JP2013042689A - Cancer cell condensation filter - Google Patents
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- G—PHYSICS
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Abstract
Description
本発明は、血中循環癌細胞（Ｃｉｒｃｕｌａｔｉｎｇ Ｔｕｍｏｒ Ｃｅｌｌ、以下、場合により「ＣＴＣ」という。）を効率良く捕獲できるフィルターに関する。 The present invention relates to a filter that can efficiently capture circulating tumor cells (hereinafter referred to as “CTC”).
癌細胞濃縮の研究・臨床的意義は極めて大きく、血液中の癌細胞を濃縮することができれば、癌の診断に応用することができる。例えば、癌の予後および治療の最も重要な要因は、初診時および処置時における癌細胞の転移の有無である。癌細胞の初期の拡散が末梢血中に及んだ場合、ＣＴＣを検出することは、癌の病状進行を判断する有用な手段である。しかしながら、血液中には、赤血球や白血球などの血液成分が圧倒的に多く存在するため、極めて少量のＣＴＣの検出は困難である。 Research and clinical significance of cancer cell concentration is extremely great, and if cancer cells in blood can be concentrated, it can be applied to cancer diagnosis. For example, the most important factor in cancer prognosis and treatment is the presence or absence of cancer cell metastasis at the first visit and treatment. When the initial spread of cancer cells reaches the peripheral blood, detecting CTC is a useful means of determining the progression of the disease state of the cancer. However, since blood components such as red blood cells and white blood cells are overwhelmingly present in blood, it is difficult to detect a very small amount of CTC.
近年、パリレンを用いたフィルターを使用することで、少量のＣＴＣを効率的に検出する方法が提案されている（例えば、特許文献１を参照）。 In recent years, a method for efficiently detecting a small amount of CTC by using a filter using parylene has been proposed (see, for example, Patent Document 1).
転移が始まった一般的な癌患者の場合、血液中１００億個の血球細胞あたり、ＣＴＣはおよそ１個存在しているに過ぎない。このような極低濃度のＣＴＣを、高効率に捕獲することは非常に困難である。 For a typical cancer patient who has started metastasis, there is only about one CTC per 10 billion blood cells in the blood. It is very difficult to capture such an extremely low concentration CTC with high efficiency.
ＣＴＣは、血液中の血球細胞、例えば赤血球や白血球、あるいは血小板などに比べてサイズが一回り大きい。したがって、理論的には、機械的濾過法を適用してこれらの血球成分を除去し、ＣＴＣを濃縮することが可能である。しかしながら、白血球の中にはＣＴＣと同じ程度のサイズを有する細胞が存在する。このため、従来のフィルターでは、濃縮したＣＴＣに多数の白血球が混入し、ＣＴＣを高精度に検出することが困難な場合があった。 CTC is one size larger than blood cells in blood, such as red blood cells, white blood cells, or platelets. Therefore, theoretically, it is possible to apply these mechanical filtration methods to remove these blood cell components and concentrate the CTC. However, some leukocytes have the same size as CTC. For this reason, in the conventional filter, many white blood cells are mixed in the concentrated CTC, and it may be difficult to detect CTC with high accuracy.
したがって、本発明は、血球成分、特に白血球の残存を低減し、ＣＴＣを高い捕獲率で濃縮することができるフィルターを提供することを目的とする。 Therefore, an object of the present invention is to provide a filter that can reduce the residual of blood cell components, particularly leukocytes, and concentrate CTC at a high capture rate.
本発明は、複数の貫通孔が形成された金属基板からなり、貫通孔の開口形状は、楕円、円、長方形、正方形及び角丸長方形からなる群より選択される１種以上の形状である、癌細胞濃縮フィルターを提供する。 The present invention comprises a metal substrate in which a plurality of through holes are formed, and the opening shape of the through holes is one or more shapes selected from the group consisting of an ellipse, a circle, a rectangle, a square, and a rounded rectangle. A cancer cell concentration filter is provided.
