JP2011163830A - Detection of circulation tumor cells using size-selective micro cavity array - Google Patents
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Abstract
Description
本発明は、サイズ選択マイクロキャビティアレイ（size-selective microcavity array）を備えた、循環腫瘍細胞（Circulating tumor cell: ＣＴＣ）を捕捉することができるマイクロ流体デバイスや、該マイクロ流体デバイスを用いたＣＴＣの検出方法に関する。 The present invention relates to a microfluidic device having a size-selective microcavity array and capable of capturing circulating tumor cells (CTC), and a CTC using the microfluidic device. It relates to a detection method.
ＣＴＣはがん患者の末梢血流を循環する腫瘍細胞と定義され、原発腫瘍又は転移腫瘍から血管中へ浸潤した腫瘍細胞である。このＣＴＣの検出は、転移性悪性腫瘍の早期発見の方法の一つとして近年注目されている（例えば、非特許文献１又は２参照）。その理由は、Ｘ線写真や血清中の腫瘍マーカー検出よりも低侵襲かつ正確に転移性悪性腫瘍の診断を行え、患者の予後予測や治療効果の指標として利用できる点にある。 CTCs are defined as tumor cells that circulate in the peripheral bloodstream of cancer patients and are tumor cells that have infiltrated into blood vessels from primary or metastatic tumors. In recent years, detection of this CTC has attracted attention as a method for early detection of metastatic malignant tumors (see, for example, Non-Patent Document 1 or 2). The reason is that metastatic malignant tumors can be diagnosed more accurately and less invasively than X-ray photographs and tumor marker detection in serum, and can be used as an indicator of patient prognosis and therapeutic effects.
ＣＴＣは非常に稀少な細胞であり、転移性がん患者の血液に含まれる１０８〜１０９個の血液細胞の内、わずか１細胞程度しか存在しないことが知られている（例えば、非特許文献３又は４参照）。このため、末梢血から稀少なＣＴＣを正確に検出するための技術開発に多大な努力が注がれている。これまでに開発されてきた主要な検出方法には、免疫組織化学法（例えば、非特許文献３参照）、ＰＣＲ法（例えば、非特許文献５参照）、フローサイトメトリー法（例えば、非特許文献６参照）などがある。しかしながら、前述したようにＣＴＣは非常に稀少な細胞であるため、血液をそのままこれらの検出方法に供することは出来ない。通常は前処理として、ＣＴＣの濃縮操作が必須であり、検出法に則したレベルまでＣＴＣ存在比を濃縮させる必要がある。 CTC is a very rare cell, and it is known that only about 1 cell is present among 10 8 to 10 9 blood cells contained in the blood of patients with metastatic cancer (for example, non-patent Reference 3 or 4). For this reason, great efforts have been put into technology development for accurately detecting rare CTCs from peripheral blood. Major detection methods that have been developed so far include immunohistochemical methods (see, for example, Non-Patent Document 3), PCR methods (see, for example, Non-Patent Document 5), and flow cytometry methods (for example, Non-Patent Documents). 6). However, since the CTC is a very rare cell as described above, blood cannot be directly used for these detection methods. Usually, as a pretreatment, a CTC concentration operation is essential, and it is necessary to concentrate the CTC abundance ratio to a level in accordance with the detection method.
ＣＴＣの濃縮方法として開発されてきた様々な手法の中で、最も広く利用されているのは、細胞表面の特異的抗原を標的とした腫瘍細胞の濃縮である。その多くは、上皮細胞接着分子（Epithelial cell adhesion molecule: ＥｐＣＡＭ）に対するモノクローナル抗体を固定化した磁気微粒子を血液と混合した後、磁石を用いて腫瘍細胞を濃縮する方法をとっている（例えば、非特許文献７参照）。しかしながら、ＥｐＣＡＭの発現量は腫瘍のタイプに依存し、大きく変動することが知られている（例えば、非特許文献８又は９参照）。その他の方法では、細胞のサイズなどの形態を基準として濃縮する手法がある。白血球に比べてサイズが大きな上皮性腫瘍細胞をフィルトレーションによって選別する方法は、ＩＳＥＴ法（Isolation by Size of Epithelial Tumor cells）と呼ばれている。ＩＳＥＴは、孔径８μｍのポリカーボネートメンブレンフィルターを用いて血液をフィルトレーションするという簡便な手法であり、安価かつユーザーフレンドリーな手法である。ここで用いられているポリカーボネートメンブレンフィルターは、重イオンを照射した後、エッチングを行うトラックエッチングという手法によって、孔が形成されている。しかし、孔が比較的低密度であり、二つ又はそれ以上の孔が重なりあったりする問題があるため、ＣＴＣの捕捉に利用した場合、その捕捉効率は５０〜６０％とされている（例えば、非特許文献１０参照）。よって、濃縮法が簡便かつ効率も良い手法は未だ開発されていない。 Among various techniques that have been developed as CTC enrichment methods, the most widely used is the enrichment of tumor cells targeting specific antigens on the cell surface. Many of them employ a method of concentrating tumor cells using a magnet after mixing magnetic microparticles immobilizing monoclonal antibodies against epithelial cell adhesion molecule (EpCAM) with blood (for example, using non-magnetic particles). (See Patent Document 7). However, the expression level of EpCAM is known to vary greatly depending on the type of tumor (see, for example, Non-Patent Document 8 or 9). As another method, there is a method of concentrating on the basis of a form such as a cell size. A method of selecting epithelial tumor cells larger in size than leukocytes by filtration is called the ISET method (Isolation by Size of Epithelial Tumor cells). ISET is a simple method of filtering blood using a polycarbonate membrane filter having a pore diameter of 8 μm, and is an inexpensive and user-friendly method. The polycarbonate membrane filter used here has holes formed by a technique called track etching in which etching is performed after irradiation with heavy ions. However, since the holes have a relatively low density and there is a problem that two or more holes overlap each other, the trapping efficiency is 50 to 60% when used for trapping CTC (for example, Non-Patent Document 10). Therefore, a method with a simple and efficient concentration method has not yet been developed.
ＣＴＣの検出を効率的かつ正確なものにするためには、濃縮と検出といった技術を首尾一貫して行うことが必要である。多段階のハンドリング操作、例えば細胞の染色、洗浄、分離、分注などの操作はＣＴＣのロスを引き起こすため、可能な限りこれらの操作を避け、一体の検出装置中で分析が一貫して行える形が望ましい。Ｃｅｌｌｓｅａｒｃｈ（VeridexTM, Warren, PA）はＣＴＣ検出装置として唯一ＦＤＡの認可を受けた装置である。この装置では、全血に対し抗ＥｐＣＡＭ抗体固定化磁気微粒子によるＣＴＣの濃縮を行い、腫瘍細胞に対して免疫染色を行った後、自動化蛍光顕微鏡を用いて腫瘍細胞の計数が行われる（例えば、非特許文献１１参照）。一方で、ＣＴＣ検出のためにマイクロ流体デバイスの開発も行われている。Tonerらが開発したＣＴＣ検出用マイクロ流体デバイスはＣＴＣ−ｃｈｉｐと呼ばれ（例えば、非特許文献１２参照）、フォトリソグラフィーによって形成されたシリコン製の流路内に、円筒状構造物（マイクロポスト）が７８０００個構成されている。このマイクロポストには、抗ＥｐＣＡＭ抗体がコーティングされており、本流路に血液を送液すると、血液中のＣＴＣがマイクロポスト上に捕捉される。捕捉されたＣＴＣに対して、上皮細胞マーカー（cytokeratin）をターゲットとした蛍光免疫染色を行い、蛍光顕微鏡を用いて腫瘍細胞の計数が行われる。本装置は、手のひらに乗る小型デバイスでありながら、５ｍＬ以上の血液をそのまま分析に供することができるという大きな利点を持っている。実際に転移性がん患者血液からＣＴＣ検出を行っており、回収したＣＴＣからチロシンキナーゼ阻害薬に対する耐性を生む変異を検出することが出来ている（例えば、非特許文献１３参照）。ＣｅｌｌｓｅａｒｃｈやＣＴＣ−ｃｈｉｐを用いたＣＴＣ検出は転移性がん患者血液などの実サンプルを用いた実験が精力的に行われ実績を挙げているが、これらの手法は抗ＥｐＣＡＭ抗体でＣＴＣを濃縮するという原理になっている。そのため、ＣＴＣ陰性又は弱陽性の腫瘍細胞は検出できないという問題点が挙げられる。 In order for CTC detection to be efficient and accurate, techniques such as enrichment and detection need to be performed consistently. Multi-stage handling operations such as cell staining, washing, separation, and dispensing cause CTC loss. Therefore, avoid these operations as much as possible, and perform analysis in an integrated detector. Is desirable. Cellsearch (Veridex ™, Warren, PA) is the only CTC detector that has received FDA approval. In this apparatus, CTC is concentrated on whole blood using anti-EpCAM antibody-immobilized magnetic microparticles, and tumor cells are immunostained, and then tumor cells are counted using an automated fluorescent microscope (for example, Non-patent document 11). On the other hand, a microfluidic device has also been developed for CTC detection. The CTC detection microfluidic device developed by Toner et al. Is called CTC-chip (see, for example, Non-Patent Document 12), and a cylindrical structure (micropost) is formed in a silicon flow path formed by photolithography. Is composed of 78000 pieces. The micropost is coated with an anti-EpCAM antibody, and when blood is fed into the channel, CTC in the blood is captured on the micropost. The captured CTC is subjected to fluorescent immunostaining targeting an epithelial cell marker (cytokeratin), and tumor cells are counted using a fluorescence microscope. Although this device is a small device placed on the palm, it has a great advantage that it is possible to use 5 mL or more of blood as it is for analysis. Actually, CTC detection is performed from the blood of metastatic cancer patients, and a mutation that produces resistance to a tyrosine kinase inhibitor can be detected from the collected CTC (see, for example, Non-Patent Document 13). CTC detection using Cellsearch and CTC-chip has been proven by vigorous experiments using actual samples such as blood from metastatic cancer patients, but these methods concentrate CTCs with anti-EpCAM antibodies. This is the principle. Therefore, there is a problem that CTC negative or weak positive tumor cells cannot be detected.
