JP2017159225A - Particle fractionation apparatus and particle fractionation method - Google Patents
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Abstract
Description
本発明は、粒子分別装置及び粒子分別方法に関し、さらに詳しくは、多数の粒子をその粒径や凝集度ならびに密度に応じて精度よく、迅速に、且つ簡易簡便に分別することのできる粒子分別装置及び粒子分別方法に関するものである。 The present invention relates to a particle sorting apparatus and a particle sorting method. More specifically, the present invention relates to a particle sorting apparatus capable of sorting a large number of particles accurately, quickly, and simply according to the particle size, degree of aggregation, and density. And a particle sorting method.
粒子を各種要因、例えば密度、凝集状態、粒径等をもって分別する技術は種々提案されている。
例えば、特許文献１には、粒子の沈降速度の相違を利用した技術として、原水１１と凝集剤とが投入される第１の凝集槽と、第１の凝集槽の下流に連通して配置された第１の凝集槽より大きな内容積を有する第２の凝集槽と、第２の凝集槽で成長したフロックを分別する沈降分離槽と、沈降分離槽で分離された処理液を沈静させる最終沈静槽の最低４個の槽を一体として形成した装置が提案されている。
また、特許文献２には、遠心力を利用した技術として、基台と、基台上で回転する円盤型容器と、懸濁液供給タンクを有し、円盤型容器は、その回転中心の周囲に周方向に等間隔で配設された複数の扇形遠心分離槽と、粒子供給筒を備え、複数の扇形遠心分離槽は、それぞれ周側壁と底壁とから成り互いに独立したくぼみとして形成されており、粒子供給筒は、前記円盤型容器の中心部から前記扇形遠心分離槽に向けて前記懸濁液を放出するように設けられており、懸濁液供給タンクから供給される懸濁液に含まれる粒子を遠心分離槽の外側壁の内面に周方向に区画され複数の粒子回収ポケットに分別するように形成された装置が提案されている。
Various techniques for separating particles according to various factors such as density, agglomeration state, particle size and the like have been proposed.
For example, in Patent Document 1, as a technique using the difference in the sedimentation speed of particles, a first flocculation tank into which raw water 11 and a flocculating agent are charged and a downstream of the first flocculation tank are arranged. A second agglomeration tank having a larger internal volume than the first agglomeration tank, a sedimentation separation tank for separating flocs grown in the second agglomeration tank, and a final calming for calming the treatment liquid separated in the sedimentation separation tank An apparatus in which at least four tanks are integrally formed has been proposed.
Patent Document 2 includes a base, a disk-shaped container that rotates on the base, and a suspension supply tank as a technique using centrifugal force, and the disk-shaped container is around the center of rotation. Are provided with a plurality of fan-shaped centrifuge tanks arranged at equal intervals in the circumferential direction and a particle supply cylinder, and each of the plurality of fan-shaped centrifuge tanks is composed of a peripheral side wall and a bottom wall, and is formed as an independent recess. And the particle supply cylinder is provided so as to discharge the suspension from the central part of the disk-shaped container toward the fan-shaped centrifuge tank. An apparatus has been proposed in which contained particles are partitioned on the inner surface of the outer wall of the centrifuge tank in the circumferential direction and separated into a plurality of particle recovery pockets.
しかしながら、特許文献１及び２における提案では、未だに分別操作が煩雑であり、また十分に要求されているレベルでの分別ができないという問題があった。
たとえば、粒子として細胞粒子の分別も産業界で強く求められている。その背景は、再生医療技術の発達や免疫研究などの進展により、骨髄や膵島、肝蔵細胞などを体内に血管などを通じて移植する細胞治療への期待が高いことによる。特に、肝細胞移植に関しては、先天的な肝臓疾患の新生児に対する治療として国内初の臨床研究報告がなされており（非特許文献１）、将来的にはＥＳ細胞・ｉＰＳ細胞利用への展開としても期待が高い。しかしながら研究用として利用されているガン細胞由来の実験用細胞に比べて、細胞移植に用いられる細胞は、非常に弱く、利用可能な細胞の量に限りがある。
特に国内では、膵島は膵臓移植不適合臓器から、肝臓実質細胞は乳幼児に対する生体肝移植時の残余肝臓から、細胞を分離し移植医療に用いられており、高機能な細胞を高効率に確保することが大きな課題となっている。このため細胞粒子を分別することが必要である。通常細胞移植用の細胞粒子は、摘出臓器内に組織分解酵素液を灌流し溶解し、分散・抽出することで取得される。しかし、この一連の工程で多くの細胞が機能を失い、また喪失され、細管を用い細胞分散液を移植先の肝臓の流入血管の一つである門脈などから輸液とともに移植する際にも非常に多くの細胞が喪失することが知られている（非特許文献2）。このような背景から、いかに細胞の機能を維持し、細胞を破壊・喪失せずに取りあつかうかが重要であると共に、機能を喪失した細胞粒子と機能的な細胞粒子とを如何にして分別するかという観点での技術開発が重要であり、これらを克服するための技術、特に、細胞分散液をやさしく、積極的に連続的に操作し、機能的な細胞粒子と非機能的な細胞粒子とを効率用分別するための基礎技術の確立が求められている。
以上のような観点から、多数の粒子をその粒径や凝集度に応じて精度よく、迅速に、且つ簡易簡便に分別することのできる粒子の分別方法の開発が要望されている。
However, the proposals in Patent Documents 1 and 2 still have a problem that the sorting operation is still complicated and the sorting at a sufficiently required level cannot be performed.