上記本発明のフィルターによれば、血球成分の残存を低減し、ＣＴＣを高い捕獲率で濃縮することができる。ここで、残存とは、細胞が、フィルターを通過せずにフィルター上に残ることをいう。金属は加工性に優れているため、フィルターの加工精度を高めることができる。これにより、血球成分の残存を低減させ、ＣＴＣの高い捕獲率を実現することができる。 According to the filter of the present invention, residual blood cell components can be reduced and CTC can be concentrated at a high capture rate. Here, remaining means that cells remain on the filter without passing through the filter. Since metal is excellent in workability, the processing accuracy of the filter can be increased. Thereby, the residual of blood cell components can be reduced and a high capture rate of CTC can be realized.
また、金属はプラスチックなどの他の材料と比べて剛直であるため、外部から力が加わってもそのサイズや形状が維持される。このため、貫通孔よりも若干大きな血液成分（特に白血球）を変形させて通過させ、高精度の分離・濃縮が可能になると考えられる。白血球の中にはＣＴＣと同じ程度のサイズを有する細胞が存在し、サイズの違いだけではＣＴＣのみを高濃度で区別できない場合がある。しかしながら、白血球は癌細胞よりも変形能が大きいため、吸引や加圧などによる外部の力により、自分より小さな孔を通過することができ、ＣＴＣと分離することが可能となる。 In addition, since metal is rigid compared to other materials such as plastic, its size and shape are maintained even when external force is applied. For this reason, it is considered that blood components (particularly white blood cells) that are slightly larger than the through-holes are deformed and allowed to pass therethrough and can be separated and concentrated with high accuracy. Among leukocytes, there are cells having the same size as CTC, and there are cases where only CTC cannot be distinguished at a high concentration only by the difference in size. However, since leukocytes are more deformable than cancer cells, they can pass through smaller pores and can be separated from CTCs by an external force such as suction or pressurization.
また、上記の貫通孔の開口形状が楕円、円、長方形、正方形、角丸長方形及び多角形からなる群より選択される１種以上の形状であることにより、細胞が貫通孔に目詰まりしにくく、血液中の血球細胞の透過性を高め、ＣＴＣの高い捕獲率を実現することができる。 Moreover, since the opening shape of said through-hole is 1 or more types selected from the group which consists of an ellipse, a circle | round | yen, a rectangle, a square, a rounded rectangle, and a polygon, a cell does not clog a through-hole easily. The permeability of blood cells in the blood can be increased, and a high capture rate of CTC can be realized.
上記の金属基板は、金、銀、銅、アルミニウム、タングステン、ニッケル、クロム、ステンレス及びこれらの合金からなる群より選択される少なくとも１種の金属を主成分とするものであることが好ましい。 The metal substrate is preferably composed mainly of at least one metal selected from the group consisting of gold, silver, copper, aluminum, tungsten, nickel, chromium, stainless steel, and alloys thereof.
これらの金属は加工性に優れており、フィルターの加工精度を更に高めることができる。これにより、血球成分の残存を低減させ、更に高いＣＴＣの捕獲率を実現することができる。 These metals are excellent in workability and can further improve the processing accuracy of the filter. As a result, the remaining blood cell components can be reduced, and a higher CTC capture rate can be realized.
上記の貫通孔の開口形状は、短辺の長さが５．０〜１５．０μｍの長方形又は角丸長方形であることが好ましい。角丸長方形とは、２つの等しい長さの長辺と２つの半円形からなる形状である。貫通孔の開口形状が、上記のサイズの長方形又は角丸長方形であることにより、フィルターの目詰まりを更に効率よく防止することができる。 The opening shape of the through hole is preferably a rectangle having a short side length of 5.0 to 15.0 μm or a rounded rectangle. A rounded rectangle is a shape composed of two long sides of equal length and two semicircles. Since the opening shape of the through hole is a rectangle or a rounded rectangle of the above size, the clogging of the filter can be prevented more efficiently.
上記の貫通孔の平均開口率は０．１〜５０％であることが好ましい。平均開口率がこの範囲にあることにより、更に高いＣＴＣの捕獲率を実現することができる。 The average aperture ratio of the through holes is preferably 0.1 to 50%. When the average aperture ratio is within this range, a higher CTC capture rate can be realized.
