JP2006501449A - 細胞分離のためのマイクロ流体デバイスおよびその使用 - Google Patents
細胞分離のためのマイクロ流体デバイスおよびその使用 Download PDFInfo
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Abstract
Description
本発明は、医学的診断およびマイクロ流体システムの分野に関する。
本発明は、1つの試料から細胞を分離する(例、母体血液から胎児赤血球を分離する)ための方法を特徴とする。本方法は、細胞を含む1つの試料を一つまたは複数の微小流路内に導入する段階から始まる。1つの態様では、本デバイスは少なくとも2つの処理段階を含む。例えば、細胞混合物が所望の細胞型の通過を選択的に許容する微小流路内に導入され、次に所望の型が濃縮された細胞集団が所望の細胞の通過を許容する第2の微小流路内に導入され、所望の型がいっそう濃縮された細胞集団が生成される。細胞の選択は、例えば、サイズ、形状、変形性、表面特性(例、細胞表面受容体または抗原および膜透過性)などのその混合物中の細胞の特性、または細胞内特性(例、特定酵素の発現)に基づく。
A.細胞溶解
本発明の1つのデバイスは、例えば母体血液などの細胞混合物中で例えば母体赤血球などの細胞集団を選択的に溶解させるために使用される。このデバイスは、ほぼ同一条件下で極めて多数の細胞を処理することができる。溶解デバイスは、望ましくはその後の処理に先立って極めて多数の細胞を除去する。例えば細胞膜およびタンパク質などの破片は、その後の何らかの処理に先立って、例えば濾過または沈降によって捕捉することができる。
本発明の溶解デバイスの設計は、図1に示されている。このデバイスにおける流路の全体的に分岐状の構造は、並列処理ネットワーク全域で等価の圧力低下を許容する。このデバイスは、機能上、4つの個別区間に分離できる。1)例えば血液、溶解試薬、および洗浄バッファーなどの流体を接合部1および2へ運搬する分散型流入流路(図2);2)2つの接合部間に存在する細胞溶解反応のための蛇行状反応チャンバー(図3);3)溶解試薬を希釈するために接合部2の下流にある希釈チャンバー(図3);および4)溶解した試料を収集用バイアルまたは別のマイクロ流体デバイスへ運搬するための分散型流出流路(図4)。
分岐状の流入および流出流路網は、各流路内への試薬の一様な分布を可能にする(8、図1に示されている)。マクロ世界をデバイスと接続するための3つのポートの直径は、典型的には1mm〜10mm、例えば2、5、6、または8mmである。ポート1、2、および3については、デバイスと一体成形された外部マニホールドにより気密シールを形成することができる(図1)。例えば血液、溶解試薬、および希釈剤などの3つの溶液バイアルはそのようなマニホールドと連結することができる。図1に示されている3種の溶液のためのポート1、2および3から反応チャンバーおよび混合チャンバーへの流入流路は、デバイスのz面(各々が1組の分布流路を備える3層、図2を参照)内で分離されていても、または外部マニホールド内に存在していてもよい。外部マニホールド内に存在する場合は、分布流路は、例えばステンレススチールから機械加工されたCNC(コンピュータによる数値制御)流路であり、直径500μmの寸法を有していてよい。マニホールドは、図1に示されている1’、2’、および3’の位置でエッチングされているポートでデバイスと気密的に連結することができる。マニホールド内に分散型流路を配置すると、デバイスの複雑性および費用が低下する。分散型流路をデバイス上に維持すると、試料間のキャリーオーバー汚染の問題を回避しながら、より小さなサイズの流路を選択できる柔軟性が高まる。各試料流入流路は別個の流出口を有していてよい、または図4に示されているように、各試料流入用の流出流路が結合されていてもよい。マニホールドの代替物として、例えばガスケットまたはニップルへの圧縮取付具によって、あるいはルアーロックなどの防水性結合具を使用して、各流体流入用または流出用の管路をデバイスに取り付けることもできる。混合接合部およびチャンバーを越えて流体を輸送するデバイス上の流路は、幅および深さが10μm〜500μmの範囲内、例えば幅および深さが大きくとも10μm、25μm、50μm、75μm、100μm、150μm、200μm、250μm、350μm、または450μmであってよい。流路の構造は、望ましくは長方形であるが、さらにまた円形、半円形、V字形、あるいは他の適切な形状であってもよい。1つの態様では、一つまたは複数の流出流路は流入流路の断面積の合計に等しい断面積を有する。