その他のグループでは、腫瘍細胞のサイズと形態を指標として、ＣＴＣを検出するマイクロ流体デバイスが開発されている。これらのデバイスでは、その流路構造内にメンブレンマイクロフィルター（例えば、非特許文献１４参照）や、三日月型の細胞捕捉ウェル（例えば、非特許文献１５参照）、４段階の細さの流路（例えば、非特許文献１６参照）を配して、血液中の血球細胞と腫瘍細胞をサイズによって選別し、腫瘍細胞を選択的に濃縮するものとしている。また、その流路を利用して、濃縮後の細胞に対して溶解などの操作を連続的に行うことが出来る。これらのデバイスを用いたモデル腫瘍細胞を用いた回収効率の評価実験においては、８０％以上のＣＴＣ回収効率を得ている。しかしながら、この評価はあくまでモデル細胞を用いた実験で行われており、実際にＣＴＣ検出時に必要となる細胞の染色操作や洗浄操作といった要素技術項目については検討されていない。さらに、がん患者血液などの実サンプルを用いた実験は行われておらず、実際にＣＴＣ検出に利用できるかどうかは明らかにされていない。 Other groups have developed microfluidic devices that detect CTCs using tumor cell size and morphology as indicators. In these devices, a membrane microfilter (see, for example, Non-Patent Document 14), a crescent-shaped cell capture well (see, for example, Non-Patent Document 15), a four-stage channel ( For example, refer to Non-Patent Document 16), and blood cells and tumor cells in blood are selected according to size, and the tumor cells are selectively concentrated. In addition, by using the flow channel, operations such as lysis can be continuously performed on the concentrated cells. In an evaluation experiment of recovery efficiency using model tumor cells using these devices, a CTC recovery efficiency of 80% or more is obtained. However, this evaluation is only conducted in an experiment using model cells, and elemental technical items such as cell staining and washing operations that are actually required for CTC detection have not been studied. Furthermore, experiments using actual samples such as cancer patient blood have not been conducted, and it has not been clarified whether they can actually be used for CTC detection.
他方、本発明者らは、試料中に含まれる細胞を１細胞レベルで捕捉することができる単一細胞捕捉用のマイクロ流路デバイスや、かかるマイクロ流路デバイスを用いた試料中に含まれる細胞を１細胞レベルで分離・捕捉する方法や、前記マイクロ流路デバイスを利用した単一細胞の遺伝子発現の定量的解析方法について提案している（例えば、特許文献１参照）。 On the other hand, the present inventors have provided a microchannel device for capturing a single cell that can capture cells contained in a sample at the level of one cell, and a cell contained in a sample using such a microchannel device. Have been proposed for a method for separating and capturing a single cell level, and a method for quantitative analysis of gene expression of a single cell using the microchannel device (for example, see Patent Document 1).
本発明の課題は、血液を前処理することなくそのまま分析できる；血球の分離から染色、洗浄までを一貫して行える；装置自体が小型で安価である；ＣＴＣの回収原理がＥｐＣＡＭなどの表面抗原に依らないこと；等の特徴を有するＣＴＣの検出技術として非常に強力なツールを提供することにある。 The problem of the present invention is that blood can be analyzed as it is without pretreatment; blood cell separation, staining, and washing can be performed consistently; the device itself is small and inexpensive; and the CTC recovery principle is a surface antigen such as EpCAM. It is to provide a very powerful tool as a CTC detection technique having the following characteristics.
上記課題を解決するため、本発明者らは鋭意検討し、従来の磁気分離法によるＣＴＣ回収に問題点が多いことから（後述の比較例参照）、腫瘍細胞の選択的な捕捉を可能とするサイズ選択マイクロキャビティアレイを開発し、血液からのＣＴＣ検出への応用を目指した。血液のフィルトレーションによってＣＴＣを検出する原理はＩＳＥＴ法と共通しているが、市販のメンブレンフィルターとは異なり、マイクロキャビティアレイの孔径、孔数、配置を正確に制御することができる、電鋳技術を用いてニッケル基板に形成されたＣＴＣ捕捉用の微細貫通孔を有するサイズ選択マイクロキャビティアレイや、レーザー技術を用いて石英基板に形成されたＣＴＣ捕捉用の微細貫通孔を有するサイズ選択マイクロキャビティアレイを構築した。また、マイクロ流体デバイス内にマイクロキャビティアレイを配することで、血液からのＣＴＣの濃縮に始まり、染色や洗浄のプロセスを一つのデバイス内で一貫して行うことが可能となることを見いだし、また、ＣＴＣが高密度に形成されたマイクロキャビティアレイ上に捕捉されるため、自動化蛍光顕微鏡などを用いて迅速に計数することができることを確認し、本発明を完成するに至った。 In order to solve the above-mentioned problems, the present inventors have intensively studied and because there are many problems in CTC recovery by the conventional magnetic separation method (see the comparative example described later), it enables selective capture of tumor cells. A size-selective microcavity array was developed with the aim of applying it to CTC detection from blood. The principle of detecting CTC by blood filtration is the same as that of the ISET method. However, unlike commercially available membrane filters, the diameter, number and arrangement of microcavity arrays can be accurately controlled. Size-selective microcavity array with fine through-holes for CTC capture formed on a nickel substrate using the technology, and size-selective microcavity with fine through-holes for CTC capture formed on the quartz substrate using the laser technology An array was constructed. In addition, by arranging a microcavity array in a microfluidic device, it has been found that it is possible to consistently perform staining and washing processes within one device, starting with the concentration of CTC from blood. Since the CTC was captured on the microcavity array formed at a high density, it was confirmed that it could be quickly counted using an automated fluorescence microscope, and the present invention was completed.
すなわち本発明は、（１）サイズ選択マイクロキャビティアレイにより血液試料中に含まれる循環腫瘍細胞（ＣＴＣ）を捕捉することができるマイクロ流体デバイスであって、試料供給口と試料排出口、及び試料供給口と試料排出口を連通するマイクロ流路が形成され、マイクロ流路の一部に相当する位置にサイズ選択マイクロキャビティアレイ用開口窓が設けられた上部部材と；前記上部部材の開口窓の下方に相当する位置に、ＣＴＣ捕捉用の孔径、孔数、配置が制御された微細貫通孔を有するサイズ選択マイクロキャビティアレイと、該サイズ選択マイクロキャビティアレイを保持する密封性シールからなるマイクロキャビティアレイ保持部と；前記サイズ選択マイクロキャビティアレイの下方に相当する位置に設けられた吸引用開口窓と、前記吸引用開口窓と吸引口を連通する吸引流路が形成された下部部材と；を備えていることを特徴とするマイクロ流体デバイスに関する。 That is, the present invention is (1) a microfluidic device capable of capturing circulating tumor cells (CTC) contained in a blood sample by a size-selective microcavity array, which comprises a sample supply port, a sample discharge port, and a sample supply An upper member in which a microchannel that connects the mouth and the sample outlet is formed, and an opening window for a size selection microcavity array is provided at a position corresponding to a part of the microchannel; below the opening window of the upper member A micro-cavity array holding comprising a size-selective microcavity array having fine through-holes controlled in diameter, number of holes, and arrangement for capturing CTC and a hermetic seal for holding the size-selected microcavity array A suction opening window provided at a position corresponding to the lower side of the size selection microcavity array A lower member suction channel is formed which communicates the suction opening window and the suction port; relates to a microfluidic device, characterized in that it comprises a.
また本発明は、（２）マイクロキャビティアレイ保持部が、２〜３枚のサイズ選択マイクロキャビティアレイと，各サイズ選択マイクロキャビティアレイを保持する密封性シールからなる多層に形成され、上層から下層に向かって、サイズ選択マイクロキャビティアレイに設けられた貫通孔の孔径が小さくなっていることを特徴とする上記（１）記載のマイクロ流体デバイスや、（３）サイズ選択マイクロキャビティアレイが、孔径５μｍ〜２０μｍから選ばれる微細貫通孔を有するマイクロキャビティアレイであることを特徴とする上記（１）又は（２）記載のマイクロ流体デバイスや、（４）サイズ選択マイクロキャビティアレイが、孔径８μｍ又は１０μｍから選ばれる微細貫通孔を有し、孔の中心間距離６０μｍ、１００×１００の１０，０００孔をアレイ状に配したマイクロキャビティアレイであることを特徴とする上記（１）〜（３）のいずれか記載のマイクロ流体デバイスや、（５）サイズ選択マイクロキャビティアレイのＣＴＣ捕捉用の微細貫通孔が、電鋳技術を用いてニッケル基板に形成されたものであることを特徴とする上記（１）〜（４）のいずれか記載のマイクロ流体デバイスや、（６）サイズ選択マイクロキャビティアレイのＣＴＣ捕捉用の微細貫通孔が、レーザー技術を用いて石英基板に形成されたものであることを特徴とする上記（１）〜（４）のいずれか記載のマイクロ流体デバイスや、（７）上部部材が、試料供給口及び試料排出口が形成され、下面に試料供給口と試料排出口を連通するマイクロ流路形成用の溝が設けられたプラスチック製平板からなる上部基板と、前記上部基板と協働してマイクロ流路を形成するとともに、マイクロ流路の一部に相当する位置にサイズ選択マイクロキャビティアレイ用開口窓が設けられたプラスチック製上部平板とから構成されていることを特徴とする上記（１）〜（６）のいずれか記載のマイクロ流体デバイスや、（８）下部部材が、サイズ選択マイクロキャビティアレイの下方に相当する位置に吸引用開口窓が設けられたプラスチック製下部平板と、表面に吸引口が形成され、上面に前記下部平板と協働して前記吸引用開口窓と吸引口を連通する吸引流路形成用の溝が設けられたプラスチック製平板からなる下部基板とから構成されていることを特徴とする上記（１）〜（７）のいずれか記載のマイクロ流体デバイスや、（９）吸引口がペリスタルティックポンプに接続されていることを特徴とする上記（１）〜（８）のいずれか記載のマイクロ流体デバイスや、（１０）上記（１）〜（９）のいずれか記載のマイクロ流体デバイスを用いることを特徴とする循環腫瘍細胞（ＣＴＣ） の検出方法や、（１１）血液を前処理することなくそのまま試料として使用することを特徴とする上記（１０）記載のＣＴＣの検出方法や、（１２）血液導入速度を２００〜１０００μＬ／ｍｉｎとすることを特徴とする上記（１０）又は（１１）記載のＣＴＣの検出方法に関する。 Further, according to the present invention, (2) the microcavity array holding portion is formed in a multilayer composed of two or three size selection microcavity arrays and a hermetic seal for holding each size selection microcavity array, and the upper layer to the lower layer. On the other hand, the microfluidic device according to the above (1), wherein (3) the size-selective microcavity array has a pore diameter of 5 μm to The microfluidic device according to the above (1) or (2), or (4) the size-selecting microcavity array, which is a microcavity array having fine through holes selected from 20 μm, is selected from 8 μm or 10 μm in pore diameter 10 through which the distance between the centers of the holes is 60 μm and 100 × 100. A microfluidic device according to any one of the above (1) to (3), wherein the microfluidic device is a microcavity array having 000 holes arranged in an array, The microfluidic device according to any one of (1) to (4) above, or (6) a size-selective microcavity array, wherein the through-hole is formed on a nickel substrate using an electroforming technique The microfluidic device according to any one of the above (1) to (4), wherein the fine through hole for capturing the CTC is formed in a quartz substrate using a laser technique, or (7) The upper member is a plastic flat plate in which a sample supply port and a sample discharge port are formed, and a groove for forming a microchannel that connects the sample supply port and the sample discharge port is provided on the lower surface. And an upper substrate made of plastic provided with a size selection microcavity array opening window at a position corresponding to a part of the microchannel while forming a microchannel in cooperation with the upper substrate. The microfluidic device according to any one of (1) to (6) above, or (8) a suction opening window at a position corresponding to the lower part of the size selection microcavity array The lower plate made of plastic is provided with a suction port on the surface, and the upper surface is provided with a groove for forming a suction channel that communicates with the suction opening window and the suction port in cooperation with the lower plate. A microfluidic device according to any one of the above (1) to (7), wherein (9) the suction port is a peristal A microfluidic device according to any one of (1) to (8), or (10) a microfluidic device according to any one of (1) to (9), wherein the microfluidic device is connected to a tick pump. A method for detecting circulating tumor cells (CTC) characterized by using, or (11) a method for detecting CTC as described in (10) above, wherein the blood is used as a sample without pretreatment, 12) The present invention relates to the CTC detection method as described in (10) or (11) above, wherein the blood introduction rate is 200 to 1000 μL / min.