For example, the separation of cell particles as particles is strongly required in the industry. This is due to the high expectations for cell therapy in which bone marrow, pancreatic islets, hepatic cells, etc. are transplanted into the body through blood vessels, etc. due to the development of regenerative medicine technology and immunological research. In particular, with regard to hepatocyte transplantation, the first clinical research report has been made in Japan as a treatment for neonates with congenital liver disease (Non-patent Document 1). Expectation is high. However, compared to experimental cells derived from cancer cells used for research purposes, the cells used for cell transplantation are very weak and the amount of cells that can be used is limited.
Especially in Japan, islets are used for transplantation by isolating pancreatic islets from incompatible organs for pancreas transplantation and liver parenchymal cells from residual liver at the time of living liver transplantation for infants, ensuring highly functional cells with high efficiency. Has become a major issue. For this reason, it is necessary to sort the cell particles. Usually, cell particles for cell transplantation are obtained by perfusing a tissue-degrading enzyme solution into an excised organ, dissolving it, and dispersing and extracting it. However, many cells lose function and are lost in this series of steps, and it is also extremely difficult to transplant a cell dispersion with infusion from the portal vein, which is one of the inflowing blood vessels of the liver of the transplant destination, using tubules. It is known that many cells are lost (Non-patent Document 2). Against this background, it is important how to maintain the function of the cell and deal with it without destroying or losing the cell, and how to classify cell particles that have lost function and functional cell particles. It is important to develop technology from the viewpoint of the above, especially technologies for overcoming these problems, especially the gentle and active operation of cell dispersions, and functional and non-functional cell particles. Establishment of basic technology for separating waste for efficiency is required.
In view of the above, there is a demand for the development of a method for separating particles that can quickly and easily separate a large number of particles with high accuracy according to the particle size and the degree of aggregation.
したがって、本発明の目的は、多数の粒子をその密度，粒径や凝集度に応じて精度よく、迅速に、且つ簡易簡便に分別することのできる粒子の分別方法及び分別装置を提供することにある。 Accordingly, an object of the present invention is to provide a particle sorting method and a sorting apparatus capable of sorting a large number of particles accurately, quickly and simply according to their density, particle size and degree of aggregation. is there.
本発明者らは、上記課題を解消すべく鋭意検討した結果、多数の細胞粒子が存在する細胞培養液などの粒子分散流体と細胞・溶液に対して密度の高い流体間に形成される液液界面を活用すれば序器課題を解消し得ると考え、この液液界面を活用する手法について種々検討した結果、液液界面を有する液液２相流体を用いて、曲がり管内に誘起される二次流れを積極的に用いれば、細胞粒子を破壊することなく、上記目的を達成し得ることを知見し、本発明を完成するに至った。
すなわち、本発明は以下の各発明を提供するものである。
１．多数の粒子を分別する粒子分別装置であって、
上記の多数の粒子と、第1の流液と、該第1の流液とは不溶性であり、且つ密度の異なる第２の流液とが投入可能であり、これらを所定割合で有する移送用液を移送するために保持する移送準備部、
上記移送用液を流通させる流通路からなり、該流通路が所定の湾曲形状を有する湾曲体である分別部、及び
上記流通路に連結されており、分別された粒子を分別区分ごとに収集する収集部
を具備する粒子分別装置。
２．多数の粒子を分別する粒子分別方法であって、
上記の多数の粒子と、第1の流液と、該第1の流液とは不溶性であり、且つ密度の異なる第２の流液とを所定割合で有する移送用液を調整する移送準備工程、
上記移送用液を流通させる流通路であって、管状であり、所定の湾曲形状を有する湾曲体である流通路に上記移送用液を流通させて粒子の分別を行う分別工程、及び
分別された粒子を分別区分ごとに収集する収集工程を具備する粒子分別方法。
３．上記粒子が細胞粒子であり、上記分別工程は、該細胞粒子を生きている細胞と死滅した細胞とに分別する工程であって、密度の差、及び／又は粒子の大きさの差により分別する工程である２記載の粒子分別方法。
As a result of intensive studies to solve the above problems, the present inventors have found that a liquid liquid formed between a particle-dispersed fluid such as a cell culture solution in which a large number of cell particles are present and a fluid having a high density relative to the cell / solution It is thought that if the interface is utilized, the introductory problem can be solved. As a result of various investigations on the method of utilizing the liquid-liquid interface, two liquid-phase fluids having a liquid-liquid interface are used to induce the two-phase induced in the bent pipe. It has been found that if the next flow is positively used, the above object can be achieved without destroying the cell particles, and the present invention has been completed.
That is, the present invention provides the following inventions.
1. A particle separation device for separating a large number of particles,
The above-mentioned many particles, the first flowing liquid, and the second flowing liquid that is insoluble in the first flowing liquid and different in density can be charged, and for transfer having these in a predetermined ratio. A transfer preparation unit for holding the liquid to transfer,
The flow path through which the transfer liquid is circulated, and the flow path is a curved portion having a predetermined curved shape, and is connected to the flow path, and collects the separated particles for each classification section. A particle sorting apparatus including a collecting unit.
2. A particle sorting method for sorting a large number of particles,
A transfer preparation step of adjusting a transfer liquid having a predetermined ratio of the above-mentioned many particles, the first flowing liquid, and the second flowing liquid insoluble in the first flowing liquid and having different densities. ,
A flow path through which the transfer liquid is circulated, a separation step for separating particles by flowing the transfer liquid through a flow path that is tubular and has a predetermined curved shape, and the separation A particle sorting method comprising a collecting step of collecting particles for each sorting section.
