WO2002073190A1 - Method of sorting particulates with different specific gravities - Google Patents

Method of sorting particulates with different specific gravities Download PDF

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Publication number
WO2002073190A1
WO2002073190A1 PCT/JP2002/002317 JP0202317W WO02073190A1 WO 2002073190 A1 WO2002073190 A1 WO 2002073190A1 JP 0202317 W JP0202317 W JP 0202317W WO 02073190 A1 WO02073190 A1 WO 02073190A1
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specific gravity
centrifuge tube
fine particles
solid
solid inclusions
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PCT/JP2002/002317
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Japanese (ja)
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Hajime Ogata
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Hajime Ogata
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material

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  • the present invention relates to a method and an apparatus for separating a mixture of two or more types of fine particles having different specific gravities into two groups of light and heavy weights simply and with high accuracy.
  • the present invention relates to a method and apparatus suitable for removing erythrocytes from blood and separating and collecting nucleated cells such as leukocytes.
  • Conventional methods for removing erythrocytes from body fluids such as blood to separate and collect nucleated cells include (1) a buffy coat-coating method, (2) a red blood cell removal method by hemolysis, and (3) a hemagglutination sedimentation. Separation method, (4) Centrifugation method using density gradient of specific gravity solution, (5) Separation method of high specific gravity using partition wall fixed in centrifuge tube, (6) Floating solid inclusion for centrifugation Techniques such as a centrifugal separation method using a method are known.
  • This method is a method performed by the present inventor and already published as Japanese Patent Application Laid-Open No. Hei 9-155552, the purpose of which is to centrifuge together two or more types of particles having different specific gravities.
  • a solid inclusion that intervenes in the specific gravity band can move up and down in the centrifuge tube by centripetal force to separate particles into two groups of light and heavy, and has a concave or sloped upper surface whose size just fits the inner wall of the centrifuge tube. It is used.
  • Such a solid inclusion has a large contact area with the wall of the centrifuge tube, and a centrifugal force is applied obliquely outward to the solid inclusion against the inner wall of the centrifuge tube during centrifugation.
  • centrifugal force and specific gravity setting conditions for intervening between the erythrocyte layer and the nucleated cell layer after centrifugation are narrow, and further improvements are expected. It is rare.
  • the present inventor has found that a fine particle group containing two or more kinds of fine particles having different specific gravities and a solid inclusion having a specific specific gravity and a shape are centrifuged together, so that the method is more efficient and more efficient than the conventional method.
  • a method for separating fine particles with different specific gravities into two groups with light inclusions in Sakai.
  • a fine particle group containing two or more types of fine particles with different specific gravities has a specific gravity located in the middle zone of the specific gravity of the fine particle group, and can move up and down while inscribed in the centrifuge tube. Centrifuge with the solid inclusions whose upper part is more convex than the contacting part, and pass the tangent line between the inner wall of the centrifuge tube and the solid inclusions to move high specific gravity fine particles below the solid inclusions or low specific gravity.
  • a method for separating fine particles having different specific gravities wherein the fine particles are moved above the solid inclusions and at the same time, the solid inclusions are moved and interposed between the high and low density two particle groups,
  • two or more kinds of fine particles having different specific gravities to be separated are fine particles.
  • Particles having a diameter of about 0.01 to 200 / zm and containing two or more kinds of fine particles with different specific gravities regardless of the type and shape of the fine particles, especially fine particles are mixed in the liquid medium It is a fine particle group in a state.
  • a typical example is a body fluid, particularly blood containing cells having various specific gravities such as red blood cells and white blood cells.
  • Other examples include colloidal liquids containing colloidal particles having different specific gravities, liquids containing micelles, emulsions, suspensions, and the like.
  • it is particularly suitable for collecting nucleated cells by separating and removing red blood cells from a cell population in a body fluid.
  • the material, size and the like of the centrifuge tube used in the present invention are not particularly limited, as long as they are used in an ordinary centrifuge. That is, it is preferable that the bottom 6 has a shape such as a hemispherical shape, a semiellipsoidal shape, or a cone shape, and a cylindrical body following the bottom 6.
  • the material of the centrifuge tube should be a material that has a gap that allows the particles to move between the wall of the centrifuge tube and the solid inclusion inscribed when a centrifugal force is applied, or such a gap. What has elasticity enough to produce Examples of the hard material include glass and metal materials, and examples of the material having elasticity include synthetic resin and rubber.
  • the inner wall of the centrifuge tube may be coated with a lubricant such as paraffin so that solid inclusions can easily move up and down the centrifuge tube during centrifugation.
  • the solid inclusion used in the present invention is, when two or more types of fine particles having different specific gravities are dispersed in a liquid, insoluble in the liquid, and when a centrifugal force is applied, the fine particles are in contact with the centrifuge tube wall.
  • a material having a gap that can move between the solid inclusions inscribed therein and a material having flexibility enough to create such a gap, that is, an elastic body is preferable.
  • Examples of the material of the solid inclusion 2 include agar, gelatin, a silicone resin, a polystyrene resin, a synthetic resin such as an ABS resin and a polyurethane resin, ceramics such as glass, proteins, and polysaccharides.
  • a cavity may exist inside the solid inclusion, and the cavity may be filled with another substance.
  • One of the features of the present invention is that the shape of the solid inclusions present at the interface of the fine particles having different specific gravities by centrifugation becomes convex above the part inscribed in the centrifuge tube. It was molded as follows.
