JP6822006B2 - Target particle recovery method and recovery device - Google Patents

Target particle recovery method and recovery device Download PDF

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JP6822006B2
JP6822006B2 JP2016159672A JP2016159672A JP6822006B2 JP 6822006 B2 JP6822006 B2 JP 6822006B2 JP 2016159672 A JP2016159672 A JP 2016159672A JP 2016159672 A JP2016159672 A JP 2016159672A JP 6822006 B2 JP6822006 B2 JP 6822006B2
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俊樹 後藤
俊樹 後藤
片山　晃治
晃治 片山
篤史 森本
篤史 森本
泰之 秋山
泰之 秋山
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本発明は、目的粒子を含む溶液から当該目的粒子を回収する方法および装置に関する。特に本発明は、目的粒子以外の夾雑粒子が多く含む溶液に対しても、目的粒子を回収可能な方法および装置に関する。 The present invention relates to a method and an apparatus for recovering the target particles from a solution containing the target particles. In particular, the present invention relates to a method and an apparatus capable of recovering target particles even in a solution containing a large amount of contaminant particles other than the target particles.

溶液中に同じ種類の目的細胞が含まれていたとしても、当該目的細胞の性質が個々に異なることが知られている（非特許文献１）。一方で、溶液中に含まれる目的細胞から通常得られる情報は、当該目的細胞個々の情報を平均化した情報となるため、当該目的細胞の情報を個々に得ることは難しい。そのため、溶液中に含まれる目的細胞を個々に解析し、当該目的細胞に関する情報を個々に得ることへの関心が高まっている。 It is known that even if the target cells of the same type are contained in the solution, the properties of the target cells are different from each other (Non-Patent Document 1). On the other hand, since the information normally obtained from the target cells contained in the solution is the averaged information of the individual target cells, it is difficult to individually obtain the information of the target cells. Therefore, there is increasing interest in individually analyzing the target cells contained in the solution and individually obtaining information on the target cells.

溶液中に含まれる目的細胞を個々に解析する例として、血液試料中に含まれる血中循環腫瘍細胞（Ｃｉｒｃｕｌａｔｉｎｇ Ｔｕｍｏｒ Ｃｅｌｌｓ、以下ＣＴＣ）の解析があげられる。ＣＴＣはガンの転移や再発に重要な役割を果たすと考えられており、ＣＴＣの解析が可能になると、ガン患者の術後診断や投薬方針を決定することができるため、治療の最適化や効率化につながると考えられる。しかしながら、ＣＴＣは未解明な点が多く、またＣＴＣが有する遺伝子の変異やコピー数変化が個々のＣＴＣで異なるという報告もあるため、ＣＴＣを個々に解析する必要がある（非特許文献２）。 An example of individually analyzing the target cells contained in the solution is the analysis of circulating tumor cells (Circulating Tumor Cells, hereinafter referred to as CTC) contained in a blood sample. CTC is thought to play an important role in the metastasis and recurrence of cancer, and when CTC can be analyzed, postoperative diagnosis and medication policy of cancer patients can be determined, so treatment optimization and efficiency It is thought that it will lead to the transformation. However, there are many unclear points about CTC, and there are reports that mutations in genes and copy number changes of CTC differ among individual CTCs, so it is necessary to analyze CTCs individually (Non-Patent Document 2).

溶液中に含まれる目的細胞を個々に解析可能な手段の一例として、特許文献１に記載の粒子保持手段があげられる。特許文献１に記載の粒子保持手段は、溶液中に含まれる目的粒子を保持可能な凹部を複数設けており、誘電泳動力を発生させることで、目的粒子を前記凹部へ個々に保持し目的粒子を回収することができる。前記粒子保持手段に設けた凹部に保持された目的粒子は、顕微鏡など当該目的粒子が有する特徴を検出可能な手段により検出することで、形態学的分析、組織分析を行なった後、検出した目的粒子をマイクロマニピュレーターなどの採取手段で取得することで、当該目的粒子に含まれる物質（目的粒子が細胞といった生体材料の場合は核酸やタンパク質など）の分析が行なえる。しかしながら、溶液中に目的細胞以外の細胞（夾雑細胞）が多く含まれる場合は、粒子保持手段に設けた凹部に目的細胞と夾雑細胞とが混在した状態で回収されるおそれがあり、目的細胞の正確な検出、取得、解析ができないおそれがあった。 As an example of a means capable of individually analyzing target cells contained in a solution, the particle holding means described in Patent Document 1 can be mentioned. The particle holding means described in Patent Document 1 is provided with a plurality of recesses capable of holding the target particles contained in the solution, and by generating a dielectrophoretic force, the target particles are individually held in the recesses and the target particles are held. Can be recovered. The target particles held in the recesses provided in the particle holding means are detected after performing morphological analysis and tissue analysis by detecting the characteristics of the target particles by a detectable means such as a microscope. By acquiring the particles by a collecting means such as a microscope, it is possible to analyze the substances contained in the target particles (nucleic acid, protein, etc. when the target particles are biomaterials such as cells). However, when a large amount of cells other than the target cells (contaminated cells) are contained in the solution, the target cells and the contaminating cells may be collected in a mixed state in the recess provided in the particle holding means, and the target cells may be collected. There was a risk that accurate detection, acquisition, and analysis could not be performed.

特開２００９−２７３４５９号公報JP-A-2009-273459

Ｇｒｏｒｉａ，Ｈ．Ｈ．，Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ，４４，２２５９−２２６５（１９８４）Gloria, H. et al. H. , Cancer Research, 44, 2259-2265 (1984) Ｍａｒｔｉｎａ，Ａ．，ｅｔ ａｌ．，Ｏｎｃｏｔａｒｇｅｔ，４，８１２−８１３（２０１３）Martina, A.M. , Et al. , Oncotarget, 4,812-813 (2013)

本発明の課題は、溶液中に含まれる目的粒子を、当該目的粒子を保持可能な凹部を設けた粒子保持手段を用いて、当該目的粒子を個別に回収する方法において、溶液中に目的粒子以外の粒子（夾雑粒子）が多く含まれている場合でも、目的粒子を個別に回収可能な方法および装置を提供することにある。 An object of the present invention is a method of individually recovering target particles contained in a solution by using a particle holding means provided with a recess capable of holding the target particles, in which the target particles are not included in the solution. It is an object of the present invention to provide a method and an apparatus capable of individually recovering target particles even when a large amount of particles (contamination particles) are contained.

上記課題を解決するために、本発明者らは鋭意検討を重ねた結果、目的粒子を保持可能な粒子保持手段の形状を工夫することで、本発明に到達した。 As a result of diligent studies in order to solve the above problems, the present inventors have arrived at the present invention by devising the shape of the particle holding means capable of holding the target particles.