3. The particle is a cell particle, and the sorting step is a step of sorting the cell particle into a living cell and a dead cell, and the sorting is performed based on a difference in density and / or a difference in particle size. 3. The method for fractionating particles according to 2, which is a process.
本発明の分別装置は、多数の粒子をその粒径や凝集度に応じて精度よく、迅速に、且つ簡易簡便に分別することのできるものである。
また、本発明の分別方法は、多数の粒子をその粒径や凝集度に応じて精度よく、迅速に、且つ簡易簡便に分別することができる。
The sorting apparatus of the present invention can sort a large number of particles quickly, simply and easily with high accuracy according to the particle size and the degree of aggregation.
Further, the separation method of the present invention can separate a large number of particles accurately, quickly, and simply according to the particle size and the degree of aggregation.
１：粒子分別装置、１０：移送準備部、２０：分別部、５０移送用液 1: Particle fractionator, 10: Transfer preparation unit, 20: Separation unit, 50 liquid for transfer
以下、本発明をその実施形態に基づいてさらに詳細に説明する。
＜全体構成＞
本実施形態の粒子分別装置は、図1に示すように、多数の粒子を分別する粒子分別装置であり、上記の多数の粒子と、第1の流液と、該第1の流液とは不溶性であり、且つ密度の異なる第２の流液とが投入可能であり、これらを所定割合で有する移送用液を移送するために保持する移送準備部、上記移送用液を流通させる流通路からなり、該流通路が所定の湾曲形状を有する湾曲体である分別部、及び上記流通路に連結されており、分別された粒子を分別区分ごとに収集する収集部を具備する。
以下さらに説明する。なお、上記粒子、上記の第1の流液及び上記の第２の流液並びに移送用液については後述する。
Hereinafter, the present invention will be described in more detail based on the embodiments.
<Overall configuration>
As shown in FIG. 1, the particle sorting apparatus of the present embodiment is a particle sorting apparatus that sorts a large number of particles. The multiple particles, the first flowing liquid, and the first flowing liquid are A second flow liquid that is insoluble and has a different density can be charged, and a transfer preparation section that holds the transfer liquid having these in a predetermined ratio, and a flow passage through which the transfer liquid flows. The flow path includes a sorting unit that is a curved body having a predetermined curved shape, and a collection unit that is connected to the flow path and collects the sorted particles for each sorting section.
This will be further described below. The particles, the first flowing liquid, the second flowing liquid, and the transfer liquid will be described later.
＜移送準備部＞
移送準備部は、上記粒子、上記の第1の流液及び上記の第２の流液を一つのチャンバー又は管路より構成される容器内に投入して、移送用液としての液液界面を有する液液２相流体を形成する部分である。
本実施形態においては、図１に示すように、第1の流液供給管と、第１の流液供給管に対して所定角度をもって配置された第２の流液供給管と、両者が同一カ所において連結された、上記の第1の流液及び上記の第２の流液を一緒に移送用液として分別部に移送する移送管とからなる。
第1の流液供給管と、第２の流液供給管と、移送管とは、それぞれ図２に示すように所定の角度をもって連結されているが、第1の流液供給管と移送管との角度θ１と第２の流液供給管と移送管との角度θ２とは、本実施形態においては同じ角度である。なお、本実施形態においては、θ１＝θ２＝θ３＝１２０°となるように構成されている。また、本実施形態においては、各管は図１における矢視した場合には一本の管に見えるように１平面上に配置されているが、これに制限されず３次元的に角度を設けてもよい。また、角度も本実施形態のように同じ角度ではなく、それぞれ異なる角度に設定することも可能である。
第1の流液供給管、第２の流液供給管及び移送管それぞれの管径は特に制限されないが、本実施形態においては、全て同じ径とされている。二次流れの速度の大きさを規定するディーン数を一致させれば移送管を太く形成することも可能であり，また流量の組み合わせにより任意の管径においても実施可能であるが、ここでは，単純化するために、全ての管径は同じにしている。なお、本実施形態ではそれぞれ内径２ｍｍとしている。
また、ここでは特に図示しないが、第1の流液供給管と第２の流液供給管とはそれぞれ第1の流液を貯蔵する容器及び第２の流液を貯蔵する容器、並びにポンプなどの移送力付加手段に連結されている。
移送管の長さは第1の流液と第２の流液とを合わせてから液液２相が安定する程度の長さを設けるのが好ましく、移送速度に応じて設定するべきであり、レイノルズ数にあわせた助走区間を確保する長さと設定するのが好ましい。なお、本実施形態においては５０ｍｍとしている。
<Transfer preparation department>
The transfer preparation unit throws the particles, the first flowing liquid, and the second flowing liquid into a container composed of one chamber or a pipe, and sets a liquid-liquid interface as a transfer liquid. It is a part which forms the liquid-liquid two-phase fluid which has.
In the present embodiment, as shown in FIG. 1, both the first flowing liquid supply pipe and the second flowing liquid supply pipe arranged at a predetermined angle with respect to the first flowing liquid supply pipe are the same. It consists of a transfer pipe that is connected at a location and transfers the first and second flowing liquids together as a transfer liquid to the separation section.
The first liquid supply pipe, the second liquid supply pipe, and the transfer pipe are connected to each other at a predetermined angle as shown in FIG. And the angle θ2 between the second fluid supply pipe and the transfer pipe are the same angle in this embodiment. In the present embodiment, the configuration is such that θ1 = θ2 = θ3 = 120 °. Further, in this embodiment, each tube is arranged on one plane so that it can be seen as a single tube when viewed in the direction of the arrow in FIG. May be. Also, the angles can be set to different angles instead of the same angle as in the present embodiment.