上記のフィルターの厚さは、３〜１００μｍであることが好ましい。この範囲の膜圧のフィルターは、取り扱いが容易であり、精密加工にも適している。 The thickness of the filter is preferably 3 to 100 μm. A filter with a membrane pressure in this range is easy to handle and suitable for precision machining.
上記のフィルターは末梢血液、腹水又は胸水中の癌細胞用であることが好ましく、小細胞肺癌又は非小細胞肺癌の癌細胞用であることが好ましい。 The filter is preferably for cancer cells in peripheral blood, ascites or pleural effusion, and preferably for cancer cells of small cell lung cancer or non-small cell lung cancer.
上記のフィルターの構造は、これらの癌細胞の濃縮に特に適している。 The filter structure described above is particularly suitable for the enrichment of these cancer cells.
本発明によれば、白血球の残存を低減し、ＣＴＣを高い捕獲率で濃縮することが可能なフィルターを提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the filter which can reduce the residual of leukocytes and can concentrate CTC with a high capture rate can be provided.
以下、場合により図面を参照しながら、好適な実施形態を説明する。なお、図面の説明において同一要素には同一符号を付し、重複する説明を省略する。また、図面は理解を容易にするため一部を誇張して描いており、寸法比率は説明のものとは必ずしも一致しない。 Hereinafter, preferred embodiments will be described with reference to the drawings as the case may be. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. In addition, the drawings are exaggerated for easy understanding, and the dimensional ratios do not necessarily match those described.
図１（Ａ）は、フィルターの一実施形態を示す概略図である。フィルター１００は、複数の貫通孔１０が形成された基板２０からなる。貫通孔１０の開口形状は角丸長方形である。貫通孔１０の配置は、図１（Ａ）のような整列配置でもよく、列毎に配置がずれた千鳥配置でもよく、任意に配置されたランダム配置であってもよい。
FIG. 1A is a schematic view showing an embodiment of a filter. The
図１（Ｂ）は、上記実施形態のフィルターの貫通孔１０の上面図である。貫通孔１０の開口形状は、短辺がａ、長辺がｂである長方形の短辺に隣接して、半径がｃである２つの半円形が結合した形状である。一実施形態において、ａ、ｂ、ｃはそれぞれ８、２２及び４μｍである。
FIG. 1B is a top view of the
基板（フィルター）の材質は金属である。金属としては、金、銀等の貴金属、銅、アルミニウム、タングステン、ニッケル、クロム等の卑金属、及びこれらの金属の合金が例示できるがこれらに限定するものではない。金属は単体で用いてもよく、機能性を付与するために他の金属との合金又は金属の酸化物として用いてもよい。価格や入手の容易さの観点から、ニッケル、銅及びこれらを主成分とする金属を用いることが好ましい。ここで、主成分とは、上記基板を形成する材料のうち５０重量％以上を占める成分をいう。これらの金属にフォトリソグラフィーなどの方法を使って、貫通孔を形成することができる。 The material of the substrate (filter) is metal. Examples of the metal include, but are not limited to, noble metals such as gold and silver, base metals such as copper, aluminum, tungsten, nickel and chromium, and alloys of these metals. The metal may be used alone, or may be used as an alloy with another metal or an oxide of a metal in order to impart functionality. From the viewpoint of price and availability, it is preferable to use nickel, copper, and metals containing these as main components. Here, the main component refers to a component occupying 50% by weight or more of the material forming the substrate. Through holes can be formed in these metals using a method such as photolithography.
貫通孔の開口形状として、楕円、円、長方形、正方形、角丸長方形、多角形等が例示できる。効率良く癌細胞を捕獲できる観点からは、円、長方形又は角丸長方形が好ましい。また、フィルターの目詰まり防止の観点からは、長方形又は角丸長方形が特に好ましい。 Examples of the opening shape of the through hole include an ellipse, a circle, a rectangle, a square, a rounded rectangle, and a polygon. From the viewpoint of efficiently capturing cancer cells, a circle, rectangle, or rounded rectangle is preferable. Further, from the viewpoint of preventing clogging of the filter, a rectangle or a rounded rectangle is particularly preferable.
