JP2010081915A - Cell recovery magnetic stand and cell recovery kit - Google Patents
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Abstract
Description
本発明は、ガン研究、移植医学、細菌学、プロテオーム、臨床化学等の細胞の分離・精製技術の分野に係り、検体中に含まれる細胞を補足した磁気ビーズを回収するための磁気スタンドおよびキットに関する。 The present invention relates to the field of cell separation / purification technology such as cancer research, transplantation medicine, bacteriology, proteome, clinical chemistry, etc., and a magnetic stand and kit for collecting magnetic beads supplemented with cells contained in a specimen About.
現在、タンパク質、核酸および細胞などの特定の物質を特異的若しくは非特異的に捕捉するように修飾した、磁気ビーズと称される磁性粒子が生化学の分野で広く用いられている。 At present, magnetic particles called magnetic beads modified so as to specifically or non-specifically capture specific substances such as proteins, nucleic acids and cells are widely used in the field of biochemistry.
また、血液、体液、糞便などから特定の細胞を回収後、回収された細胞から核酸を抽出し、一塩基多型などを調べることにより、ガン検診、薬剤の効果予想などを行うことが研究されている。例えば特許文献１では糞便中から上皮系の細胞を回収する方法が提案されている。 In addition, after collecting specific cells from blood, body fluids, stool, etc., nucleic acid is extracted from the collected cells and examined for single nucleotide polymorphisms, etc., to conduct cancer screenings, predict drug effects, etc. ing. For example, Patent Document 1 proposes a method for recovering epithelial cells from feces.
磁気ビーズは遠心分離等の煩雑な操作を伴わずに、スタンドに磁石が内包された磁気スタンドにより簡便に分離・捕集することができる。これまで、様々な磁気スタンドが考案されている。例えば特許文献２には、前方の弓状のアームと、背板に設けた溝により容器を保持し、背板内に埋め込んだ平板状の永久磁石により磁性粒子を捕集する磁気スタンドが開示されている。 The magnetic beads can be easily separated and collected by a magnetic stand in which a magnet is included in the stand without complicated operations such as centrifugation. Various magnetic stands have been devised so far. For example, Patent Document 2 discloses a magnetic stand that holds a container by a front arcuate arm and a groove provided in a back plate and collects magnetic particles by a flat permanent magnet embedded in the back plate. ing.
磁気スタンドの磁石部には希土類磁石が広く用いられる。例えば特許文献３では表面磁束密度が０.３５Ｔ以上である。また短時間に磁気分離を行う特許文献４では１.８Ｔの高強度の磁界を発現する磁石部をもつものが提案されている。 Rare earth magnets are widely used for the magnet part of the magnetic stand. For example, in Patent Document 3, the surface magnetic flux density is 0.35 T or more. Further, Patent Document 4 that performs magnetic separation in a short time proposes a magnet having a magnetic part that expresses a high-intensity magnetic field of 1.8 T.
ところで、細胞を用いて行われる検査では細胞を生きたまま回収することが望まれる。しかし、例えば特許文献５のように磁気ビーズを用いての細胞回収では細胞死が起きることが知られている。 By the way, in a test performed using cells, it is desired to collect the cells alive. However, it is known that cell death occurs in cell recovery using magnetic beads as in Patent Document 5, for example.
さらに、近年医療の現場では患者のそばでおこなう臨床検査として定義されるＰＯＣ検査への要求が高まっている。そのため検査の短時間化が求められており、目的細胞の回収、つまり磁気スタンドによる磁気ビーズの捕集時間の短縮が求められている。 Furthermore, in recent years, there has been an increasing demand for a POC test defined as a clinical test performed by a patient in the medical field. Therefore, it is required to shorten the inspection time, and it is required to collect target cells, that is, to shorten the time for collecting magnetic beads by a magnetic stand.
従って、本発明の目的は検体中の目的細胞を細胞死を抑制しつつ、短時間で回収することができる細胞回収用磁気スタンドを提供することである。 Accordingly, an object of the present invention is to provide a magnetic stand for cell recovery that can recover target cells in a specimen in a short time while suppressing cell death.