  • Specific shapes of the solid inclusion include those formed in a line with the inner wall of the centrifuge tube, such as a true sphere, an oval sphere, a foot pole, a raindrop, a spindle, and an abacus. Among them, particularly preferred is a true sphere or ellipsoid (the ratio of the major axis to the minor axis is preferably within 1: 2).
  • the specific gravity of the solid inclusions can be appropriately selected depending on the purpose of separating fine particles.
  • a substance having a specific gravity that falls between the specific gravities of two fine particles to be separated for example, silica, clay
  • the specific gravity can be adjusted arbitrarily by mixing the required amount of ceramic powder, metal powder, etc., making the interior hollow, and enclosing other substances in the cavity.
  • a spherical solid inclusion 2 is placed in a centrifugal tube 1 having a hemispherical bottom 6 and a cylindrical body 7 following the same so as to inscribe the centrifugal tube 1.
  • Blood 10 containing two kinds of microparticles having different specific gravities of red blood cells 3 (high-density microparticles) and nucleated cells 4 (low-density microparticles) is placed in this centrifuge tube 1 (Fig. 4), and the centrifuge tube is centrifuged. .
  • red blood cells 3 having a high specific gravity gather at the part 5 where the inclined surface of the solid inclusion and the inner wall of the centrifugal tube are asymptotic, and when the centrifugal force is further applied, the red blood cells 3
  • the solid inclusions 2 move downward through the solid inclusions 5 while being pushed up by the negative centrifugal force generated by the specific gravity difference from the red blood cells.
  • most of the red blood cells 3 move below the solid inclusions, and the solid inclusions 2 intervene at the interface 9 where the nucleated cells 4 and the red blood cells 3 are in contact (FIG. 6).
  • a mixture of two or more types of fine particles having different specific gravities for example, blood, may be arranged below the solid inclusion.
  • a mixture of two or more kinds of fine particles having different specific gravities is put into a centrifuge tube, and a solid inclusion formed thereon is introduced.
  • the shape of the solid inclusion is preferably spherical or elliptical because of its easy insertion.
  • the microparticles antagonize the narrow space 5 formed by the asymptotics between the surface 8 of the solid inclusion and the inner wall of the centrifuge tube while leaving a small specific gravity gradient. Even if there is a lot difference, the desired low specific gravity fine particles 4 can be stably separated in a state where other fine particles are not mixed.
  • the liquid phase containing the low-specific-gravity particles above the solid inclusions can be easily separated by tilting the centrifuge tube or by using a suction tool such as a pipette to separate the high and low groups of fine particles. it can.
  • a polystyrene sphere having a diameter of 10.6 Oram (specific gravity: 1.05) was molded and inserted into a glass tube centrifuge tube having an inner diameter of 10.75 mm and a length of 75 mm.
  • 4 ml of human peripheral blood (specific gravity: about 1.056) was poured into the centrifuge tube, and the mixture was centrifuged at 50 OG for 20 minutes in a centrifuge.
  • Erythrocytes (specific gravity: 1.0964) that settled on the inner tangent to the solid inclusions moved down the solid inclusions along the inner wall of the centrifuge tube, and simultaneously the solid inclusions were pushed upward. Eventually, a fraction containing serum and leukocytes remained above the solid inclusions, and red blood cells accumulated below.
  • a fine particle group containing two or more types of fine particles having different specific gravities can be converted into two groups with high and low specific gravities with high accuracy by simply performing a conventional centrifugal separation operation using a relatively simple apparatus. Can be fractionated.
  • this method is very suitable for removing red blood cells from blood and collecting leukocytes, which are nucleated cells.

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Abstract

A method of easily and accurately sorting the groups of particulates containing two or more types of particulates with different specific gravities into two groups, i.e., a high specific gravity group and a low specific gravity group, comprising the step of centrifugally separating the particulate groups together with solid inclusions having a specific gravity positioned in the intermediate zone of the specific gravity of the particulate groups, vertically movable while inscribing in a centrifugal tube, and having an upper part from a portion inscribing in the centrifugal tube formed in projected shape to move high specific gravity particulates to the lower side of the solid inclusions through the tangential line of the centrifugal tube inside wall to the solid inclusions or move low specific gravity particulates to the upper side of the solid inclusions and, at the same time, move the solid inclusions to the intermediate position of two high and low specific gravity groups of particulates, whereby the method can be utilized favorably for separating leucocytes from blood.

Description

明 細 書 比重の異なる微粒子の分別法 技術分野  Description Separation method of fine particles with different specific gravity
本発明は、 比重の異なる二種類以上の微粒子の混合物を、 簡便かつ高い精度で 軽重二群に分別する方法およびその装置に関する。 特に血液から赤血球を除去し て白血球などの有核細胞を分離採取するのに適した方法並びに装置に関する。 背景技術  The present invention relates to a method and an apparatus for separating a mixture of two or more types of fine particles having different specific gravities into two groups of light and heavy weights simply and with high accuracy. In particular, the present invention relates to a method and apparatus suitable for removing erythrocytes from blood and separating and collecting nucleated cells such as leukocytes. Background art
血液などの体液から赤血球を除去して有核細胞を分離採取する方法には、 従来 から (1 ) バフィ一 ·コート採取法、 (2 ) 溶血による赤血球除去法、 (3 ) 赤血 球凝集沈降分離法、 (4 ) 比重溶液の密度勾配を利用した遠心分離法、 ( 5 ) 遠心 管内に固定された隔壁を用いた高比重分画除去法、 (6 ) 遠沈分離用遊動式固形 介在物を用いた遠心分離法などの手法が知られている。  Conventional methods for removing erythrocytes from body fluids such as blood to separate and collect nucleated cells include (1) a buffy coat-coating method, (2) a red blood cell removal method by hemolysis, and (3) a hemagglutination sedimentation. Separation method, (4) Centrifugation method using density gradient of specific gravity solution, (5) Separation method of high specific gravity using partition wall fixed in centrifuge tube, (6) Floating solid inclusion for centrifugation Techniques such as a centrifugal separation method using a method are known.