The diameters of the first flow liquid supply pipe, the second flow liquid supply pipe, and the transfer pipe are not particularly limited, but are all the same in this embodiment. If the Dean number that defines the magnitude of the secondary flow speed is matched, it is possible to make the transfer pipe thick, and it is possible to implement it at any pipe diameter by combining the flow rates. For simplicity, all tube diameters are the same. In the present embodiment, the inner diameter is 2 mm.
Although not specifically shown here, the first and second fluid supply pipes are a container for storing the first fluid and a container for storing the second fluid and a pump, respectively. It is connected to the transfer force adding means.
It is preferable that the length of the transfer pipe should be long enough to stabilize the two liquid phases after combining the first and second liquids, and should be set according to the transfer speed. It is preferable to set the length to ensure a run-up section that matches the Reynolds number. In this embodiment, it is 50 mm.
＜分別部＞
分別部２０は、図1に示すように、半円状に湾曲した流通路である主部２１と、主部２１の末端から直線状に延びて、収集部（図示せず）に連結されている直線状の末端部２２とからなる。
本実施形態においては、主部２１及び末端部２２はいずれも管状部材からなり、これらの管内径はいずれも移送管１３の内径と同じである。また、主部２１の曲率半径９８ｍｍで、半円（θ＝１８０°）である。また、末端部２２の長さは移送準備部１０における移送管１３の長さと同じ理由から同じ長さにしてある。
主部２１の形状は、本実施形態においては半円形状としたが、これに制限されるものではなく、種々形状とすることが可能である。たとえばＳ字状、螺旋状等である。要するに、主部は、後述する２次流れを作ることができる、湾曲した形状であれば特に制限されない。
<Separation Department>
As shown in FIG. 1, the separation unit 20 includes a main part 21 that is a semicircular curved flow path, and extends linearly from the end of the main part 21 and is connected to a collection part (not shown). And a straight end portion 22.
In the present embodiment, both the main portion 21 and the end portion 22 are made of tubular members, and the inner diameters of these tubes are the same as the inner diameter of the transfer tube 13. The main portion 21 has a radius of curvature of 98 mm and a semicircle (θ = 180 °). Further, the length of the end portion 22 is the same as the length of the transfer pipe 13 in the transfer preparation unit 10 for the same reason.
The shape of the main portion 21 is a semicircular shape in the present embodiment, but is not limited to this, and can be various shapes. For example, an S shape, a spiral shape, or the like. In short, the main part is not particularly limited as long as it has a curved shape capable of creating a secondary flow described later.
＜収集部＞
収集部については特に図示しないが、公知の液液回収装置を特に制限なく採用することができる。たとえば、マイクロ矩形管を用いた装置や、２つに分離している第１の流液と第２の流液とのうち密度の差を利用して、末端部からの移送用液を２相に分離した状態で回収貯蔵するプールと、該プールのうち密度の軽い第２の流液を連続的に吸引回収する吸引装置とにより構成することができる。また、この他に吸引するのではなく、移送用液の流れを利用して回収用の流路を液液界面領域と管壁周辺領域とに別個に設置し、それぞれの流路に移送用液の流れそのままに各相の液を流入させることで、液液界面に存在する粒子群と管壁周辺域に存在する粒子群とをそれぞれ分けて回収することができる。
<Collection Department>
The collecting unit is not particularly shown, but a known liquid-liquid recovery device can be employed without any particular limitation. For example, a device using a micro rectangular tube or a two-phase transfer liquid from the end portion using a difference in density between the first and second flowing liquids separated into two. And a pool that is collected and stored in a separated state and a suction device that continuously sucks and collects the second flowing liquid having a low density in the pool. In addition to this, instead of suctioning, recovery flow paths are separately installed in the liquid-liquid interface area and the pipe wall peripheral area using the flow of the transfer liquid, and the transfer liquid is placed in each flow path. By allowing the liquid of each phase to flow in as it is, the particle group existing at the liquid-liquid interface and the particle group existing around the tube wall can be separately collected.
＜粒子分別方法＞
本実施形態の粒子分別方法は、多数の粒子を分別する粒子分別方法であって、上述の粒子分別装置を用いて行うことができる。
そして、上記の多数の粒子と、第1の流液と、該第1の流液とは不要性であり、且つ密度の異なる第２の流液とを所定割合で有する移送用液を調整する移送準備工程、
上記移送用液を流通させる流通路であって、管状であり、所定の湾曲形状（上述の湾曲体である主部の形状）を有する湾曲体である流通路に上記移送用液を流通させて粒子の分別を行う分別工程、及び
分別された粒子を分別区分ごとに収集する収集工程を行うことにより実施することができる。
<Particle separation method>
The particle sorting method of the present embodiment is a particle sorting method for sorting a large number of particles, and can be performed using the above-described particle sorting apparatus.
Then, the transfer liquid having the predetermined number of the second flowing liquids which are unnecessary in the first flowing liquid and the first flowing liquid and have different densities is prepared. Transfer preparation process,
A flow passage through which the transfer liquid is circulated, and is tubular and has a predetermined curved shape (the shape of the main part, which is the above-described curved body). It can be carried out by carrying out a sorting step for sorting particles and a collecting step for collecting the sorted particles for each sorting section.