溶解および希釈のためには、2つの流体の流れが結合され、チャンバー内を通過させられる。チャンバーは直線状または蛇行状流路であってよい。図1に示されているデバイスでは、試料および溶解バッファーが接合部1で結合され、そして溶解した試料および希釈剤が接合部2で結合される。反応チャンバーおよび希釈チャンバーの蛇行状構造は、デバイスのための合理的な全体的設置面積を維持しながら、拡散または他の受動的機序により適正に混合するために2種の反応溶液の十分な滞留時間を可能にする(図3)。蛇行状流路は、例えばデバイスの全長を減少させるために、または混合を強化するためのカオス的移流を導入するために、二次元または三次元で構築することができる。滞留時間を短くするためには、直線状チャンバーが望ましいことがある。代表的な滞留時間には、少なくとも1秒間、5秒間、10秒間、30秒間、60秒間、90秒間、2分間、5分間、30分間、1時間、または1時間以上が含まれる。反応/希釈チャンバー内の流体流量は、最適な処理スループットを可能にしながら2種の試薬の十分な混合を可能にするために、流路の幅、深さ、および有効長を制御することによって正確に制御することができる。1つの態様では、細胞を溶解させる(反応チャンバー)および溶解した試料を希釈する(希釈チャンバー)ための蛇行状混合チャンバーは各〜26μLの流体量を有する。他の例の反応/希釈チャンバー量の範囲は、例えば多くとも20、50、100、または150μLなどの10〜200μLである。一部の態様では、反応および希釈チャンバーの幅および深さは流入および流出流路と同一の、すなわち10〜500μmの範囲を有する。または、これらのチャンバーは、デバイスを通過する一様な流動速度を保証するために、あらゆる流入(または流出流路)の結合断面積に等しい断面積を有していてもよい。1つの例では、チャンバーは100μm×100μmの流路である。チャンバーの全長は、少なくとも1cm、5cm、10cm、20cm、30cm、40cm、または50cmであってよい。
1つの態様では、このデバイスは例えばシリンジポンプ、蠕動ポンプ、吸引器、または真空ポンプを使用する負圧ポンピングを利用する。負圧は、流路内に未処理試料を残すことなく、完全量の臨床血液試料を処理することを可能にする。デバイスを通して試料をポンピングするためには、例えばシリンジポンプ、蠕動ポンプ、容積式ポンプ、流体カラム、または他の流体ポンプからの陽圧もまた使用できる。陽圧ポンピングに関連する死容積問題に起因する試料消失は、バッファーを用いて残留試料を追い出すことによって克服できる。ポンプは、例えばシリコン製ガスケットを使用して、典型的には気密シールによってデバイスへ接続される。
本発明のデバイスを作製するためには様々な技術を利用することができ、利用される技術は一部には最適な材料に基づいて選択される。本発明のデバイスを作製するための代表的材料には、ガラス、シリコン、スチール、ニッケル、ポリメタクリル酸メチル(PMMA)、ポリカーボネート、ポリスチレン、ポリエチレン、ポリオレフィン、シリコン類(例、ポリジメチルシロキサン)、およびそれらの組み合わせが含まれる。その他の材料は、当技術分野において知られている。これらの材料で流路を作製する方法は当技術分野において知られている。これらの方法には、フォトリソグラフィー(例、立体リソグラフィーまたはX線フォトリソグラフィー)、モールディング法、エンボス加工法、シリコン微細加工法、湿式もしくは乾式化学エッチング法、ミリング法、ダイアモンド切削法、リソグラフィーによる電気めっきおよび成形法(LIGA)および電気めっき法が含まれる。例えば、ガラスについては、後に湿式(KOH)または乾式エッチング(フッ素またはその他の反応ガスを用いた反応性イオンエッチング)を実施する伝統的なフォトリソグラフィーによるシリコン作製法を利用できる。レーザー微細加工法などの技術は、高度の光子吸収効率を備えるプラスチック材料のために採用できる。この技術は、この工程が連続的種類であるため、低スループット作製のために適合する。大量生産されるプラスチック製デバイスには、熱可塑性射出成形法、および圧縮成形法が適合する。本発明のデバイスを作製するためには、(サブミクロンで機能の忠実度を保存する)コンパクトディスクの大量生産に使用される従来的な熱可塑性射出成形法もまた利用されてよい。例えば、デバイスの機能は従来型のフォトリソグラフィーによってガラスマスター上で複製される。ガラスマスターを電気鋳造すると、頑丈で耐熱衝撃性、熱伝導性かつ硬質の型が作り出される。この型は、機能をプラスチック製デバイスに成形する射出成形法または圧縮成形法のためのマスターテンプレートとして機能する。デバイスを作製するために使用されるプラスチック材料並びに光学的品質および最終製品のスループットに関する要件に依存して、製造方法として圧縮成形法または射出成形法を選択できる。圧縮成形法(ホットエンボス加工法またはリリーフインプリンティング法とも呼ばれる)には、小型構造にとって卓越しているが高縦横比構造を複製する際に使用するのは困難でサイクル時間が長い高分子量ポリマーと適合する利点がある。射出成形法は、高縦横比構造とも良好に作用するが、低分子量ポリマーにとって最も適合する。
細胞または溶解した細胞から放出される化合物が流路壁上へ非特異的に吸着するのを減少させるために、一つまたは複数の流路壁は非付着性または反発性となるように化学修飾することができる。これらの壁は、ヒドロゲルを形成するために使用される試薬などの、市販の非付着性試薬による薄膜コーティング(例、単層)でコーティングできる。流路壁を修飾するために使用できる追加の化学種の例には、オリゴエチレングリコール、フッ化ポリマー、オルガノシラン、チオール、ポリエチレングリコール、ヒアルロン酸、ウシ血清アルブミン、ポリビニルアルコール、ムチン、ポリ-HEMA、メタクリル化PEG、およびアガロースが含まれる。反対に荷電した種に反発するためには、荷電ポリマーもまた利用されてよい。反発に使用される化学種の型および流路壁へ付着させる方法は反発される種の性質ならびに付着される壁および種の性質に依存するであろう。そのような表面修飾技術は、当技術分野において周知である。壁は、デバイスが組立てられる前、または後に機能化することができる。
