound)
The copolymerizable compound that can be contained in the photosensitive resin composition is a radical polymerizable compound having at least one unsaturated double bond and not having a polyethynylene glycol structure. Are, for example, monocarboxylic acids such as (meth) acrylates such as methyl methacrylate, methyl acrylate, etc., acrylic acid, methacrylic acid, crotonic acid; dicarboxylic acids such as maleic acid, fumaric acid, citraconic acid, mesaconic acid, itaconic acid A methacrylic acid derivative having a carboxyl group and an ester bond such as an acid, 2-succinoloylethyl methacrylate, 2-maleynoloylethyl methacrylate, 2-hexahydrophthaloylethyl methacrylate, and cyclohexyl (meth) acrylate; Methylcyclohexyl (Meth) acrylate, dicyclopentanyloxyethyl (meth) acrylate, isobornyl (meth) acrylate, dicyclopentanyl (meth) acrylate, styrene, α-methylstyrene, α-methylstyrene, p-chlorostyrene, divinylbenzene, Examples thereof include aromatic vinyl compounds such as vinyl pyridine and others.
[0048]
The proportion of each component constituting the photosensitive resin composition is, for example, 20 to 90% by mass of the alkali-soluble polymer component, 10 to 50% by mass of the polyethylene glycol-modified radically polymerizable compound component, and 0 to 10% by mass of the copolymerizable compound. It is preferably 50% by mass.
[0049]
The amount of the photopolymerization initiator to be used is preferably 1 to 50 parts by weight, particularly preferably 10 to 40 parts by weight, based on 100 parts by weight of the alkali-soluble polymer component. When the proportion of the photopolymerization initiator is less than 1 part by weight, the sensitivity is reduced due to the susceptibility to the deactivation of radicals by oxygen. There is a tendency that the compatibility is deteriorated and the storage stability of the photosensitive resin composition is reduced.
[0050]
(Preparation of photosensitive resin composition solution)
The above-mentioned photosensitive resin composition is dissolved in an appropriate solvent to prepare a composition solution, the composition solution is applied on the surface of the substrate 10, and the solvent is removed by heating to obtain a desired photosensitive film. 12 are formed.
[0051]
〔Organic solvent〕
As a solvent for preparing the photosensitive resin composition solution, an appropriate organic solvent can be used according to the type of each composition component.
The organic solvent may be one that dissolves each component of the photosensitive resin composition or one that uniformly disperses the components. Specific examples of the organic solvent include, for example, methyl ethyl ketone, methyl isobutyl ketone, 2-heptanone, cyclohexanone, ethylene glycol monomethyl ether, propylene glycol monomethyl ether, propylene glycol monomethyl ether acetate, methyl 3-methoxypropionate, and 3-ethoxypropionic acid. Examples thereof include ethyl, ethyl lactate, and γ-butyrolactone, and at least one selected from these can be used.
[0052]
Among these organic solvents, ethylene glycol monomethyl ether, propylene glycol monomethyl ether, propylene glycol monomethyl ether acetate, methyl 3-methoxypropionate, cyclohexanone, and 3-ethoxy from the viewpoints of properties and uniformity in obtaining uniformity of the coating film. Ethyl propionate, ethyl lactate, γ-butyrolactone and the like can be preferably used.
[0053]
The amount of the organic solvent used may be such that the solution or dispersion of the photosensitive resin composition has a desired viscosity or concentration.
[0054]
(Formation of photosensitive film)
The photosensitive film is formed by applying the photosensitive resin composition solution to the surface of the substrate 10.
As a method of applying the photosensitive resin composition solution to the surface of the substrate 10, for example, a method such as a spin coating method, a roll coating method, a screen printing method, and an applicator method can be used.
The drying conditions of the coating film with the composition solution vary depending on the type of each component in the composition, the mixing ratio, the coating film thickness, and the like, but it is usually about 70 to 120 ° C for about 5 to 20 minutes, preferably 80 to 100 ° C. If the drying time is too short, the state of adhesion during development will be poor, while if it is too long, the resolution will decrease due to heat fogging.
