JP2008005749A - 細胞分離チップおよびこれを使用した細胞培養方法 - Google Patents
細胞分離チップおよびこれを使用した細胞培養方法 Download PDFInfo
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Abstract
【解決手段】細胞を含む緩衝液を流下させる流路を備え、流路の途中で細胞を検出し、且つ、所定の条件を満足するか否かを判別して下流域で複数の流路に振り分ける。所定の条件を満足する細胞を収集する培養槽の上面には半透膜を張っておき、細胞分離中の汚染を防止する。分離の終わった段階で、所定の条件を満足する細胞を収集する培養槽に連なる流路を閉鎖するとともに、培養槽を分離装置から切り離して、所定の培地を含む培養器に入れて培養する。
【選択図】 図1
Description
1)目視による形態学的な細胞分類:たとえば尿中に出現する異型細胞検査による膀胱がんや尿道のがんなどの検査や血中の異型細胞分類、組織中における細胞診によるがん検査などをあげることができる、
2)蛍光抗体法による細胞表面抗原(マーカー)染色による細胞分類:一般にCDマーカと呼ばれる細胞表面抗原を、それに特異的な蛍光標識抗体で染色するもので、セルソーターによる細胞分離やフローサイトメーターや組織染色によるがん検査などに用いられている。もちろんこれらは、医療面のみならず、細胞生理研究用や、工業的な細胞利用の上でも多用されている。
3)あるいは、幹細胞の分離に関しては、細胞内に取り込まれる形の蛍光色素をレポーターとして幹細胞を含む細胞を大まかに分離し、更にその後で実際に培養を行うことで目的の幹細胞を分離する例がある。これは、幹細胞の有効なマーカーがまだ確立されていないので、実際に培養し、分化誘導したもののみを利用することで、実質的に目的細胞を分離しているのである。
1)レーザーなどを流路上の検出部に照射し、細胞が横切るときの散乱光や細胞を蛍光染色できる場合はその蛍光を検出する、
2)検出部に電極を設け、細胞が電極を横切るときのインピーダンスやコンダクタンスの変化を検出する、
3)CCDカメラなどを用い、細胞を画像として検出する。
2)の方法はやはり高速な処理が可能であるが、検出後の細胞の移動速度が測定できないことと、分取機構と組み合わせるのが難しいため、一般的には細胞分類に用いるフローサイトメーターに採用されている。
3)の方法は、一見、簡便のようであるが、流路中を常に移動する複数の細胞を取り扱う必要があるので、セルソーターでは画像処理の負荷が大きくなるため一般的には用いられていない。
図1は、本発明の細胞分離培養装置のシステム構成の1例を模式的に示す平面図である。細胞分離培養装置100はチップ基板101により構成されている。チップ基板101の下面に流路を、上面にこの流路に連通する開口を設け、試料や必要な緩衝液(培地)の供給口とする。また、十分な緩衝液の供給と各流路での流量の調整のためにリザーバを設ける。流路の作成はPMMAなどのプラスチックを金型に流し込むいわゆる射出成型で作成することができる。チップ基板101全体のサイズは20×30×1mm(t)である。チップ基板101の下面に刻まれた溝や貫通穴を流路やウェルの形状とするために、溝が刻まれた下面側に0.1mm厚のラミネートフィルムを熱圧着してある。開口数1.4、倍率100倍の対物レンズを用いて、0.1mmのラミネートフィルムを通して流路内を流れる細胞を観察できる。プラスチックを透光性の高いものとすれば、チップ基板101の上面側からも観測できる。
マイナス電極液:1%トレハロース、0.25M NaCl,0.296M リン酸ナトリウム(pH6.0)、1%アガロース、
プラス電極液:1%トレハロース、0.25M NaCl,0.282Mリン酸ナトリウム(pH8.0)、1%アガロース、
上記のように調製したゲル電極付細胞分離培養装置の表面を粘着性テープでシールする。多孔質プラスチックタオルであるプラセームに水を浸し、しぼった2cm角の断片をゲル電極付細胞分離培養装置とともに30mm×40mmのプラスチックバックにいれシーラーを用いてバックの口をふさぐ。
ここでは、細胞を蛍光染料により修飾あるいは金微粒子または金微粒子以外のナノ粒子により修飾して、蛍光あるいは散乱光により細胞を検出するために、アプタマーを利用する例を説明する。細胞を識別したり分離したりしようとすると、何らかの指標に従い区別する必要があるが、ここでは、表面抗原の標識物質にマイルドな条件で分解可能な物質を用いるとともに、表面抗原の標識物質を生理的な条件の下で、細胞に影響の無いように分解して除去する例を説明する。具体的には、標識物質に種々立体構造を形成することのできるポリヌクレオチドを利用する。このポリヌクレオチドは一般的にアプタマーと呼ばれる概念のものである。たとえば、全長を80塩基とし、3’末端側と5’末端側の20塩基は制御された既知の塩基配列とし、中央部の40塩基はランダムな配列の多種類の合成ポリヌクレオチドを用意する。これらの合成ポリヌクレオチドを、内面に分離したい細胞の表面抗原を固定したカラムに通す。その結果、カラム内面には分離したい細胞の表面抗原にアフィニティーがある配列のポリヌクレオチドが捕捉される。このカラムをアルカリ処理して、捕捉されたポリヌクレオチドを分離回収し、PCR増幅することにより、細胞表面抗原に特異的に結合するポリヌクレオチドを得ることができる。すなわち、マイルドな条件で分解可能な表面抗原の標識物質としてのアプタマーを得ることができる。