本発明の細胞回収用磁気スタンドは、容器内の検体中に分散した磁気ビーズを捕集するための磁気スタンドであって、磁気ビーズを捕集するための外部磁界の発生源として多極磁石を用い、前記多極磁石の表面磁束密度が０.３０Ｔ以下であることを特徴とする。かかる構成により、細胞死を抑制できる。また、表面磁束密度を低くしても多極磁石を用いることにより短時間に細胞を回収することが可能である。 The magnetic stand for cell recovery of the present invention is a magnetic stand for collecting magnetic beads dispersed in a specimen in a container, and a multipolar magnet is used as a source of an external magnetic field for collecting the magnetic beads. The multi-pole magnet has a surface magnetic flux density of 0.30 T or less. With this configuration, cell death can be suppressed. Moreover, even if the surface magnetic flux density is lowered, cells can be collected in a short time by using a multipolar magnet.
また、前記細胞回収用磁気スタンドにおいて、前記多極磁石の表面磁束密度が０.２１Ｔ以上であることが好ましい。多極磁石において０.２１Ｔ以上とすれば、広く用いられている表面磁束密度が０.３５Ｔ〜０.４０Ｔの平板上の磁石部から構成される磁気スタンドと同等、もしくはより短時間で細胞回収が行えるので好ましい。 In the magnetic stand for cell recovery, the surface magnetic flux density of the multipolar magnet is preferably 0.21 T or more. If it is 0.21 T or more in a multipolar magnet, cell recovery can be performed in a time equivalent to or shorter than that of a magnetic stand composed of a magnet part on a flat plate having a surface magnetic flux density of 0.35 T to 0.40 T. Is preferable.
さらに、前記細胞回収用磁気スタンドにおいて、前記多極磁石が４極以上の磁極を有することが好ましい。４極以上の磁極を設けることによって、それにより磁気ビーズが分散して捕集されるため、細胞の壊死を抑制することができる。 Furthermore, in the magnetic stand for cell recovery, it is preferable that the multipolar magnet has four or more magnetic poles. By providing four or more magnetic poles, magnetic beads are dispersed and collected thereby, so that cell necrosis can be suppressed.
さらに、前記細胞回収用磁気スタンドにおいて、前記多極磁石の形状がリング状であることが好ましい。生化学や分子生物学分野で広く用いられている容器の形状は円筒型であり、多極磁石の形状がリング状であることにより容器と密着でき、磁気捕集を短時間で出来好ましい。すなわちリング状磁石を用いれば、その内側（穴部）に円筒状または円錐状の容器を密接する状態で収容できるため、効率よく磁気ビーズを回収できる。 Furthermore, in the magnetic stand for cell recovery, it is preferable that the multipolar magnet has a ring shape. The shape of the container widely used in the fields of biochemistry and molecular biology is a cylindrical shape, and since the shape of the multipolar magnet is a ring shape, the container can be brought into close contact with the container and magnetic collection can be performed in a short time. That is, if a ring-shaped magnet is used, a cylindrical or conical container can be accommodated in close contact with the inner side (hole), so that magnetic beads can be efficiently recovered.
前記細胞回収用磁気スタンドにおいて、前記検体が、血液、血液の成分、糞便、糞便の希釈液、組織片の溶解液のいずれかであることが好ましい。多極磁石を用いた磁気スタンドは、血液、血液の成分、糞便、糞便の希釈液、組織片の溶解液などの粘度が高い検体を用いる場合に好適である。 In the cell stand for cell recovery, it is preferable that the specimen is any one of blood, blood components, stool, stool diluent, and tissue fragment lysate. A magnetic stand using a multipolar magnet is suitable when a specimen having a high viscosity such as blood, blood components, stool, stool diluent, or tissue fragment lysate is used.
本発明の細胞回収用キットは、容量が１５ｍＬ以上である容器と、前記いずれかの磁気スタンドとを備えていることを特徴とする。多極磁石を用いた磁気スタンドを備えた構成は、１５ｍＬ以上の大容量の容器中から目的細胞を回収する場合に好適である。 The cell collection kit of the present invention comprises a container having a capacity of 15 mL or more and any one of the magnetic stands. A configuration including a magnetic stand using a multipolar magnet is suitable for collecting target cells from a large-capacity container of 15 mL or more.