まず、本発明の法上に用いられる粒子や流液について説明する。
（粒子、分別対象）
本発明において分別の対象として用いられる粒子としては、細胞粒子（さらに具体的には肝臓細胞、膵島，幹細胞ならびに分化した細胞等）、各種ポリマー粒子（さらに具体的には、スチレン粒子、マイクロチャネル法により製造されるポリマー粒子等）、微生物（藻類、ミドリムシ等）、高分子カプセル化粒子、金属粒子
等が挙げられる。
これらの粒子の粒径は特に制限されず、種々粒径のものを分別することができる。
また、本発明による分別は、流液中に存在する粒子の集合単位ごとに、かかる集合単位の大きさや密度をもって行うことができる。すなわち、粒子の集合単位ごとの大きさや密度が分別の対象であると言える。ここで集合単位とは、粒子が１つのみで存在すればその一つが、粒子が複数個凝集して一つのクラスターを構成して存在する場合には当該クラスターが、それぞれ該当する。
（第１の流液）
第１の流液は任意に設定することができるが、上記の粒子と親和性の高いものとするのが好ましい。たとえば粒子として細胞粒子を用いる場合には、培養液（水を主成分とし、細胞培養成分を含有するもの）等を用いることができ、ポリマー粒子の場合には該ポリマー粒子の原料ポリマーの良溶媒となる液体を用いることができる。
（第２の流液）
第２の流液は、第１の流液に対して不溶性で、第１の流液ならびに粒子よりも密度が高いものであるが、密度は特に制限されるものではなく、第１の流体と液液界面を形成し２相の流体を形成する程度に第１の流液と第２の流液とで差を設けることができればよい。
また第２の流液の密度は、用いる粒子の密度よりも高いのが好ましい。第２の流液の具体例としては、たとえば粒子として細胞粒子を用いる場合には、細胞より密度の高いフッ素不活性液体（商品名「フロリナート」３Ｍ社製等の市販品を用いることもできる）を挙げることができる。
First, the particles and liquid used in the method of the present invention will be described.
(Particles, separation target)
In the present invention, the particles used as objects of separation include cell particles (more specifically, liver cells, pancreatic islets, stem cells and differentiated cells), various polymer particles (more specifically, styrene particles, microchannel method). Polymer particles, etc.), microorganisms (algae, Euglena, etc.), polymer-encapsulated particles, metal particles, and the like.
The particle size of these particles is not particularly limited, and those having various particle sizes can be separated.
In addition, the separation according to the present invention can be performed for each aggregate unit of particles present in the flowing liquid with the size and density of the aggregate unit. That is, it can be said that the size and density for each aggregate unit of the particles are objects to be sorted. Here, the collective unit corresponds to one of the particles if only one particle is present, and the cluster corresponds to the case where a plurality of particles are aggregated to form one cluster.
(First flowing liquid)
Although the first flowing liquid can be set arbitrarily, it is preferable that the first flowing liquid has high affinity with the above particles. For example, when cell particles are used as the particles, a culture solution (water as a main component and containing cell culture components) can be used. In the case of polymer particles, a good solvent for the polymer material of the polymer particles Can be used.
(Second flowing liquid)
The second flowing liquid is insoluble in the first flowing liquid and has a higher density than the first flowing liquid and the particles, but the density is not particularly limited. It is only necessary that a difference can be provided between the first flowing liquid and the second flowing liquid to such an extent that a liquid-liquid interface is formed to form a two-phase fluid.
Moreover, it is preferable that the density of a 2nd liquid is higher than the density of the particle to be used. As a specific example of the second flowing liquid, for example, when cell particles are used as the particles, a fluorine inert liquid having a higher density than the cells (commercially available product such as “Florinato” manufactured by 3M Corporation may be used). Can be mentioned.
（移送用液）
移送用液は、上述の第１の流液と第２の流液とで２相が積層された状態となっている流液であり、図３（ａ）に示すような構成となっている。すなわち、図３に示すように、移送用液５０は、第１の流液５１と第１の流液５１の下層に位置する第２の流液５３とからなり、両者により液液界面５５が形成されている状態の流液である。
(Transfer liquid)
The transfer liquid is a flowing liquid in which two phases are laminated by the first flowing liquid and the second flowing liquid, and has a configuration as shown in FIG. . That is, as shown in FIG. 3, the transfer liquid 50 includes a first flowing liquid 51 and a second flowing liquid 53 positioned below the first flowing liquid 51, and the liquid-liquid interface 55 is formed by both. It is the flowing liquid of the state formed.
以下、各工程について説明する。
（移送準備工程）
まず、第１の流液を第１の流液供給管１１で移送管１３に供給し、且つ第２の流液を第２の流液供給管１２で移送管１３に供給し、移送管１３の入り口において第１の流液と第２の流液とを衝突させて２液を合わせ、図３（ａ）に示すような２相に分離し液液界面の存在する移送用液を形成する。この際、第１の流液の供給速度と第２の流液の供給速度とは、それぞれ第１及び第２の流液が混合しない程度であり、且つ粒子の損壊、細胞粒子の場合には細胞の死滅が生じないような速度とするのが好ましい。
例えば、二次流れ（各相内における螺旋状の流れ）と粒子の浮力、抗力のバランスが重要であるため、二次流れの大きさをディーン数により見積もり、また粒子密度から浮力（重力）を、大きさ、粘度から抗力を測り、これらのバランスが最適となるように速度を決定することができる。
Hereinafter, each step will be described.
(Transfer preparation process)
First, the first flowing liquid is supplied to the transfer pipe 13 by the first flowing liquid supply pipe 11, and the second flowing liquid is supplied to the transfer pipe 13 by the second flowing liquid supply pipe 12, and the transfer pipe 13 is supplied. The first liquid and the second liquid are collided at the entrance to the two liquids, and the two liquids are combined to separate into two phases as shown in FIG. 3A to form a transfer liquid having a liquid-liquid interface. . At this time, the supply speed of the first flowing liquid and the supply speed of the second flowing liquid are such that the first and second flowing liquids are not mixed, and in the case of particle breakage or cell particles, It is preferable that the speed is such that cell death does not occur.