本発明では、例えば母体血液などの細胞の試料が一つまたは複数の微小流路内に導入される。試料内の細胞集団を選択的に溶解させるための試薬を含有する溶解バッファーは、次に血液試料と混合される。望ましくは、混合は、例えば拡散またはカオス的移流などの受動的手段によって行われるが、能動的手段が利用されてもよい。追加の受動的および能動的ミキサーは、当技術分野において知られている。溶解反応は、所望の時間にわたり持続させられる。この時間の長さは、例えば流路の長さまたは流体の流速によって制御することができる。さらに、流路内で混合される溶液の容積は、例えば流路のサイズまたは流動速度を変化させることにより、溶液の相対的容積流量を変化させることによって制御することが可能である。流速は、いずれかの望ましい時間にわたり、減速させる、上昇させる、または停止させることができる。溶解が発生した後、溶解試薬および溶解した細胞から放出されたいずれかの潜在的に有害な種(例、エンドソーム酵素)の濃度を低下させるために希釈剤を流路内に導入することができる。希釈剤は溶解試薬を中和する、またはさもなければ例えばpHもしくは粘度などの流体環境を変化させる種を含有していてよい、あるいは細胞の表面または細胞内標識化のための試薬を含有していてよい。希釈剤は、例えば吸光度測定値などの一定の検出スキームにとって重要なことがある溶液の光学密度をさらに低下させることができる。
本発明のまた別のデバイスは、細胞をデバイスの表面へ結合させることによる混合物からの全細胞の枯渇を含む。そのようなデバイスの表面は、例えば細胞表面受容体に対する抗体またはリガンドなどの、細胞の特定小集団へ結合する物質を含む。本方法におけるこの段階は、所望の細胞がデバイスへ結合させられるポジティブ選択、所望の細胞がデバイスを通過させられるネガティブ選択を利用することができる。いずれの場合でも、分析またはその後の処理のために所望の細胞を含有する細胞集団が収集される。
このデバイスは、混合物中で、例えば特異的表面分子を発現する障害物などの、細胞集団へ結合できる様々な形状の障害物の配列を含有するマイクロ流体システムである。結合した細胞は、デバイス上で直接分析できる、または例えばその後の分析もしくは処理のためにデバイスから取り除かれてよい。または、障害物へ結合していない細胞は、例えばその後の処理または分析のために収集されてよい。
本発明のデバイスを作製するための代表的方法は、図18に要約されている。この実施例では、標準的フォトリソグラフィーを使用してシリコン・オン・インシュレーター(SOI)ウエハー上で障害物のフォトレジストパターンが作製される。SOIウエハーは、厚さ500μmのSi(100)ウエハー、その上の厚さ1μmのSiO2層、およびその上の厚さ100μmのSi(100)層から構成される。フォトレジスト接着を最適化するために、SOIウエハーはフォトレジストコーティングを行う前にヘキサメチルジシラザンの高温蒸気に暴露させられてよい。UV感受性フォトレジストは、ウエハー上にスピンコーティングされ、90℃で30分間焼成され、クロム接触マスクを通して300秒間にわたりUV光線へ暴露させられ、現像液中で5分間現像され、そして90℃で30分間後焼成される。工程パラメーターは、フォトレジストの性質および厚さに依存して変化させてよい。クロム接触マスクのパターンがフォトレジストに移されると、障害物の形状を決定する。
本発明の方法は、細胞混合物をマイクロ流体デバイスの表面と接触させる段階を含む。血液などの複雑な細胞混合物中の細胞集団は、次にデバイスの表面へ結合する。望ましくは、デバイスの表面へ結合できる細胞の少なくとも60%、70%、80%、90%、95%、98%、または99%が混合物から取り除かれる。表面コーティングは、望ましくは細胞の非特異的結合を最小限に抑えるように設計される。例えば、結合成分へ結合できない細胞の少なくとも99%、98%、95%、90%、80%、または70%はデバイスの表面へ結合しない。デバイスにおける選択的結合は、細胞混合物中から特定の生きた細胞集団の分離を生じさせる。障害物は、障害物を含有するチャンバー内で結合の可能性が増加するように、細胞がそれと相互作用する表面積を増加させるためにデバイス内に存在する。流動条件は、細胞が障害物間内に進入するために機械的に変形させられる必要なく、細胞がデバイス内で極めて穏やかに取り扱われるような条件である。細胞が本発明のマイクロ流体デバイスを流入および流出するように細胞を輸送するためには、陽圧もしくは負圧ポンピングまたは流体カラムからの流動を利用できる。また別の態様では、細胞は、例えば非生物学的物質(例、ビーズ)、非生育性細胞破片(例、膜分画)または分子(例、タンパク質、核酸、もしくは細胞溶解物)などの非細胞性物質から分離される。