本発明の磁気スタンドを用いることにより目的細胞の細胞死を抑制しつつ、短時間で磁気ビーズを回収することができる。 By using the magnetic stand of the present invention, magnetic beads can be recovered in a short time while suppressing cell death of the target cells.
（１）磁気スタンド
本発明に係る磁気スタンドは、容器内の検体中に分散した磁気ビーズを捕集するための磁気スタンドであり、磁気ビーズを捕集するための外部磁界の発生源として多極磁石を用いる。多極磁石を用いていれば形状などが特に限定されるものではないが、多極磁石および、それを埋め込んだ磁気スタンドの形状は、例えばリング状（円筒形）、円錐形であることが好ましい。磁気分離用に広く普及している容器は通常円筒形であるため、前記形状を採用することで磁石等の表面と容器外壁が密着でき、磁気ビーズの捕集が短時間に行える。このうち、容器を保持する形に成形された樹脂や金属などに多極磁石が埋め込まれた構造は、容器の保持が容易であり好ましい。また、容器の保持を爪で行う構造になっていることも好ましい。さらに、容器を保持する部分が複数個一体化された構造であっても構わない。また、多極磁石が露出した構造を用いることもできるし、容器を保持する部分が一つである構造を用いることもできる。
(1) Magnetic stand A magnetic stand according to the present invention is a magnetic stand for collecting magnetic beads dispersed in a specimen in a container, and is a multipole as a source of an external magnetic field for collecting magnetic beads. Use a magnet. The shape is not particularly limited as long as a multipolar magnet is used, but the shape of the multipole magnet and the magnetic stand in which it is embedded is preferably, for example, a ring shape (cylindrical shape) or a conical shape. . Since the container widely used for magnetic separation is usually cylindrical, by adopting the shape, the surface of the magnet or the like and the outer wall of the container can be in close contact, and the magnetic beads can be collected in a short time. Of these, a structure in which a multipolar magnet is embedded in a resin or metal molded to hold the container is preferable because the container can be easily held. It is also preferable that the container is held by a nail. Furthermore, a structure in which a plurality of portions for holding the container are integrated may be used. In addition, a structure in which the multipolar magnet is exposed can be used, and a structure in which the container is held by one part can also be used.
多極磁石の表面磁束密度は０.３０Ｔ以下である。但し、磁気分離能発現のために、０Tは含まない。磁石の表面磁束密度が０.３０Ｔ以下であれば細胞死を起こさずに細胞が回収できる。また、磁石の表面磁束密度が０.２１Ｔ以上（すなわち０.２１Ｔ〜０.３０Ｔ）が好ましい。磁気ビーズを捕集するための外部磁界の発生源として用いる多極磁石の表面磁束密度が０.２１Ｔ以上であれば、広く用いられている表面磁束密度が０.３５Ｔ〜０.４０Ｔの平板上の磁石部から構成される磁気スタンドと同等、もしくはより短時間で細胞回収が行うことができる。多極磁石は一体成形を行った成形体に多極着磁を行っても、複数個の磁石により多極構造を組むように配置しても構わない。この場合、磁石の形状または複数個の磁石の配置は、円筒形が好ましい。円筒形または円筒形状に配置された磁石の磁極の向き、すなわち着磁形態としては、例えば、ラジアル異方性や極異方性の形態のものを適用することができる。また、短時間で磁気ビーズを回収する観点から、多極磁石は４極以上の磁極を有することが好ましい。磁石の種類は限定しないが、例えばネオジウム鉄ホウ素磁石、サマリウムコバルト磁石などの希土類磁石や、成形性に優れたボンド磁石を使用できる。また、多極磁石として電磁石を用いることもできるが、簡易な磁気スタンドを構成するためには、永久磁石がより好ましい。なお、本発明に係る磁気スタンドおよびキットは細胞回収用のみならず、磁気ビーズを回収する磁気スタンドおよびキットとして広く適用できるものである。 The surface magnetic flux density of the multipolar magnet is 0.30 T or less. However, 0T is not included for the magnetic separation ability expression. If the surface magnetic flux density of the magnet is 0.30 T or less, the cells can be recovered without causing cell death. Further, the surface magnetic flux density of the magnet is preferably 0.21 T or more (that is, 0.21 T to 0.30 T). If the surface magnetic flux density of the multipolar magnet used as the source of the external magnetic field for collecting the magnetic beads is 0.21 T or more, the widely used surface magnetic flux density is from 0.35 T to 0.40 T on the flat plate. Cell recovery