本発明によると、血液を前処理することなくそのまま分析できる；血球の分離から染色、洗浄までを一貫して行える；装置自体が小型で安価である；ＣＴＣの回収原理がＥｐＣＡＭなどの表面抗原に依らないこと；等の特徴を有する、ＣＴＣを捕捉することができるサイズ選択マイクロキャビティアレイを備えたマイクロ流体デバイスや、該マイクロ流体デバイスを用いたＣＴＣの検出方法を提供することができる。 According to the present invention, blood can be analyzed as it is without pretreatment; blood cell separation, staining, and washing can be performed consistently; the device itself is small and inexpensive; the CTC recovery principle is applied to surface antigens such as EpCAM. It is possible to provide a microfluidic device having a size-selective microcavity array capable of capturing CTC, and a method for detecting CTC using the microfluidic device.
本発明のマイクロ流体デバイスとしては、試料供給口と試料排出口、及び試料供給口と試料排出口を連通するマイクロ流路が形成され、マイクロ流路の一部に相当する位置にサイズ選択マイクロキャビティアレイ用開口窓が設けられた上部部材と；前記上部部材の開口窓の下方に相当する位置に、ＣＴＣ捕捉用の孔径、孔数、配置が制御された微細貫通孔を有するサイズ選択マイクロキャビティアレイと、該サイズ選択マイクロキャビティアレイを保持する密封性シールからなるマイクロキャビティアレイ保持部と；前記サイズ選択マイクロキャビティアレイの下方に相当する位置に設けられた吸引用開口窓と、前記吸引用開口窓と吸引口を連通する吸引流路が形成された下部部材と；を備え、サイズ選択マイクロキャビティアレイにより血液試料中に含まれるＣＴＣを捕捉することができる装置であれば特に制限されないが、前記上部部材とマイクロキャビティアレイ保持部と下部部材とが装脱着可能に構成され、かつ、前記サイズ選択マイクロキャビティアレイが交換可能に構成されているマイクロ流体デバイスを好適に例示することができる。ここでサイズ選択マイクロキャビティアレイを備えたＣＴＣ検出の原理を図１に示す。 The microfluidic device of the present invention includes a sample supply port and a sample discharge port, and a microchannel that connects the sample supply port and the sample discharge port, and a size selection microcavity at a position corresponding to a part of the microchannel. A size-selective microcavity array having an upper member provided with an array opening window; and a fine through hole in which the hole diameter, the number of holes, and the arrangement for capturing CTC are controlled at a position corresponding to the lower part of the opening window of the upper member. A microcavity array holding portion comprising a hermetic seal for holding the size selection microcavity array; a suction opening window provided at a position corresponding to the lower side of the size selection microcavity array; and the suction opening window And a lower member having a suction flow path communicating with the suction port; and blood by a size-selective microcavity array The device is not particularly limited as long as it can capture CTC contained in a sample, but the upper member, the microcavity array holding portion, and the lower member are configured to be detachable, and the size selection microcavity array A microfluidic device configured to be replaceable can be suitably exemplified. Here, the principle of CTC detection provided with a size selection microcavity array is shown in FIG.
上記マイクロキャビティアレイ保持部を単層で構成し（図２参照）、孔径の異なるサイズ選択マイクロキャビティアレイを順次交換してＣＴＣを検出することもできるが、マイクロキャビティアレイ保持部を、上層から下層に向かって、サイズ選択マイクロキャビティアレイに設けられた貫通孔の孔径が、例えば１５μｍ，１０μｍ，５μｍへと、あるいは１０μｍから８μｍへと順次小さくなっている２〜３枚のサイズ選択マイクロキャビティアレイと，各サイズ選択マイクロキャビティアレイを保持する密封性シールからなる多層に形成した積層マイクロ流体デバイスを好適に例示することができる（図３参照）。かかる多層に形成したマイクロ流体デバイスを用いたＣＴＣ検出の概念図を図４として示す。 Although the microcavity array holding part can be configured as a single layer (see FIG. 2) and the CTC can be detected by sequentially exchanging size-selecting microcavity arrays with different hole diameters, the microcavity array holding part is changed from the upper layer to the lower layer. Two to three size-selective microcavity arrays in which the hole diameters of the through-holes provided in the size-selective microcavity array are successively reduced from, for example, 15 μm, 10 μm, 5 μm, or from 10 μm to 8 μm, A multilayer microfluidic device formed in multiple layers composed of hermetic seals holding each size-selective microcavity array can be suitably exemplified (see FIG. 3). A conceptual diagram of CTC detection using such a microfluidic device formed in multiple layers is shown in FIG.
上記サイズ選択マイクロキャビティアレイとしては、電鋳技術を用いてニッケル基板に形成されたＣＴＣ捕捉用の微細貫通孔を有するものや、レーザー技術を用いてガラス基板、好ましくは石英基板に形成されたＣＴＣ捕捉用の微細貫通孔を有するものを好適に例示することができる。かかるサイズ選択マイクロキャビティアレイ（ニッケル基板）の模式図を図５に、また、孔径サイズが１０μｍのサイズ選択マイクロキャビティアレイ（ニッケル基板）の顕微鏡写真を図６に示す。電鋳技術を用いてニッケル基板に微細貫通孔を形成させることにより、ＣＴＣ捕捉用の孔径、孔数、配置が制御された微細貫通孔を形成することができる。ＣＴＣ捕捉用の孔径としては、５μｍ，８μｍ，１０μｍ，１５μｍ，２０μｍなど通常孔径５μｍ〜２０μｍから選ぶことができ、孔数としては１０００個以上、好ましくは３０００個以上、より好ましくは５０００個以上、中でも１００×１００の１０，０００孔から１００，０００孔程度が特に好ましい。また、配置としては等間隔に設けることが好ましく、孔の中心間距離は２０μｍ以上とすることができ、例えば、孔径が８μｍや１０μｍの場合、孔の中心間距離を６０μｍ程度とすることができる。 As the above-mentioned size selection microcavity array, those having fine through holes for capturing CTC formed on a nickel substrate using electroforming technology, or CTC formed on a glass substrate, preferably a quartz substrate using laser technology. What has the fine through-hole for capture | acquisition can be illustrated suitably. A schematic diagram of such a size selection microcavity array (nickel substrate) is shown in FIG. 5, and a photomicrograph of the size selection microcavity array (nickel substrate) having a pore size of 10 μm is shown in FIG. By forming the fine through hole in the nickel substrate using the electroforming technique, it is possible to form the fine through hole in which the hole diameter, the number of holes, and the arrangement for capturing the CTC are controlled. The hole diameter for capturing CTC can be selected from 5 to 20 μm, such as 5 μm, 8 μm, 10 μm, 15 μm, and 20 μm. The number of holes is 1000 or more, preferably 3000 or more, more preferably 5000 or more, Of these, 100 × 100 10,000 holes to 100,000 holes are particularly preferable. Further, the arrangement is preferably provided at equal intervals, and the distance between the centers of the holes can be 20 μm or more. For example, when the hole diameter is 8 μm or 10 μm, the distance between the centers of the holes can be about 60 μm. .
上記密封性シールとしては、サイズ選択マイクロキャビティアレイと上部部材や下部部材との間、あるいはサイズ選択マイクロキャビティアレイ同士を密封しうるものであれば特に制限されず、ゴム製やプラスチック製の市販のシール部材を用いることができる。 The sealing seal is not particularly limited as long as it can seal between the size selection microcavity array and the upper member or the lower member, or between the size selection microcavity arrays, and is commercially available made of rubber or plastic. A seal member can be used.
上記上部部材としては、試料供給口及び試料排出口が形成され、下面に試料供給口と試料排出口を連通するマイクロ流路形成用の溝が設けられたプラスチック製平板からなる上部基板と、前記上部基板と協働してマイクロ流路を形成するとともに、マイクロ流路の一部に相当する位置にサイズ選択マイクロキャビティアレイ用開口窓が設けられたプラスチック製上部平板とから構成されている部材を、製作や組立ての容易さから好適に挙げることができる。 As the upper member, a sample supply port and a sample discharge port are formed, and an upper substrate made of a plastic flat plate provided with a groove for forming a microchannel that connects the sample supply port and the sample discharge port on the lower surface; A member composed of a plastic upper plate provided with a size selection microcavity array opening window at a position corresponding to a part of the microchannel while forming a microchannel in cooperation with the upper substrate. From the viewpoint of ease of production and assembly, it can be preferably mentioned.