すなわち本発明の第一の態様は、
目的粒子を含む溶液を、当該目的粒子を保持可能な凹部を複数設けた粒子保持手段に導入する工程と、
前記凹部に前記目的粒子を保持させる工程とを含む、目的粒子の回収方法であって、
粒子保持手段に設ける凹部は、連通部を介して隣接した凹部と連通しており、
前記連通部は、溶液中に含まれる目的粒子は保持できない一方、溶液中に含まれる当該目的粒子以外の夾雑粒子は保持可能であり、
凹部に目的粒子を保持させる工程を、誘電泳動力を用いて行なう、
前記回収方法である。 That is, the first aspect of the present invention is
A step of introducing a solution containing the target particles into a particle holding means provided with a plurality of recesses capable of holding the target particles, and a step of introducing the solution.
A method for recovering target particles, which comprises a step of holding the target particles in the recesses.
The recess provided in the particle holding means communicates with the adjacent recess via the communication portion.
While the communication portion cannot retain the target particles contained in the solution, it can retain contaminant particles other than the target particles contained in the solution.
The step of holding the target particles in the recesses is performed using dielectrophoretic force.
This is the recovery method.

また本発明の第二の態様は、粒子保持手段に設ける凹部の上面形状が多角形であり、連通部を当該多角形の頂点のうちの少なくとも一つと隣接した凹部における当該多角形の頂点のうちの少なくとも一つとが連通するよう設けた、前記第一の態様に記載の目的粒子の回収方法である。 Further, in the second aspect of the present invention, the upper surface shape of the recess provided in the particle holding means is a polygon, and the communication portion is among the vertices of the polygon in the recess adjacent to at least one of the vertices of the polygon. The method for recovering target particles according to the first aspect, which is provided so as to communicate with at least one of the above.

また本発明の第三の態様は、目的粒子を含む溶液が血液試料であり、目的粒子が腫瘍細胞であり、夾雑粒子が白血球、赤血球、血小板、小胞、細胞デブリから選ばれるいずれか１つ以上である、前記第一または第二の態様に記載の目的粒子の回収方法である。 Further, in the third aspect of the present invention, the solution containing the target particles is a blood sample, the target particles are tumor cells, and the contaminating particles are selected from leukocytes, erythrocytes, platelets, vesicles, and cell debris. The above is the method for recovering target particles according to the first or second aspect.

また本発明の第四の態様は、前記第一から第三の態様のいずれかに記載の方法で回収した目的粒子を、当該目的粒子が有する特徴に基づき検出する、目的粒子の検出方法である。 A fourth aspect of the present invention is a method for detecting target particles, which detects target particles recovered by the method according to any one of the first to third aspects based on the characteristics of the target particles. ..

また本発明の第五の態様は、前記第四の態様で検出した目的粒子を、当該目的粒子を採取する手段で取得する、目的粒子の取得方法である。 A fifth aspect of the present invention is a method for obtaining target particles, in which the target particles detected in the fourth aspect are acquired by means for collecting the target particles.

さらに本発明の第六の態様は、
溶液中に含まれる目的粒子を保持可能な凹部を複数設け、当該凹部は連通部を介して隣接した凹部と連通しており、当該連通部は前記目的粒子は保持できない一方、当該目的粒子以外の夾雑粒子は保持可能な、粒子保持手段と、
前記目的粒子および前記夾雑粒子を保持させるための誘電泳動力を発生させる手段と、
を備えた、目的粒子の回収装置である。 Furthermore, the sixth aspect of the present invention is
A plurality of recesses capable of holding the target particles contained in the solution are provided, and the recesses communicate with the adjacent recesses via a communication portion, and the communication portion cannot hold the target particles, but other than the target particles. Particle retention means that can retain contaminant particles,
A means for generating a dielectrophoretic force for holding the target particles and the contaminant particles, and
It is a target particle recovery device equipped with.

以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.

本発明において粒子とは、溶液中に単独または凝集状態で分散する不溶性物質のことをいう。具体例としては、ビーズ、粉砕用ボール、液晶用スペーサー、クロマトグラフィー用分離剤、吸着剤といった工業材料からなる粒子や、細胞、ウイルス、オルガネラ、小胞といった生体材料からなる粒子があげられる。特に本発明は、前記生体材料からなる粒子の回収に好ましい方法および装置である。 In the present invention, the particles refer to insoluble substances that are dispersed alone or in an aggregated state in a solution. Specific examples include particles made of industrial materials such as beads, crushing balls, liquid crystal spacers, chromatographic separators, and adsorbents, and particles made of biomaterials such as cells, viruses, organelles, and vesicles. In particular, the present invention is a preferred method and apparatus for recovering particles made of the biomaterial.

目的粒子が前述した生体材料からなる粒子である場合の、本発明における目的粒子を含む溶液の一例としては、全血、希釈血液、血清、血漿、髄液、臍帯血、成分採血液、尿、唾液、***、糞便、痰、羊水、腹水、腹腔洗浄液などの生体試料や、肝臓、肺、脾臓、腎臓、皮膚、腫瘍、リンパ節などの組織の一片を懸濁させた組織懸濁液や、前記生体試料または前記組織懸濁液より分離して得られる、前記生体試料または前記組織由来の細胞を含む画分や、あらかじめ単離した細胞の培養液、があげられる。このうち生体試料または組織由来の細胞を含む画分の一例として、生体試料や組織懸濁液を密度勾配形成用媒体の上に重層後、密度勾配遠心することで得られる画分があげられる。 When the target particle is a particle made of the above-mentioned biological material, examples of the solution containing the target particle in the present invention include whole blood, diluted blood, serum, plasma, spinal fluid, umbilical cord blood, component blood sampling, urine, and the like. Biological samples such as saliva, semen, feces, sputum, sheep water, ascites, and peritoneal lavage fluid, and tissue suspensions in which pieces of tissue such as liver, lung, spleen, kidney, skin, tumor, and lymph nodes are suspended. Examples thereof include a fraction containing cells derived from the biological sample or tissue obtained by separating from the biological sample or tissue suspension, and a culture solution of cells isolated in advance. Among these, as an example of a fraction containing cells derived from a biological sample or tissue, there is a fraction obtained by layering a biological sample or tissue suspension on a medium for forming a density gradient and then centrifuging the density gradient.

目的粒子を含む溶液が血液試料である場合の、目的粒子の一例としては、血液循環腫瘍細胞（ＣＴＣ）などの腫瘍細胞、循環血液内皮細胞（ＣＥＣ）、循環血管内皮細胞（ＣＥＰ）、循環胎児細胞（ＣＦＣ）、抗原特異的Ｔ細胞、各種幹細胞があげられる。一方、夾雑粒子は前述した目的粒子以外の粒子であり、具体的には、血液試料中に含まれる細胞である白血球、赤血球、血小板および小胞、ならびにこれら細胞または前述した目的粒子由来のデブリがあげられる。なお本発明における血液試料は、全血、血清、血漿、臍帯血、成分採血液といった血液検体に限らず、当該血液検体を生理食塩水などで希釈した試料や、当該血液検体より分離して得られる、前記血液検体由来の細胞を含む画分も、血液試料に含まれる。 When the solution containing the target particles is a blood sample, examples of the target particles include tumor cells such as blood circulating tumor cells (CTC), circulating blood endothelial cells (CEC), circulating vascular endothelial cells (CEP), and circulating fetuses. Examples include cells (CFC), antigen-specific T cells, and various stem cells. On the other hand, the contaminating particles are particles other than the above-mentioned target particles, and specifically, leukocytes, erythrocytes, platelets and vesicles, which are cells contained in the blood sample, and debris derived from these cells or the above-mentioned target particles. can give. The blood sample in the present invention is not limited to blood samples such as whole blood, serum, plasma, umbilical cord blood, and component blood samples, but can be obtained by diluting the blood sample with physiological saline or the like or by separating it from the blood sample. A fraction containing cells derived from the blood sample is also included in the blood sample.