細胞結合デバイスのまた別の態様は、ポリビニルアルコール、ポリメタクリル酸ヒドロキシメチル、ポリアクリルアミド、またはポリエチレングリコールなどであるがそれらに限定されない緩く架橋結合したヒドロゲル内に取り込まれた、化学的に誘導されたガラス/プラスチック製ビーズを利用する。化学的に誘導されたビーズは、この態様での障害物として機能する。細胞混合物は、2つの全く正反対に対向する流入口を通して細胞枯渇デバイス内に方向付けられる。陽圧(例えば、注入ポンプまたは流体カラムから)または負圧(例えば、プルモードのシリンジポンプ、真空ポンプ、または吸引器から)は、ヒドロゲルを通して液体を駆動する。試料中の細胞とヒドロゲルの三次元容積中に分散した化学的に誘導されたビーズとの相互作用は、例えば白血球などの細胞の枯渇(ネガティブ選択)または例えば胎児赤血球などの細胞の捕捉(ポジティブ選択)のいずれかを生じさせる。ビーズによる当該細胞の最高の相互作用および捕捉を可能にするために、分子量、架橋密度、ビーズ密度、ならびに分布および流動速度を最適化できる。高含水量ヒドロゲルは、試料を通る容易な流動を許容しながらビーズを捕捉する構造を提供する。試料は、次に2つの正反対に対向する流出口を通って収集される。分岐した流入/流出流路の設計は、ヒドロゲルの容積を通る試料の最高に均質な分布を保証する。
このデバイスでは、典型的には望ましくない細胞が枯渇させられている細胞混合物がマイクロ流体デバイス内でアレイ化される。この段階のための代表的デバイスは、国際公開番号WO 01/35071に記載されている。アレイ内の細胞は次に、所望の細胞の位置を決定するために、例えば顕微鏡または比色アッセイによって分析される。所望の細胞は、例えば溶解、次にPCRによってアレイ上で分析されてよい、または細胞は例えば光学的ピンセットなどの様々な機構によってアレイから収集されてよい。WO 01/35071に記載された代表的デバイスでは、細胞はアレイ化デバイス内に導入され、デバイス内に機械加工された穴の中に受動的に沈殿できる。または、陽圧または負圧を利用すると細胞をアレイ内の穴へ方向付けることができる。細胞が穴の中に配置されると、選択された細胞は例えば気泡作動ポンプなどのアクチュエーターによってアレイから個別に放出させることができる。アレイ化デバイスには、さらに例えば誘電泳動トラッピング法などの細胞を固定化および放出するための他の方法が使用されてもよい。アレイから放出されると、細胞を収集し分析に供することができる。例えば、胎児赤血球はアレイ内で同定され、次に遺伝的異常について分析される。胎児赤血球は形態学的に、または特異的分子マーカー(例、胎児ヘモグロビン、転移受容体(CD71)、トロンボスポンジン受容体(CD36)、またはグリコホリンA(GPA))によって同定できる。
また別のデバイスは、サイズ、形状または変形性に基づいて選択的に粒子の通過を許容するふるいの使用に基づいて粒子を分離するためのデバイスである。ふるい内の孔のサイズ、形状、または変形性がそのふるいを通過できる細胞の型を決定する。例えばサイズが連続的に増加する細胞を除去するためには、2つ以上のふるいを直列または並列で配列できる。
1つの態様では、ふるいには間隔をあけて離れている一連の障害物が含まれる。本発明のデバイスでは、様々な障害物のサイズ、形状、および配列を使用できる。ふるいでは、例えば円形、四角形、長方形、楕円形、または三角形の断面を備える障害物などの障害物の様々な形状を使用できる。迅速かつ効率的な濾過を保証するためには、障害物間の間隙サイズおよび障害物の形状を最適化できる。例えば、RBCのサイズ範囲は約5〜8μmであり、血小板のサイズ範囲は約1〜3μmである。全WBCのサイズは10μmより大きい。障害物間の大きな間隙はRBCおよび血小板がふるいを通過する速度を上昇させるが、大きな間隙はさらにWBCが消失するリスクを増加させる。間隙のサイズが小さいと、WBCのより効率的な捕捉を保証するが、RBCおよび血小板の通過速度もまた緩徐になる。用途の型に依存して、様々な形状を使用できる。
本発明のデバイスは、例えば血液からより大きなWBCおよび胎児RBCを濾過する、連続フローセルソーターである。このデバイス内のふるいの位置は、同時に目詰まりを回避して分離後の粒子の回収を許容しながら、最大数の粒子がふるいと接触することを保証するために選択される。一般には、粒子は典型的にはマイクロメートルサイズであるデバイス内の流路内の極度に低いレイノルズ数のために維持されているそれらの層流ラインを越えて移動させられる。
例えば本発明のデバイスの流路およびふるいを作製するためには、ポリジメチルシロキサン(PDMS)ソフトリソグラフィー、ポリマーキャスティング(例、エポキシ樹脂、アクリル樹脂、またはウレタン)、射出成形法、ポリマーホットエンボス加工法、レーザー微細加工法、薄膜表面微細加工法、ガラスおよびシリコン両方のディープエッチング法、電気鋳造法および立体リソグラフィーなどの三次元加工法のような単純な微細加工技術を使用できる。上記に列挙した工程の大多数は、極微細機能を複製するためにフォトマスクを使用する。5μmを越える機能サイズのためには、透明性に基づくエマルジョンマスクを使用できる。2〜5μmの機能サイズにはガラスに基づくクロム製フォトマスクを必要とする。より小さな機能のためには、ガラスに基づく電子線直接書込みマスクを使用できる。これらのマスクは次に、例えば次にPDMS、エポキシ樹脂、およびアクリル樹脂のようなポリマー材料を複製成形するためのマスターとして使用できるSU-8フォトレジストを使用して、シリコンまたはガラスの場合にはエッチングするためのフォトレジストパターンを画定するため、またはネガ複製を画定するためのどちらかに使用される。加工された流路は、次にデバイスを完成させるためのガラスのような剛性基質上に接着させることができる。その他の作製方法は、当技術分野において知られている。本発明のデバイスは、単一材料または材料の組み合わせから作製されてよい。