前記上部基板としては、表面（上面又は側面）に試料供給口及び試料送出口が形成され、下面に試料供給口と試料送出口を連通するマイクロ流路形成用の溝が設けられたプラスチック製の平板を例示することができ、上面は多少曲面となっていてもよく、また、下面は前記上部平板の上面と液密に積層しうる限り多少曲面となっていてもよいが、上下面とも水平面からなる平板が加工の簡便さの点で好ましい。マイクロ流路形成用の溝は、細胞の付着防止の点から、曲面状の内面となる溝、例えば半円筒状（断面半円弧状）の内面溝や断面が矩形の角形溝を好適に例示することができる。上記試料供給口に試料を供給するため、試料供給ポンプ等の試料供給手段が試料供給ラインを介して接続されている。さらに試料供給ラインのうち試料供給ポンプの上流には、試料供給バルブ及び試料貯留槽が上流側から下流側に向って順次接続しておくこともできる。また、上記試料送出口には、試料送出ポンプ等の試料送出手段が試料送出ラインを介して接続しておいてもよく、単に試料送出バルブを取付けておくこともできる。上記試料供給ポンプや試料送出ポンプとしては、チューブポンプもしくはプランジャポンプ等の微小流量を制御可能なマイクロポンプを具体的に例示することができる。 As the upper substrate, a sample supply port and a sample delivery port are formed on the front surface (upper surface or side surface), and a plastic channel provided with a groove for forming a microchannel that communicates the sample supply port and the sample delivery port on the lower surface. A flat plate can be exemplified, and the upper surface may be somewhat curved, and the lower surface may be somewhat curved as long as it can be liquid-tightly laminated with the upper surface of the upper flat plate, but both the upper and lower surfaces are horizontal surfaces. The flat plate which consists of is preferable at the point of the simplicity of a process. From the viewpoint of preventing cell adhesion, the micro-channel forming groove is preferably exemplified by a groove that is a curved inner surface, for example, a semi-cylindrical (semi-circular arc) inner groove or a rectangular groove having a rectangular cross section. be able to. In order to supply a sample to the sample supply port, sample supply means such as a sample supply pump is connected via a sample supply line. Furthermore, a sample supply valve and a sample storage tank can be sequentially connected from the upstream side to the downstream side upstream of the sample supply pump in the sample supply line. Moreover, sample delivery means such as a sample delivery pump may be connected to the sample delivery outlet via a sample delivery line, or a sample delivery valve can be simply attached. Specific examples of the sample supply pump and the sample delivery pump include a micro pump capable of controlling a minute flow rate, such as a tube pump or a plunger pump.
前記上部平板としては、上記の上部基板と協働してマイクロ流路を形成するとともに、マイクロ流路の一部に相当する位置にサイズ選択マイクロキャビティアレイ用開口窓が設けられたプラスチック製上部平板を例示することができ、上部基板と協働してマイクロ流路を形成するため、上部基板に設けられたマイクロ流路形成用の溝に相当する上面位置に、マイクロ流路形成用の溝、例えば半円筒状（断面半円弧状）の内面溝を設けることもできるが、上面を平面としてマイクロ流路を形成することもできる。また、サイズ選択マイクロキャビティアレイ用開口窓は、サイズ選択マイクロキャビティアレイの全面が露出する大きさのものが好ましく、窓の形状は円形、正方形等の矩形、三角形、楕円、角丸四角形などを具体的に例示することができる。 As the upper flat plate, a plastic upper plate in which a microchannel is formed in cooperation with the upper substrate and a size selection microcavity array opening window is provided at a position corresponding to a part of the microchannel. In order to form the microchannel in cooperation with the upper substrate, the microchannel forming groove is formed on the upper surface corresponding to the microchannel forming groove provided on the upper substrate, For example, an inner surface groove having a semi-cylindrical shape (a semicircular cross section) can be provided, but a microchannel can also be formed with the upper surface as a plane. In addition, the size selection microcavity array opening window is preferably a size that exposes the entire surface of the size selection microcavity array, and the shape of the window may be a circle, a rectangle such as a square, a triangle, an ellipse, a rounded rectangle, etc. Can be exemplified.
上記下部部材としては、サイズ選択マイクロキャビティアレイの下方に相当する位置に吸引用開口窓が設けられたプラスチック製下部平板と、表面に吸引口が形成され、上面に前記下部平板と協働して前記吸引用開口窓と吸引口を連通する吸引流路形成用の溝が設けられたプラスチック製平板からなる下部基板とから構成されている部材を、製作や組立ての容易さから好適に挙げることができる。 The lower member includes a plastic lower flat plate provided with a suction opening window at a position corresponding to a lower portion of the size selection microcavity array, a suction port formed on the surface, and an upper surface in cooperation with the lower flat plate. A member composed of the suction opening window and a lower substrate made of a plastic flat plate provided with a groove for forming a suction flow path communicating with the suction port is preferably mentioned from the viewpoint of ease of production and assembly. it can.
前記プラスチック製下部平板としては、サイズ選択マイクロキャビティアレイの下方に相当する位置に吸引用開口窓が設けられ、前記下部基板と協働して吸引流路を形成しうる平板であれば特に制限されず、吸引用開口窓の形状も特に制限されないが、通常円形として構成される。この下部平板は、減圧吸引条件下に、前記サイズ選択マイクロキャビティアレイと下部基板との隙間を気密に維持しうる平板形状に構成されている。 The plastic lower flat plate is not particularly limited as long as it is provided with a suction opening window at a position corresponding to the lower side of the size selection microcavity array and can form a suction flow path in cooperation with the lower substrate. The shape of the suction opening window is not particularly limited, but is usually configured as a circle. The lower flat plate is configured in a flat plate shape that can keep the gap between the size selection microcavity array and the lower substrate airtight under reduced pressure suction conditions.
前記下部基板としては、表面に吸引口が形成され、上面に前記下部平板と協働して前記吸引用開口窓と吸引口を連通する吸引流路形成用の溝が設けられたプラスチック製平板からなるものであれば特に制限されず、吸引口は減圧吸引手段に連結され、サイズ選択マイクロキャビティアレイ上の試料液は、微細貫通孔、吸引用開口窓、吸引流路、吸引口を順次経由して排出される。吸引口にはペリスタルティックポンプに接続しておくことが好ましい。この吸引時には、前記上部基板の試料送出口の下流の試料送出バルブを閉塞しておくこともできる。また、下部基板の片側に、下から順次下部平板、サイズ選択マイクロキャビティアレイ、上部平板、上部基板を積層したときと同じ高さとなるような段差部を設けておくことが好ましい。このような段差部を設けることにより、減圧吸引時に下部基板上に下部平板、サイズ選択マイクロキャビティアレイ、上部平板、上部基板を安定に積層載置することができ、吸引圧によりこれら平板を互いにボルト等により固着することなく、しっかりと固定することができ、逆に、減圧解放時には簡便にこれら平板を互いにとり外すことができる。 As the lower substrate, a suction port is formed on the surface, and a plastic flat plate provided with a suction channel forming groove on the upper surface in communication with the suction opening window and the suction port in cooperation with the lower flat plate. The suction port is connected to the vacuum suction means, and the sample liquid on the size selection microcavity array sequentially passes through the fine through hole, the suction opening window, the suction flow path, and the suction port. Discharged. The suction port is preferably connected to a peristaltic pump. At the time of this suction, the sample delivery valve downstream of the sample delivery port of the upper substrate can be closed. Further, it is preferable to provide a stepped portion on one side of the lower substrate so as to have the same height as when the lower flat plate, the size selection microcavity array, the upper flat plate, and the upper substrate are laminated sequentially from the bottom. By providing such a stepped portion, the lower plate, the size selection microcavity array, the upper plate, and the upper substrate can be stably stacked on the lower substrate during vacuum suction, and these plates are bolted to each other by suction pressure. The flat plates can be firmly fixed without being fixed by each other, and conversely, these plates can be easily detached from each other when the pressure is released.
前記上部基板、上部平板、下部平板、下部基板の材質としては、硬質のプラスチックが好ましく、これらは同じ材質でも異なる材質でも特に制限されず、ＰＤＭＳ（poly-dimethylsiloxisane）、ＰＭＭＡ（Poly(methyl methacrylate)）、ＰＣ、硬質ポリエチレン製等のプラスチック平板を例示することができるが、密封性シールとの密着性の点でＰＤＭＳが好ましい。 The material of the upper substrate, the upper plate, the lower plate, and the lower substrate is preferably a hard plastic, and these are not particularly limited by the same material or different materials. PDMS (poly-dimethylsiloxisane), PMMA (Poly (methyl methacrylate) ), A plastic flat plate made of PC, hard polyethylene or the like can be exemplified, but PDMS is preferable in terms of adhesion with a hermetic seal.
前記マイクロ流路の内面、好ましくはマイクロ流路の内面に加えてサイズ選択マイクロキャビティアレイの上面を非イオン性界面活性剤であらかじめ処理しておくことが、マイクロ流路内面への細胞の付着を防除する上で好ましい。この非イオン性界面活性剤処理に先だって、表面プラズマ処理をすることがより好ましい。陰イオン界面活性剤を用いて表面を被覆することにより、表面電位が負に帯電している微生物の吸着を防除することはできるが、細胞は十分に防除することができず、非イオン性界面活性剤で処理する必要がある。かかる非イオン性界面活性剤としては、ブロックポリマー型エーテル、ポリオキシエチレン硬化ヒマシ油、ショ糖脂肪酸エステル（シュガーエステル）、ポリオキシエチレンソルビタン脂肪酸エステル、ショ糖脂肪酸エステルを例示することができるが、ブロックポリマー型エーテル、特にポリオキシエチレンポリオキシプロピレンブロックコポリマー型非イオン性界面活性剤が好ましい。上記ブロックポリマー型エーテルとしては、例えば、ポリオキシエチレン（１９６）ポリオキシプロピレン（６７）グリコール（プルロニックＦ１２７）、ポリオキシエチレン（１６０）ポリオキシプロピレン（３０）グリコール（プルロニックＦ６８）、ポリオキシエチレン（４２）ポリオキシプロピレン（６７）グリコール（プルロニックＰ１２３）、ポリオキシエチレンオキシプロピレンセチルエーテル（２０Ｅ．Ｏ ４Ｐ．Ｏ）を挙げることができる。ポリオキシエチレン硬化ヒマシ油としては、例えば、水素添加ヒマシ油ポリオキシエチレンエーテル、ポリオキシエチレン水素添加ヒマシ油等を挙げることができる。ポリオキシエチレンソルビタン脂肪酸エステルとしては、例えば、ポリソルベート４０（ツイーン４０）、ポリソルベート６０（ツイーン６０）、ポリソルベート６５、ポリソルベート８０（ツイーン８０）、モノラウリン酸ポリオキシエチレンソルビタン（２０Ｅ．Ｏ）を挙げることができる。例えば、プルロニックＦ１２７などのポリオキシエチレンポリオキシプロピレンブロックコポリマー型非イオン性界面活性剤を用いる場合、その濃度は０．５〜１０％、好ましくは１〜５％程度である。 In addition to the inner surface of the microchannel, preferably the upper surface of the size-selective microcavity array in addition to the inner surface of the microchannel, the cell adhesion to the inner surface of the microchannel is preliminarily treated with a nonionic surfactant. It is preferable in controlling. It is more preferable to perform a surface plasma treatment prior to the nonionic surfactant treatment. By coating the surface with an anionic surfactant, it is possible to control the adsorption of microorganisms whose surface potential is negatively charged, but the cells cannot be sufficiently controlled, and the nonionic interface It needs to be treated with an activator. Examples of such nonionic surfactants include block polymer type ether, polyoxyethylene hydrogenated castor oil, sucrose fatty acid ester (sugar ester), polyoxyethylene sorbitan fatty acid ester, and sucrose fatty acid ester. Block polymer type ethers, particularly polyoxyethylene polyoxypropylene block copolymer type nonionic surfactants are preferred. Examples of the block polymer type ether include polyoxyethylene (196) polyoxypropylene (67) glycol (Pluronic F127), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68), polyoxyethylene ( 42) Polyoxypropylene (67) glycol (Pluronic P123) and polyoxyethyleneoxypropylene cetyl ether (20E.O 4P.O) can be mentioned. Examples of the polyoxyethylene hydrogenated castor oil include hydrogenated castor oil polyoxyethylene ether, polyoxyethylene hydrogenated castor oil, and the like. Examples of the polyoxyethylene sorbitan fatty acid ester include polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 65, polysorbate 80 (Tween 80), and polyoxyethylene sorbitan monolaurate (20EO). it can. For example, when a polyoxyethylene polyoxypropylene block copolymer type nonionic surfactant such as Pluronic F127 is used, its concentration is about 0.5 to 10%, preferably about 1 to 5%.