本発明において粒子保持手段に設ける凹部の上面形状（平面図における形状）は、目的粒子を保持できれば特に限定はなく、円形であってもよく、楕円形であってもよく、正方形、ひし形、平行四辺形、六角形、八角形といった多角形であってもよい。また、凹多角形や、凸多角形も前記多角形に含まれる。なお前記凹部を構成する壁面は直線形状または凹形状（例えば、前記凹多角形）とすると、夾雑粒子を後述する連通部へ速やかに効率よく誘導し、前記凹部への夾雑粒子の混入を抑制できるため、好ましい。
前記凹部の大きさは、回収対象である目的粒子の大きさや形状に応じ適宜選択すればよいが、前記凹部の大きさを前記目的粒子が一つだけ保持可能な大きさとすると、その後の目的粒子の形態学的分析（例えば、高速フーリエ変換）、組織型分析、核酸分析、タンパク質分析などの解析が容易に行なえる点で好ましい。例えば目的粒子がＣＴＣ（直径：１０から２５μｍ）の場合は、前記凹部を直径２５μｍから３０μｍの粒子が一つだけ保持可能な大きさとすると好ましい。 In the present invention, the upper surface shape (shape in the plan view) of the concave portion provided in the particle holding means is not particularly limited as long as the target particles can be held, and may be circular, elliptical, square, rhombus, or parallel. It may be a polygon such as a quadrilateral, a hexagon, or an octagon. Further, a concave polygon and a convex polygon are also included in the polygon. When the wall surface forming the concave portion has a linear shape or a concave shape (for example, the concave polygonal shape), the contaminating particles can be quickly and efficiently guided to the communication portion described later, and the contamination of the condensing particles in the concave portion can be suppressed. Therefore, it is preferable.
The size of the recess may be appropriately selected according to the size and shape of the target particle to be collected. However, if the size of the recess is such that only one target particle can be held, the subsequent target particle It is preferable in that analysis such as morphological analysis (for example, fast Fourier transform), histological analysis, nucleic acid analysis, and protein analysis can be easily performed. For example, when the target particle is CTC (diameter: 10 to 25 μm), it is preferable that the recess has a size capable of holding only one particle having a diameter of 25 μm to 30 μm.

本発明において粒子保持手段に設ける凹部は、連通部を介して隣接した凹部と連通している。なお前記凹部の上面形状が多角形の場合、前記連通部は当該多角形の頂点のうちの少なくとも一つと隣接した凹部における当該多角形の頂点のうちの少なくとも一つとが連通するよう設けると好ましい。当該好ましい連通部の具体例として、前記多角形の頂点のうちの一つと隣接した凹部における前記多角形の頂点のうちの一つとが連通した連通部や、前記多角形の頂点のうちの二以上または全ての頂点と隣接した凹部における前記多角形の頂点のうちの一つとが連通した連通部や、前記多角形の頂点のうちの二以上または全ての頂点と隣接した凹部における前記多角形の頂点のうちの二以上または全ての頂点とが連通した連通部があげられる。前記連通部の幅は、溶液中に含まれる目的粒子は保持できない一方、溶液中に含まれる当該目的粒子以外の夾雑粒子は保持可能な幅とすればよい。一例として、溶液が血液試料で、目的粒子がＣＴＣ（直径：１０から２５μｍ）で、夾雑粒子が赤血球（直径：７から８μｍ）、白血球（直径：大半は６から１５μｍ）および血小板（直径：１から４μｍ）の場合、連通部の幅は５から１５μｍまでの範囲とするとよく、８から１２μｍまでの範囲とするとより好ましい。前記連通部の長さは特に限定はないものの、長さを長くすると夾雑粒子の前記連通部への保持量が増大する一方、夾雑粒子の前記凹部への混入量は減少するため、好ましいといえる。 In the present invention, the recess provided in the particle holding means communicates with the adjacent recess via the communication portion. When the upper surface shape of the recess is polygonal, it is preferable that the communication portion is provided so that at least one of the vertices of the polygon and at least one of the vertices of the polygon in the adjacent recess communicate with each other. As a specific example of the preferable communication portion, a communication portion in which one of the vertices of the polygon and one of the vertices of the polygon in an adjacent recess communicate with each other, or two or more of the vertices of the polygon. Alternatively, a communication portion in which one of the vertices of the polygon communicates with all the vertices in the recess adjacent to the vertices, or the vertices of the polygon in the recess adjacent to two or more or all the vertices of the polygon. There is a communication part in which two or more or all of the vertices are communicated with each other. The width of the communication portion may be such that the target particles contained in the solution cannot be retained, while the contaminant particles other than the target particles contained in the solution can be retained. As an example, the solution is a blood sample, the target particles are CTC (diameter: 10 to 25 μm), and the contaminant particles are red blood cells (diameter: 7 to 8 μm), leukocytes (diameter: most 6 to 15 μm) and platelets (diameter: 1). In the case of (to 4 μm), the width of the communicating portion is preferably in the range of 5 to 15 μm, more preferably in the range of 8 to 12 μm. Although the length of the communication portion is not particularly limited, it can be said that it is preferable to increase the length because the holding amount of the contaminating particles in the communicating portion increases and the amount of the contaminating particles mixed in the recesses decreases. ..

また、夾雑粒子が前記連通部への保持量が増大する理由は、本発明における粒子保持手段に設ける凹部には、一様でない不均一な電界（電気力線）が与えられるからである。従って、凹部よりも連通部の方がより面積が小さいため、当該目的粒子は電界の集中する方向（電気力線が密な方向）、つまり凹部よりも連通部の方向へ引き寄せられる。言い換えると、連通部は凹部よりも電界が集中することになり、目的粒子よりも小さい夾雑粒子はより連通部に粒子が引き寄せられやすくなる。 Further, the reason why the amount of the contaminating particles held in the communicating portion increases is that a non-uniform electric field (line of electric force) is applied to the recess provided in the particle holding means in the present invention. Therefore, since the area of the communicating portion is smaller than that of the concave portion, the target particle is attracted in the direction in which the electric field is concentrated (the direction in which the lines of electric force are dense), that is, in the direction of the communicating portion rather than the concave portion. In other words, the electric field is concentrated in the communicating portion rather than the concave portion, and the contaminating particles smaller than the target particles are more likely to be attracted to the communicating portion.