前記公知の手法には、 それぞれ次のような問題点があった。  Each of the known methods has the following problems.
( 1 ) バフィ一'コート採取法  (1) Buffy's coat collection method
この方法は、 採取しょうとする有核細胞への大量の赤血球の混入が不可避で、 また有核細胞の回収率も手技を施す人の熟練度に大きく左右される。  In this method, it is inevitable that a large amount of red blood cells is mixed into the nucleated cells to be collected, and the recovery rate of the nucleated cells largely depends on the skill of the person performing the procedure.
( 2 ) 溶血による赤血球除去法  (2) Erythrocyte removal by hemolysis
この方法は、 溶血に用いる細胞毒性の試薬の使用が不可避であり、 また有核細 胞の回収率および純度は、 試薬の濃度や混合比率および反応時間といった条件に 左右される。 しかも大容量の検体処理には不向きである。  In this method, the use of cytotoxic reagents used for hemolysis is inevitable, and the recovery and purity of nucleated cells depend on conditions such as the reagent concentration, the mixing ratio, and the reaction time. Moreover, it is not suitable for processing a large amount of sample.
( 3 ) 赤血球凝集沈降分離法  (3) Hemagglutination sedimentation method
この方法によって満足しうる回収率を得るためには、 試料を多量の等張液で希 釈し凝集沈降手技を数回反復する必要がある。 従って、 分画後に浮遊細胞を再度 遠沈洗浄してデキストランやへタスターチなどの凝集薬を除去し多量の溶液中に 拡散した状態で存在する有核細胞を回収する必要があり、 作業工程が多く煩雑で ある。 ( 4 ) 比重溶液の密度勾配を利用した遠心分離法 In order to obtain a satisfactory recovery by this method, it is necessary to dilute the sample with a large amount of isotonic solution and repeat the coagulation sedimentation procedure several times. Therefore, after fractionation, it is necessary to centrifuge and wash the suspended cells again to remove flocculants such as dextran and hestastarch, and to collect nucleated cells present in a state of being dispersed in a large amount of solution. It is complicated. (4) Centrifugation using density gradient of specific gravity solution
この方法は、 溶液の重層や目的とする比重帯の分離手技等に慎重な操作と熟練 が要求される。 また、 分画後に浮遊細胞を再度遠沈洗浄して、 たとえばフイコー ル (Ficoll)、 パーコール (Percoll)等の比重溶液を除去する必要があり、 作業 工程が多く煩雑である。  This method requires careful operation and skill in separating the solution and the target specific gravity band. In addition, it is necessary to centrifuge and wash the suspended cells again after the fractionation to remove a specific gravity solution such as Ficoll and Percoll.
( 5 ) 遠心管内に固定された隔壁を用いた赤血球を含む高比重分画除去法 この方法は、 遠心管内に固定された隔壁の下に比重溶液を入れ、 血液等の試料 の重層や遠心分離後の回収操作を容易にするものであるが、 遠心分離後に除去さ れた高比重分画の体積に等しい容積の比重溶液が隔壁上に移動するために、 (4 ) と同じく、 比重溶液を除去する必要があり、 作業工程が多く煩雑である。  (5) Method for removing high-density fractions containing red blood cells using a septum fixed in a centrifuge tube This method involves placing a specific gravity solution under the septum fixed in a centrifuge tube, and overlaying or centrifuging a sample such as blood. This facilitates the subsequent recovery operation.However, since the specific gravity solution having a volume equal to the volume of the high specific gravity fraction removed after centrifugation moves onto the partition wall, the specific gravity solution is removed as in (4). It is necessary to remove it, and the work process is many and complicated.
( 6 ) 遠沈分離用遊動式固形介在物を用いた遠心分離法  (6) Centrifugation method using floating solid inclusion for centrifugation
この方法は、 本発明者によってなされ、 既に特開平 9— 1 5 5 2 3 4号として 公開されている方法であるが、 二種類以上の異なった比重の粒子を共に遠心する ことにより目的とする比重帯に介在し、 軽重二群に粒子を分離するために求心力 により遠心管中で上下に遊動可能で、 遠心管の内壁に丁度はまる大きさの上面が 凹または斜面に成形した固形介在物を用いるものである。 し力、し、 このような形 状の固形介在物は、 遠心管壁との接触面積が大きく、 かつ遠心時遠心管内壁に対 して固形介在物に斜め外方に遠心力がかかるために、 遠心力が高いほど固形介在 物が遊動し難くなる。 したがって、 そこに具体的に記載されている形状では、 遠 心分離後に赤血球層と有核細胞層との間に介在させるための遠心力や比重設定条 件の幅が狭く、 更なる改良が望まれている。  This method is a method performed by the present inventor and already published as Japanese Patent Application Laid-Open No. Hei 9-155552, the purpose of which is to centrifuge together two or more types of particles having different specific gravities. A solid inclusion that intervenes in the specific gravity band, can move up and down in the centrifuge tube by centripetal force to separate particles into two groups of light and heavy, and has a concave or sloped upper surface whose size just fits the inner wall of the centrifuge tube. It is used. Such a solid inclusion has a large contact area with the wall of the centrifuge tube, and a centrifugal force is applied obliquely outward to the solid inclusion against the inner wall of the centrifuge tube during centrifugation. The higher the centrifugal force, the more difficult it is for solid inclusions to move. Therefore, with the shape specifically described there, the centrifugal force and specific gravity setting conditions for intervening between the erythrocyte layer and the nucleated cell layer after centrifugation are narrow, and further improvements are expected. It is rare.