また、本発明のＣＴＣの検出方法としては、上記本発明のマイクロ流体デバイスを用いてＣＴＣの検出を行う方法であれば特に制限されず、血液を前処理することなくそのまま試料として使用することが好ましい。また、血液導入速度を２００〜１０００μＬ／ｍｉｎとすることがＣＴＣ回収率を高める点で好ましい。サイズ選択マイクロキャビティアレイ上に捕捉されたＣＴＣに対する検出は、ＦＩＴＣやＰＥで標識された抗ＥｐＣＡＭ抗体等のＣＴＣ特異的な抗体を用いて蛍光染色して蛍光顕微鏡等により観察することにより行うことができる。また、光学顕微鏡を用いて明視野観察を行う場合には、パパニコロウ染色やギムザ染色を行うことで細胞内の核、細胞質等の形態的特徴を指標としてＣＴＣ検出を行うことが出来る。特に、捕捉したＣＴＣを長期的に観察する際には、光学顕微鏡を用いて明視野観察することが望ましい。 Further, the CTC detection method of the present invention is not particularly limited as long as it is a method of detecting CTC using the microfluidic device of the present invention, and it can be used as a sample without pretreatment of blood. preferable. Further, the blood introduction rate is preferably 200 to 1000 μL / min from the viewpoint of increasing the CTC recovery rate. Detection of CTC captured on the size-selective microcavity array can be performed by fluorescent staining using a CTC-specific antibody such as anti-EpCAM antibody labeled with FITC or PE and observing with a fluorescence microscope or the like. it can. In addition, when performing bright field observation using an optical microscope, CTC detection can be performed using morphological features such as intracellular nuclei and cytoplasm as an index by performing Papanicolaou staining or Giemsa staining. In particular, when observing the captured CTC for a long period of time, it is desirable to perform bright field observation using an optical microscope.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
（ニッケル基板）
［試薬及び器具］
培地としてＲＰＭＩ−１６４０，ＤＭＥＭ，ＭＥＭをＳＩＧＭＡより購入した。これに、５６℃で３０分間加熱処理することで非動化した１０％ウシ胎仔血清（Fetal Bovine Serum）、１％ペニシリン−ストレプトマイシンを抗生物質として添加し培養に用いた。ＭＥＭには１０％ウシ胎仔血清、０．１ｍＭ非必須アミノ酸、２ｍＭ Ｌ−グルタミン、１ｍＭピルビン酸ナトリウム、０．０１ｍｇ／ｍＬウシのインシュリン、１．５ｍｇ／ｍＬ重炭酸｛じゅうたんさん｝ナトリウムを添加し培養に用いた。細胞の染色に用いたＦＩＴＣ標識−抗ＣＤ４５抗体及びＰＥ標識−抗ＥｐＣＡＭ抗体はＢＤ bioscienceより購入した。Ｈｏｅｃｈｓｔ ３３３４２はMolecular probeより購入したものを利用した。実験には蒸留水又は超純水を用い、その他の試薬類は、すべて市販特級品又はそれに準じたものを使用した。細胞の懸濁，洗浄にはＰＢＳ（２ｍＭ ＥＤＴＡ，０．５％ ＢＳＡ）を用いた。
(Nickel substrate)
[Reagents and instruments]
RPMI-1640, DMEM, and MEM were purchased from SIGMA as the medium. To this, 10% fetal bovine serum (Fetal Bovine Serum), which was immobilized by heat treatment at 56 ° C. for 30 minutes, was added as an antibiotic and used for culture. MEM was supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acid, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.01 mg / mL bovine insulin, 1.5 mg / mL sodium bicarbonate. Used for culture. FITC-labeled anti-CD45 antibody and PE-labeled anti-EpCAM antibody used for cell staining were purchased from BD bioscience. Hoechst 33342 was purchased from Molecular probe. Distilled water or ultrapure water was used for the experiment, and all other reagents were commercially available special grades or equivalents. PBS (2 mM EDTA, 0.5% BSA) was used for cell suspension and washing.
［サイズ選択マイクロキャビティアレイの作製］
マイクロキャビティアレイを加工するための基板としてニッケル基板（１８×１８ｍｍ，厚さ３５μｍ）を使用した。マイクロキャビティアレイを介して血液を直接送液し、血中のＣＴＣのみを選択的にマイクロキャビティ上に捕捉するため、血球成分のサイズとフィルトレーションに関する文献等の値から、マイクロキャビティアレイの孔径サイズは８〜１１μｍとした。ニッケル基板の加工に関しては、オプトニクス精密株式会社に依頼し、電鋳技術を用いてニッケル基板に貫通孔を加工した。孔の中心間距離を６０μｍとし、合計で１００×１００の１０，０００孔をアレイ状に配したものを設計した。図５にはサイズ選択マイクロキャビティアレイの模式図を、図６には孔径サイズが１０μｍのサイズ選択マイクロキャビティアレイの顕微鏡写真（×１０００）をそれぞれ示す。さらに、ＰＤＭＳマイクロ流路によりマイクロキャビティアレイ基板を挟み込む形式でのＣＴＣ回収装置を設計した。シリコンチューブ（０．５×１ｍｍ）からなるインレット（試料供給口），アウトレット（試料排出口）を配した上部流路と、吸引ラインを配した下部流路を作製した。これらのＰＤＭＳマイクロ流路をマイクロキャビティアレイに対して、ｉｎ ｓｉｔｕ ＰＣＲ ｓｌｉｄｅ ｓｅａｌ（TAKARABIO）を介して貼り付けた。これらのＰＤＭＳからなる構造体は、その型をＣＡＤ−ＣＡＭＭを用いてＰＭＭＡを切削することにより鋳型を作製し、この型にＰＤＭＳを注入し、加熱硬化させたものを使用した。ＣＴＣ回収装置は、シリンジを加工したリザーバーにインレットを接続し、下部流路をぺリスタルティックポンプに接続し、コンピューター制御式電動ステージを配した正立顕微鏡下に設置して使用した。図２には、サイズ選択マイクロキャビティアレイを配したＣＴＣを回収するマイクロ流体デバイスの概要が示され、図２ａはＣＴＣ回収マイクロ流体デバイスの模式図が、図２ｂには血液導入の様子が、それぞれ示されている。
[Production of size-selective microcavity array]
A nickel substrate (18 × 18 mm, thickness 35 μm) was used as a substrate for processing the microcavity array. Since the blood is directly fed through the microcavity array and only the CTC in the blood is selectively captured on the microcavity, the pore size of the microcavity array can be determined from literature values regarding the size and filtration of blood cell components. The size was 8-11 μm. Regarding the processing of the nickel substrate, we requested Optonix Precision Co., Ltd. to process through holes in the nickel substrate using electroforming technology. The distance between the centers of the holes was set to 60 μm, and a total of 100 × 100 10,000 holes were arranged in an array. FIG. 5 shows a schematic diagram of a size selection microcavity array, and FIG. 6 shows a micrograph (× 1000) of the size selection microcavity array having a pore size of 10 μm. Furthermore, a CTC recovery device was designed in such a manner that the microcavity array substrate is sandwiched between PDMS microchannels. An upper flow path having an inlet (sample supply port) and an outlet (sample discharge port) made of a silicon tube (0.5 × 1 mm) and a lower flow path having a suction line were prepared. These PDMS microchannels were affixed to the microcavity array via in situ PCR slide seal (TAKARABIO). As the structure composed of these PDMS, a mold was prepared by cutting PMMA using CAD-CAMM, and PDMS was injected into this mold and heat cured. The CTC recovery device was used by connecting an inlet to a reservoir in which a syringe was processed, connecting a lower flow path to a peristaltic pump, and placing it under an upright microscope equipped with a computer-controlled electric stage. FIG. 2 shows an outline of a microfluidic device that collects CTCs with a size-selective microcavity array, FIG. 2a shows a schematic diagram of the CTC collecting microfluidic device, and FIG. 2b shows a state of blood introduction. It is shown.
本発明のマイクロ流体デバイスを用いたＣＴＣ回収の手順は以下の通りである。１）血液を（適宜希釈し）マイクロキャビティアレイを配したＣＴＣ回収装置に導入する。２）血球細胞はマイクロキャビティアレイを通り抜けさせ、サイズが大きな腫瘍細胞のみをマイクロキャビティアレイ上に捕捉する。３）染色液を同じようにマイクロキャビティアレイを介して送液し、マイクロキャビティアレイ上の腫瘍細胞又は白血球を蛍光免疫染色する。４）マイクロキャビティアレイ上の細胞の蛍光観察像を取得して、免疫表現型から腫瘍細胞を同定し、その数をカウントする。これらの操作を連続的に行うためにマイクロキャビティアレイに接続するマイクロ流路を作製した。上部流路のインレットは、シリンジを加工したリザーバーに接続し、このリザーバーに順次、サンプルや試薬を投入していくことで、細胞の捕捉や染色・洗浄を連続的に容易に行えるようにした。 The procedure for CTC recovery using the microfluidic device of the present invention is as follows. 1) Introduce blood (appropriately diluted) into a CTC recovery device with a microcavity array. 2) Blood cells pass through the microcavity array and only large tumor cells are captured on the microcavity array. 3) The staining solution is similarly fed through the microcavity array, and the tumor cells or leukocytes on the microcavity array are fluorescently immunostained. 4) Obtain fluorescence observation images of cells on the microcavity array, identify tumor cells from the immunophenotype, and count the number. In order to perform these operations continuously, a microchannel connected to the microcavity array was prepared. The inlet of the upper channel was connected to a reservoir where a syringe was processed, and samples and reagents were sequentially introduced into this reservoir, so that cells could be easily captured, stained and washed continuously.