can be performed in the same or shorter time than a magnetic stand composed of the magnet part. The multipolar magnet may be arranged so as to form a multipolar structure with a plurality of magnets, even if the molded body that has been integrally molded is subjected to multipolar magnetization. In this case, the shape of the magnet or the arrangement of the plurality of magnets is preferably cylindrical. As the direction of the magnetic pole of the magnet arranged in a cylindrical shape or a cylindrical shape, that is, a magnetized form, for example, a radial anisotropic or polar anisotropic form can be applied. From the viewpoint of collecting magnetic beads in a short time, the multipolar magnet preferably has four or more magnetic poles. Although the kind of magnet is not limited, For example, rare earth magnets, such as a neodymium iron boron magnet and a samarium cobalt magnet, and a bond magnet excellent in moldability can be used. Moreover, although an electromagnet can be used as a multipolar magnet, a permanent magnet is more preferable for constituting a simple magnetic stand. The magnetic stand and kit according to the present invention can be widely applied not only for cell recovery but also as a magnetic stand and kit for recovering magnetic beads.
（２）容器およびキット
液体の検体が入っている容器の容量は特に限定するものではないが、例えば従来の磁気スタンドでは磁気ビーズの捕集に長時間を要していた、容量が１５ｍＬ以上、さらには５０ｍＬ以上、１００ｍＬ以上の遠沈管、ビーカー等の容器を用いることが出来る。本発明の磁気スタンドを用いることで短時間に効率よく細胞の回収をすることができる。また、容器の形状は円筒型若しくは円錐型であることが好ましい。かかる形状の容器は、円筒形の磁石や円弧形の磁石を円状に配置した磁石集合体と、隙間無く密着することが可能であり、好ましい。中央に検体が入らないデッドゾーンを有する容器、すなわち検体が入る部分が円環状になっている容器を用いると、検体が円筒形の磁石や円弧形の磁石を円状に配置した磁石集合体に近接した位置に配置されることになるので、回収効率がいっそう向上する。また円筒形（カップ状）の容器を用いるとともに、その中央に棒状のダミー部材を挿入するキットまたは方法によっても、同様に回収効率を向上させることができる。なお、かかる構成は、磁石の表面磁束密度の大きさに関係なく、磁気ビーズの回収キット、回収方法として広く適用できる。
(2) The capacity of the container and the container containing the liquid specimen of the kit is not particularly limited. For example, in the conventional magnetic stand, it took a long time to collect the magnetic beads, the capacity is 15 mL or more, Furthermore, containers such as centrifuge tubes and beakers of 50 mL or more and 100 mL or more can be used. By using the magnetic stand of the present invention, cells can be efficiently collected in a short time. Further, the shape of the container is preferably a cylindrical shape or a conical shape. Such a container is preferable because it can be in close contact with a magnet assembly in which cylindrical magnets or arc-shaped magnets are arranged in a circle without any gaps. When using a container with a dead zone that does not contain a specimen in the center, that is, a container in which the specimen enters an annular shape, the specimen is a magnet assembly in which cylindrical magnets or arc-shaped magnets are arranged in a circle. As a result, the collection efficiency is further improved. The recovery efficiency can be similarly improved by using a cylindrical or cup-shaped container and a kit or method in which a rod-shaped dummy member is inserted in the center thereof. Such a configuration can be widely applied as a magnetic bead recovery kit and recovery method regardless of the magnitude of the surface magnetic flux density of the magnet.