一般的なＣＴＣの大きさは直径１０μｍ以上である。ここで、細胞の直径とは、顕微鏡で観察した場合の細胞の輪郭上の任意の２点を結ぶ直線のうち最も長い直線の長さをいう。そこで、血球成分の透過性とＣＴＣの捕捉性能の観点から、貫通孔の平均孔径は、５〜１５μｍであることが好ましく、６〜１２μｍであることがより好ましく、７〜１０μｍであることが特に好ましい。本明細書において、開口形状が楕円、長方形、多角形等の円以外の形状における孔径とは、それぞれの貫通孔を通過できる球の直径の最大値とする。貫通孔の孔径は、例えば開口形状が長方形又は角丸長方形の場合には、その長方形又は角丸長方形の短辺の長さとなり、開口形状が多角形の場合には、その多角形の内接円の直径となる。開口形状が長方形や角丸長方形の場合、ＣＴＣや白血球が貫通孔に補足された場合であっても、開口部において、開口形状の長辺方向に隙間ができる。この隙間を通して液体が通過可能であるため、フィルターの目詰まりを防止することが可能になる。 A typical CTC has a diameter of 10 μm or more. Here, the diameter of a cell means the length of the longest straight line connecting two arbitrary points on the outline of the cell when observed with a microscope. Therefore, from the viewpoint of blood cell component permeability and CTC capture performance, the average pore diameter of the through holes is preferably 5 to 15 μm, more preferably 6 to 12 μm, and particularly preferably 7 to 10 μm. preferable. In this specification, the hole diameter when the opening shape is other than a circle such as an ellipse, rectangle, or polygon is the maximum value of the diameter of a sphere that can pass through each through hole. For example, when the opening shape is a rectangle or a rounded rectangle, the diameter of the through hole is the length of the short side of the rectangle or the rounded rectangle. The diameter of the circle. When the opening shape is a rectangle or a rounded rectangle, a gap is formed in the long side direction of the opening shape even when CTC or white blood cells are captured by the through-hole. Since the liquid can pass through this gap, it is possible to prevent clogging of the filter.
フィルターの貫通孔の平均開口率は０．１〜５０％が好ましく、０．５〜４０％がより好ましく、１〜３０％が特に好ましく、１〜１０％が最も好ましい。ここで、開口率とは、フィルター上の所定の領域において、当該領域の面積に対する貫通孔が占める面積をいう。平均開口率とは、フィルター全体の面積に対する貫通孔が占める面積をいう。平均開口率は、目詰まり防止の観点から、大きいほど好ましいが、５０％を超えると、フィルターの強度が低下したり、加工が困難になる場合がある。また０．１％より小さいと目詰まりを発生しやすくなるため、フィルターの癌細胞濃縮性能が低下する場合がある。 The average aperture ratio of the through holes of the filter is preferably 0.1 to 50%, more preferably 0.5 to 40%, particularly preferably 1 to 30%, and most preferably 1 to 10%. Here, the aperture ratio refers to the area occupied by the through hole with respect to the area of the predetermined area on the filter. The average aperture ratio refers to the area occupied by the through holes with respect to the area of the entire filter. The average aperture ratio is preferably as large as possible from the viewpoint of preventing clogging. However, if it exceeds 50%, the strength of the filter may be lowered, or processing may be difficult. Moreover, since it will become easy to generate | occur | produce clogging if it is less than 0.1%, the cancer cell concentration performance of a filter may fall.