本発明のデバイスは、単独で、またはいずれかの組み合わせで使用できる。さらに、本明細書に記載の方法の段階はあらゆる順序で利用できる。胎児赤血球を検出かつ分離するための組み合わせデバイスの略図は図25に示されている。1つの実施例では、試料は細胞溶解段階を使用して処理することができ、次に所望の細胞を細胞結合デバイス内で捕捉することができる。捕捉された細胞が十分に純粋である場合は、その後の処理段階は必要とされない。または、アレイ化の前に溶解または結合の1つの段階だけが利用されてもよい。また別の実施例では、細胞混合物に溶解、サイズに基づく分離、結合、およびアレイ化を供させることができる。
本発明の一つまたは複数のデバイスによって濃縮させた後、細胞を収集して、例えば核酸分析などの様々な方法によって分析することができる。試料は、さらに分析の前に処理されてもよい。1つの実施例では、細胞は蛍光インサイチュー・ハイブリダイゼーション(FISH)用の平面基質上で収集され、続いて細胞の固定およびイメージングが実施されてよい。そのような分析を使用すると、ダウン症候群、エドワード症候群、パトー症候群、クラインフェルター症候群、ターナー症候群、鎌状赤血球貧血、デュシェンヌ型筋ジストロフィー、および嚢胞性線維症などの胎児異常を検出することができる。分析は、さらに例えば性別などの胎児の特定形質を決定するために実施することもできる。
上記の明細書で言及した全ての出版物、特許、および特許出願は参照として本明細書に組み入れられる。本明細書に記載の方法およびシステムの様々な修飾および変形は、本発明の範囲および精神から逸脱せずに当業者には明白であろう。本発明を特定の態様と結び付けて記載してきたが、要求された本発明はこのような特定の態様へ不当に限定されるべきではないと理解されたい。実際に、本発明を実施するための当業者には明白である記載したモードの様々な修飾は、本発明の範囲内に含まれることが意図されている。
Claims (67)
- 少なくとも3つの細胞型の細胞混合物から第1の細胞型が濃縮された集団を生成する方法であって、
(a)少なくとも第1、第2、および第3の細胞型を含む細胞混合物を提供する段階と;
(b)該第2の細胞型と比較して該第1の細胞型が濃縮された第1の細胞集団を生成するために、該第1の細胞型の通過を選択的に可能にする第1の微小流路(microfluidiv channel)と該混合物を接触させる段階;ならびに
(c)該第2および第3の細胞型と比較して該第1の細胞型が濃縮された第2の細胞集団を生成するために、該第1の細胞型の通過を選択的に可能にする第2の微小流路と該第1細胞集団を接触させ、それにより第1の細胞型が濃縮された集団を生成する段階
を含む方法。 - 段階(b)が、第2の細胞型と比較して第1の細胞型の相対的集団を少なくとも100倍、1000倍、10,000倍、100,000倍、または1,000,000倍増加させる、請求項1記載の方法。
- 段階(c)が、第3の細胞型と比較して第1の細胞型の相対的集団を少なくとも100倍、1000倍、10,000倍、100,000倍、または1,000,000倍増加させる、請求項1記載の方法。
- 第2の細胞集団をアレイ化する段階(d)をさらに含む、請求項1記載の方法。
- 第1の細胞型が胎児赤血球であり、第2の細胞型が母体赤血球であり、かつ第3の細胞型が母体白血球である、請求項1記載の方法。
- 試料から一つまたは複数の所望の細胞を分離するためのマイクロ流体システムであって、
(a)(i)少なくとも2つの流入流路;
(ii)反応チャンバー;および
(iii)流出流路を含み、
該流入流路が該反応チャンバーを通して該流出流路へ接続されている、溶解デバイス;
(b)微小流路内に配置された障害物を含む細胞結合デバイスであって、該障害物が第2の細胞型と比較して第1の細胞型へ優先的に結合する、細胞結合デバイス;ならびに
(c)個別の細胞を封じ込めるための場所の二次元アレイを含むアレイ化デバイス
を含むシステム。 - 試料から一つまたは複数の所望の細胞を分離するためのマイクロ流体システムであって、
(a)(i)少なくとも2つの流入流路;
(ii)反応チャンバー;および
(iii)流出流路を含み、
該流入流路が該反応チャンバーを通して該流出流路へ接続されている、溶解デバイス;ならびに
(b)微小流路内に配置された障害物を含む細胞結合デバイスであって、該障害物が第2の細胞型と比較して第1の細胞型へ優先的に結合する、細胞結合デバイス
を含むシステム。 - 試料から所望の細胞を分離するためのマイクロ流体システムであって、
(a)(i)少なくとも2つの流入流路;
(ii)反応チャンバー;および
(iii)流出流路を含み、
該流入流路が該反応チャンバーを通して該流出流路へ接続されている、溶解デバイス;ならびに
(b)個別の細胞を封じ込めるための場所の二次元アレイを含むアレイ化デバイス
を含むシステム。 - 試料から一つまたは複数の所望の細胞を分離するためのマイクロ流体システムであって、
(a)微小流路内に配置された障害物を含む細胞結合デバイスであって、該障害物が第2の細胞型と比較して第1の細胞型へ優先的に結合する、細胞結合デバイス;および
(b)個別の細胞を封じ込めるための場所の二次元アレイを含むアレイ化デバイス
を含むシステム。 - 障害物が、抗CD71抗体、抗CD36抗体、抗GPA抗体、もしくは抗CD45抗体、またはそれらの組み合わせでコーティングされている、請求項6、7、および9のいずれか一項記載のマイクロ流体システム。