（３）検体
検体は液体であれば限定するものではなく、細胞を目的物質とする検体であればよい。例えば、血液、血清・血漿などの血液の成分、糞便またはその希釈液、組織片の溶解液など高粘度の液体も対象とすることが出来る。また、目的物質は細胞であれば特に限定しないが、例えば血液細胞、上皮/上皮系細胞、ガン細胞などである。
(3) The specimen is not limited as long as it is a liquid, and may be any specimen that uses cells as the target substance. For example, blood, blood components such as serum and plasma, feces or a diluted solution thereof, and a high-viscosity liquid such as a solution of tissue fragments can also be targeted. The target substance is not particularly limited as long as it is a cell, and examples thereof include blood cells, epithelial / epithelial cells, cancer cells and the like.
（４）磁気ビーズ
磁気ビーズは特に限定するものではないが細胞表面抗体に対する特異抗体が固定されていることが好ましい。磁気ビーズの粒子径は特に限定しないが、３．０μｍ以上であることが好ましい。磁気ビーズの平均粒子径が３．０μｍ未満の場合、血液から細胞を回収するときに磁気スタンドで捕集した磁気ビーズがヒブリンなどと絡まって容器内壁に固着し再分散しなくなることがある。平均粒子径が３．０μｍ以上であれば、固着せず再分散性が高いため、不純物を除くために行う洗浄の効率が向上する。また、磁気ビーズの磁性成分はこれを特に限定するものではないが、Ｆｅなどの磁性金属を用いることが好ましい。この場合、該磁性金属の核を酸化物等で被覆したものを用いる。飽和磁化は８０A・ｍ２/ｋｇ以上であることが好ましい。血液、血清・血漿などの血液の成分、糞便またはその希釈液、組織片の溶解液など高粘度の液体を検体とする場合、飽和磁化が８０A・ｍ２/ｋｇ以上であると磁気ビーズの捕集時間が短くなり、効率よい細胞の回収・精製を行うことができる。さらに、磁性金属を用いた１００A・ｍ２/ｋｇ以上の飽和磁化を有する磁気ビーズを用いれば、磁性酸化物では達成できない、効率よい細胞の回収・精製を行うことができる。
(4) Magnetic beads Although magnetic beads are not particularly limited, it is preferable that a specific antibody against a cell surface antibody is immobilized. The particle size of the magnetic beads is not particularly limited, but is preferably 3.0 μm or more. When the average particle size of the magnetic beads is less than 3.0 μm, when collecting cells from blood, the magnetic beads collected by the magnetic stand may become entangled with the hybrin and adhere to the inner wall of the container and may not be redispersed. If the average particle size is 3.0 μm or more, the particles are not fixed and the redispersibility is high, so that the efficiency of cleaning performed to remove impurities is improved. Further, the magnetic component of the magnetic beads is not particularly limited, but it is preferable to use a magnetic metal such as Fe. In this case, the magnetic metal core coated with an oxide or the like is used. The saturation magnetization is preferably 80 A · m 2 / kg or more. When using high-viscosity liquids such as blood, blood components such as serum and plasma, feces or diluted solutions thereof, and tissue fragment lysates, the magnetic beads can be captured when the saturation magnetization is 80 A · m 2 / kg or more. Collection time is shortened, and efficient cell recovery and purification can be performed. Furthermore, if magnetic beads using a magnetic metal and having a saturation magnetization of 100 A · m 2 / kg or more are used, efficient cell collection and purification that cannot be achieved with magnetic oxides can be performed.
以下、本発明に係る実施例を詳細に説明する。ただし、これら実施例によって必ずしも本発明が限定されるわけではない。 Hereinafter, embodiments according to the present invention will be described in detail. However, the present invention is not necessarily limited by these examples.