また、 血清分離用には既にゾル状の介在物を用いて、 血球等の分離を行う方法 も知られてはいるが、 細胞分離用として用いるためには、 介在物の厳密な比重調 整が必要で、 かつ検体の比重分布の個体差に影響されるため、 安定した結果が得 られない。 発明の開示  Also, a method of separating blood cells and the like using a sol-like inclusion for serum separation is already known, but strict specific gravity adjustment of the inclusion is required for use for cell separation. Stable results cannot be obtained because they are necessary and are affected by individual differences in the specific gravity distribution of specimens. Disclosure of the invention
本発明者は、 比重の異なる 2種以上の微粒子を含む微粒子群と特定の比重と形 状を有する固体介在物を一緒に遠心することにより、 従来法より、 より効率的且 つ簡便に、 比重の異なる微粒子群を固体介在物を堺に互いに軽重二群に分離する 方法を見つけた。 The present inventor has found that a fine particle group containing two or more kinds of fine particles having different specific gravities and a solid inclusion having a specific specific gravity and a shape are centrifuged together, so that the method is more efficient and more efficient than the conventional method. In a simple and convenient way, we found a method for separating fine particles with different specific gravities into two groups with light inclusions in Sakai.
すなわち、 本発明は、  That is, the present invention
(1) 比重の異なる二種以上の微粒子を含む微粒子群を、 その微粒子群の比重の 中間帯に位置する比重を持ち、 遠心管に内接しながら上下に移動可能であり、 遠 心管に内接している部分より上部が凸状に成形された固体介在物と共に遠心し て、 遠心管内壁と固体介在物との接線を通過して高比重微粒子を固体介在物の下 方に、 または低比重微粒子を固体介在物の上方に移動させると同時に固体介在物 を比重の高低二粒子群の中間に移動、 介在させることを特徴とする比重の異なる 微粒子の分別法、  (1) A fine particle group containing two or more types of fine particles with different specific gravities has a specific gravity located in the middle zone of the specific gravity of the fine particle group, and can move up and down while inscribed in the centrifuge tube. Centrifuge with the solid inclusions whose upper part is more convex than the contacting part, and pass the tangent line between the inner wall of the centrifuge tube and the solid inclusions to move high specific gravity fine particles below the solid inclusions or low specific gravity. A method for separating fine particles having different specific gravities, wherein the fine particles are moved above the solid inclusions and at the same time, the solid inclusions are moved and interposed between the high and low density two particle groups,
(2) 個体介在物の形状が、 遠心管内壁に対して線で接する形状に成形されたも のであることを特徴とする (1) 記載の方法、  (2) The method according to (1), wherein the solid inclusion is formed into a shape that is in contact with the inner wall of the centrifuge tube by a line.
(3) 固体介在物の形状が、 真球状、 楕円球状、 フットボール状、 雨滴状、 紡錘 状またはそろばん珠状であることを特徴とする (1) または (2) 記載の方法、 (3) The method according to (1) or (2), wherein the shape of the solid inclusion is a true sphere, an ellipsoid, a football, a raindrop, a spindle or an abacus.
(4) 微粒子が粒径 0. 01~200 jitmの範囲内にあるものであることを特徴 とする (1) 〜 (3) のいずれかに記載の方法、 (4) The method according to any one of (1) to (3), wherein the fine particles have a particle size in the range of 0.01 to 200 jitm.
(5) 微粒子が液体中に懸濁しているかまたはコロイド状として存在しているこ とを特徴とする (1) 〜 (4) のいずれかに記載の方法、  (5) The method according to any one of (1) to (4), wherein the fine particles are suspended in a liquid or exist as a colloid.
(6) 微粒子を含む液体が、 体液であることを特徴とする (1) 〜 (5) のいず れかに記載の方法、  (6) The method according to any one of (1) to (5), wherein the liquid containing fine particles is a body fluid.
(7) 固体介在物が、 寒天、 ゼラチン、 合成樹脂、 セラミタス、 タンパク質また は多糖類であることを特徴とする (1) 〜 (6) のいずれかに記載の方法、 (7) The method according to any one of (1) to (6), wherein the solid inclusion is agar, gelatin, synthetic resin, ceramitas, protein or polysaccharide.
(8) 遠心管がガラスまたは合成樹脂製であることを特徴とする (1) 〜 (7) のいずれかに記載の方法、 および (8) The method according to any one of (1) to (7), wherein the centrifuge tube is made of glass or synthetic resin, and
(9) 遠心管内と、 その遠心管に内接しながら上下に移動可能であり、 遠心管に 内接している部分より上部が凸状に成形された、 固体介在物からなることを特徴 とする (1) 〜 (8) のいずれかに記載の方法を実施するための装置、  (9) It is made of solid inclusions that can move up and down inside the centrifuge tube while being inscribed in the centrifuge tube, and the upper part of the portion inscribed in the centrifuge tube is formed in a convex shape. 1) an apparatus for performing the method according to any of (8),
あ 。  Ah .