[0055]
(Exposure processing)
The light applied to the photosensitive film 12 via the pattern mask 14 is, for example, ultraviolet light or visible light having a wavelength of 300 to 500 nm. As a light source of such light, a low-pressure mercury lamp, a high-pressure mercury lamp, an ultra-high-pressure mercury lamp, a metal halide lamp, an argon gas laser, or the like can be used. Further, instead of ultraviolet rays or visible rays, far ultraviolet rays, X-rays, electron beams, or the like can be used, and these are also effective as light for exposure processing.
The irradiation amount of light in the exposure treatment varies depending on the type and blending amount of each component in the photosensitive resin composition, the film thickness of the coating film, and the like. For example, when an ultra-high pressure mercury lamp is used, 100 to 500 mJ / cm²It is.
[0056]
(Development processing)
After the exposure process, the photosensitive film 12 is subjected to a development process. In the development processing, an alkaline aqueous solution is used as a developer to dissolve and remove a portion that is soluble in the developer, that is, a film part in a region that has not been irradiated with light in the exposure process, and leaves only a light-irradiated region (exposed region). Thereby, the cell culture region partitioning film 16 is formed by the remaining photosensitive coating, and at the same time, the target partitioned cell culture region S is formed.
[0057]
Examples of the developer include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium silicate, sodium metasilicate, aqueous ammonia, ethylamine, n-propylamine, diethylamine, di-n-propylamine, triethylamine, methyldiethylamine, Dimethylethanolamine, triethanolamine, tetramethylammonium hydroxide, tetraethylammonium hydroxide, pyrrole, piperidine, 1,8-diazabicyclo [5,4,0] -7-undecene, 1,5-diazabicyclo [4,3, Aqueous solutions of alkalis such as [0] -5-nonane can be used. Further, an aqueous solution obtained by adding a suitable amount of a water-soluble organic solvent such as methanol or ethanol or a surfactant to the aqueous solution of the above alkalis can also be used as the developer.
[0058]
The development time, that is, the time of contact with the developer, varies depending on the type of each component in the composition, the mixing ratio, and the dry film thickness of the composition, but is usually 30 to 360 seconds, and the development method is a puddle method, Any of a dipping method, a paddle method, and a spray developing method may be used. After the development, washing with running water is performed, for example, for 30 to 90 seconds, followed by air drying using an air gun or the like or drying in an oven.
[0059]
[Post-processing]
The above-mentioned photosensitive resin composition can be sufficiently cured only by the above-described exposure treatment, but is further cured by additional exposure treatment (hereinafter, referred to as “post-exposure”) or heating depending on the application. be able to. The post-exposure can be performed in the same manner as in the above-described exposure treatment, and the amount of light irradiation is not particularly limited. However, when a high-pressure mercury lamp is used, 100 to 1000 mJ / cm²It is preferred to be carried out under the following conditions. The heating method is performed using a heating apparatus such as a hot plate or an oven at a predetermined temperature, for example, 100 to 200 ° C. for a predetermined time, for example, 5 to 60 minutes when using a hot plate, and 30 to 30 minutes in an oven. Heat treatment may be performed for 90 minutes.
By performing this post-treatment, a patterned cured product having more favorable characteristics can be obtained.
[0060]
[Other components]
If necessary, the photosensitive resin composition may contain additional components other than those described above as long as the effects are not impaired. Examples of such additive components include those which can be used as additives for conventionally known negative photoresist compositions.
[0061]
According to the present invention, since the cell culture region S is formed by the cell culture region partition film 16 formed by the photolithography technique, the cell culture region S is patterned as a minute region having an intended area and shape. It can be formed in a shape. Therefore, it is easy to make the area and shape of the cell culture region S appropriate according to the type of the target adhesion-dependent cell, and it is possible to ensure the adhesion of the intended adhesion-dependent cell and its growth. A cell culture bed chip that can be performed at a low temperature can be reliably obtained.
[0062]
For the above-mentioned reason, by using the cell culture bed chip, the desired adhesion-dependent cell adhesion can be extremely advantageously performed in the cell culture region S. That is, not only the cell culture region S has suitable conditions for the adhesion-dependent cells, but also the surface of the cell culture region partition membrane 16 has an adhesion inhibitory property for the adhesion-dependent cells. Therefore, if the adhesion-dependent cells are seeded on the entire surface of the cell culture bed chip, the adhesion-dependent cells can be automatically adhered to only the cell culture region S, so to speak. This is because the adhesion of the adhesion-dependent cells on the surface other than the cell culture region S is inhibited by the characteristics of the cell culture region partition film 16.