TAATACGACT CACTATAGGG AGACAA:(配列番号1)
40塩基からなるランダムな配列の3’側に導入する配列を配列番号2に示す。NTTCGACAGG AGGCTCACAA CAGG:(配列番号2)である。
図13は、細胞培養器の例を説明する概略図である。350は培養器である。細胞分離培養装置のチップ基板101の培養槽213,214の部分をチップ基板101ごと切り取って、培養器350の中に入れた状態を示す。培養槽213,214は、ラック351に載せられて培養器350内に置かれる。図では5段の架台を備えたラックの例を示す。培養器350には、CO25%を含む空気の供給パイプ354と培地352の供給パイプ355が設けられる。それぞれのパイプには開閉弁が設けられている。培養槽213,214を培養器350の中に入れて分離された細胞の培養をするためには、ラック351が必要と言うわけではなく、培養槽213,214が、適宜投入された形でも良い。
図15は、細胞検出領域221の光学系の概要を示す概念図である。細胞分離培養装置のチップ基板101の上面に細胞に光を照射するための光源25とフイルタ26を配置する。チップ基板101の下面に、細胞に照射された照射光の検出系を示す。ここでは、図2を参照して分るように、細胞検出領域221から細胞分離領域222を経由して流路218への流路に沿った断面として示す。細胞は、図10で説明したように蛍光染料、あるいは、細胞を金粒子またはナノ粒子で修飾される。
[配列表]
SEQUENCE LISTING
<110> Onchip Cellomics Consortium
<120> Cell segregating method, cell identifying method, and cell checking method
<130> NT06P0677
<160> 2
<210> 1
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223> Synthesized DNA
<400> 1
taatacgact cactataggg agacaa 26
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<221>
<223> Synthesized DNA
<400> 1
nttcgacagg aggctcacaa cagg 24
Claims (11)
- 基板と、
該基板の一面に構成される細胞を含む緩衝液を流下させるための流路と、
該流路を流下する細胞の情報を流路の所定の領域で検出する細胞情報検出領域と、
前記細胞情報検出領域の下流で検出された細胞の情報に応じて該細胞を前記流路に連絡する複数の分岐流路のいずれかに流下させる細胞分離領域と、
前記複数の分岐流路の下流に設けられ、それぞれの分岐流路を流下する細胞を含む緩衝液を保持する複数の培養槽と、
を備え、
前記細胞情報検出領域で検出された細胞の情報が所定の条件を満足するものであるときに前記細胞分離領域で該所定の条件を満足する細胞が流下させられる分岐流路の下流に設けられた培養槽の上面には該培養槽内に細菌等の流入を防止するための半透膜が設けられたことを特徴とする細胞分離培養チップ。 - 前記基板が金型による射出成型で作成されたプラスチック基板であり、前記流路が前記プラスチック基板の一面に作成された溝とこれを覆うラミネートフィルムで形成された請求項1記載の細胞分離培養チップ。
- 前記細胞情報検出領域で検出する細胞の情報が細胞の画像情報から得られるものである請求項1記載の細胞分離培養チップ。
- 前記流路を緩衝液とともに流下する細胞がアプタマーを介して所定の蛍光材料によって修飾されたものであり、情報検出領域で検出する細胞の情報が細胞を修飾している蛍光材料による蛍光の輝度情報から得られるものである請求項1記載の細胞分離培養チップ。
- 前記流路を緩衝液とともに流下する細胞がアプタマーを介して所定の金粒子またはナノ粒子によって修飾されたものであり、情報検出領域で検出する細胞の情報が細胞を修飾している金粒子またはナノ粒子による散乱光情報から得られるものである請求項1記載の細胞分離培養チップ。
- 前記細胞分離領域は、前記流路を細胞が緩衝液とともに流下する流路の両側に対向し、かつ、緩衝液の流れに対して位置をずらして配置された電解質を含むゲルからなる複数のゲル電極の開口部が設けられ、前記細胞分離領域において複数のゲル電極間に所定の電流を流した場合と流さない場合に応じて、細胞が前記細胞分離領域で複数の分岐流路のいずれかに分配される請求項1記載の細胞分離培養チップ。
- 前記半透膜は前記アプタマーを分解するためのリボザイムが通過できる大きさの穴を有するものである請求項4記載の細胞分離培養チップ。
- 前記培養槽の内部の底面が塗布されたコラーゲン、ポリリジン、あるいは、フィブロネクチンの層で覆われ、あるいは、これらの塗布に代えて、疎水性処理されている請求項1記載の細胞分離培養チップ。