［動物細胞の培養及び蛍光染色］
ＮＣＩ−Ｈ３５８細胞、ＡＧＳ細胞、ＳＷ６２０細胞はＲＰＭＩ−１６４０培地を用いて３７℃，５％ＣＯ２濃度下で静置培養した。培養３〜４日後、培地をピペッティングにより除去し、ＰＢＳ（ｐＨ７．２）で洗浄を行った後、０．２％トリプシン／０．０２％ＥＤＴＡを添加し、ＣＯ２インキュベーター内で３〜５分間インキュベートした。検鏡し細胞がディッシュから剥離していることを確認した後に、血清入り培地を加えトリプシン作用を止めてピペッティングし、細胞を回収した。回収した細胞は遠心後（１２００ｒｐｍ，３ｍｉｎ）、上清を除去して新鮮培地に再懸濁し、培地をピペッティングにより全て回収した。トリパンブルーを用いた色素排除法により生細胞数を計数し、１×１０５ｃｅｌｌｓ／ｍＬに調製後、新しいディッシュに播種した。ＭＣＦ−７細胞はＭＥＭ培地を用いて３７℃，５％ＣＯ２濃度下で静置培養した。培養後の操作は他の細胞と同様である。
[Animal cell culture and fluorescent staining]
NCI-H358 cells, AGS cells, and SW620 cells were statically cultured using RPMI-1640 medium at 37 ° C. and 5% CO 2 concentration. After 3 to 4 days of culture, the medium was removed by pipetting, washed with PBS (pH 7.2), 0.2% trypsin / 0.02% EDTA was added, and 3-5 in a CO 2 incubator. Incubated for minutes. After microscopic examination and confirming that the cells were detached from the dish, serum-containing medium was added to stop trypsin action, and pipetting was performed to collect the cells. The collected cells were centrifuged (1200 rpm, 3 min), the supernatant was removed and resuspended in a fresh medium, and all the medium was collected by pipetting. The number of viable cells was counted by a dye exclusion method using trypan blue, adjusted to 1 × 10 5 cells / mL, and then seeded on a new dish. MCF-7 cells were statically cultured in MEM medium at 37 ° C. and 5% CO 2 concentration. The operation after culturing is the same as other cells.
また、細胞捕捉実験に用いる細胞として、１．５ｍＬチューブに１ｍＬの培地を回収した。回収した細胞はリン酸緩衝液（ＰＢＳ，ｐＨ７．４）で遠心洗浄（２００ｇ，５ｍｉｎ）を３回繰り返した後にＰＢＳに再懸濁した。その後、５μＭ ＣｅｌｌＴｒａｃｋｅｒ Ｒｅｄ ＣＭＴＰＸ（Molecular Probes）を添加し、３０分間インキュベーションすることにより細胞を蛍光染色した。染色後、上記と同様の方法で遠心洗浄を行い、過剰な蛍光色素を取り除き、細胞捕捉実験に用いる蛍光染色細胞を調製した。また、モデル腫瘍細胞の回収効率評価実験には、ヘパリン含有真空採血管に採血した健常者血液に１０〜１００個の腫瘍細胞を混合させたサンプルを用いた。 Moreover, 1 mL culture medium was collect | recovered to the 1.5 mL tube as a cell used for a cell capture | acquisition experiment. The collected cells were resuspended in PBS after repeating centrifugal washing (200 g, 5 min) three times with a phosphate buffer (PBS, pH 7.4). Thereafter, 5 μM CellTracker Red CMTPX (Molecular Probes) was added, and the cells were fluorescently stained by incubation for 30 minutes. After staining, centrifugal washing was performed in the same manner as described above to remove excess fluorescent dye, and fluorescent stained cells used for cell capture experiments were prepared. In addition, in the experiment for evaluating the recovery efficiency of model tumor cells, a sample in which 10 to 100 tumor cells were mixed with blood of a healthy person collected in a heparin-containing vacuum blood collection tube was used.
［サイズ選択マイクロキャビティアレイを用いた細胞捕捉・循環腫瘍細胞検出操作］
リザーバーにＰＢＳ（２ｍＭ ＥＤＴＡ，０．５％ ＢＳＡ）１ｍＬを導入し、マイクロキャビティアレイ上をＰＢＳで満たしてから、ぺリスタルティックポンプを介して２００μＬ／ｍｉｎで送液を開始した。約５分後、リザーバーに１ｍＬの血液サンプルを導入した。約５分後、血球成分を流しきるために、リザーバーに２ｍＬのＰＢＳ（２ｍＭ ＥＤＴＡ，０．５％ ＢＳＡ）を導入した。約１０分後、ポンプ流速を２０μＬ／ｍｉｎに変更し、リザーバーに６００μＬの細胞染色液（ＰＥ標識抗ＥｐＣＡＭ抗体、ＦＩＴＣ標識抗ＣＤ４５抗体、Ｈｏｅｃｈｓｔ ３３３４２（０．５μｇ／ｍＬ））を導入した。マイクロキャビティアレイ上に捕捉された細胞に対して３０分間染色を行った後、リザーバーに１ｍＬのＰＢＳ（２ｍＭ ＥＤＴＡ，０．５％ ＢＳＡ）を導入して細胞の洗浄を行った。１５分間以上の洗浄を行った後に、蛍光顕微鏡観察を行った。
[Cell capture and circulating tumor cell detection using size-selective microcavity array]
After introducing 1 mL of PBS (2 mM EDTA, 0.5% BSA) into the reservoir and filling the microcavity array with PBS, liquid feeding was started at 200 μL / min via a peristaltic pump. After about 5 minutes, a 1 mL blood sample was introduced into the reservoir. About 5 minutes later, 2 mL of PBS (2 mM EDTA, 0.5% BSA) was introduced into the reservoir in order to completely drain blood cell components. After about 10 minutes, the pump flow rate was changed to 20 μL / min, and 600 μL of cell staining solution (PE-labeled anti-EpCAM antibody, FITC-labeled anti-CD45 antibody, Hoechst 33342 (0.5 μg / mL)) was introduced into the reservoir. After the cells captured on the microcavity array were stained for 30 minutes, 1 mL of PBS (2 mM EDTA, 0.5% BSA) was introduced into the reservoir to wash the cells. After washing for 15 minutes or more, fluorescence microscope observation was performed.
マイクロキャビティアレイ上に捕捉・回収された全ての細胞を観察するためにアレイ全体の撮像を行った。これにはコンピューター制御式電動ステージ、冷却デジタルカメラ（ＤＰ７０；Olympus）を装備した蛍光顕微鏡（ＢＸ６１; Olympus）を用い、ＤＡＰＩ、ＦＩＴＣ、ＰＥ由来の蛍光を観察するためにＷＵ、ＮＩＢＡ及びＣｙ３フィルターを用いてそれぞれ画像を取得した。画像取得及び解析ソフトウェアにはＬｕｍｉｎａ Ｖｉｓｉｏｎ（Mitani Corporation）を用いた。 The entire array was imaged to observe all cells captured and collected on the microcavity array. This uses a computer-controlled electric stage and a fluorescence microscope (BX61; Olympus) equipped with a cooled digital camera (DP70; Olympus), and uses WU, NIBA, and Cy3 filters to observe fluorescence derived from DAPI, FITC, and PE. Images were acquired using each. Lumina Vision (Mitani Corporation) was used as image acquisition and analysis software.
［サイズ選択マイクロキャビティアレイを用いた循環腫瘍細胞回収の検討結果］
はじめに、作製したサイズ選択マイクロキャビティアレイを用いて、１ｍＬの血液を目詰まりなく送液することが出来るかを検討した。流速は、２００μＬ／ｍｉｎとした。これまで用いてきた孔径２μｍのマイクロキャビティアレイを用いて、血液を送液した場合、すぐさま目詰まりが起こった。一方、サイズ選択マイクロキャビティアレイを用いた場合では、スムーズな送液を行うことができ、血液１ｍＬを送液した後、バッファーを送液することで、マイクロキャビティアレイ上を綺麗に洗い流すことができた。
[Results of investigating circulating tumor cells using size-selective microcavity array]
First, it was examined whether 1 mL of blood could be sent without clogging using the produced size selection microcavity array. The flow rate was 200 μL / min. When blood was fed using a microcavity array having a pore size of 2 μm that had been used so far, clogging occurred immediately. On the other hand, when using a size-selective microcavity array, smooth liquid feeding can be performed, and after feeding 1 mL of blood, the buffer can be sent to cleanly wash the microcavity array. It was.
次に、孔径を大きくしたサイズ選択マイクロキャビティアレイを用いて、腫瘍細胞を通り抜けさせずにマイクロキャビティアレイ上に捉えられるかを検討した。ここでは、ＮＣＩ−Ｈ３５８細胞をモデル細胞として用い、細胞１００個をＰＢＳ１ｍＬに懸濁したものをサンプルとした。ポンプ流速を変化させた場合に、どれだけ細胞をマイクロキャビティアレイ上に回収できるかを検討した。血液導入速度とＣＴＣ回収率の関係性を図７に示す。流速は２００〜２０００μＬ／ｍｉｎまで変化させたが、１０００μＬ／ｍｉｎ以下の流速であれば、９０％近い回収効率を得られることが分かった。ポンプ流速をこれ以上に上げると、マイクロキャビティアレイ上に気泡が発生するなどの問題が起こることがあり、回収率は減少した。また、試行間誤差が大きくなるという問題もあることが分かった。よって、以後の実験では、１０００μＬ／ｍｉｎ以下の流速で血液導入と腫瘍細胞回収を行うこととした。 Next, using a size-selective microcavity array with a large pore size, it was examined whether it could be captured on the microcavity array without passing through tumor cells. Here, NCI-H358 cells were used as model cells, and 100 cells suspended in 1 mL of PBS were used as samples. We examined how much cells could be collected on the microcavity array when the pump flow rate was varied. FIG. 7 shows the relationship between the blood introduction rate and the CTC recovery rate. Although the flow rate was changed from 200 to 2000 μL / min, it was found that a recovery efficiency close to 90% could be obtained at a flow rate of 1000 μL / min or less. When the pump flow rate was increased further, problems such as generation of bubbles on the microcavity array sometimes occurred, and the recovery rate decreased. Moreover, it turned out that there also exists a problem that the error between trials becomes large. Therefore, in subsequent experiments, blood introduction and tumor cell recovery were performed at a flow rate of 1000 μL / min or less.