本発明の方法で溶液から回収した目的粒子の検出は、当該目的粒子が有する特徴に基づき行なえばよい。一例として、明視野像、蛍光画像、化学発光画像といった目的粒子の光学的特徴に基づき検出する場合は光学検出器や光学顕微鏡などの光学測定器を用いて検出すればよく、目的粒子の弾性や粘性といった特徴に基づき検出する場合は超音波顕微鏡などの超音波測定器を用いて検出すればよく、放射性同位元素を標識した目的粒子など目的粒子の放射化学的特徴に基づき検出する場合はシンチレーション検出器などの放射線検出器を用いて検出すればよく、目的粒子の熱応答性や熱物性に基づき検出する場合は当該熱応答性や熱物性を検出可能な装置を用いて検出すればよい。具体例として、目的粒子がＣＴＣなどの細胞であり、当該細胞の検出を光学測定器を用いて行なう場合、前記目的細胞を含む溶液を、前記細胞を保持可能な凹部を有した粒子保持手段に導入し、誘電泳動力を用いて前記凹部に前記細胞を保持した後、顕微鏡や光学検出器などの光学測定器で観察すればよい。その際前記凹部の下面は、前記細胞を固定可能な材料（例えば、ポリ−Ｌ−リジン）で被覆してもよい。 The target particles recovered from the solution by the method of the present invention may be detected based on the characteristics of the target particles. As an example, when detecting based on the optical characteristics of the target particle such as a bright field image, a fluorescent image, or a chemical emission image, the detection may be performed using an optical detector such as an optical detector or an optical microscope, and the elasticity of the target particle may be detected. When detecting based on characteristics such as viscosity, detection may be performed using an ultrasonic measuring instrument such as an ultrasonic microscope, and when detecting based on radiochemical characteristics of target particles such as target particles labeled with radioactive isotopes, scintillation detection is performed. The detection may be performed using a radiation detector such as a device, and when detecting based on the thermal responsiveness and thermal physical characteristics of the target particle, the detection may be performed using a device capable of detecting the thermal responsiveness and thermal physical characteristics. As a specific example, when the target particle is a cell such as CTC and the cell is detected by using an optical measuring instrument, the solution containing the target cell is used as a particle holding means having a recess capable of holding the cell. After introducing the cells and holding the cells in the recesses using dielectrophoretic force, the cells may be observed with an optical measuring instrument such as a microscope or an optical detector. At that time, the lower surface of the recess may be coated with a material (for example, poly-L-lysine) capable of immobilizing the cells.

前述した方法で検出した目的粒子は、光ピンセット、誘電泳動力を発生させる手段、マイクロマニピュレーションなどの採取手段を用いることで、当該目的粒子を取得できる。目的粒子が細胞などの生体材料の場合、前記取得した目的粒子は、当該目的粒子中の核酸やタンパク質などの分析が行なえる。 The target particles detected by the above-mentioned method can be obtained by using optical tweezers, a means for generating a dielectrophoretic force, a collecting means such as micromanipulation, or the like. When the target particle is a biomaterial such as a cell, the obtained target particle can be analyzed for nucleic acids, proteins, and the like in the target particle.

本発明は、目的粒子を含む溶液を当該目的粒子を保持可能な凹部を複数設けた粒子保持手段に導入する工程と、前記凹部に前記目的粒子を保持させる工程とを含む、目的粒子の回収方法において、粒子保持手段に設ける凹部が連通部を介して隣接した凹部と連通しており、前記連通部が溶液中に含まれる目的粒子は保持できない一方、溶液中に含まれる当該目的粒子以外の夾雑粒子は保持可能であり、凹部に目的粒子を保持させる工程を誘電泳動力を用いて行なうことを特徴としており、溶液中に含まれる目的粒子を、夾雑粒子の影響を受けることなく、粒子保持手段に設けた凹部に保持させることができる。従って、個々の目的粒子の正確な検出、取得、解析が行なえる。 The present invention is a method for recovering target particles, which comprises a step of introducing a solution containing the target particles into a particle holding means provided with a plurality of recesses capable of holding the target particles, and a step of holding the target particles in the recesses. In the above, the recess provided in the particle holding means communicates with the adjacent recess via the communicating portion, and the communicating portion cannot hold the target particles contained in the solution, while the contamination other than the target particles contained in the solution. The particles can be held, and the step of holding the target particles in the recesses is performed by using a dielectricing force, and the target particles contained in the solution are held by the particle holding means without being affected by the contaminating particles. It can be held in the recess provided in. Therefore, accurate detection, acquisition, and analysis of individual target particles can be performed.

特に本発明は、溶液中に含まれる目的粒子数が少なく、かつ夾雑粒子数が当該目的粒子数と比較して極めて多い場合に有用であり、例えば、本発明を血液中に含まれる血中循環腫瘍細胞（ＣＴＣ）の検出に適用することで、ＣＴＣの有無の判断結果に対する信頼性が向上し、精度高くガンを診断することができる。 The present invention is particularly useful when the number of target particles contained in the solution is small and the number of contaminant particles is extremely large as compared with the number of target particles. For example, the present invention is contained in blood for blood circulation. By applying it to the detection of tumor cells (CTC), the reliability of the determination result of the presence or absence of CTC is improved, and cancer can be diagnosed with high accuracy.

本発明の粒子回収装置の一態様を示した図（分解図）である。ただし、凹部同士を連通する連通部の記載は省略している。It is a figure (exploded view) which showed one aspect of the particle recovery apparatus of this invention. However, the description of the communication portion that communicates the recesses with each other is omitted. 図１に示す粒子回収装置の正面図である。It is a front view of the particle recovery device shown in FIG. 図１に示す粒子回収装置に備える粒子保持手段の一態様を示した図である。（Ａ）は平面図であり、（Ｂ）は（Ａ）のうちａ）の領域を拡大した図である。It is a figure which showed one aspect of the particle holding means provided in the particle recovery apparatus shown in FIG. (A) is a plan view, and (B) is an enlarged view of the area a) in (A).

以下、図面を用いて本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to the drawings.

本発明の粒子回収装置の一態様を図１に示す。また図１に示した粒子回収装置の正面図を図２に示す。なお図１および図２では、凹部１６１同士を連通する連通部の記載は省略している。 One aspect of the particle recovery device of the present invention is shown in FIG. The front view of the particle recovery device shown in FIG. 1 is shown in FIG. Note that in FIGS. 1 and 2, the description of the communication portion that communicates the recesses 161 with each other is omitted.