フィルターの厚さは３〜１００μｍであることが好ましく、５〜５０μｍであることがより好ましく、１０〜３０μｍであることが特に好ましい。基板の厚さが３μｍより薄いと、フィルターの強度が低下し、取り扱いが困難になる場合がある。また、基板の厚さが１００μｍを超えると、必要以上の材料を消費したり、加工に長時間かかったりするため、コスト的に不利になったり、精密加工そのものが困難になる場合がある。 The thickness of the filter is preferably 3 to 100 μm, more preferably 5 to 50 μm, and particularly preferably 10 to 30 μm. If the thickness of the substrate is less than 3 μm, the strength of the filter may be reduced and handling may be difficult. On the other hand, if the thickness of the substrate exceeds 100 μm, more materials than necessary are consumed and processing takes a long time, which may be disadvantageous in cost and may make precision processing itself difficult.
続いて、本実施形態のフィルターの製造方法を説明する。フィルターの製造方法は特に制限されず、例えば、フォトリソグラフィー法を利用して、エッチングや電気めっきを施すことによる製造方法が挙げられる。フォトリソグラフィー法等を利用した製造方法を以下に説明する。まず、ステンレス等からなる支持体上に感光性のレジストフィルム（感光層）を貼り合わせる。次に、フィルターの貫通孔の開口形状のパターンを有するマスクを感光層上に固定する。続いて、マスク上から光（活性光線）を照射する。光照射後、感光層上に支持体がある場合にはこれを除去し、アルカリ性水溶液、水系現像液及び有機溶剤等の現像液によるウエット現像、又はドライ現像等で未露光部を除去することによって現像し、レジストパターンを形成する。続いて、現像されたレジストパターンをマスクとして、マスクされずに露出している基板上にめっきを行う。めっきの方法としては、例えば、銅めっき、はんだめっき、ニッケルめっき、金めっきなどが挙げられる。めっき処理後、めっき層を支持体及び感光層から剥離すると、めっき層が得られる。このめっき層がフィルターである。得られたフィルターの表面を粗化処理してもよい。粗化処理の方法としては、酸性又は塩基性水溶液等による化学エッチング、サンドブラスト等の物理的処理等が挙げられる。 Then, the manufacturing method of the filter of this embodiment is demonstrated. The manufacturing method of the filter is not particularly limited, and examples thereof include a manufacturing method by performing etching or electroplating using a photolithography method. A manufacturing method using a photolithography method or the like will be described below. First, a photosensitive resist film (photosensitive layer) is bonded onto a support made of stainless steel or the like. Next, a mask having a pattern of the opening shape of the through hole of the filter is fixed on the photosensitive layer. Subsequently, light (active light) is irradiated from above the mask. After light irradiation, if there is a support on the photosensitive layer, it is removed and the unexposed part is removed by wet development with a developer such as an alkaline aqueous solution, aqueous developer and organic solvent, or dry development. Development is performed to form a resist pattern. Subsequently, plating is performed on the exposed substrate without being masked using the developed resist pattern as a mask. Examples of the plating method include copper plating, solder plating, nickel plating, and gold plating. After the plating treatment, the plating layer is obtained by peeling the plating layer from the support and the photosensitive layer. This plating layer is a filter. You may roughen the surface of the obtained filter. Examples of the roughening treatment include chemical treatment with an acidic or basic aqueous solution, physical treatment such as sandblasting, and the like.
一実施形態において、フィルターを流路内に組み込み、流路に血液を導入することによりＣＴＣを濃縮することができる。流路への血液の導入には、流路の入り口方向からの加圧を用いる方法、流路の出口方向からの減圧を用いる方法、ぺリスタルティックポンプを使用する方法等が例示できる。また、使用するフィルターの面積は、例えば１ｍＬの血液からＣＴＣを濃縮する場合、１．０〜１０．０ｃｍ２が適している。 In one embodiment, the CTC can be concentrated by incorporating a filter in the channel and introducing blood into the channel. Examples of the introduction of blood into the flow path include a method using pressurization from the flow path entrance direction, a method using pressure reduction from the flow path exit direction, and a method using a peristaltic pump. In addition, the area of the filter to be used is suitably 1.0 to 10.0 cm 2 when CTC is concentrated from 1 mL of blood, for example.