これまでに報告されているマイクロ流体デバイス型のＣＴＣ検出装置では、血液の送液速度は１０〜３００μＬ／ｍｉｎとされている（Nagrath et al. 2007; Tan et al. 2009）。よって、サイズ選択マイクロキャビティアレイが可能とするサンプル導入速度はこれらに比較して圧倒的に早いということが言える。血液サンプルの状態は採血時から時間が経つほど悪化していくことが考えられ、ＣＴＣの回収及び検出に与える影響も懸念される。よって、迅速な処理は検出精度を上げる目的でも重要な役割を果たすと考えられ、これは本装置の大きな利点と考えられる。 In the microfluidic device type CTC detection apparatus reported so far, the blood feeding speed is 10 to 300 μL / min (Nagrath et al. 2007; Tan et al. 2009). Therefore, it can be said that the sample introduction speed that the size selection microcavity array enables is overwhelmingly faster than these. It is conceivable that the state of the blood sample deteriorates over time from the time of blood collection, and there is a concern about the effect on the recovery and detection of CTC. Therefore, rapid processing is considered to play an important role for the purpose of increasing detection accuracy, which is considered to be a great advantage of this apparatus.
［モデル腫瘍細胞の回収効率の評価］
サイズ選択マイクロキャビティアレイを用いたＣＴＣの回収効率を評価するために、健常者血液１ｍＬに１０，２５，５０，１００個の各ＮＣＩ−Ｈ３５８細胞を添加したサンプルを調製し、各サンプルにおける回収細胞数を評価した。図８はサイズ選択マイクロキャビティアレイ上に捉えられたＮＣＩ−Ｈ３５８細胞及び白血球細胞の蛍光顕微鏡写真である。図８に示されるように、回収された全ての細胞が核を有している。腫瘍細胞はＥｐＣＡＭを発現しているが、ＣＤ４５を発現していない。正常な血球細胞はＣＤ４５を発現しているが、ＥｐＣＡＭを発現していない。このように、染色液を導入することにより、細胞の染色が可能であり、その表現型の違いと細胞のサイズの違いから腫瘍細胞と正常白血球は容易に判別できた。処理後にサイズ選択マイクロキャビティアレイ上に残存する正常白血球数はおよそ１０００個ほどであったが、ＣＴＣのカウントに際しては妨げにならない量であった。また、ＣＴＣに関しては微細孔に捕捉されているため、アレイ領域だけのスキャニングで全回収細胞数をカウントできた。このモデルＣＴＣの検出効率評価に関する結果を図９に示す。この結果から１０〜１００個のＣＴＣ存在数において、９６％以上の回収率で安定的に細胞を回収し検出できることが示された。
[Evaluation of recovery efficiency of model tumor cells]
In order to evaluate the recovery efficiency of CTC using a size-selective microcavity array, samples were prepared by adding 10, 25, 50, and 100 NCI-H358 cells to 1 mL of healthy human blood, and the recovered cells in each sample Number was evaluated. FIG. 8 is a fluorescence micrograph of NCI-H358 cells and white blood cells captured on a size-selected microcavity array. As shown in FIG. 8, all the collected cells have nuclei. Tumor cells express EpCAM but not CD45. Normal blood cells express CD45 but do not express EpCAM. Thus, by introducing a staining solution, cells can be stained, and tumor cells and normal leukocytes were easily discriminated from the difference in phenotype and cell size. Although the number of normal white blood cells remaining on the size-selected microcavity array after the treatment was about 1000, it was an amount that would not interfere with the CTC count. Moreover, since CTC was trapped in the micropores, the total number of recovered cells could be counted by scanning only the array region. The results regarding the detection efficiency evaluation of this model CTC are shown in FIG. From this result, it was shown that cells can be recovered and detected stably at a recovery rate of 96% or more when the number of CTCs is 10 to 100.
［細胞サイズと循環腫瘍細胞回収の関係性の評価］
ヒトの血液１ｍＬ中には約５×１０９個の赤血球、約７×１０６個の白血球、約３×１０８個の血小板が存在している。それぞれのおおよその大きさは、赤血球が７〜８μｍ、血小板が２〜３μｍ、白血球は細胞によってサイズに幅があり、リンパ球などでは６〜１５μｍ、大きな単球では２０μｍになる。一方、上皮系の腫瘍細胞は多くが１０μｍ以上の直径を有している。また、血液細胞は毛細血管を巡り、体内を循環するという性質から上皮系の細胞などと比較して変形能が高いと考えられている。よって、数種の細胞株を用いた検出実験を行い、回収率を評価した後、これらのモデル腫瘍細胞のサイズを測定し、細胞サイズが回収効率に与える影響について考察した。
[Evaluation of relationship between cell size and circulating tumor cell recovery]
In 1 mL of human blood, there are about 5 × 10 9 red blood cells, about 7 × 10 6 white blood cells, and about 3 × 10 8 platelets. The approximate size of each cell is 7-8 μm for red blood cells, 2-3 μm for platelets, and white blood cells vary in size, ranging from 6-15 μm for lymphocytes and 20 μm for large monocytes. On the other hand, most epithelial tumor cells have a diameter of 10 μm or more. In addition, blood cells are considered to have high deformability compared to epithelial cells and the like due to the nature of circulating in the body through blood capillaries. Therefore, after conducting detection experiments using several cell lines and evaluating the recovery rate, the size of these model tumor cells was measured, and the influence of the cell size on the recovery efficiency was discussed.
ＭＣＦ−７，ＮＣＩ−Ｈ３５８，ＡＧＳ，ＳＷ６２０の各細胞株を用いて行ったＣＴＣ検出実験の結果を図１０に、ＭＣＦ−７，ＮＣＩ−Ｈ３５８，ＡＧＳ，ＳＷ６２０の各細胞のサイズ分布を図１１に示す。１０μｍ径のサイズ選択マイクロキャビティアレイを用いた場合、それぞれの細胞株の平均回収率は、ＭＣＦ−７：９８％，ＮＣＩ−Ｈ３５８：９６％，ＡＧＳ：７４％，ＳＷ６２０：３８％であった。また、用いた細胞のサイズを計測したところ、ヒト細気管支肺胞上皮癌ＮＣＩ−Ｈ３５８の場合、細胞のサイズは９〜３０μｍにまたがり、平均直径は約１８μｍであった。ヒト乳癌細胞ＭＣＦ−７は平均直径が２２μｍと最も大きく、サイズに１５〜４０μｍまでのバリエーションがあった。ＳＷ６２０細胞は、７〜２０μｍの範囲で１１μｍ径前後の細胞が最も多かった。ヒト胃癌ＡＧＳ細胞は、８〜２４μｍの範囲で１５μｍ径前後の細胞が最も多かった。これらの結果を見てみると、細胞サイズが１０μｍ以下までまたがる細胞については、回収率が低下していることが分かった。腫瘍細胞のサイズ分布は約８μｍ以上であることが示されている。このため、サイズ選択マイクロキャビティアレイの孔径を８μｍに変更して、ＳＷ６２０細胞の回収率を行った。その結果、回収率は９１％まで向上した。微細貫通孔の孔径や形状を最適化することによって、様々なタイプの腫瘍細胞を効率的に捕捉できるのではないかと考えられた。 FIG. 10 shows the results of a CTC detection experiment conducted using each cell line of MCF-7, NCI-H358, AGS, and SW620, and FIG. 11 shows the size distribution of each cell of MCF-7, NCI-H358, AGS, and SW620. Shown in When a 10 μm diameter size selection microcavity array was used, the average recovery rate of each cell line was MCF-7: 98%, NCI-H358: 96%, AGS: 74%, SW620: 38%. Moreover, when the size of the used cell was measured, in the case of human bronchioloalveolar carcinoma NCI-H358, the cell size spanned 9-30 μm and the average diameter was about 18 μm. The human breast cancer cell MCF-7 had the largest average diameter of 22 μm, and the size varied from 15 to 40 μm. SW620 cells had the largest number of cells around 11 μm in the range of 7-20 μm. Human gastric cancer AGS cells had the largest number of cells around 15 μm in the range of 8-24 μm. Looking at these results, it was found that the recovery rate of cells having a cell size of 10 μm or less was reduced. It has been shown that the size distribution of tumor cells is about 8 μm or more. For this reason, the pore size of the size selection microcavity array was changed to 8 μm, and the recovery rate of SW620 cells was performed. As a result, the recovery rate improved to 91%. It was thought that various types of tumor cells could be efficiently captured by optimizing the pore diameter and shape of the fine through-holes.
［磁気分離を用いた循環腫瘍細胞回収との比較］
本発明のマイクロ流体デバイスを用いたＣＴＣ回収の有効性を評価するために、比較として一般的に用いられている磁気分離法を利用したＣＴＣ回収を行った。ここではMiltenyi Biotec社のＣＤ３２６（ＥｐＣＡＭ）マイクロビーズと磁気分離カラムを用いた。健常者血液１ｍＬにモデル腫瘍細胞ＮＣＩ−Ｈ３５８を１００個添加したサンプルを調製し、腫瘍細胞の検出を試みた。磁気分離法は広く用いられている方法であるが、その処理には多段階の操作が必要である。この実験においても、はじめに、赤血球を取り除くために溶血操作を行った。溶血操作の後、磁気ビーズへの非特異吸着を抑制するためのブロッキング剤を添加し、続いてＣＤ３２６（ＥｐＣＡＭ）マイクロビーズと反応させた。続いて各蛍光標識抗体を添加して細胞を染色した後、サンプルを磁気分離カラムに移して、洗浄することにより目的外の白血球などを洗い流し、最後にカラムを磁石からはずして、目的細胞を溶出させた。以上のように、磁気分離法はサンプルの移し変えや洗浄操作が非常に多いため、目的のＣＴＣをロスする危険性が非常に多い。結果として、１００個の細胞導入時の磁気分離による回収細胞数は５０個であった。サイズ選択マイクロキャビティアレイを用いた場合の回収細胞数は９６％であるので、磁気分離法と比較して、その回収効率は大幅に上回っていることが示された。磁気分離法における回収率は操作条件によって大きく影響を受けるが、ＥｐＣＡＭを標的とした場合の回収効率は９〜９０％と報告されており、各方法によって大きく異なっている（Lara et al. 2004）。また、標的としてもＥｐＣＡＭ以外にサイトケラチンを標的としてＣＴＣを特異的に分離する手法もある（Deng et al. 2008）。また、ＣＤ４５を標的として血液細胞をサンプルから除いてＣＴＣを濃縮する手法も報告されている（Allard et al. 2004）。しかし、どの抗原を標的としても、もともと大過剰に存在している血球細胞の混入は避けられないため、最終的なＣＴＣカウントに際して、疑陽性の細胞が検出される可能性を否定できない。また、多段階にわたる検出までのプロセスは煩雑であり、実験間誤差を生む要因となると考えられる。よって、操作の簡易性と再現性の高さといった観点からもサイズ選択マイクロキャビティアレイを用いた本発明方法は有用な方法であるといえる。
[Comparison with circulating tumor cell recovery using magnetic separation]
In order to evaluate the effectiveness of CTC recovery using the microfluidic device of the present invention, CTC recovery using a magnetic separation method generally used as a comparison was performed. Here, Miltenyi Biotec CD326 (EpCAM) microbeads and a magnetic separation column were used. A sample was prepared by adding 100 model tumor cells NCI-H358 to 1 mL of healthy human blood, and detection of tumor cells was attempted. The magnetic separation method is a widely used method, but the process requires multistage operation. Also in this experiment, first, hemolysis was performed to remove red blood cells. After the hemolysis operation, a blocking agent for suppressing non-specific adsorption to the magnetic beads was added, followed by reaction with CD326 (EpCAM) microbeads. Subsequently, each fluorescently labeled antibody is added to stain the cells, and then the sample is transferred to a magnetic separation column and washed to wash away undesired leukocytes, and finally the column is removed from the magnet to elute the target cells. I let you. As described above, the magnetic separation method has a great risk of losing the target CTC because the sample transfer and washing operations are very frequent. As a result, the number of recovered cells by magnetic separation at the time of introducing 100 cells was 50. Since the number of recovered cells was 96% when using the size-selective microcavity array, it was shown that the recovery efficiency was significantly higher than that of the magnetic separation method. The recovery rate in the magnetic separation method is greatly influenced by the operating conditions, but the recovery efficiency when EpCAM is targeted is reported to be 9 to 90%, which varies greatly depending on each method (Lara et al. 2004). . In addition to EpCAM, there is also a method for specifically separating CTC using cytokeratin as a target (Deng et al. 2008). In addition, a method for concentrating CTC by removing blood cells from a sample targeting CD45 has been reported (Allard et al. 2004). However, even if any antigen is targeted, it is unavoidable that blood cells that are originally present in a large excess are unavoidable, and therefore, the possibility of false positive cells being detected in the final CTC count cannot be denied. In addition, the process up to multi-stage detection is complicated and is considered to be a factor causing errors between experiments. Therefore, it can be said that the method of the present invention using the size-selective microcavity array is also a useful method from the viewpoint of easy operation and high reproducibility.