本発明において、 分別の対象となる比重の異なる二種以上の微粒子とは、 微粒 子径が 0 . 0 1〜2 0 0 /z m程度のもので、 微粒子の種類、 形状を問わず、 比重 の異なる二種以上の微粒子を含む微粒子群、 特に微粒子が液体媒体中混在してい る状態の微粒子群である。 その代表的なものは、 体液、 特に赤血球、 白血球など の種々の比重の異なる細胞を含む血液である。 その他、 比重の異なるコロイド微 粒子を含むコロイド液、 ミセルを含む液、 エマルシヨン、 サスペンジョンなども 含まれる。 本発明においては、 特に体液中の細胞集団から赤血球を分離除去して 有核細胞を採取するのに適している。 In the present invention, two or more kinds of fine particles having different specific gravities to be separated are fine particles. Particles having a diameter of about 0.01 to 200 / zm and containing two or more kinds of fine particles with different specific gravities regardless of the type and shape of the fine particles, especially fine particles are mixed in the liquid medium It is a fine particle group in a state. A typical example is a body fluid, particularly blood containing cells having various specific gravities such as red blood cells and white blood cells. Other examples include colloidal liquids containing colloidal particles having different specific gravities, liquids containing micelles, emulsions, suspensions, and the like. In the present invention, it is particularly suitable for collecting nucleated cells by separating and removing red blood cells from a cell population in a body fluid.
本発明に用いられる遠心管は、 材質、 大きさ等は、 特に限定されるものではな く、 通常の遠心分離器に用いられるものであればよい。 即ち、 半球状、 半楕円体 状、 コーン状などの形状を有する底部 6と、 それに続く円筒状の胴部からなるも のがよい。 遠心管の材質は、 遠心力が加わったとき ί敷粒子が遠心管器壁と内接し ている固体介在物との間を移動しうる隙間を有しているものか、 または、 そのよ うな間隙を生む程度の弾性を有しているものがよい。 硬質のものとしては、 ガラ ス、 金属製のもの等が挙げられ、 弾性を有するものとしては、 合成樹脂、 ゴム等 が用いられる。 それらの中でも、 ガラスまたはポリプロピレン、 塩化ビュル等の 合成樹脂製のもの等が好適に使用される。 遠心管内壁には、 遠心した際、 固体介 在物が遠心管の上下に移動しやすくするために、 パラフィン等の滑剤を塗布して おくこともできる。  The material, size and the like of the centrifuge tube used in the present invention are not particularly limited, as long as they are used in an ordinary centrifuge. That is, it is preferable that the bottom 6 has a shape such as a hemispherical shape, a semiellipsoidal shape, or a cone shape, and a cylindrical body following the bottom 6. The material of the centrifuge tube should be a material that has a gap that allows the particles to move between the wall of the centrifuge tube and the solid inclusion inscribed when a centrifugal force is applied, or such a gap. What has elasticity enough to produce Examples of the hard material include glass and metal materials, and examples of the material having elasticity include synthetic resin and rubber. Among them, those made of glass or synthetic resin such as polypropylene and chloride chloride are preferably used. The inner wall of the centrifuge tube may be coated with a lubricant such as paraffin so that solid inclusions can easily move up and down the centrifuge tube during centrifugation.
本発明に用いられる固体介在物は、 比重の異なる二種以上の微粒子が、 液体に 分散されている場合は、 その液体に不溶のもので、 遠心力が加わったとき微粒子 が遠心管器壁と内接している固体介在物との間を移動しうる隙間を有しているも のか、または、そのような間隙を生む程度の柔軟性を有しているもの、すなわち、 弾性体がよい。  The solid inclusion used in the present invention is, when two or more types of fine particles having different specific gravities are dispersed in a liquid, insoluble in the liquid, and when a centrifugal force is applied, the fine particles are in contact with the centrifuge tube wall. A material having a gap that can move between the solid inclusions inscribed therein and a material having flexibility enough to create such a gap, that is, an elastic body is preferable.
固体介在物 2の材質は、 たとえば、 寒天、 ゼラチン、 シリコン樹脂、 ポリスチ レン樹脂、 A B S樹脂, ポリウレタン樹脂等の合成樹脂、 ガラスなどのセラミク ス、 タンパク質、 多糖類等が挙げられる。 固体介在物の内部は空洞が存在してい てもよく、 その空洞に他の物質が充填されてもよい。  Examples of the material of the solid inclusion 2 include agar, gelatin, a silicone resin, a polystyrene resin, a synthetic resin such as an ABS resin and a polyurethane resin, ceramics such as glass, proteins, and polysaccharides. A cavity may exist inside the solid inclusion, and the cavity may be filled with another substance.
本発明の特徴の一つは、 遠心することにより比重の異なる微粒子群の界面に存 在させる固体介在物の形状を、 遠心管と内接している部分より上部が凸状となる ように成形したことにある。 One of the features of the present invention is that the shape of the solid inclusions present at the interface of the fine particles having different specific gravities by centrifugation becomes convex above the part inscribed in the centrifuge tube. It was molded as follows.