（実施例１）
（フィルターの作製）
レジストフィルム（フォテックＨ−Ｙ９２０、２０μｍ厚、日立化成工業株式会社製）を１０ｍｍ角のステンレス板（ＳＵＳ３０４、仕上げ３／４Ｈ、厚さ１００μｍ、日新製鋼（株）製）の片面に貼り合わせた。貼り合わせの条件は、ロール温度１０５℃、圧力０．５ＭＰａ、ラインスピード１ｍ／分であった。次いで、光の透過部が８×３０μｍの角丸長方形で、そのピッチが短軸方向６０μｍ、長軸方向６０μｍになるように形成したネガフィルムを、ステンレス板のレジストフィルムを貼り合わせた面上に静置した。この角丸長方形は、図１（Ｂ）に示す形状であり、図１（Ｂ）におけるａ、ｂ及びｃがそれぞれ８、２２及び４μｍである形状である。ここで、長軸方向とは、角丸長方形の長辺方向を指し、短軸方向とは、長軸方向に直交する方向を指す。本実施例においては、同一の方向を向いた角丸長方形が、長軸方向及び短軸方向に一定のピッチで整列したネガフィルムを用いた。
Example 1
(Preparation of filter)
A resist film (Photec H-Y920, 20 μm thickness, manufactured by Hitachi Chemical Co., Ltd.) was bonded to one side of a 10 mm square stainless steel plate (SUS304, finishing 3 / 4H,
続いて、６００ｍｍＨｇ以下の真空下において、ネガフィルムを載置したステンレス板の上部から、紫外線照射装置を用いて、紫外線を１００ｍＪ／ｃｍ２照射した。続いて、１％炭酸ナトリウム水溶液で現像を行い、ステンレス板の上にレジスト層を形成した。このレジストフィルム付き基材に、ｐＨが４．５になるように調整したニッケルめっき液中５０℃で約２０分間ニッケルめっきを実施した。ニッケルめっき液の組成を表１に示す。 Subsequently, under a vacuum of 600 mmHg or less, 100 mJ / cm 2 of ultraviolet rays were irradiated from above the stainless steel plate on which the negative film was placed using an ultraviolet irradiation device. Subsequently, development was performed with a 1% aqueous sodium carbonate solution to form a resist layer on the stainless steel plate. The substrate with the resist film was subjected to nickel plating at 50 ° C. for about 20 minutes in a nickel plating solution adjusted to have a pH of 4.5. Table 1 shows the composition of the nickel plating solution.
得られたニッケルめっき層を基材のステンレス板から剥離し、レジストフィルムをアルカリ剥離液で剥離し、厚さが２０μｍ、平均開口率が６．７％である実施例１のフィルターを得た。 The obtained nickel plating layer was peeled off from the stainless steel plate of the base material, and the resist film was peeled off with an alkali peeling solution to obtain a filter of Example 1 having a thickness of 20 μm and an average aperture ratio of 6.7%.
（実施例２）
直径８μｍの円形の光の透過部が、１００００個／ｃｍ２の密度で等間隔に配置するように形成したネガフィルムを使用してレジスト層を形成した以外は実施例１と同様にして、厚さが２０μｍ、平均開口率が１．４％である実施例２のフィルターを作製した。
(Example 2)
Except that a resist layer was formed using a negative film formed so that circular light transmission portions having a diameter of 8 μm were arranged at equal intervals at a density of 10,000 / cm 2 , A filter of Example 2 having a thickness of 20 μm and an average aperture ratio of 1.4% was produced.