図１に示す粒子回収装置１００は、
貫通部１１１ａを有した平板上の遮光部材１１１と、貫通部１１２ａを有した平板上の絶縁体１１２から構成される凹部壁面部材１１０と、
凹部壁面部材１１０の上部に密着して設けた、導入口１２１、排出口１２２および貫通部１２３を有した平板上のスペーサ１２０と、
凹部壁面部材１１０の下部およびスペーサ１２０の上部を密着して挟むよう設けた電極１３１・１３２と、
電極１３１・１３２同士を接続する導線１４０と、
電極１３１・１３２に信号を印加する信号発生器１５０と、
を備えている。遮光部材１１１が有する貫通部１１１ａと絶縁体１１２が有する貫通部１１２ａとは互いに同一の寸法および形状であり、かつそれぞれの貫通部の位置が一致するよう遮光部材１１１および絶縁体１１２を設けている。貫通部１１１ａ、貫通部１１２ａおよび遮光部材１１１の下部に密着して設けた電極１３１により凹部１６１が構成され、導入口１２１から目的粒子２００を含む液体を導入すると、貫通部１２３を通じて凹部１６１へ目的粒子２００が導入される。電極１３２はスペーサ１２０上部に密着して設けており、導入口１２１から導入した、粒子を含む液体の飛散や蒸発を防止している。なお凹部１６１に保持した粒子の回収を容易にするため、電極１３２はスペーサ１２０から取り外し可能な構造となっている。 The particle recovery device 100 shown in FIG. 1 is
A light-shielding member 111 on a flat plate having a penetrating portion 111a, a recessed wall surface member 110 composed of an insulator 112 on a flat plate having a penetrating portion 112a, and
A spacer 120 on a flat plate having an introduction port 121, a discharge port 122, and a penetration portion 123, which are provided in close contact with the upper portion of the recessed wall surface member 110.
Electrodes 131 and 132 provided so as to closely sandwich the lower portion of the recessed wall surface member 110 and the upper portion of the spacer 120.
The lead wire 140 that connects the electrodes 131 and 132 to each other,
A signal generator 150 that applies a signal to the electrodes 131 and 132, and
Is equipped with. The light-shielding member 111 and the insulator 112 are provided so that the penetrating portion 111a of the light-shielding member 111 and the penetrating portion 112a of the insulator 112 have the same dimensions and shapes and the positions of the respective penetrating portions are the same. .. A recess 161 is formed by an electrode 131 provided in close contact with the penetrating portion 111a, the penetrating portion 112a, and the lower portion of the light-shielding member 111. When a liquid containing the target particles 200 is introduced from the introduction port 121, the target is introduced into the recess 161 through the penetrating portion 123. Particle 200 is introduced. The electrode 132 is provided in close contact with the upper portion of the spacer 120 to prevent scattering and evaporation of the liquid containing particles introduced from the introduction port 121. The electrode 132 has a structure that can be removed from the spacer 120 in order to facilitate the recovery of the particles held in the recess 161.

図１に示す粒子回収装置１００のうち、電極１３１、遮光部材１１１および絶縁体１１２から構成される粒子保持手段１６０の一態様を図３に示す。図３に示す粒子保持手段１６０には、目的粒子を保持可能な凹部１６１と、目的粒子は保持できない一方、夾雑粒子は保持可能な、凹部１６１同士を連通する連通部１６２とを設けている（図３（Ｂ））。凹部１６１の上面形状（平面図における形状）は一辺３０μｍの正方形を４５度回転した形状であり、その深さは４０μｍである。凹部１６１同士の間隔（隣接する凹部の中心間距離）は５０μｍである。なお連通部１６２の幅は１０μｍである。 Of the particle recovery device 100 shown in FIG. 1, one aspect of the particle holding means 160 composed of the electrode 131, the light-shielding member 111, and the insulator 112 is shown in FIG. The particle holding means 160 shown in FIG. 3 is provided with a recess 161 capable of holding the target particles and a communication portion 162 communicating the recesses 161 with each other so that the target particles cannot be held while the contaminant particles can be held. FIG. 3 (B). The upper surface shape (shape in the plan view) of the recess 161 is a shape obtained by rotating a square having a side of 30 μm by 45 degrees, and the depth thereof is 40 μm. The distance between the recesses 161 (distance between the centers of adjacent recesses) is 50 μm. The width of the communication portion 162 is 10 μm.

次に本発明の粒子回収装置を用いた目的粒子の回収方法の一例を説明する。 Next, an example of a method for recovering target particles using the particle recovery device of the present invention will be described.

図１に示す粒子回収装置１００に設けた導入口１１３ａから目的粒子２００を含む液体を導入し、誘電泳動力を利用して目的粒子２００を凹部１６１へ導入させる。具体的には、信号発生器１５０から電極１３１・１３２へ交流電圧を印加することで誘電泳動力を発生させ、凹部１６１へ目的粒子２００を導入する。なお目的粒子２００以外の粒子（夾雑粒子）のうち、目的粒子よりも径が小さい粒子は連通部１６２へ優先的に導入される。その理由は、連通部１６２に働く誘電泳動力は凹部１６１に働く誘電泳動力よりも大きい一方、連通部１６２の幅は目的粒子２００よりも狭く、目的粒子２００が連通部１６２に導入される可能性は低いためである。一方、目的粒子よりも径が大きい夾雑粒子は凹部１６１および連通部１６２には導入されないため、その後行なう洗浄工程により粒子回収装置１００系外へ排出される。 A liquid containing the target particles 200 is introduced from the introduction port 113a provided in the particle recovery device 100 shown in FIG. 1, and the target particles 200 are introduced into the recess 161 by utilizing the dielectrophoretic force. Specifically, an AC voltage is applied from the signal generator 150 to the electrodes 131 and 132 to generate a dielectrophoretic force, and the target particle 200 is introduced into the recess 161. Among the particles (contamination particles) other than the target particles 200, the particles having a diameter smaller than the target particles are preferentially introduced into the communication portion 162. The reason is that the dielectrophoretic force acting on the communicating portion 162 is larger than the dielectrophoretic force acting on the recess 161 while the width of the communicating portion 162 is narrower than that of the target particle 200, so that the target particle 200 can be introduced into the communicating portion 162. This is because the sex is low. On the other hand, since the contaminating particles having a diameter larger than the target particles are not introduced into the recess 161 and the communicating portion 162, they are discharged to the outside of the particle recovery device 100 system by the subsequent cleaning step.

粒子が細胞である場合、図１に示す粒子回収装置１００に導入する目的細胞を含む液体は、誘電泳動力で目的細胞が移動できるよう懸濁された液であればよく、例えば、マンニトール、グルコース、スクロース等の糖類を含んだ水溶液や、当該水溶液に塩化カルシウム、塩化マグネシウム等の電解質、および／またはＢＳＡ（ウシ血清アルブミン）等のタンパク質をさらに含んだ水溶液に、目的細胞を含んだ試料を懸濁させた液体があげられる。特に目的細胞を含む液体として、マンニトールを含む水溶液に細胞を含んだ試料を懸濁させた液体を用いると、細胞へのダメージが少なくなる点で好ましい。添加するマンニトールの濃度は等張液となる濃度とすればよく、具体的には２５０ｍＭから３５０ｍＭの間とするとよい。信号発生器１５０から電極１３１・１３２へ印加する交流電圧は、凹部１６１に保持された目的細胞の充放電が周期的に繰り返される波形を有した交流電圧とすると好ましく、周波数を１００ｋＨｚから３ＭＨｚまでの間とし、電界強度を１×１０５から５×１０５Ｖ／ｍまでの間とすると特に好ましい（ＷＯ２０１１／１４９０３２号および特開２０１２−０１３５４９号公報参照）。 When the particles are cells, the liquid containing the target cells to be introduced into the particle recovery device 100 shown in FIG. 1 may be a liquid suspended so that the target cells can move by dielectrophoretic force, for example, mannitol and glucose. , A sample containing the target cells in an aqueous solution containing saccharides such as sucrose, or an aqueous solution containing an electrolyte such as calcium chloride or magnesium chloride and / or a protein such as BSA (bovine serum albumin). A turbid liquid can be mentioned. In particular, as the liquid containing the target cells, it is preferable to use a liquid in which a sample containing the cells is suspended in an aqueous solution containing mannitol, because damage to the cells is reduced. The concentration of mannitol to be added may be a concentration that produces an isotonic solution, and specifically, it may be between 250 mM and 350 mM. The AC voltage applied from the signal generator 150 to the electrodes 131 and 132 is preferably an AC voltage having a waveform in which charging and discharging of the target cells held in the recess 161 are periodically repeated, and the frequency is from 100 kHz to 3 MHz. It is particularly preferable that the electric field strength is between 1 × 10 5 and 5 × 10 5 V / m (see WO2011 / 149032 and JP2012-013549).