[0063]
Furthermore, by using a cell culture bed chip having cells partitioned by such a cell culture region partition membrane, a specific biological reaction can be performed on the adhesion-dependent cells. Various tests and diagnoses can be advantageously performed.
[0064]
【Example】
Hereinafter, specific examples of the present invention will be described, but the present invention is not limited to the following examples.
[0065]
Example 1
According to the examples of FIGS. 1 to 3, a cell culture bed chip was produced under the following conditions.
(1) Preparation of photosensitive resin composition solution
A photosensitive resin composition solution was prepared using the following types and amounts of each component, and an organic solvent.
・ Alkali-soluble polymer component
Polyethylene glycol (molecular weight 3,000), 70 parts by mass
・ Polyethylene glycol-modified radical polymerizable compound
Nano ethylene glycol diacrylate, 13 parts by mass
・ Copolymerizable compound
Methacrylic acid, 17 parts by mass
・ Photopolymerization initiator
2-methyl [4- (methylthio) phenyl] -2-morpholino-1-propanone, 12 parts by mass
·Organic solvent
Ethyl lactate, 250 parts by mass
[0066]
(2) Formation of photosensitive film
A photosensitive resin composition solution is applied to the surface of a substrate made of a glass plate having a length of 75 mm, a width of 25 mm and a thickness of 1 mm by a spin coating method, dried at a temperature of 90 ° C. for 5 minutes and dried to a thickness of 2 μm. A photosensitive coating was formed.
[0067]
(3) Exposure processing
An ultraviolet ray having a wavelength of 350 nm obtained from an ultra-high pressure mercury lamp was applied to the formed photosensitive film through a pattern mask at 100 mJ / cm.²Irradiation.
[0068]
(4) Development processing
The photosensitive film after the exposure treatment is developed for 60 seconds by a dipping method using a developing solution composed of an aqueous solution of tetramethylammonium hydroxide (TMAH) having a concentration of 2.4% by mass, and developed with running water. This was performed for 60 seconds, and air-dried using an air gun to dissolve and remove the unexposed region of the film. This resulted in a pattern in which circular cell culture regions (S) each having a diameter of 100 μm were arranged 225 vertically and 110 horizontally and 110 horizontally. A cell culture bed chip partitioned by a cell culture region partitioning membrane (16) was prepared.
[0069]
(5) Post-processing
To complete the curing of the dried cell culture bed chip, light from a high pressure mercury lamp was applied at 100 mJ / cm.²For 20 minutes.
[0070]
(6) Culture test
After the cell culture bed chip obtained as described above was sterilized with ethylene oxide gas, the liver parenchyma cells obtained by collagenase treatment of the rat liver were placed in a clean bench, and DMEM containing 10% fetal calf serum in a Petri dish was used. 5 × 10 per chip in medium⁶The seeds were inoculated in a number of pieces and cultured at 37 ° C. in an incubator in a 5% carbon dioxide gas atmosphere.
After a lapse of 24 hours, the cell culture bed chip was taken out of the incubator and observed with a microscope. As a result, almost no cells were found on the cell culture region partition membrane. It was confirmed that all of the cells were neatly arranged in the partitioned cell culture region as cell clumps having a two-dimensional uniform spread of about 80 μm in diameter.
[0071]
As described above, according to the cell culture bed chip, in each of the cell culture regions partitioned and arranged in accordance with the intended pattern, the hepatic parenchymal cells adhere to the cell culture region while being uniformly spread. However, hepatocytes are not present in other regions, and therefore, a state where the adhesion regions of the hepatocytes are arranged in a pattern according to the pattern of the cell culture region can be realized.
[0072]
【The invention's effect】
In the cell culture bed chip of the present invention, a cell culture region partition membrane made of a material having an adhesion-inhibiting property to adhesion-dependent cells is formed on a surface of a substrate having a cell adhesive surface, and the cell culture region partition membrane Where the cell culture region is formed by partitioning, the cell culture region is preferably formed by removing a part of the cell culture region partition film by a so-called photolithography technique, so that it is preferably used. In each cell culture area, the cell surface exhibits sufficient cell adhesiveness due to the surface of the substrate, and therefore has sufficient cell adhesiveness. Can be.