For example, the balance between secondary flow (spiral flow in each phase), particle buoyancy, and drag is important, so the size of the secondary flow is estimated by the Dean number, and buoyancy (gravity) is calculated from the particle density. The drag can be measured from the size and viscosity, and the speed can be determined so that the balance between them is optimal.
（分別工程）
分別工程は、上述の分別部２０における、管状の主部２１により構成される流通路に上記移送用液を流通させることにより行う。
この際の流通方向は図１の矢印方向であり、その速度は、主部の内径、第１の流液及び第２の流液の粘度、更には粒子の粒径や密度により任意に設定されるものである。
また、上記粒子が細胞粒子である場合には、上記分別工程は、該細胞粒子を１つのみの粒子（正常な細胞粒子）と粒子が複数個凝集してなる集合物（死滅した細胞粒子）とに分別する。正常な細胞粒子はそれぞれ独立して一つずつ存在するが、死滅した細胞粒子は凝集してクラスターを形成することが知られているが、本発明の分別方法における分別工程では、このクラスターの形成が効率よく行われるようにし、且つ分別収集しやすい状態で正常な細胞粒子と死滅した細胞粒子とを分離した状態で位置づけることができる。
このように分別できるメカニズムについて説明すると、粒子をともなう曲がり管内においては遠心力が流体、粒子に誘起され、さらに管内には、主流に合わせて断面内に二次流れとして上下対称な一対の渦（Dean渦）が形成される。
一般的に曲がり管内ではＤｅａｎ数が遠心力による流れの不安定性を表す無次元数として下記式で定義され、Ｄｅａｎ渦の強さはＤｅａｎ数の大きさに依存する。
そして、主部内に移送された移送用液５０は、主部である流通用の管内を図１の矢印方向に進むと共に、２相それぞれの相内で図３（ｂ）（ｃ）に示す矢印方向のＤｅａｎ渦が発生して各粒子は主流方向（図１の矢印方向）のみならず図３（ｂ）（ｃ）に示すＤｅａｎ渦の影響をも受ける。しかも、主部２１が湾曲していることにより、液液仮面５３が（ｂ）に示すように傾き、また（ｃ）に示すように波打つ現象が生じ、また２相の両流液位（水位に相当する流液の位置）にも（ａ）の場合のような湾曲による延伸力のかかっていない状況とは異なる状態が発生する。これらの相乗作用により、密度や粒径が小さい粒子（図３における正常細胞）はＤｅａｎ渦の流れに乗り主部２１の流路内をらせん状に進むこととなるが、一方密度や粒径が大きい粒子（図３における死滅細胞）はＤｅａｎ渦の流れに乗らず外壁側にとどまったまま流路を進むこととなる。このため、末端部においては（ｄ）に示すように、液液界面に密度や粒径が小さい粒子（図３における正常細胞）が位置し、末端部を構成する管の壁側に密度や粒径が大きい粒子（図３における死滅細胞）が位置することとなる。
(Separation process)
The separation step is performed by causing the transfer liquid to flow through the flow passage formed by the tubular main portion 21 in the separation portion 20 described above.
The flow direction in this case is the arrow direction in FIG. 1, and the speed is arbitrarily set according to the inner diameter of the main part, the viscosity of the first and second flowing liquids, and the particle size and density of the particles. Is.
In addition, when the particles are cell particles, the sorting step includes an aggregate (dead cell particles) formed by agglomerating only one particle (normal cell particle) and a plurality of particles. Sort into and. Although normal cell particles exist one by one independently, it is known that dead cell particles aggregate to form a cluster. In the separation step of the separation method of the present invention, this cluster formation is performed. Can be performed efficiently and can be positioned in a state where normal and dead cell particles are separated in a state where separation and collection are easy.
The mechanism that can be separated in this way is explained. Centrifugal force is induced in the fluid and particles in the bent tube with particles, and in the tube, a pair of vortices that are vertically symmetrical as a secondary flow in the cross section in accordance with the main flow ( Dean vortex) is formed.
In general, in a bent pipe, the Dean number is defined by the following formula as a dimensionless number representing the instability of the flow due to centrifugal force, and the strength of the Dean vortex depends on the magnitude of the Dean number.
Then, the transfer liquid 50 transferred into the main portion proceeds in the direction of the arrow in FIG. 1 through the flow pipe as the main portion, and the arrows shown in FIGS. 3B and 3C in the respective phases of the two phases. A direction Dean vortex is generated and each particle is affected not only by the main flow direction (the direction of the arrow in FIG. 1) but also by the Dean vortex shown in FIGS. In addition, since the main portion 21 is curved, the liquid-liquid temporary surface 53 is inclined as shown in (b), and a wave-like phenomenon occurs as shown in (c). A position different from the situation in which no stretching force is applied due to the bending as in (a) occurs. Due to these synergistic actions, particles having a small density and particle size (normal cells in FIG. 3) ride on the flow of the Dean vortex and advance spirally in the flow path of the main portion 21, while the density and particle size are small. Large particles (dead cells in FIG. 3) travel along the flow path while staying on the outer wall side without riding on the flow of the Dean vortex. For this reason, as shown in (d), at the end portion, particles (normal cells in FIG. 3) having a small density and particle size are located at the liquid-liquid interface, and the density and particles are located on the wall side of the tube constituting the end portion Particles having a large diameter (dead cells in FIG. 3) will be located.