固体介在物の具体的な形状としては、 真球状、 楕円球状、 フットポール状、 雨 滴状、 紡錘状、 そろばん珠状など、 遠心管内壁と線で接するよう形成されたもの が挙げられる。 これらの中で、 特に好ましいものは、 真球状又は楕円体状 (長軸 と短軸との比が 1 : 2以内のものが望ましい。) である。  Specific shapes of the solid inclusion include those formed in a line with the inner wall of the centrifuge tube, such as a true sphere, an oval sphere, a foot pole, a raindrop, a spindle, and an abacus. Among them, particularly preferred is a true sphere or ellipsoid (the ratio of the major axis to the minor axis is preferably within 1: 2).
固体介在物の比重は、微粒子分別の目的に応じて適宜選択することができる力 たとえば分別しようとする二つの微粒子の比重の間にくるように適当な比重を有 する物質、 たとえば、 シリカ、 クレー、 セラミクスパウダー、 金属粉末などを必 要量を混入したり、 内部を空洞にしたり、 空洞内に他の物質を封入することで、 その比重を任意に調節することができる。  The specific gravity of the solid inclusions can be appropriately selected depending on the purpose of separating fine particles.For example, a substance having a specific gravity that falls between the specific gravities of two fine particles to be separated, for example, silica, clay The specific gravity can be adjusted arbitrarily by mixing the required amount of ceramic powder, metal powder, etc., making the interior hollow, and enclosing other substances in the cavity.
次に本発明の代表的な実施方法について、 図面を参照しながら説明する。 まず、 図 3に示される通り、 半球状の底部 6とそれに続く円筒状胴部 7を有する 遠心管 1に、 真球状固体介在物 2を、 遠心管 1に内接するように配置する。 この 遠心管 1に赤血球 3 (高比重微粒子) と有核細胞 4 (低比重微粒子) の比重の異 なる二種の微粒子を含む血液 1 0を入れ (図 4 )、 遠心管を遠心機にかける。 遠 心を始めると、 高比重である赤血球 3が固体介在物傾斜面と遠心管内壁が漸近し てくる部分 5に集まり、 さらに遠心力が加わるとその赤血球は、 遠心管内壁と固 体介在物との間 5を擦り抜けて固体介在物の下方に移動し、一方固形介在物 2は、 赤血球との比重差により生じる負の遠心力により押し上げられて上方に移動す る。 遠心終了時点では、 固体介在物の下方に大部分の赤血球 3が移動し、 有核細 胞 4と赤血球 3とが接する界面 9に固体介在物 2が介在する (図 6 )。  Next, a typical embodiment of the present invention will be described with reference to the drawings. First, as shown in FIG. 3, a spherical solid inclusion 2 is placed in a centrifugal tube 1 having a hemispherical bottom 6 and a cylindrical body 7 following the same so as to inscribe the centrifugal tube 1. Blood 10 containing two kinds of microparticles having different specific gravities of red blood cells 3 (high-density microparticles) and nucleated cells 4 (low-density microparticles) is placed in this centrifuge tube 1 (Fig. 4), and the centrifuge tube is centrifuged. . At the start of centrifugation, red blood cells 3 having a high specific gravity gather at the part 5 where the inclined surface of the solid inclusion and the inner wall of the centrifugal tube are asymptotic, and when the centrifugal force is further applied, the red blood cells 3 The solid inclusions 2 move downward through the solid inclusions 5 while being pushed up by the negative centrifugal force generated by the specific gravity difference from the red blood cells. At the end of the centrifugation, most of the red blood cells 3 move below the solid inclusions, and the solid inclusions 2 intervene at the interface 9 where the nucleated cells 4 and the red blood cells 3 are in contact (FIG. 6).
比重の異なる二種以上の微粒子の混合物、 例えば血液は、 固体介在物の下方に 配置してもよい。 この場合は、 まず、 遠心管に、 比重の異なる二種以上の微粒子 の混合物を入れ、 その上に成形された固体介在物を揷入する。 この場合の固体介 在物の形状は, 球形または楕円形のものが、 揷入が容易であることにおいて好ま しい。 この比重の異なる二種以上の微粒子の混合物と固体介在物を収容した遠心 管を遠心すると低比重微粒子が遠心管内壁と固体介在物との接触線を擦り抜けて 固体介在物の上方に移動し、 高比重粒子が固体介在物の下方に留まる。 その際、 固体介在物は、 低比重粒子が上方に移動した分だけ下方に移動する。 遠心する場 合の遠心力と時間は、 分別しようとする微粒子の種類や量により適宜決めればよ い。 A mixture of two or more types of fine particles having different specific gravities, for example, blood, may be arranged below the solid inclusion. In this case, first, a mixture of two or more kinds of fine particles having different specific gravities is put into a centrifuge tube, and a solid inclusion formed thereon is introduced. In this case, the shape of the solid inclusion is preferably spherical or elliptical because of its easy insertion. When a centrifuge tube containing a mixture of two or more types of fine particles having different specific gravities and solid inclusions is centrifuged, the low-density particles move above the solid inclusions through the contact line between the inner wall of the centrifuge tube and the solid inclusions. High specific gravity particles stay below solid inclusions. At that time, the solid inclusions move downward by the amount of the low specific gravity particles moving upward. Centrifuge place The centrifugal force and the time may be appropriately determined depending on the type and amount of the fine particles to be separated.