（小細胞肺癌細胞株の調製）
小細胞肺癌細胞株であるＮＣＩ−Ｈ６９細胞を１０％ウシ胎児血清（ＦＢＳ）を含むＲＰＭＩ−１６４０培地にて、３７℃・５％ＣＯ２条件下で静置培養した。トリプシン処理により培養皿から細胞を剥離させて回収し、リン酸緩衝液（Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ、ＰＢＳ）を用いて洗浄した後に、１０μＭ ＣｅｌｌＴｒａｃｋｅｒ Ｒｅｄ ＣＭＴＰＸ（ライフテクノロジーズジャパン株式会社）にて３７℃、３０分間静置させる事でＮＣＩ−Ｈ６９細胞を染色した。その後、ＰＢＳにて洗浄し、トリプシン処理にて３７℃にて３分間静置させることで細胞塊を解離させた。その後、培地にてトリプシン処理を停止させ、ＰＢＳにて洗浄後、２ｍＭＥＤＴＡ及び０．５％ウシ血清アルブミン（ＢＳＡ）を含むＰＢＳ（以下「２ｍＭ ＥＤＴＡ−０．５％ ＢＳＡ−ＰＢＳ」という。）に懸濁した。
(Preparation of small cell lung cancer cell lines)
NCI-H69 cells, which are small cell lung cancer cell lines, were statically cultured in an RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37 ° C. and 5% CO 2 . Cells were detached from the culture dish by trypsin treatment and recovered, washed with phosphate buffered saline (PBS), and then washed with 10 μM CellTracker Red CMTPX (Life Technologies Japan, Inc.) at 37 ° C. for 30 minutes. NCI-H69 cells were stained by allowing to stand. Thereafter, the cells were washed with PBS and allowed to stand at 37 ° C. for 3 minutes by trypsin treatment to dissociate the cell mass. Thereafter, the trypsin treatment is stopped in the medium, washed with PBS, and then washed with PBS containing 2 mM EDTA and 0.5% bovine serum albumin (BSA) (hereinafter referred to as “2 mM EDTA-0.5% BSA-PBS”). Suspended.
（血液サンプル中のＣＴＣの濃縮）
実施例のフィルターをセットしたＣＴＣ回収装置を組み立てた。ＣＴＣ回収装置は、血液サンプルや試薬を導入するための流路を備えており、流路の入り口は、シリンジを加工して作製したリザーバーに接続した。このリザーバーに、血液サンプルや試薬を順次投入していくことで、ＣＴＣの捕捉、染色、洗浄などの操作を連続的に容易に行えるようにした。
(Concentration of CTC in blood sample)
A CTC recovery device in which the filter of the example was set was assembled. The CTC recovery device includes a flow channel for introducing a blood sample and a reagent, and the inlet of the flow channel is connected to a reservoir produced by processing a syringe. By sequentially putting blood samples and reagents into this reservoir, operations such as CTC capture, staining, and washing can be performed easily and continuously.
ＣＴＣ回収装置に血液サンプルを導入して癌細胞を濃縮した。血液サンプルとして、エチレンジアミン四酢酸ジナトリウム（ＥＤＴＡ）含有真空採血管に採血した健常者血液に、血液１ｍＬあたり１０００個の癌細胞を混合させたサンプルを用いた。癌細胞としては、上記のヒト小細胞肺癌細胞株ＮＣＩ−Ｈ６９を使用した。 A blood sample was introduced into the CTC recovery device to concentrate the cancer cells. As a blood sample, a sample obtained by mixing 1000 cancer cells per mL of blood into blood of a healthy person collected in a vacuum blood collection tube containing disodium ethylenediaminetetraacetate (EDTA) was used. As a cancer cell, the above-mentioned human small cell lung cancer cell line NCI-H69 was used.
まず、リザーバーに、２ｍＭ ＥＤＴＡ−０．５％ ＢＳＡ−ＰＢＳ １ｍＬを導入し、フィルター上を満たした。続いて、ペリスタルティックポンプを使用して流速２００μＬ／分で送液を開始した。約５分後、リザーバーに１ｍＬの血液サンプルを導入した。これにより、癌細胞をフィルター上に捕捉した。 First, 1 mL of 2 mM EDTA-0.5% BSA-PBS was introduced into the reservoir to fill the filter. Subsequently, liquid feeding was started at a flow rate of 200 μL / min using a peristaltic pump. After about 5 minutes, a 1 mL blood sample was introduced into the reservoir. This captured the cancer cells on the filter.