（収集工程）
収集工程では、上述の分別工程において、図３（ｄ）に示すように分別された粒子又は粒子群（クラスター）を、常法にしたがってそれぞれ回収する。
回収方法は、それぞれの相における外壁側と液液界面とを別に吸引し、それぞれ別の容器に回収する方法などを挙げることができる。
(Collection process)
In the collection step, the particles or particle groups (clusters) separated in the above-described separation step as shown in FIG.
Examples of the recovery method include a method in which the outer wall side and the liquid-liquid interface in each phase are separately sucked and recovered in separate containers.
＜効果、用途＞
本発明の分別装置及び分別方法は、粒子を破壊することがほとんどなく、粒子の大きさや凝集状態で分離し回収することができる。
これにより、例えば細胞粒子に適用することにより、細胞移植や細胞医療に重要な細胞の生死分離を、遠心分離機などを用いずに、連続的かつ低侵襲で行うことが可能となる。さらに、細胞移植に重要な適切なサイズの細胞クラスターを選別し、抽出することが可能となる。
さらには培養空間として用いることにより、酸素供給ならびに各種液性因子環境を管理可能であり、底面接触などの物理的な刺激も低減可能な培養環境の供給が可能となる。
<Effects and uses>
The fractionation apparatus and the fractionation method of the present invention hardly destroy particles, and can be separated and recovered in the size or aggregated state of the particles.
Thereby, for example, by applying to cell particles, it becomes possible to perform life-and-death separation of cells important for cell transplantation and cell medicine continuously and less invasively without using a centrifuge or the like. Furthermore, it becomes possible to select and extract a cell cluster of an appropriate size important for cell transplantation.
Furthermore, by using it as a culture space, it is possible to manage the oxygen supply and various liquid factor environments, and to supply a culture environment that can reduce physical stimulation such as bottom contact.
本発明は上述した実施形態に何ら制限されるものではなく、本発明の趣旨を逸脱しない範囲で種々変形可能である。
たとえば、上述の実施形態では各管の形状として断面円形状の管を用いて説明したが、液液二層界面が形成される矩形管や楕円管、その他形状を最適化した管を用いてもよい。
また、液液界面を形成することができ、二次流れを形成可能であれば、管のような閉鎖型の流路でなくとも、断面コの字状やＵ字状の開放型の流路により、流通路を形成することができる。
The present invention is not limited to the embodiment described above, and various modifications can be made without departing from the spirit of the present invention.
For example, in the above-described embodiment, the description has been given using a circular cross-sectional tube as the shape of each tube, but a rectangular tube or an elliptical tube in which a liquid-liquid two-layer interface is formed, or a tube whose shape is optimized may be used. Good.
In addition, if a liquid-liquid interface can be formed and a secondary flow can be formed, an open channel having a U-shaped cross section or a U-shape can be used instead of a closed channel such as a tube. Thus, a flow path can be formed.
以下、本発明について実施例及び比較例を示してさらに具体的に説明するが本発明はこれらに何ら制限されるものではない。 EXAMPLES Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated more concretely, this invention is not restrict | limited to these at all.
〔実施例１〕
図１及び２に示す装置を作製した。各管の内径はそれぞれr = 2 mmとし、曲率半径はR=47ｍｍ、98ｍｍ、142ｍｍの３種類を用意し用いた。
第１の流液として精製水を、第２の流液としてフッ素不活性液体（商品名「Fluorinert FC-40」3M社製）を用い、粒子として、蛍光粒子を用いた。使用粒子は粒子のクラスターと単独粒子とが混在する条件と同様にするために、中心粒径dp=70 μｍ（密度1500 kg/m3）と、dp=31 μｍ（密度1050 kg/m3）とを等量の重量部で混合して用いた。
まず、両粒子を精製水に分散して粒子0.01wt%の懸濁液を調製し、そのうち20mlをシリンジに充填し、またフッ素不活性液体も同様なシリンジに同量充填し、それぞれのシリンジを第１の流液供給管及び第２の流液供給管に連結して、シリンジポンプによりそれぞれの供給管に送液した。
検査領域は曲がり流路入り口部をθ=0°とし、θ=0°（図１の（ａ））、90°（図１の（ｂ））、180°（図１の（ｃ））の3か所で確認した。また、液液界面が安定して形成可能なDe=20、 35、 50、 65、 80の条件で実験を行った。観察はズームレンズとデジタルカメラ（SONY ILCE-QX1）により、流路上面を動画撮影して流路内の粒子分布を計測し、連続画像から画像処理により粒子分布を計測した。
粒径dp=70μｍ、曲率半径R=98mm の場合の各位置における粒子分布の結果グラフを図４（a） に示す。
主部２１に流入直後（θ=0°）においては、左右対称の分布となっており、流速の早い中央において局所流速に対応し粒子数が多く示されている。
一方、主部２１の中央（θ=90°）においては非対称な分布となり、多くの粒子が曲管外周側壁面近傍に集まっており、流路外側に粒子が大きく偏在しており、0.8≦a/r≦1の範囲に約50%の粒子が集中している。これはDean流れの効果が強く示されたものと考えられる。
さらに下流側であるθ=180°の位置においては壁面から少し離れた位置に(a/ r =0.8)に最大値を示し、これらの結果から、粒子の分離できていることがわかる。
また、図４（ｂ）に、粒子として粒径dp=31のものと70μｍのものとを用い、曲率半径R=98mmの主部を用い、De=50における結果を示す。図４（ｂ）に示す結果から明らかなように、dp=70μｍ（粒径の大きな粒子）においては粒子が0.6≦a/r≦1すなわち管の内壁近傍側に偏在しているのに対し、dp=31μｍ（粒径の小さな粒子）においては内側に若干の集中が示されているものの粒子は特定箇所への偏在が認められなかった。この結果から、本発明の分別装置及び分別方法は、粒子の分別に有用であることが分かった。
[Example 1]
The apparatus shown in FIGS. 1 and 2 was produced. The inner diameter of each tube was r = 2 mm, and the radius of curvature was three types of R = 47 mm, 98 mm, and 142 mm.