- 細胞結合デバイスが、第4の細胞型と比較して第3の細胞型へ優先的に結合する障害物をさらに含む、請求項6、7、および9のいずれか一項記載のマイクロ流体システム。
- アレイ化デバイスが個別の細胞の選択的放出のためのアクチュエーターをさらに含む、請求項6、8、および9のいずれか一項記載のマイクロ流体システム。
- 反応チャンバーが蛇行状流路を含む、請求項6から8のいずれか一項記載のマイクロ流体システム。
- 溶解デバイスが希釈チャンバーおよび第3の流入流路をさらに含み、反応チャンバーが該希釈チャンバーを通じて流出流路へ接続されており、該第3の流入口が該反応チャンバーと該希釈チャンバーとの間に配置されている、請求項6から8のいずれか一項記載のマイクロ流体システム。
- 希釈チャンバーが蛇行状流路を含む、請求項14記載のマイクロ流体デバイス。
- 試料から一つまたは複数の第2の細胞型を分離するための方法であって、
(a)一つまたは複数の微小流路内に(i)少なくとも第1および第2の細胞型を含む試料、および(ii)第1の細胞型を優先的に溶解させる溶液を導入して、第2の細胞型と比較して第1の細胞型のより高度な溶解を引き起こす段階;
(b)段階(a)の生成物を微小流路内に配置された障害物を含むマイクロ流体デバイスと接触させる段階であって、該障害物が該第2の細胞型へ優先的に結合する段階と;
(c)該障害物へ結合した細胞を収集し、それにより該第2の細胞型が濃縮された細胞集団を生成する段階;
(d)該第2の細胞型が濃縮された該細胞集団をアレイ化する段階;
(e)該第2の細胞型が濃縮された該集団中で一つまたは複数の該第2の細胞型を同定する段階;ならびに
(f)該一つまたは複数の第2の細胞型を収集し、それにより該試料から該一つまたは複数の第2の細胞型を分離する段階
を含む方法。 - 試料から一つまたは複数の第2の細胞型を分離するための方法であって、
(a)一つまたは複数の微小流路内に(i)少なくとも第1、第2、および第3の細胞型を含む試料、および(ii)第1の細胞型を優先的に溶解させる溶液を導入して、第2の細胞型と比較して第1の細胞型のより高度な溶解を引き起こす段階;
(b)段階(a)の生成物を微小流路内に配置された障害物を含むマイクロ流体デバイスと接触させる段階であって、該障害物が該第2の細胞型と比較して該第3の細胞型へ優先的に結合する段階;
(c)該障害物へ結合していない細胞を収集し、それにより該第2の細胞型が濃縮された細胞集団を生成する段階;
(d)該第2の細胞型が濃縮された該細胞集団をアレイ化する段階;
(e)該第2の細胞型が濃縮された該細胞集団中で一つまたは複数の該第2の細胞型を同定する段階;ならびに
(f)該一つまたは複数の第2の細胞型を収集し、それにより該試料から該一つまたは複数の第2の細胞型を分離する段階
を含む方法。 - 第2の細胞型が濃縮された細胞集団を生成するための方法であって、
(a)一つまたは複数の微小流路内に(i)少なくとも第1および第2の細胞型を含む試料、および(ii)第1の細胞型を優先的に溶解させる溶液を導入して、第2の細胞型と比較して第1の細胞型のより高度な溶解を引き起こす段階;
(b)段階(a)の生成物を微小流路内に配置された障害物を含むマイクロ流体デバイスと接触させる段階であって、該障害物が該第2の細胞型へ優先的に結合する段階;ならびに
(c)該障害物へ結合した細胞を収集し、それにより該第2の細胞型が濃縮された細胞集団を生成する段階
を含む方法。 - 第2の細胞型が濃縮された細胞集団を生成するための方法であって、
(a)一つまたは複数の微小流路内に(i)少なくとも第1、第2、および第3の細胞型を含む試料、および(ii)第1の細胞型を優先的に溶解させる溶液を導入して、第2の細胞型と比較して第1の細胞型のより高度な溶解を引き起こす段階;
(b)段階(a)の生成物を微小流路内に配置された障害物を含むマイクロ流体デバイスと接触させる段階であって、該障害物が該第2の細胞型と比較して該第3の細胞型へ優先的に結合する段階;ならびに
(c)該障害物へ結合していない細胞を収集し、それにより該第2の細胞型が濃縮された細胞集団を生成する段階
を含む方法。 - 試料から一つまたは複数の第2の細胞型を分離するための方法であって、
(a)一つまたは複数の微小流路内に(i)少なくとも第1および第2の細胞型を含む試料、および(ii)第1の細胞型を優先的に溶解させる溶液を導入して、第2の細胞型と比較して第1の細胞型のより高度な溶解を引き起こす段階;
(b)段階(a)の生成物をアレイ化する段階;
(c)該一つまたは複数の第2の細胞型を同定する段階;ならびに
(d)該一つまたは複数の第2の細胞型を収集し、それにより該試料から該一つまたは複数の第2の細胞型を分離する段階
を含む方法。 - 試料から一つまたは複数の第2の細胞型を分離するための方法であって、
(a)少なくとも第1および第2の細胞型を含む試料を微小流路内に配置された障害物を含むマイクロ流体デバイスと接触させる段階であって、該障害物が該第2の細胞型と比較して該第1の細胞型へ優先的に結合する段階;
(b)該障害物へ結合していない細胞を収集し、それにより枯渇した細胞集団を生成する段階;
(c)該枯渇した細胞集団をアレイ化する段階;
(d)該枯渇した集団中で該一つまたは複数の第2の細胞型を同定する段階;および
(e)該一つまたは複数の該第2の細胞型を収集し、それにより該試料から該一つまたは複数の第2の細胞型を分離する段階
を含む方法。 - 試料から一つまたは複数の第2の細胞型を分離するための方法であって、