５分後、血球成分を洗浄するために、リザーバーに２ｍＬの２ｍＭ ＥＤＴＡ−０．５％ ＢＳＡ−ＰＢＳを導入した。 After 5 minutes, 2 mL of 2 mM EDTA-0.5% BSA-PBS was introduced into the reservoir to wash blood cell components.
さらに１０分後、ポンプ流速を２０μＬ／分に変更し、リザーバーに６００μＬの細胞染色液（Ｈｏｅｃｈｓｔ ３３３４２ ０．５μｇ／ｍＬ）を導入し、フィルター上の癌細胞又は白血球を蛍光染色した。フィルター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２ｍＭ ＥＤＴＡ−０．５％ ＢＳＡ−ＰＢＳを導入して細胞の洗浄を行った。 After another 10 minutes, the pump flow rate was changed to 20 μL / min, 600 μL of cell staining solution (Hoechst 33342 0.5 μg / mL) was introduced into the reservoir, and the cancer cells or leukocytes on the filter were fluorescently stained. The cells captured on the filter were stained for 30 minutes, and then 1 mL of 2 mM EDTA-0.5% BSA-PBS was introduced into the reservoir to wash the cells.
続いて、コンピューター制御式電動ステージ及び冷却デジタルカメラ（ＤＰ７０、オリンパス株式会社）を装備した蛍光顕微鏡（ＢＸ６１、オリンパス株式会社）を使用してフィルターを観察し、フィルター上の癌細胞及び白血球の数をカウントした。Ｈｏｅｃｈｓｔ ３３３４２及びＣｅｌｌＴｒａｃｋｅｒ Ｒｅｄ ＣＭＴＰＸ由来の蛍光を観察するために、それぞれＷＵ及びＷＩＧフィルター（オリンパス株式会社）を用いて画像を取得した。画像取得及び解析ソフトウェアにはＬｕｍｉｎａ Ｖｉｓｉｏｎ（三谷商事株式会社）を用いた。結果を表２に示す。実験は各３回行い、結果は平均値±標準偏差で表記した。癌細胞回収率（捕獲率）は、次の計算式により計算した。
癌細胞回収率（％）＝フィルター上に回収された癌細胞数／血液サンプルに混合した癌細胞数×１００
Subsequently, the filter was observed using a fluorescence microscope (BX61, Olympus Corporation) equipped with a computer-controlled electric stage and a cooling digital camera (DP70, Olympus Corporation), and the number of cancer cells and white blood cells on the filter was determined. I counted. In order to observe fluorescence derived from Hoechst 33342 and CellTracker Red CMTPX, images were acquired using WU and WIG filters (Olympus Corporation), respectively. Lumina Vision (Mitani Corporation) was used as image acquisition and analysis software. The results are shown in Table 2. Each experiment was performed three times, and the results were expressed as mean ± standard deviation. The cancer cell recovery rate (capture rate) was calculated by the following formula.
Cancer cell recovery rate (%) = number of cancer cells recovered on filter / number of cancer cells mixed in blood sample × 100
適切な開口形状を有する金属フィルターを用いることによって、癌細胞に混入して回収される白血球の数が減少した。また癌細胞の回収率も向上した。ＮＣＩ−Ｈ６９株以外の癌細胞を用いても同様の結果が得られた。円形よりも角丸長方形の開口形状のフィルターのほうが、残存白血球数がより少なく、癌細胞の回収率も高かった。 By using a metal filter having an appropriate opening shape, the number of leukocytes collected and collected in cancer cells was reduced. Also, the recovery rate of cancer cells was improved. Similar results were obtained using cancer cells other than the NCI-H69 strain. The rounded rectangular filter had a smaller number of residual white blood cells and a higher recovery rate of cancer cells than the round shape.
１０…貫通孔、２０…基板、３０…面、１００…フィルター、ａ…短辺、ｂ…長辺、ｃ…半径。
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| JP (1) | JP5854456B2 (en) |
| CN (1) | CN202730123U (en) |
| DE (1) | DE202012003212U1 (en) |
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| JP5854456B2 (en) | 2016-02-09 |
| DE202012003212U1 (en) | 2012-07-11 |
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