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WO2006001196A1 - Séparateur de cellule et procédé de séparation de cellule - Google Patents

Séparateur de cellule et procédé de séparation de cellule Download PDF

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Publication number
WO2006001196A1
WO2006001196A1 PCT/JP2005/010876 JP2005010876W WO2006001196A1 WO 2006001196 A1 WO2006001196 A1 WO 2006001196A1 JP 2005010876 W JP2005010876 W JP 2005010876W WO 2006001196 A1 WO2006001196 A1 WO 2006001196A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluid
cell separation
dividing means
opening
micromixer
Prior art date
Application number
PCT/JP2005/010876
Other languages
English (en)
Japanese (ja)
Inventor
Nobuhide Kasagi
Naoki Shikazono
Yuji Suzuki
Wei Heong Tan
Original Assignee
The University Of Tokyo
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Tokyo filed Critical The University Of Tokyo
Publication of WO2006001196A1 publication Critical patent/WO2006001196A1/fr

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/432—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction with means for dividing the material flow into separate sub-flows and for repositioning and recombining these sub-flows; Cross-mixing, e.g. conducting the outer layer of the material nearer to the axis of the tube or vice-versa
    • B01F25/4321—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction with means for dividing the material flow into separate sub-flows and for repositioning and recombining these sub-flows; Cross-mixing, e.g. conducting the outer layer of the material nearer to the axis of the tube or vice-versa the subflows consisting of at least two flat layers which are recombined, e.g. using means having restriction or expansion zones
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/433—Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/433—Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
    • B01F25/4331—Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