最終的な定常状態においては、 固体介在物の面 8と遠心管内壁との漸近により 構成される狭い空間 5に微粒子群は少量の比重勾配を残したまま拮抗するので、 微粒子群自体の比重にロット差がある場合でも、 目的とする低比重微粒子 4を他 の微粒子の混在が少ない状態で安定して分別することができる。  In the final steady state, the microparticles antagonize the narrow space 5 formed by the asymptotics between the surface 8 of the solid inclusion and the inner wall of the centrifuge tube while leaving a small specific gravity gradient. Even if there is a lot difference, the desired low specific gravity fine particles 4 can be stably separated in a state where other fine particles are not mixed.
遠心後は、 固体介在物より上にある低比重微粒子を含む液相を遠心管の傾斜に より、 又は、 ピペットなどの吸引具により取り出すことにより、 容易に高低二群 の微粒子を分別することができる。 図面の簡単な説明  After centrifugation, the liquid phase containing the low-specific-gravity particles above the solid inclusions can be easily separated by tilting the centrifuge tube or by using a suction tool such as a pipette to separate the high and low groups of fine particles. it can. BRIEF DESCRIPTION OF THE FIGURES
図 1  Figure 1
真球状固体介在物の平面図 Plan view of a spherical solid inclusion
図 2  Figure 2
楕円球状固体介在物の垂直断面図 Vertical cross section of elliptical spherical solid inclusion
図 3  Fig. 3
真球状の固体介在物を配置した遠心管の垂直断面図 Vertical sectional view of a centrifuge tube with a spherical solid inclusion
図 4  Fig. 4
真球状固体介在物を収容した遠心管垂直断面の模式図 Schematic diagram of a vertical cross section of a centrifuge tube containing a spherical solid inclusion
図 5  Fig 5
真球状固体介在物を収容した遠心管垂直断面の模式図 Schematic diagram of a vertical cross section of a centrifuge tube containing a spherical solid inclusion
図 6  Fig. 6
遠心終了時の傾斜した真球状固体介在物を収容した遠心管垂直断面の模式図 符号の説明 Schematic drawing of the vertical cross section of a centrifuge tube containing the inclined solid spherical inclusions at the end of centrifugation
1 . 遠心管  1. Centrifuge tube
2 . 固体介在物  2. Solid inclusions
3 . 赤血球  3. Red blood cells
4 . 有核細胞 (白血球)) 5. 真球状個体介在物と遠心管内壁が漸近する部分 4. Nucleated cells (white blood cells) 5. Part where the spherical inclusions and the inner wall of the centrifuge tube are asymptotic
6. 遠心管底部  6. Centrifuge tube bottom
7. 遠心管胴部  7. Centrifuge tube body
8. 固体介在物の微粒子群と接触する面  8. Surface of solid inclusions in contact with fine particles
9. 低比重微粒子と高比重微粒子とが接する線  9. Line where low specific gravity particles and high specific gravity particles touch
10. 血液 発明を実施するための最良の形態  10. BEST MODE FOR CARRYING OUT THE INVENTION
実施例 Example
実施例 1  Example 1
直径 10. 6 Oramのポリスチレン製真球 (比重 1 · 05) を成形し、 内径 1 0. 75 mm長さ 75 mmのガラス管製遠心管に挿入した。 この遠心管にヒト末 梢血 (比重:約 1. 056) 4m 1を注ぎ、 遠心装置にかけて 50 OGで、 20 分間遠心した。 固体介在物の内接線に沈降した赤血球 (比重: 1. 0964) が 遠心管内壁を伝つて固体介在物の下方に移動し、 同時に固体介在物が上方に押し 上げられた。最終的には固体介在物の上方に血清および白血球を含む画分が残り、 下方に赤血球が集積した。  A polystyrene sphere having a diameter of 10.6 Oram (specific gravity: 1.05) was molded and inserted into a glass tube centrifuge tube having an inner diameter of 10.75 mm and a length of 75 mm. 4 ml of human peripheral blood (specific gravity: about 1.056) was poured into the centrifuge tube, and the mixture was centrifuged at 50 OG for 20 minutes in a centrifuge. Erythrocytes (specific gravity: 1.0964) that settled on the inner tangent to the solid inclusions moved down the solid inclusions along the inner wall of the centrifuge tube, and simultaneously the solid inclusions were pushed upward. Eventually, a fraction containing serum and leukocytes remained above the solid inclusions, and red blood cells accumulated below.
この実験の結果を表 1に纏めた。  Table 1 summarizes the results of this experiment.
実施例 2 Example 2
固体介在物の遠心管内壁と接するポリスチレン樹脂製の楕円球 (短軸対長軸比 ヽ 1 : 1. 2のもので比重 1. 05) を用い、 600 Gで 15分遠心した外は 実施例と同様に血液を処理した。 その結果も 〔表 1〕 に纏めた。 Using a polystyrene resin elliptical sphere (short axis to long axis ratio ヽ 1: 1.2, specific gravity 1.05) in contact with the inner wall of the solid inclusion centrifuge tube, centrifuged at 600 G for 15 minutes. Blood was processed as in. The results are also summarized in [Table 1].