(a)少なくとも第1および第2の細胞型を含む試料を微小流路内に配置された障害物を含むマイクロ流体デバイスと接触させる段階であって、該障害物が該第1の細胞型と比較して該第2の細胞型へ優先的に結合する段階;
(b)該障害物へ結合した細胞を収集し、それにより枯渇した細胞集団を生成する段階;
(c)該枯渇した細胞集団をアレイ化する段階;
(d)該枯渇した集団中で該一つまたは複数の第2の細胞型を同定する段階;および
(e)該一つまたは複数の第2の細胞型を収集し、それにより該試料から該一つまたは複数の該第2の細胞型を分離する段階
を含む方法。 - 第2の細胞型が胎児赤血球である、請求項16から22のいずれか一項記載の方法。
- 段階(a)中の溶液がNaHCO3およびアセタゾルアミドを含む、請求項16から20のいずれか一項記載の方法。
- 段階(a)の後に、一つまたは複数の微小流路内で段階(a)の生成物を希釈剤で希釈する段階をさらに含む、請求項16から20のいずれか一項記載の方法。
- 障害物の各々が結合成分でコーティングされている、請求項16から19、21、および22のいずれか一項記載の方法。
- 結合成分が、抗CD71抗体、抗CD36抗体、抗GPA抗体、もしくは抗CD45抗体、またはそれらの組み合わせを含む、請求項16記載の方法。
- 試料中の一つまたは複数の第2の細胞型の少なくとも75%、80%、90%、95%、98%、または99%がアレイ化デバイス内でアレイ化される、請求項16、17、および20から22のいずれか一項記載の方法。
- 試料中の第2の細胞型の少なくとも75%、80%、90%、95%、98%、または99%が収集される、請求項18から19のいずれか一項記載の方法。
- 第2の細胞型が濃縮された細胞集団を生成するための方法であって、
(a)少なくとも第1および第2の細胞型を含む試料を提供する段階;
(b)一つまたは複数の微小流路内に(i)該試料、および(ii)第1の細胞型を優先的に溶解させる溶解バッファーを導入して、第2の細胞型と比較して第1の細胞型のより高度な溶解を引き起こし、該濃縮された集団を生成する段階
を含む方法。 - 段階(b)の後に、希釈した試料を生成するために、一つまたは複数の微小流路内で濃縮された集団を希釈剤で希釈する段階(c)をさらに含む、請求項30記載の方法。
- 段階(c)の後に、希釈した試料を収集する段階(d)をさらに含む、請求項31記載の方法。
- 試料が2つまたはそれ以上の微小流路内に導入される、請求項30記載の方法。
- 第1の細胞型が母体赤血球であり、第2の細胞型が胎児赤血球である、請求項30記載の方法。
- 溶解バッファーがNaHCO3およびアセタゾルアミドを含む、請求項34記載の方法。
- 溶解バッファーが少なくとも30秒間試料と接触させられる、請求項34記載の方法。
- 第2の細胞型の少なくとも98%が溶解されない、請求項30記載の方法。
- (a)(i)少なくとも3つの流入流路;
(ii)蛇行状流路を含む反応チャンバー;
(iii)蛇行状流路を含む希釈チャンバー;および
(iv)流出流路
を含むマイクロ流体デバイス;ならびに
(b)他の細胞型より1つの細胞型を優先的に溶解させる溶解バッファー
を含むキット。 - (c)希釈剤をさらに含む、請求項38記載のキット。
- 溶解バッファーがNaHCO3およびアセタゾルアミドを含む、請求項38記載のキット。
- 流入流路および流出流路が10から500μmの直径寸法を有する、請求項38記載のキット。
- (ii)および(iii)の蛇行状流路が各々少なくとも10μLの容積を有する、請求項41記載のキット。
- (ii)および(iii)の蛇行状流路が各々少なくとも20μLの容積を有する、請求項42記載のキット。
- 枯渇した細胞集団を生成する方法であって、
(a)少なくとも第1および第2の細胞型を含む試料を、該第2の細胞型と比較して該第1の細胞型へ優先的に結合する障害物を含むマイクロ流体デバイスと接触させる段階;
(b)該障害物へ結合した細胞または該障害物へ結合していない細胞を収集し、それにより枯渇した細胞集団を生成する段階
を含む方法。 - 障害物が第1の細胞型の表面へ結合する結合成分でコーティングされている、請求項44記載の方法。
- 第1の細胞型が胎児赤血球である、請求項44記載の方法。
- 第2の細胞型が胎児赤血球である、請求項44記載の方法。
- 試料内の第1の細胞型の少なくとも60%が障害物へ結合する、請求項44記載の方法。
- 試料内の第2のの細胞型の少なくとも70%が障害物へ結合しない、請求項44記載の方法。
- 障害物が二次元アレイで規則化されている、請求項44記載の方法。
- その各々が結合成分でコーティングされている、微小流路内に配置された障害物を含むマイクロ流体デバイス。
- 障害物が二次元アレイで規則化されている、請求項51記載のマイクロ流体デバイス。
- 障害物の各々の高さが50から500μmである、請求項51記載のマイクロ流体デバイス。
- 障害物の各々が、高さに直交する5から500μmの範囲内の寸法を有する、請求項51のマイクロ流体デバイス。
- 障害物の各々が他の障害物から少なくとも10から1000μm離れて配置されている、請求項51記載のマイクロ流体デバイス。
- 結合成分が、抗CD71抗体、抗CD36抗体、抗GPA抗体、もしくは抗CD45抗体、またはそれらの組み合わせを含む、請求項51記載のマイクロ流体デバイス。