先ず、細胞を回収する場合の生細胞率に与える影響を以下のようにして調べた。検体として１ｍＬのＰＢＳ（リン酸バッファ）に培養した大腸がん細胞ＨＴ−２９細胞を１００万細胞懸濁させた細胞懸濁液を用いた。磁気ビーズとしては、表１に記載の平均粒子径をもつ飽和磁化１２０A・ｍ２/ｋｇのシリカ被覆鉄粒子にＶＵ―ＩＤ９抗体を固定化した抗体固定化磁気ビーズを用いた。前記検体を２ｍＬマイクロチューブに入れ、前記磁気ビーズを４ｍｇ加え３０分室温で攪拌した。マイクロチューブを表面磁束密度が０.４０Ｔの磁気スタンドに立て、２０秒間放置し、磁石と接する壁面に磁気ビーズを回収・保持させ、非磁性成分（磁気ビーズと結合していない成分）を除去した。更に、マイクロチューブを磁気スタンドより外し、５００μＬのＰＢＳを加え攪拌した。ついでマイクロチューブを磁気スタンドに立て、２０秒間放置し、磁石と接する壁面に磁気ビーズを回収・保持させ、非磁性成分を除去することで、洗浄を行った。この洗浄工程を、計２回行ない細胞回収を行った。回収した細胞を１００μＬのＰＢＳに分散させた細胞懸濁液を作成し、該細胞懸濁液にトリパンブルー１００μＬを加えて染色した。血球計算板にカバーガラスを載せ、血球計算板とカバーガラスの隙間に、染色した細胞懸濁液を入れた。血球計算板を位相差顕微鏡に載せ、８区画の染色されていない生細胞数と、染色された死細胞数とを数えた。１区画当たりの生細胞と死細胞数の平均値を求め生細胞数/（生細胞数+死細胞数）を１００倍することにより生細胞率を求めた。結果を表２に示す。また、磁気ビーズの磁気特性は、最大印加磁界を１．６ＭＡ／ｍとしてＶＳＭ（振動型磁力計）により測定した。 First, the influence on the viable cell rate when cells were collected was examined as follows. A cell suspension obtained by suspending 1 million cells of colon cancer cells HT-29 cells cultured in 1 mL of PBS (phosphate buffer) was used as a specimen. As the magnetic beads, antibody-immobilized magnetic beads in which VU-ID9 antibody was immobilized on silica-coated iron particles having a saturation magnetization of 120 A · m 2 / kg having an average particle diameter shown in Table 1 were used. The specimen was placed in a 2 mL microtube, 4 mg of the magnetic beads were added, and the mixture was stirred at room temperature for 30 minutes. The microtube was placed on a magnetic stand with a surface magnetic flux density of 0.40T and left for 20 seconds to collect and hold the magnetic beads on the wall in contact with the magnet to remove non-magnetic components (components not bonded to the magnetic beads). . Further, the microtube was removed from the magnetic stand, and 500 μL of PBS was added and stirred. Next, the microtube was placed on a magnetic stand and allowed to stand for 20 seconds. The magnetic beads were collected and held on the wall surface in contact with the magnet, and the nonmagnetic component was removed to perform washing. This washing step was performed twice in total to collect cells. A cell suspension was prepared by dispersing the collected cells in 100 μL of PBS, and 100 μL of trypan blue was added to the cell suspension for staining. A cover glass was placed on the hemocytometer, and the stained cell suspension was placed in the gap between the hemocytometer and the cover glass. A hemocytometer was placed on a phase-contrast microscope, and the number of viable unstained cells in 8 sections and the number of dead cells stained were counted. The average value of the number of living cells and dead cells per compartment was determined, and the number of living cells / (number of living cells + number of dead cells) was multiplied by 100 to determine the viable cell rate. The results are shown in Table 2. The magnetic properties of the magnetic beads were measured with a VSM (vibrating magnetometer) with a maximum applied magnetic field of 1.6 MA / m.
細胞死は、ビーズとビーズとの間や、ビーズと容器の内壁との間に細胞が挟まれて圧力が加わり、細胞が潰されるまたは過剰な刺激を受けることにより生じると考えられる。前記圧力は、飽和磁化と体積と磁気勾配に比例し、体積は平均粒子径の３乗に比例し、磁気勾配は表面磁束密度と比例すると考えられることから、前記圧力は式１で便宜的に定義するストレス係数Ｃｓに比例すると考えられる。
Ｃｓ＝飽和磁化(A・ｍ２/ｋｇ)×平均粒子径(μm)３×表面磁束密度（T）：（式１）
Cell death is considered to occur when cells are sandwiched between beads or between the beads and the inner wall of the container, pressure is applied, and the cells are crushed or subjected to excessive stimulation. The pressure is proportional to the saturation magnetization, the volume, and the magnetic gradient, the volume is proportional to the cube of the average particle diameter, and the magnetic gradient is proportional to the surface magnetic flux density. It is considered that it is proportional to the stress coefficient Cs to be defined.