[0073]
According to the method for producing a cell culture bed chip of the present invention, the cell culture region is formed by removing a part of the cell culture region partition membrane by a so-called photolithography technique, so that the cell culture region has a suitable size. In each cell culture region, cell adhesion is exhibited by the surface of the substrate, so that sufficient cell adhesion can be obtained. Floor chips can be manufactured.
[0074]
According to the cell culture method of the present invention, in the cell culture bed chip, the cell culture region is a desired minute surface region, and the other region is an adhesion-inhibiting region. By using a floor chip, the desired culture of adhesion-dependent cells can be advantageously performed.
[0075]
Then, by using a cell culture bed chip on which the adhesion-dependent cells are cultured as described above, a specific biological reaction can be caused to the adhesion-dependent cells, so that a target diagnosis or test can be performed. Can be performed advantageously.
[Brief description of the drawings]
FIG. 1 is an explanatory cross-sectional view showing an example of a substrate used for producing a cell culture bed chip according to the present invention.
FIG. 2 is a sectional view for explaining an exposure process for a photosensitive film formed on a substrate shown in FIG. 1;
FIG. 3 is an explanatory sectional view showing an example of the configuration of the cell culture bed chip of the present invention.
[Explanation of symbols]
10 Substrate
12 Photosensitive coating
14 Mask
16 Cell culture area compartment membrane
S cell culture area

Claims (8)

A substrate having a cell-adhesive surface, provided to cover the surface of the substrate, comprising a cell culture region partition membrane made of a material exhibiting an adhesion-inhibiting property to adhesion-dependent cells,
The cell culture region partition film is formed by exposing a photosensitive film made of a photosensitive resin composition and exposing the photosensitive film to remove a part of the photosensitive film, and a plurality of minute cells exposing the surface of the substrate. A cell culture bed chip for partitioning a culture region. The cell culture floor chip according to claim 1, wherein the cell culture region partition membrane has an adhesion-dependent cell adhesion rate of 10% or less on the surface of the cell culture region partition membrane. The substrate is an untreated substrate made of a cell-adhesive substance, a surface-treated substrate whose surface has been made cell-adhesive by being subjected to surface treatment, or a surface-coated substrate having a surface made of a film made of a cell-adhesive substance The cell culture bed chip according to claim 1 or 2, wherein A photosensitive coating of a photosensitive resin composition, which is provided so as to cover the surface of the substrate having a cell-adhesive surface and is cured and exhibits adhesion-inhibiting properties to adhesion-dependent cells, was selectively exposed to light. A step of forming a cell culture region partitioning film for partitioning a plurality of minute cell culture regions exposing the surface of the substrate by removing a part of the substrate after development is performed,
A method for producing a cell culture bed chip, wherein the cell culture bed chip according to claim 1 or 2 is produced. The substrate is an untreated substrate made of a cell-adhesive substance, a surface-treated substrate whose surface has been made cell-adhesive by being subjected to surface treatment, or a surface-coated substrate having a surface made of a film made of a cell-adhesive substance The method for producing a cell culture bed chip according to claim 4, wherein The photosensitive resin composition for forming a photosensitive film for the cell culture region partition membrane is an alkali-soluble polymer component, a photopolymerization initiator that generates radicals by light, and the alkali-soluble polymer component by radical polymerization. A crosslinkable monomer component that forms a crosslinked copolymer insoluble in a developer consisting of an aqueous alkali solution by copolymerization,
The method according to claim 4 or 5, wherein the crosslinkable monomer component comprises a polyethylene glycol-modified monomer having a plurality of unsaturated double bonds. A cell obtained by using the cell culture bed chip according to any one of claims 1 to 3 and seeding adhesion-dependent cells on the entire outer surface of the cell culture bed chip on which a cell culture region partition membrane is formed. A cell culture method, comprising a step of fixing to a culture region, wherein the adhesion-dependent cell is cultured in the cell culture region. A biological reaction method, wherein a specific biological reaction is caused to the adhesion-dependent cells by using a cell culture bed chip on which the adhesion-dependent cells are cultured by the cell culture method according to claim 7.

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