実施例 1 実施例 2 Example 1 Example 2
心目 [J'  Heart [J '
珠 楕円球 遠心力 G X分 0 500X20 600X 15 容量 (m l) 4 1.8 1.7 赤数 (X 104/^ 1) 556 30 50 血 ヘモグロビン (g/d 1) υ , 0 11 · A ¾ 球 へマトクリット (%) 52.6 3.4 6.0 白 数 (/ 1 ) 6,900 14,000 14,200 血顆粒球数 (/ 1 ) 2,300 4, 100 4,400 球 リン Λ°球数 (/ 1 ) 4, 600 9,900 9,800 回 赤血球 (%) 100 2.4 3.8 収 Oval oval sphere Centrifugal force GX min 0 500X20 600X 15 Volume (ml) 4 1.8 1.7 Red number (X10 4 / ^ 1) 556 30 50 Blood hemoglobin (g / d 1) ,, 011 · A ¾ %) 52.6 3.4 6.0 White number (/ 1) 6,900 14,000 14,200 Number of blood granulocytes (/ 1) 2,300 4, 100 4,400 Income
率 白血球 〈%) 100 91.3 87.5  Rate Leukocyte <%) 100 91.3 87.5
産業上の利用可能性 Industrial applicability
本発明の方法は、 比較的簡単な装置を用いて、 従来法の遠心分離操作を行うだ けで、 比重の異なる二種以上の微粒子を含む微粒子群を、 比重の高低二群に高い 精度で分画することができる。 特に、 血液中から赤血球を除去し、 有核細胞であ る白血球を採取するのに極めて適した方法である。  In the method of the present invention, a fine particle group containing two or more types of fine particles having different specific gravities can be converted into two groups with high and low specific gravities with high accuracy by simply performing a conventional centrifugal separation operation using a relatively simple apparatus. Can be fractionated. In particular, this method is very suitable for removing red blood cells from blood and collecting leukocytes, which are nucleated cells.

Claims

請 求 の 範 囲 The scope of the claims
1. 比重の異なる二種以上の微粒子を含む微粒子群を、 その微粒子群の比重の 中間帯に位置する比重を持ち、 遠心管に内接しながら上下に移動可能であり、 遠 心管に内接している部分より上部が凸状に成形された固体介在物と共に遠心し て、 遠心管内壁と固体介在物との接線を通過して高比重微粒子を固体介在物の下 方に、 または低比重微粒子を固体介在物の上方に移動させると同時に固体介在物 を比重の高低二粒子群の中間に移動、 介在させることを特徴とする比重の異なる 微粒子の分別法。 1. A fine particle group containing two or more types of fine particles with different specific gravities has a specific gravity located in the middle zone of the specific gravity of the fine particle group, and can move up and down while inscribed in a centrifuge tube. Centrifuged together with the solid inclusions whose upper part is convex, and passed through the tangent line between the inner wall of the centrifuge tube and the solid inclusions to move the high specific gravity fine particles below the solid inclusions or low specific gravity fine particles. A method for separating fine particles having different specific gravities, wherein the particles are moved above the solid inclusions and at the same time, the solid inclusions are moved and interposed between the two groups of high and low specific gravity particles.
2. 個体介在物の形状が、 遠心管内壁に対して線で接する形状に成形されたも のであることを特徴とする請求の範囲 1. 記載の方法。  2. The method according to claim 1, wherein the shape of the solid inclusion is shaped to be in contact with the inner wall of the centrifuge tube by a line.
3. 固体介在物の形状が、 真球状、 楕円球状、 フットボール状、 雨滴状、 紡錘 状またはそろばん珠状であることを特徴とする請求の範囲 1. または 2. 記載の 方法。  3. The method according to claim 1, wherein the shape of the solid inclusion is a true sphere, an ellipsoid, a football, a raindrop, a spindle, or an abacus.
4. 微粒子が粒径 0. 01〜200 μπιの範囲内にあるものであることを特徴 とする請求の範囲 1. 〜3. のいずれかに記載の方法。  4. The method according to any one of claims 1 to 3, wherein the fine particles have a particle size in the range of 0.01 to 200 μπι.
5. 微粒子が液体中に懸濁しているかまたはコロイド状として存在しているこ とを特徴とする請求の範囲 1. 〜4. のいずれかに記載の方法。  5. The method according to any one of claims 1 to 4, wherein the fine particles are suspended in a liquid or exist as a colloid.
6. 微粒子を含む液体が、 体液であることを特徴とする請求の範囲 1. 〜5. のいずれかに記載の方法。  6. The method according to any one of claims 1 to 5, wherein the liquid containing fine particles is a body fluid.
7. 固体介在物が、 寒天、 ゼラチン、 合成樹脂、 セラミタス、 タンパク質また は多糖類であることを特徴とする請求の範囲 1.〜6.のいずれかに記載の方法。  7. The method according to any one of claims 1 to 6, wherein the solid inclusion is agar, gelatin, synthetic resin, ceramitas, protein or polysaccharide.
8. 遠心管がガラスまたは合成樹脂製であることを特徴とする請求の範囲 1. 〜7. のいずれかに記載の方法。 8. The method according to any one of claims 1 to 7, wherein the centrifuge tube is made of glass or synthetic resin.
9. 遠心管内と、 その遠心管に内接しながら上下に移動可能であり、 遠心管に 内接している部分より上部が凸状に成形された、 固体介在物からなることを特徴 とする請求の範囲 1. 〜8. のいずれかに記載の方法を実施するための装置。  9. A solid inclusion that is movable up and down inside the centrifuge tube while being inscribed in the centrifuge tube, and formed in a convex shape above the part inscribed in the centrifuge tube. An apparatus for performing the method according to any one of the ranges 1. to 8.
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