Purified water was used as the first flowing liquid, a fluorine inert liquid (trade name “Fluorinert FC-40” manufactured by 3M) was used as the second flowing liquid, and fluorescent particles were used as the particles. The particle size used is the same as the condition where a cluster of particles and single particles are mixed, so that the center particle size is dp = 70 μm (density 1500 kg / m 3 ) and dp = 31 μm (density 1050 kg / m 3 ). Were mixed in equal parts by weight.
First, disperse both particles in purified water to prepare a 0.01 wt% particle suspension, 20 ml of which is filled into a syringe, and the same amount of fluorine inert liquid is filled into a similar syringe. It connected with the 1st flowing liquid supply pipe and the 2nd flowing liquid supply pipe, and sent to each supply pipe with the syringe pump.
The inspection region has a bent flow path inlet at θ = 0 °, and θ = 0 ° (FIG. 1 (a)), 90 ° (FIG. 1 (b)), 180 ° (FIG. 1 (c)). Confirmed in 3 places. In addition, the experiment was performed under the conditions of De = 20, 35, 50, 65, and 80 where the liquid-liquid interface can be stably formed. For observation, a zoom lens and a digital camera (SONY ILCE-QX1) were used to measure the particle distribution in the flow path by taking a video of the upper surface of the flow path, and the particle distribution was measured by continuous image processing.
FIG. 4 (a) shows a result graph of the particle distribution at each position when the particle diameter dp = 70 μm and the curvature radius R = 98 mm.
Immediately after flowing into the main portion 21 (θ = 0 °), the distribution is symmetrical, and a large number of particles are shown corresponding to the local flow velocity at the center where the flow velocity is fast.
On the other hand, at the center of the main portion 21 (θ = 90 °), the distribution is asymmetric, many particles are gathered in the vicinity of the outer peripheral side wall surface of the curved pipe, and the particles are greatly unevenly distributed outside the flow path, 0.8 ≦ a Approximately 50% of the particles are concentrated in the range of / r ≦ 1. This is thought to be a strong indication of the effect of Dean flow.
Further, at the position of θ = 180 ° on the downstream side, the maximum value is shown at (a / r = 0.8) at a position slightly away from the wall surface. From these results, it can be seen that the particles can be separated.
FIG. 4B shows the results at De = 50, using particles having a particle size of dp = 31 and particles having a particle diameter of dp = 31 and a particle having a radius of curvature of R = 98 mm. As is clear from the results shown in FIG. 4B, in dp = 70 μm (particles having a large particle diameter), the particles are unevenly distributed in the vicinity of 0.6 ≦ a / r ≦ 1, that is, on the side near the inner wall of the tube. In dp = 31 μm (particles with a small particle size), although some concentration was shown on the inside, the particles were not unevenly distributed to specific places. From this result, it was found that the separation apparatus and the separation method of the present invention are useful for the separation of particles.
Claims (3)
上記の多数の粒子と、第1の流液と、該第1の流液とは不溶性であり、且つ密度の異なる第２の流液とが投入可能であり、これらを所定割合で有する移送用液を移送するために保持する移送準備部、
上記移送用液を流通させる流通路からなり、該流通路が所定の湾曲形状を有する湾曲体である分別部、及び
上記流通路に連結されており、分別された粒子を分別区分ごとに収集する収集部
を具備する粒子分別装置。 A particle separation device for separating a large number of particles,
The above-mentioned many particles, the first flowing liquid, and the second flowing liquid that is insoluble in the first flowing liquid and different in density can be charged, and for transfer having these in a predetermined ratio. A transfer preparation unit for holding the liquid to transfer,
The flow path through which the transfer liquid is circulated, and the flow path is a curved portion having a predetermined curved shape, and is connected to the flow path, and collects the separated particles for each classification section. A particle sorting apparatus including a collecting unit.
上記の多数の粒子と、第1の流液と、該第1の流液とは不溶性であり、且つ密度の異なる第２の流液とを所定割合で有する移送用液を調整する移送準備工程、
上記移送用液を流通させる流通路であって、管状であり、所定の湾曲形状を有する湾曲体である流通路に上記移送用液を流通させて粒子の分別を行う分別工程、及び
分別された粒子を分別区分ごとに収集する収集工程を具備する粒子分別方法。 A particle sorting method for sorting a large number of particles,
A transfer preparation step of adjusting a transfer liquid having a predetermined ratio of the above-mentioned many particles, the first flowing liquid, and the second flowing liquid insoluble in the first flowing liquid and having different densities. ,
A flow path through which the transfer liquid is circulated, a separation step for separating particles by flowing the transfer liquid through a flow path that is tubular and has a predetermined curved shape, and the separation A particle sorting method comprising a collecting step of collecting particles for each sorting section.
The particle is a cell particle, and the sorting step is a step of sorting the cell particle into a living cell and a dead cell, and the sorting is performed based on a difference in density and / or a difference in particle size. The method for fractionating particles according to claim 2, which is a step.
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