- (a)微小流路内に配置された障害物の、流体流路を規定する第1の領域であって、該第1の領域内の該障害物が第2の細胞型と比較して第1の細胞型へ優先的に結合する、第1の領域;および
(b)該微小流路内に配置された障害物の第2の領域であって、該第2の領域内の該障害物が第4の細胞型と比較して第3の細胞型へ優先的に結合する、第2の領域
を含む(ただし、該第1および第3の細胞型が同一ではない)、マイクロ流体デバイス。 - 第2の細胞型および第4の細胞型が同一である、請求項57記載のマイクロ流体デバイス。
- 第2の細胞型および第3の細胞型が同一である、請求項58記載のマイクロ流体デバイス。
- 第1の領域および第2の領域が、微小流路内で流体の流れに対して直列に配列されている、請求項57記載のマイクロ流体デバイス。
- 第1の領域および第2の領域が微小流路内で流体の流れに対して並列に配列されている、請求項57記載のマイクロ流体デバイス。
- 第1の領域内の障害物が第2の領域内の該障害物間に散在している、請求項61記載のマイクロ流体デバイス。
- (a)微小流路内に配置された障害物の、流体流路を規定する第1の領域であって、該第1の領域内の該障害物が、第2の細胞型と比較して第1の細胞型へ優先的に結合し、該障害物が少なくとも2つの実質的に平行な行で、かつ少なくとも2つの実質的に平行な列で配列されている、第1の領域;および
(b)該微小流路内に配置された障害物の第2の領域であって、該第2の領域内の該障害物が、第4の細胞型と比較して第3の細胞型へ優先的に結合し、該障害物が少なくとも2つの実質的に平行な行で、かつ少なくとも2つの実質的に平行な列で配列されている第2の領域を含む、マイクロ流体デバイスであって、第1および第2の領域が微小流路内で相互に隣接して配置されており、かつ第2の領域内の行が第1の領域内の行に対して第1の領域内の行間の距離より短い距離に配置されている、
を含むマイクロ流体デバイス。 - 第1の領域内の障害物が、抗CD71抗体、抗CD36抗体、抗GPA抗体、もしくは抗CD45抗体、またはそれらの組み合わせでコーティングされている、請求項63記載のマイクロ流体デバイス。
- 第2の領域内の障害物が、抗CD71抗体、抗CD36抗体、抗GPA抗体、もしくは抗CD45抗体、またはそれらの組み合わせでコーティングされている、請求項63記載のマイクロ流体デバイス。
- 第1の領域内の列間の距離に対する第1の領域内の行間の距離の比率が約√3である、請求項63記載のマイクロ流体デバイス。
- 第2の領域内の列間の距離に対する第2の領域内の行間の距離の比率が約√3である、請求項63記載のマイクロ流体デバイス。
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US20170197214A1 (en) | 2017-07-13 |
US20190022653A1 (en) | 2019-01-24 |
US11052392B2 (en) | 2021-07-06 |
WO2004029221A3 (en) | 2004-05-13 |
US20070264675A1 (en) | 2007-11-15 |
HK1079960A1 (en) | 2006-04-21 |
EP1569510B1 (en) | 2011-11-02 |
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US20070231851A1 (en) | 2007-10-04 |
ATE531257T1 (de) | 2011-11-15 |
AU2010212376B2 (en) | 2011-10-13 |
JP2010075191A (ja) | 2010-04-08 |
US8372579B2 (en) | 2013-02-12 |
US20070259424A1 (en) | 2007-11-08 |
US20060134599A1 (en) | 2006-06-22 |
AU2003277153A1 (en) | 2004-04-19 |
CA2500392A1 (en) | 2004-04-08 |
US20120006760A1 (en) | 2012-01-12 |
ES2375724T3 (es) | 2012-03-05 |
US8304230B2 (en) | 2012-11-06 |
US8986966B2 (en) | 2015-03-24 |
EP2359689B1 (en) | 2015-08-26 |
US20070172903A1 (en) | 2007-07-26 |
US10081014B2 (en) | 2018-09-25 |
US20150260711A1 (en) | 2015-09-17 |
AU2010212376A1 (en) | 2010-09-09 |
US20210370298A1 (en) | 2021-12-02 |
US8895298B2 (en) | 2014-11-25 |
WO2004029221A2 (en) | 2004-04-08 |
CA2500392C (en) | 2012-11-27 |
EP2359689A1 (en) | 2011-08-24 |
EP1569510A4 (en) | 2006-08-30 |
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