Cs = saturation magnetization (A · m 2 / kg) × average particle diameter (μm) 3 × surface magnetic flux density (T): (Formula 1)
表１に示すように飽和磁化１２０A・ｍ２/ｋｇの磁気ビーズと表面磁束密度が０.４０の平板上の磁石を用いた細胞回収において、ストレス係数Ｃｓが６.６×１０２（平均粒子径が２.４μｍ）であれば９５％以上という高い生細胞率で細胞を回収できることがわかる。すなわち、ストレス係数Ｃｓが６.６×１０２以下になれば高い生細胞率での目的細胞の回収が期待できる。 As shown in Table 1, in cell recovery using magnetic beads with a saturation magnetization of 120 A · m 2 / kg and a magnet on a flat plate with a surface magnetic flux density of 0.40, the stress coefficient Cs is 6.6 × 10 2 (average particle If the diameter is 2.4 μm), it can be seen that the cells can be recovered at a high viable cell rate of 95% or more. In other words, when the stress coefficient Cs is 6.6 × 10 2 or less, it is possible to expect collection of target cells at a high viable cell rate.
一方、目的細胞を血液から回収するときには、磁気ビーズがヒブリンなどと絡まって容器内壁に固着し再分散しなくなるという不具合を回避し、高い洗浄効率を維持する観点からは、平均粒子径が３．０μｍ以上であることが好ましい。また、高粘度の液体から目的細胞を捕捉した磁気ビーズを捕集するには８０A・ｍ２/ｋｇ以上の飽和磁化を持つことが望ましい。９５％以上の生細胞率を確保するためにストレス係数を６.６×１０２以下にするためには、平均粒子径が３．０μｍ以上、飽和磁化が８０A・ｍ２/ｋｇ以上の磁気ビーズを用いる場合、式１より表面磁束密度の値は０.３０Ｔ以下である必要がある。また、飽和磁化が１００A・ｍ２/ｋｇ以上の磁気ビーズを用いる場合には、式１より表面磁束密度の値は０.２４Ｔ以下であることが好ましい。 On the other hand, when collecting the target cells from the blood, the average particle size is 3. from the viewpoint of avoiding the problem that the magnetic beads are entangled with hibrin and the like and adhere to the inner wall of the container and are not redispersed. It is preferably 0 μm or more. Further, in order to collect magnetic beads that have captured target cells from a highly viscous liquid, it is desirable to have a saturation magnetization of 80 A · m 2 / kg or more. To 6.6 × 10 2 or less stress coefficient in order to ensure cell viability of 95% or more, average particle diameter of 3.0μm or more, magnetic beads saturation magnetization is not less than 80A · m 2 / kg Is used, the value of the surface magnetic flux density needs to be 0.30 T or less from Equation 1. In addition, when magnetic beads having a saturation magnetization of 100 A · m 2 / kg or more are used, the value of the surface magnetic flux density is preferably 0.24 T or less from Equation 1.