- 基板と、該基板の一面に構成される細胞を含む緩衝液を流下させるための流路と、該流路を流下する細胞の情報を流路の所定の領域で検出する細胞情報検出領域と、前記細胞情報検出領域の下流で検出された細胞の情報に応じて該細胞を前記流路に連絡する複数の分岐流路のいずれかに流下させる細胞分離領域と、前記複数の分岐流路の下流に設けられ、それぞれの流路を流下する細胞を含む緩衝液を保持する複数の培養槽と、を備えた細胞分離培養チップにより、前記細胞情報検出領域で検出された細胞の情報が所定の条件を満足するものであるときに前記細胞分離領域で該所定の条件を満足する細胞が流下させられる一つの分岐流路の下流に設けられた上面に細菌等の流入を防止するための半透膜が設けられた培養槽に前記所定の条件を満足する細胞を収集すること、
前記培養槽に前記所定の条件を満足する細胞を収集した後、前記培養槽と連通する流路を閉鎖するとともに前記培養槽を前記細胞分離培養チップから分離すること、
前記培養槽を所定の培地を有する培養器に入れて前記培養槽に収集された細胞を培養すること、
を特徴とする細胞培養方法。 - 前記培養槽の前記細胞分離培養チップからの分離は、前記培養槽を含む領域を基板ごと熱的に分離するものである請求項9記載の細胞培養方法。
- 前記培養槽の内部の底面が塗布されたコラーゲン、ポリリジン、あるいは、フィブロネクチンの層で覆われ、あるいは、これらの塗布に代えて、疎水性処理されている細胞分離培養チップを使用する請求項9記載の細胞培養方法。
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WO2013146972A1 (ja) * | 2012-03-29 | 2013-10-03 | 公益財団法人神奈川科学技術アカデミー | 細胞機能制御方法 |
CN103718018A (zh) * | 2011-08-15 | 2014-04-09 | 西门子公司 | 借助磁性通流测量进行的分析物的动态状态确定 |
JP2016102728A (ja) * | 2014-11-28 | 2016-06-02 | 株式会社東芝 | マイクロ分析チップ |
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US7858506B2 (en) | 2008-06-18 | 2010-12-28 | Micron Technology, Inc. | Diodes, and methods of forming diodes |
JP2010038866A (ja) * | 2008-08-08 | 2010-02-18 | Sony Corp | マイクロチップ、微小粒子分取装置及び送流方法 |
EP2490020A1 (en) * | 2011-02-18 | 2012-08-22 | Koninklijke Philips Electronics N.V. | Measurement chip, microfluidic device and method of measurement chip manufacture |
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TWI614500B (zh) * | 2016-11-21 | 2018-02-11 | 國立清華大學 | 細胞檢測晶片的影像定位與拼接方法及影像檢測系統 |
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DE60135092D1 (de) * | 2000-01-31 | 2008-09-11 | Univ Texas | Tragbare vorrichtung mit einer sensor-array-anordnung |
US7452713B2 (en) * | 2000-02-29 | 2008-11-18 | Stmicroelectronics S.R.L. | Process for manufacturing a microfluidic device with buried channels |
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CN103718018A (zh) * | 2011-08-15 | 2014-04-09 | 西门子公司 | 借助磁性通流测量进行的分析物的动态状态确定 |
WO2013146972A1 (ja) * | 2012-03-29 | 2013-10-03 | 公益財団法人神奈川科学技術アカデミー | 細胞機能制御方法 |
JP2016102728A (ja) * | 2014-11-28 | 2016-06-02 | 株式会社東芝 | マイクロ分析チップ |
US10337976B2 (en) | 2014-11-28 | 2019-07-02 | Kabushiki Kaisha Toshiba | Microanalysis chip |
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