（石英基板）
石英基板上にフェムト秒レーザを用いて、孔径８μｍのサイズ選択マイクロキャビティアレイ（孔数１０，０００個）を作製し、腫瘍細胞の捕捉について検討した。ＮＣＩ−Ｈ３５８細胞をモデル細胞として用い、細胞１００個をＰＢＳ１ｍＬに懸濁したものをサンプルとした。ポンプ流速は２００μＬ／ｍｉｎとした。石英基板のサイズ選択マイクロキャビティアレイを用いてアレイ上に補足したＮＣＩ−Ｈ３５８細胞（肺がん細胞）の光学顕微鏡写真を図１２に示す。
(Quartz substrate)
Using a femtosecond laser on a quartz substrate, a size-selected microcavity array (with 10,000 holes) having a pore diameter of 8 μm was prepared, and the capture of tumor cells was examined. NCI-H358 cells were used as model cells, and 100 cells suspended in 1 mL of PBS were used as samples. The pump flow rate was 200 μL / min. FIG. 12 shows an optical micrograph of NCI-H358 cells (lung cancer cells) captured on the array using a quartz substrate size-selective microcavity array.
本発明においては、マイクロキャビティアレイによる細胞集積化技術をＣＴＣの濃縮及び検出という目的に特化させて改良し、サイズ選択マイクロキャビティアレイを作り上げた。サイズ選択マイクロキャビティアレイは、細胞サイズの違いによって、上皮系腫瘍細胞と血球細胞を分離し、血中のＣＴＣを回収する有効な手段である。実施例に示されているとおり、腫瘍細胞モデルとしてＮＣＩ−Ｈ３５８細胞を用いた場合、血液１ｍＬ中にＣＴＣが１０〜１００細胞個の条件で９６％以上の回収効率を得ることができた。また、複数の細胞株を用いた検討実験においては、細胞サイズが回収率に与える影響を考察したが、１０μｍ径のサイズ選択マイクロキャビティアレイではサイズの小さな腫瘍細胞を透過してしまう可能性が示唆された。これについては、より小さな孔径のサイズ選択マイクロキャビティアレイを利用又は併用することで、回収率を高められると考えられる。既存の方法との比較として、ＥｐＣＡＭマイクロビーズによる磁気回収法と比べたところ、本発明方法が回収率に優ることが明らかにされた。 In the present invention, a cell-selection technique using a microcavity array has been specially improved for the purpose of CTC concentration and detection, and a size-selective microcavity array has been created. The size selection microcavity array is an effective means for separating epithelial tumor cells and blood cells and recovering CTC in blood according to the difference in cell size. As shown in the Examples, when NCI-H358 cells were used as a tumor cell model, a recovery efficiency of 96% or more could be obtained under the condition of 10 to 100 CTC in 1 mL of blood. Moreover, in the examination experiment using a plurality of cell lines, the effect of the cell size on the recovery rate was considered. However, it is suggested that a size-selected microcavity array having a diameter of 10 μm may permeate small tumor cells. It was done. In this regard, it is considered that the recovery rate can be increased by using or using a size-selective microcavity array having a smaller pore size. As a comparison with the existing method, it was revealed that the method of the present invention is superior in the recovery rate when compared with the magnetic recovery method using EpCAM microbeads.
本発明は以下に述べる特徴を有しており、ＣＴＣの検出技術の一つとして非常に強力なツールとなる。（１）血液を前処理することなくそのまま分析できることが挙げられる。従来法では、分析の妨げとなる赤血球を溶血させたり、白血球を除いたりといった操作が必須であったが、これらの処理を一切必要とせず、分析装置に直接導入することができる。（２）血球の分離から染色、洗浄までを一貫して行えることにある。本発明のマイクロ流体デバイスのような装置設計でなければ、試験管内の細胞に対して繰り返し染色と洗浄操作を行うことになるため、目的細胞のロスの低減を実現する目的で有効に働く。（３）腫瘍細胞が規則的に加工されたマイクロキャビティアレイ上に捕捉されることが挙げられる。従来のメンブレンフィルターを用いた血球フィルトレーション法などでは、孔径が均一ではなく、分散して存在しているため、捕捉の安定性やスキャニング時間に問題があった。本発明のマイクロ流体デバイスでは、均一な孔径で安定した細胞捕捉を実現し、アレイ領域のみのスキャニングによって、回収したＣＴＣをカウントすることができる。（４）本発明のマイクロ流体デバイスは、装置自体が小型で安価であるということが挙げられる。従来は、煩雑な作業を自動化するために、磁気分離やスキャニングといった要素技術を詰め込んだ大型装置（Ｃｅｌｌｓｅａｒｃｈなど）を用いなければ多検体の処理は困難であった。しかし、ポンプと蛍光顕微鏡さえあれば使用でき、同時並行に検体を処理できるうえに、熟練した技術も必要としない。（５）ＣＴＣの回収原理がＥｐＣＡＭなどの表面抗原に依らないことである。前述したように、ＥｐＣＡＭの発現量は検体毎に異なっており、これらを指標とした磁気分離法では、ＥｐＣＡＭ陰性の腫瘍細胞などは回収できない。これに対し、本発明では、細胞サイズという異なる概念で腫瘍細胞を回収でき、その後の解析等に供することもできる。上記実施例では、ＥｐＣＡＭ陽性細胞をＣＴＣとしてカウントしたが、異なるマーカーを標的とした染色操作も同じように実行でき、前述したｏｎ−ｃｈｉｐ ＦＩＳＨを利用することでＣＴＣ特異的ｍＲＮＡの検出も可能であると考えられる。 The present invention has the following features, and is a very powerful tool as one of CTC detection techniques. (1) It is mentioned that blood can be analyzed as it is without pretreatment. In the conventional method, operations such as lysing red blood cells which hinder analysis or removing white blood cells are essential. However, these processes are not required at all, and can be directly introduced into the analyzer. (2) It is possible to perform consistently from separation of blood cells to staining and washing. Unless the device design is like the microfluidic device of the present invention, the cells in the test tube are repeatedly stained and washed, which works effectively for the purpose of reducing the loss of target cells. (3) It is mentioned that tumor cells are captured on a regularly processed microcavity array. In the conventional blood cell filtration method using a membrane filter and the like, the pore diameter is not uniform, but is present in a dispersed manner, and thus there is a problem in capture stability and scanning time. In the microfluidic device of the present invention, stable cell capture with a uniform pore diameter can be realized, and the recovered CTC can be counted by scanning only the array region. (4) In the microfluidic device of the present invention, the apparatus itself is small and inexpensive. Conventionally, in order to automate complicated operations, it has been difficult to process multiple samples without using a large apparatus (such as Cellsearch) packed with elemental technologies such as magnetic separation and scanning. However, it can be used with only a pump and a fluorescence microscope, and it can process samples in parallel and does not require skilled techniques. (5) The CTC recovery principle does not depend on surface antigens such as EpCAM. As described above, the expression level of EpCAM varies from sample to sample, and EpCAM-negative tumor cells and the like cannot be recovered by magnetic separation using these as indices. In contrast, in the present invention, tumor cells can be collected based on a different concept of cell size, and can be used for subsequent analysis. In the above example, EpCAM positive cells were counted as CTC, but staining operations targeting different markers can be performed in the same manner, and CTC-specific mRNA can be detected by using the above-described on-chip FISH. It is believed that there is.
Claims (12)
試料供給口と試料排出口、及び試料供給口と試料排出口を連通するマイクロ流路が形成され、マイクロ流路の一部に相当する位置にサイズ選択マイクロキャビティアレイ用開口窓が設けられた上部部材と；
前記上部部材の開口窓の下方に相当する位置に、ＣＴＣ捕捉用の孔径、孔数、配置が制御された微細貫通孔を有するサイズ選択マイクロキャビティアレイと、該サイズ選択マイクロキャビティアレイを保持する密封性シールからなるマイクロキャビティアレイ保持部と；
前記サイズ選択マイクロキャビティアレイの下方に相当する位置に設けられた吸引用開口窓と、前記吸引用開口窓と吸引口を連通する吸引流路が形成された下部部材と；
を備えていることを特徴とするマイクロ流体デバイス。 A microfluidic device capable of capturing circulating tumor cells (CTC) contained in a blood sample by a size-selective microcavity array,
A micro-channel that connects the sample supply port and the sample discharge port, and the sample supply port and the sample discharge port are formed, and an upper part is provided with an opening window for a size-selective microcavity array at a position corresponding to a part of the micro-channel With members;
A size-selective microcavity array having fine through-holes controlled in diameter, number of holes, and arrangement for capturing CTC at a position corresponding to a position below the opening window of the upper member, and a seal that holds the size-selected microcavity array A microcavity array holder made of a conductive seal;
A suction opening window provided at a position corresponding to the lower side of the size selection microcavity array, and a lower member in which a suction flow path communicating the suction opening window and the suction port is formed;
A microfluidic device comprising:
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