（実施例１）
２００ｍＬビーカー（ニッコー社製ＴＰＸビーカー）にＰＢＳ（リン酸緩衝化生理食塩水）１００ｍＬを加え、さらに磁気ビーズ５０ｍｇを加え攪拌した。前記磁気ビーズは、コア粒子を鉄とし、ＴｉＯ２、更にはシリカで被覆した、粒子径３．８μｍのシリカ被覆鉄磁気ビーズである。磁気捕集には、図２に示す、表面磁束密度０.２１Ｔ、外径７４ｍｍ、内径６９ｍｍ、高さ２３ｍｍの円筒形の、８極の極異方性多極磁石を用いた。図中の矢印が磁束の向きを示す。磁気捕集前、１０秒間、２０秒間、３０秒間、４０秒間、５０秒間、および６０秒間の捕集時間後に、ビーカーの中心付近の溶液を０．３ｍＬ採取した。なお、平均粒径には、堀場製作所社製レーザ回折/散乱式粒度分布測定装置ＬＡ−９２０を用い、該測定方法におけるメジアン径ｄ５０値を用いた。また、表面磁束密度は東陽テクニカ製ハンディ・ガウスメータ４０４８型で測定した。
Example 1
To a 200 mL beaker (Nikko TPX beaker) was added 100 mL of PBS (phosphate buffered saline), and 50 mg of magnetic beads were further added and stirred. The magnetic beads are silica-coated iron magnetic beads having a particle diameter of 3.8 μm, in which the core particles are iron and are coated with TiO 2 and further with silica. For magnetic collection, a cylindrical, 8-pole polar anisotropic multipolar magnet having a surface magnetic flux density of 0.21 T, an outer diameter of 74 mm, an inner diameter of 69 mm, and a height of 23 mm shown in FIG. 2 was used. The arrows in the figure indicate the direction of the magnetic flux. Before magnetic collection, 0.3 mL of the solution near the center of the beaker was collected after 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, and 60 seconds of collection time. In addition, the median diameter d50 value in this measuring method was used for the average particle diameter using the laser diffraction / scattering type particle size distribution measuring apparatus LA-920 by Horiba. The surface magnetic flux density was measured with a handy gauss meter 4048 manufactured by Toyo Technica.
採取した液体を粒子が擾乱している状態でバイオ光学計（日立ハイテクノロジーズ社製日立ダイオードアレー型バイオ光度計Ｕ-００８０Ｄ）を用い５５０ｎｍの吸光度を測定した。各々の捕集時間後に採取した溶液の吸光度を捕集前に採取した溶液の吸光度で割り、１からその値を引いた値を捕集率とし求めた。その結果を図１に示す。 Absorbance at 550 nm was measured using a bio-optical meter (Hitachi diode array type biophotometer U-0080D manufactured by Hitachi High-Technologies Corporation) in a state where particles were disturbed in the collected liquid. The absorbance of the solution collected after each collection time was divided by the absorbance of the solution collected before collection, and a value obtained by subtracting the value from 1 was obtained as the collection rate. The result is shown in FIG.
（比較例１）
多極磁石の代わりに表面磁束密度が０.４０Ｔの平板上の磁石を用いた以外は実施例１と同様の方法で磁気捕集を行った。その結果を図１に示す。
(Comparative Example 1)
Magnetic collection was performed in the same manner as in Example 1 except that a magnet on a flat plate having a surface magnetic flux density of 0.40 T was used instead of the multipolar magnet. The result is shown in FIG.
図１おいて明らかなように、実施例１は比較例１と比較し短時間に磁気ビーズの捕集が可能であることがわかる。つまり、本発明の細胞回収用磁気スタンドを用いることにより目的細胞を捕捉した磁気ビーズを短時間で捕集できることが示された。 As is clear from FIG. 1, it can be seen that the magnetic beads can be collected in Example 1 in a shorter time than in Comparative Example 1. That is, it was shown that the magnetic beads capturing the target cells can be collected in a short time by using the magnetic stand for cell recovery of the present invention.
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| CN107012198A (en) * | 2017-05-25 | 2017-08-04 | 杭州富集生物科技有限公司 | A kind of replaceable magnetic frame that pathogen is enriched with for paramagnetic particle method |
| WO2019088106A1 (en) | 2017-10-31 | 2019-05-09 | 大研医器株式会社 | Magnetic particle collection method and test set |
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| CN107012198A (en) * | 2017-05-25 | 2017-08-04 | 杭州富集生物科技有限公司 | A kind of replaceable magnetic frame that pathogen is enriched with for paramagnetic particle method |
| CN107012198B (en) * | 2017-05-25 | 2023-12-01 | 杭州富集生物科技有限公司 | Replaceable magnetic rack for enriching pathogens by magnetic bead method |
| WO2019088106A1 (en) | 2017-10-31 | 2019-05-09 | 大研医器株式会社 | Magnetic particle collection method and test set |
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