以下、実施例および比較例を用いて本発明をさらに詳細に説明するが、本発明は当該例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these examples.

実施例１
（１）一方の末端がメトキシ基であり、もう一方の末端がＮ−ヒドロオキシスクシンイミドエステル基である、分子量５０００のポリエチレングリコール（ｍＰＥＧ−ＮＨＳ）と、ウシ血清アルブミン（ＢＳＡ）（３００ｍｇ、０．３ｍｍｏｌ）とを、炭酸水素ナトリウム緩衝液（０．１Ｍ、１５ｍＬ）に溶解後、当該溶液を室温で３時間撹拌することでポリエチレングリコールを結合したＢＳＡ（ＰＥＧ−ＢＳＡ）を調製した。なお調製する際、ｍＰＥＧ−ＮＨＳとＢＳＡとのモル比（ｍＰＥＧ−ＮＨＳ／ＢＳＡ）を２となるようにした。調製後、分画分子量１００００の透析膜を用いて、純水への溶液置換を３日間行なった。
（２）イミダゾリジニル尿素２ｇ、分子量６０００のポリエチレングリコール（ＰＥＧ）２ｇ、エチレンジアミン四酢酸（ＥＤＴＡ）１００ｍｇ、および塩化ナトリウム６００ｍｇを、超純水１００ｍＬに溶解し、得られた溶液を安定化剤として用いた。
（３）インフォームドコンセントを得た健常人から血液をＥＤＴＡ−２Ｋ採血管（ＶＰ−ＤＫ０５０Ｋ、テルモ社製）に３ｍＬ採血後、前記採血管に（２）で調製した安定化剤３ｍＬを添加し、得られた溶液を保存処理した希釈血液試料とした。
（４）保存処理した希釈血液試料を室温で１０分放置し、７５μＬの白血球・赤血球結合剤（ＲｏｓｅｔｔｅＳｅｐ、ＳｔｅｍＣｅｌｌ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）を添加した後、チューブ内で密度１．０８６ｇ／ｍＬの密度勾配溶液上に重層し、室温で２０００×ｇで１０分間遠心した。
（５）遠心後、目的粒子の位置する画分を含む溶液を５０ｍＬ容量の容器に回収した。
（６）回収後数分以内に０．９％（ｗ／ｖ）塩化アンモニウムと０．１％（ｗ／ｖ）炭酸水素カリウムとを含む溶血液で３０ｍＬまでメスアップ後、３００×ｇで１０分間、室温で遠心分離した。当該操作により上清に混入した赤血球が破壊され、分離回収した粒子の観察が良好になる。
（７）遠心後の上清を除去した後、粒子ペレットを、（１）に記載の方法で調製したＰＥＧ−ＢＳＡ（ＢＳＡとして０．１％（ｗ／ｖ））および３００ｍＭマンニトールを含む溶液３０ｍＬで再懸濁した。
（８）再懸濁液を３００×ｇで５分間、室温で遠心分離後、上清を除去し、再度、粒子ペレットを、ＰＥＧ−ＢＳＡ（ＢＳＡとして０．１％（ｗ／ｖ））および３００ｍＭマンニトールを含む溶液３０ｍＬで再懸濁した。当該操作は、血液成分を除去し、目的粒子（本実施例ではＣＴＣなどの腫瘍細胞（直径：１０から２５μｍ））を濃縮するための操作である。
（９）（８）で上清を除去した粒子懸濁液を、図１および図２に示す粒子回収装置１００に導入し、信号発生器１５０から電極１３１・１３２へ交流電圧（電圧２０Ｖｐｐ、周波数１ＭＨｚ、矩形波）を３分間印加することで粒子回収装置１００が有する凹部１６１に粒子を保持させた。図１および図２に示す粒子回収装置１００に備える粒子保持手段は図３に示す粒子保持手段１６０を用いた。具体的には、上面形状（平面図における形状）が一辺が３０μｍの正方形を４５度回転したひし形であり深さが４０μｍである凹部１６１を上下左右５０μｍの間隔（中心間距離）で格子状に配置し、当該ひし形の頂点同士が連通するよう幅１０μｍ、深さ４０μｍの連通部１６２を設けた。また粒子回収装置１００に備えるスペーサー１２０の厚さは１ｍｍとした。
（１０）粒子が保持された凹部数を計測し、全体の凹部数で除することで凹部への粒子の導入率を算出した。なお本実施例で用いた血液試料は健常人由来の試料であり、目的粒子である腫瘍細胞（ＣＴＣ）は含まれていない。従って、本実施例で凹部に保持された粒子は目的粒子以外の夾雑粒子（具体的には、前記（４）から（８）に示す前処理操作を行なっても残存する、白血球（直径：大半は６から１５μｍ）、赤血球（直径：７から８μｍ）、血小板（直径：１から４μｍ）、小胞、細胞デブリといった血液試料中に含まれる夾雑細胞）であり、算出した導入率は夾雑粒子の凹部への混入率となる。 Example 1
(1) Polyethylene glycol (mPEG-NHS) having a molecular weight of 5000, which has a methoxy group at one end and an N-hydrooxysuccinimid ester group at the other end, and bovine serum albumin (BSA) (300 mg, 0. 3 mmol) was dissolved in sodium hydrogen carbonate buffer (0.1 M, 15 mL), and the solution was stirred at room temperature for 3 hours to prepare BSA (PEG-BSA) to which polyethylene glycol was bound. At the time of preparation, the molar ratio of mPEG-NHS to BSA (mPEG-NHS / BSA) was set to 2. After the preparation, solution substitution with pure water was carried out for 3 days using a dialysis membrane having a molecular weight cut off of 10000.
(2) 2 g of imidazolidinyl urea, 2 g of polyethylene glycol (PEG) having a molecular weight of 6000, 100 mg of ethylenediaminetetraacetic acid (EDTA), and 600 mg of sodium chloride were dissolved in 100 mL of ultrapure water, and the obtained solution was used as a stabilizer. ..
(3) After collecting 3 mL of blood from a healthy person who gave informed consent to an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Co., Ltd.), 3 mL of the stabilizer prepared in (2) was added to the blood collection tube. The obtained solution was used as a preserved diluted blood sample.
(4) The diluted blood sample that has been preserved is left at room temperature for 10 minutes, 75 μL of a leukocyte / erythrocyte binder (RosetteSep, manufactured by StemCell Technologies) is added, and then a density gradient solution having a density of 1.086 g / mL is added in a tube. It was layered on top and centrifuged at 2000 xg for 10 minutes at room temperature.
(5) After centrifugation, the solution containing the fraction in which the target particles were located was collected in a container having a capacity of 50 mL.
(6) Within a few minutes after recovery, scalpel up to 30 mL with dissolved blood containing 0.9% (w / v) ammonium chloride and 0.1% (w / v) potassium hydrogen carbonate, and then add 10 at 300 xg. Centrifuge at room temperature for minutes. By this operation, the red blood cells mixed in the supernatant are destroyed, and the observation of the separated and collected particles is improved.
(7) After removing the supernatant after centrifugation, the particle pellets are 30 mL of a solution containing PEG-BSA (0.1% (w / v) as BSA) and 300 mM mannitol prepared by the method described in (1). Resuspended in.
(8) After centrifuging the resuspension at 300 × g for 5 minutes at room temperature, the supernatant was removed, and the particle pellets were again subjected to PEG-BSA (0.1% (w / v) as BSA) and. It was resuspended in 30 mL of solution containing 300 mM mannitol. This operation is an operation for removing blood components and concentrating target particles (tumor cells such as CTC (diameter: 10 to 25 μm) in this example).
(9) The particle suspension from which the supernatant was removed in (8) was introduced into the particle recovery device 100 shown in FIGS. 1 and 2, and the AC voltage (voltage 20 Vpp, frequency) was introduced from the signal generator 150 to the electrodes 131 and 132. The particles were held in the recess 161 of the particle recovery device 100 by applying 1 MHz) (rectangular wave) for 3 minutes. As the particle holding means provided in the particle collecting device 100 shown in FIGS. 1 and 2, the particle holding means 160 shown in FIG. 3 was used. Specifically, the top shape (shape in the plan view) is a rhombus formed by rotating a square having a side of 30 μm by 45 degrees, and the recesses 161 having a depth of 40 μm are formed in a grid pattern at intervals of 50 μm (distance between centers). A communication portion 162 having a width of 10 μm and a depth of 40 μm was provided so that the vertices of the rhombus could communicate with each other. The thickness of the spacer 120 provided in the particle recovery device 100 was set to 1 mm.
(10) The number of recesses in which the particles were held was measured and divided by the total number of recesses to calculate the introduction rate of the particles into the recesses. The blood sample used in this example is a sample derived from a healthy person and does not contain tumor cells (CTC), which is a target particle. Therefore, the particles held in the recesses in this embodiment are contaminated particles other than the target particles (specifically, leukocytes (diameter: most) that remain even after the pretreatment operations shown in (4) to (8) above. Is 6 to 15 μm), erythrocytes (diameter: 7 to 8 μm), platelets (diameter: 1 to 4 μm), vesicles, contaminating cells contained in blood samples such as cell debris), and the calculated introduction rate is that of contaminating particles. It is the mixing rate in the recess.

比較例１
実施例１（９）において、交流電圧を印加しない他は、実施例１と同様な方法で、（夾雑）粒子が保持された凹部数を計測し、全体の凹部数で除することで凹部への粒子の導入率（夾雑粒子の混入率）を算出した。 Comparative Example 1
In Example 1 (9), the number of recesses in which (contaminated) particles are held is measured by the same method as in Example 1 except that no AC voltage is applied, and the number of recesses is divided by the total number of recesses to reach the recesses. The introduction rate of particles (mixture rate of contaminant particles) was calculated.

比較例２
実施例１（９）において、粒子保持手段を連通部を設けない手段（具体的には、図３に示す粒子保持手段１６０のうち連通部１６２を除いた手段）とした他は、実施例１と同様な方法で、（夾雑）粒子が保持された凹部数を計測し、全体の凹部数で除することで凹部への粒子の導入率（夾雑粒子の混入率）を算出した。 Comparative Example 2
In Example 1 (9), except that the particle holding means is a means not provided with a communicating portion (specifically, a means of the particle holding means 160 shown in FIG. 3 excluding the communicating portion 162), the first embodiment The number of recesses in which (contamination) particles were retained was measured by the same method as in the above method, and the rate of introduction of particles into the recesses (rate of contamination of contaminant particles) was calculated by dividing by the total number of recesses.

実施例１ならびに比較例１および２での凹部への粒子の導入率（夾雑粒子の混入率）の結果をまとめて表１に示す。実施例１の条件での夾雑粒子の混入率１１．４％と低い結果だが、誘電泳動力を発生しない条件（比較例１）では、混入率５３．９％と高い結果となった。このことから、誘電泳動力によって、連通部へより強く夾雑粒子が引き寄せられ、夾雑粒子の凹部への混入率が減少したことがわかる。また粒子保持手段に連通部を設けない場合（比較例２）も、混入率が４４．５％と高い結果となり、連通部を設けないと、凹部に多く夾雑粒子が混入することがわかる。 Table 1 summarizes the results of the introduction rate of particles (mixing rate of contaminant particles) into the recesses in Example 1 and Comparative Examples 1 and 2. The mixing rate of the contaminant particles was as low as 11.4% under the condition of Example 1, but the mixing rate was as high as 53.9% under the condition of not generating dielectrophoretic force (Comparative Example 1). From this, it can be seen that the dielectrophoretic force attracted the contaminating particles more strongly to the communicating portion, and the mixing rate of the contaminating particles into the recesses decreased. Further, even when the communication portion is not provided in the particle holding means (Comparative Example 2), the mixing rate is as high as 44.5%, and it can be seen that if the communication portion is not provided, a large amount of contaminant particles are mixed in the recesses.

Figure 0006822006
実施例２
（１）目的粒子としてヒト乳がん細胞（ＳＫＢＲ３）を、５％ＣＯ２環境下、１０％ＦＢＳ（Ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍ）を含むＲＰＭＩ−１６４０培地を用いて３７℃で２４から９６時間培養後、０．２５％トリプシン／１ｍＭ ＥＤＴＡを用いて培地から剥離し、チューブに回収した。回収後、１０００ｒｐｍで５分間遠心した。
（２）（１）のＳＫＢＲ３細胞数が約１００から１５０個となる様に調整後、実施例１（２）の安定化剤を等量添加し、得られた溶液を保存処理した目的粒子懸濁液とした。
（３）保存処理した目的粒子懸濁液を室温で１０分放置し、１０００ｒｐｍで５分間、室温で遠心分離後、上清を除去し、粒子ペレットを、実施例１（１）のＰＥＧ−ＢＳＡ（ＢＳＡとして０．１％（ｗ／ｖ））および３００ｍＭマンニトールを含む溶液１ｍＬで再懸濁した。
（４）（３）で上清を除去した粒子懸濁液を用いる他は、実施例１（９）と同様の方法で粒子回収装置に導入した。
（５）ＰＥＧ−ＢＳＡ（ＢＳＡとして０．１％（ｗ／ｖ））および３００ｍＭマンニトールを含む溶液を、図１および図２に示す粒子回収装置１００（粒子保持手段１４０は図３に示す手段を使用）へ２回導入した。
（６）凹部に保持されたＳＫＢＲ３細胞数を計測し、粒子保持装置へ導入した目的粒子懸濁液中に含まれるＳＫＢＲ３細胞数で除することで、凹部へのＳＫＢＲ３細胞（目的粒子）の保持率を算出した。
Figure 0006822006
 Example 2
(1) Human breast cancer cells (SKBR3) as target particles were cultured in RPMI-1640 medium containing 10% FBS (Fetal bovine serum) in a 5% CO 2 environment at 37 ° C. for 24 to 96 hours, and then 0. It was stripped from the medium using 25% trypsin / 1 mM EDTA and collected in tubes. After recovery, it was centrifuged at 1000 rpm for 5 minutes.
(2) After adjusting the number of SKBR3 cells in (1) to about 100 to 150, an equal amount of the stabilizer of Example 1 (2) was added, and the obtained solution was preserved and treated. It was made into a turbid liquid.
(3) The target particle suspension that had been preserved was left at room temperature for 10 minutes, centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was removed, and the particle pellets were subjected to PEG-BSA of Example 1 (1). Resuspended in 1 mL of solution containing (0.1% (w / v) as BSA) and 300 mM mannitol.
(4) The particle suspension was introduced into the particle recovery apparatus in the same manner as in Example 1 (9) except that the particle suspension from which the supernatant was removed in (3) was used.
(5) A solution containing PEG-BSA (0.1% (w / v) as BSA) and 300 mM mannitol, and the particle recovery device 100 shown in FIGS. 1 and 2 (the particle holding means 140 is the means shown in FIG. 3). Introduced twice to use).
(6) Retention of SKBR3 cells (target particles) in the recesses by measuring the number of SKBR3 cells held in the recesses and dividing by the number of SKBR3 cells contained in the target particle suspension introduced into the particle retention device. The rate was calculated.

比較例３
（１）実施例２（５）において、粒子回収装置１００に備える粒子保持手段１６０を連通部を設けない手段（具体的には、図３に示す粒子保持手段１６０のうち連通部１６２を除いた手段）とした他は、実施例２と同様な方法で、凹部に保持されたＳＫＢＲ３細胞数を計測し、粒子保持装置へ導入した目的粒子懸濁液中に含まれるＳＫＢＲ３細胞数で除することで、凹部へのＳＫＢＲ３細胞（目的粒子）の保持率を算出した。 Comparative Example 3
(1) In Example 2 (5), the particle holding means 160 provided in the particle recovery device 100 is provided with no communicating portion (specifically, the communicating portion 162 is excluded from the particle holding means 160 shown in FIG. 3). The number of SKBR3 cells held in the recess is measured by the same method as in Example 2 and divided by the number of SKBR3 cells contained in the target particle suspension introduced into the particle holding device. Then, the retention rate of SKBR3 cells (target particles) in the recess was calculated.

実施例２および比較例３でのＳＫＢＲ３細胞（目的粒子）の保持率の結果を表２に示す。実施例２の条件での保持率は１００％と、比較例３の条件での保持率９９．０％と同様に高い保持率となっており、粒子保持手段に連通部を設けても目的粒子であるＳＫＢＲ３細胞の凹部への保持率に変化は見られなかった。 Table 2 shows the results of the retention rates of SKBR3 cells (target particles) in Example 2 and Comparative Example 3. The retention rate under the condition of Example 2 is 100%, which is as high as the retention rate of 99.0% under the condition of Comparative Example 3, and even if the particle retention means is provided with a communication portion, the target particles There was no change in the retention rate of SKBR3 cells in the recesses.

Figure 0006822006
Figure 0006822006

１００：粒子回収装置
１１０：凹部壁面部材
１１１：遮光部材
１１２：絶縁体
１１１ａ、１１２ａ：貫通部
１２０：スペーサ
１２１：導入口
１２２：排出口
１２３：貫通部
１３１、１３２：電極
１４０：導線
１５０：信号発生器
１６０：粒子保持手段
１６１：凹部
１６２：連通部
２００：目的粒子 100: Particle recovery device 110: Recessed wall surface member 111: Light-shielding member 112: Insulator 111a, 112a: Penetration part 120: Spacer 121: Introduction port 122: Discharge port 123: Penetration part 131, 132: Electrode 140: Lead wire 150: Signal Generator 160: Particle holding means 161: Recessed 162: Communication part 200: Target particle

Claims (6)

目的粒子を含む溶液を、当該目的粒子を保持可能な凹部を複数設けた粒子保持手段に導入する工程と、
前記凹部に前記目的粒子を保持させる工程とを含む、目的粒子の回収方法であって、
粒子保持手段に設ける凹部は、連通部を介して隣接した凹部と連通しており、前記連通部は、溶液中に含まれる目的粒子は保持できない一方、溶液中に含まれる当該目的粒子以外の夾雑粒子は保持可能であり、
前記凹部に目的粒子を保持させる工程を、誘電泳動力を用いて行ない、
前記連通部に夾雑粒子を、誘電泳動力を用いて保持させることを特徴とする、
前記回収方法。 A step of introducing a solution containing the target particles into a particle holding means provided with a plurality of recesses capable of holding the target particles, and a step of introducing the solution.
A method for recovering target particles, which comprises a step of holding the target particles in the recesses.
The recess provided in the particle holding means communicates with the adjacent recess via the communicating portion, and the communicating portion cannot hold the target particles contained in the solution, but is contaminated with particles other than the target particles contained in the solution. Particles can be retained and
The step of holding the target particles in the recess, the line of the stomach using a dielectrophoretic force,
It is characterized in that the contaminating particles are held in the communicating portion by using a dielectrophoretic force.
The collection method. 粒子保持手段に設ける凹部の上面形状が多角形であり、連通部を当該多角形の頂点のうちの少なくとも一つと隣接した凹部における当該多角形の頂点のうちの少なくとも一つとが連通するよう設けた、請求項１に記載の目的粒子の回収方法。 The upper surface shape of the recess provided in the particle holding means is a polygon, and the communication portion is provided so that at least one of the vertices of the polygon and at least one of the vertices of the polygon in the adjacent recess communicate with each other. , The method for recovering target particles according to claim 1. 目的粒子を含む溶液が血液試料であり、目的粒子が腫瘍細胞であり、夾雑粒子が白血球、赤血球、血小板、小胞、細胞デブリから選ばれるいずれか１つ以上である、請求項１または２に記載の目的粒子の回収方法。 According to claim 1 or 2, the solution containing the target particles is a blood sample, the target particles are tumor cells, and the contaminating particles are any one or more selected from leukocytes, erythrocytes, platelets, vesicles, and cell debris. The method for recovering the target particles according to the description. 請求項１から３のいずれか一項に記載の方法で回収した目的粒子を、当該目的粒子が有する特徴に基づき検出する、目的粒子の検出方法。 A method for detecting target particles, wherein the target particles recovered by the method according to any one of claims 1 to 3 are detected based on the characteristics of the target particles. 請求項４に記載の方法で検出した目的粒子を、当該目的粒子を採取する手段で取得する、目的粒子の取得方法。 A method for acquiring target particles, wherein the target particles detected by the method according to claim 4 are acquired by means for collecting the target particles. 溶液中に含まれる目的粒子を保持可能な凹部を複数設け、当該凹部は連通部を介して隣接した凹部と連通しており、当該連通部は前記目的粒子は保持できない一方、当該目的粒子以外の夾雑粒子は保持可能な、粒子保持手段を、
前記目的粒子および前記夾雑粒子を保持させるための誘電泳動力を発生させる電極で挟み込んでいることを特徴とする、目的粒子の回収装置。 A plurality of recesses capable of holding the target particles contained in the solution are provided, and the recesses communicate with the adjacent recesses via a communication portion, and the communication portion cannot hold the target particles, but other than the target particles. A particle holding means that can hold contaminant particles,
A device for recovering target particles, which is sandwiched between electrodes that generate a dielectrophoretic force for holding the target particles and the contaminant particles.
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