Definitions

  • the present invention relates to a cell separation device and a cell separation method.
  • US5536475 As a conventional cell separation apparatus using magnetic beads, for example, US5536475 is intended to extract a large amount of sample force and desired hematopoietic cells. In addition to mixing the cells, sufficient incubation time was required for the magnetic beads and the cells to be joined by molecular diffusion.
  • stem cells and the like are capable of self-proliferation, if even a very small sample force can be extracted, it is sufficient to observe subsequent growth.
  • the Reynolds number of the flow decreases, so the mixing of magnetic beads and cells is suppressed, and the target cells cannot be extracted. there were.
  • the present invention provides a cell separation method using magnetic beads, regardless of the Reynolds number, sufficiently mixing the magnetic beads and cells, attaching the cells to the magnetic beads, and separating the cells.
  • the purpose is to carry out simply and reliably.
  • the present invention provides:
  • a first fluid dividing means for dividing a fluid containing magnetic beads and predetermined cells into left and right; a second fluid dividing means for dividing the fluid divided into the left and right upward;
  • a micromixer having third fluid dividing means for dividing the divided fluid downward, and fluid merging means for joining the fluid divided upward and the fluid divided downward
  • the magnetic beads in the fluid after passing through the micromixer and the front A separator for separating the cells attached to the magnetic beads from the remainder of the fluid;
  • the present invention also provides:
  • a first fluid dividing means provided in the micromixer, a first step of dividing a fluid containing magnetic beads and predetermined cells into right and left;
  • the second step of dividing the fluid divided into the left and right upwards Using the second fluid dividing means provided in the micromixer, the second step of dividing the fluid divided into the left and right upwards;
  • the fluid containing the magnetic beads and the predetermined cells is divided in the left-right direction, and further, the divided fluid is divided in the vertical direction, and then the divided fluids are joined.
  • the cross section of the fluid divided in the vertical direction can be rotated by a predetermined angle with respect to the cross section of the first fluid, and the fluid divided in the vertical direction is joined. In this case, it can be rotated again by a predetermined angle with respect to the cross section of the first fluid. Therefore, through the above process, the first fluid is divided, and the divided fluid is rotated by a predetermined angle, and then joined, so that the fluid is sufficiently stirred. Will be mixed.
  • the magnetic beads and the cells contained in the fluid are also sufficiently agitated and mixed with each other, so that the cells are sufficiently attached to the magnetic beads.
  • the fluid is stirred and mixed by dividing the fluid and rotating the fluid cross section! Therefore, regardless of the Reynolds number of the fluid, stirring and mixing of the magnetic beads and the cells can be sufficiently promoted to attach the cells to the magnetic beads.
  • the magnetic beads and cells are sufficiently mixed for cell separation using magnetic beads, regardless of the Reynolds number.
  • the cells can be attached to the beads and the cells can be separated easily and reliably.
  • FIG. 1 is a block diagram showing an example of a cell separation device of the present invention.
  • FIG. 2 is an exploded configuration diagram showing an example of a micromixer in the cell separation apparatus shown in FIG. 1.
  • FIG. 3 is a diagram showing a state of fluid when flowing in the micromixer shown in FIG. 2.
  • FIG. 4 is a diagram showing the state of fluid when flowing in the micromixer shown in FIG.
  • FIG. 5 is an enlarged view showing a peripheral portion including a separator in the cell separation device shown in FIG. 1.
  • FIG. 6 is an enlarged view showing a peripheral portion including the purification separation device in the cell separation device shown in FIG. 1.
  • FIG. 7 is a configuration diagram showing a modification of the cell separation device shown in FIG. 1.
  • FIG. 1 is a configuration diagram showing an example of the cell separation device of the present invention.
  • a cell separation apparatus 10 shown in FIG. 1 includes a micromixer unit 20 in which a plurality of micromixers 21 are connected in series, a separator unit 30 in which a plurality of separators 31 are arranged in series, and magnetic beads and cells.
  • a flow path 40 is provided for flowing a fluid containing the fluid and passing it through the micromixer section 20 and the separator section 30 so that the fluid is subjected to cell separation according to the steps described in detail below.
  • a fluid containing magnetic beads is introduced from the inlet 11, and a fluid containing stem cells such as bone marrow fluid or blood, or blood cells is introduced from the inlet 12 to flow.
  • Mix in path 40 Next, the fluid obtained by mixing is introduced into the micromixer part 20 and stirred and mixed in each micromixer 21 as described in detail below.
  • FIG. 2 is an exploded configuration diagram showing an example of the micromixer 21 in the cell separation device 10 shown in FIG.
  • a micromixer 21 shown in FIG. 2 includes a first plate-like member 22, a second plate-like member 23 and a fourth plate-like member 25 that are sequentially provided above the first plate-like member 22, A third plate-like member 24 and a fifth plate-like member 26 are sequentially provided below the first plate-like member 22.
  • the first plate-like member 22 is formed with a T-shaped first opening 221 and an I-shaped fourth opening 222, and ends 221A and 222A of these openings are respectively First plate member 22 Are opened at opposite side ends.
  • the second plate-like member 23 is formed with an L-shaped second opening 231, and one end 231 A thereof is the first opening in the first plate-like member 22.
  • the end of the upper side opening of 221 is continuous with 11B.
  • the other end 231 B of the second opening 231 is continuous with the front end 222 B of the fourth opening 222 in the first plate-like member 22.
  • the third plate-like member 24 is similarly formed with an L-shaped third opening 241, and one end 241 A thereof is the first opening 221 in the first plate-like member 22.
  • the edge of the upper side opening 2 is continuous with 21C.
  • the other end 241 B of the third opening 241 is continuous with the front end 222 B of the fourth opening 222 in the first plate-like member 22.
  • the fourth plate member 25 and the fifth plate member 26 are provided as lids so as to seal the first opening 221 to the fourth opening 222, respectively. This is to make the micromixer shown in Fig. 2 function as an actual device.
  • 3 and 4 are diagrams showing the state of the fluid when flowing in the micromixer 21 shown in FIG.
  • a case of a multilayer fluid in which a fluid containing magnetic beads and a fluid containing cells form layers in the upward and downward directions will be described.
  • the multilayer fluid S 1 is introduced into the first opening 221 in the first plate-like member 22 of the micromixer 21. At this time, the multilayer fluid S1 is divided in the left-right direction at the upper side of the first opening 221. Next, the multilayer fluid S1 divided in the left-right direction is continuous with the first opening 221 and the second opening 231 in the second plate member 23 and the second fluid in the third plate member 24, respectively. It is introduced into the opening 241 of 3, and as a result, it is divided in the vertical direction. At this time, since the multilayer fluid S1 is rotated 90 degrees with respect to the flow direction, the cross section thereof is rotated 90 degrees.
  • the multilayer fluid S1 is introduced into the fourth opening 222 of the first plate-like member 22 that is continuous with the second opening 231 and the third opening 241 and is divided in the vertical direction.
  • the multilayer fluid S1 joins to become multilayer fluid S2.
  • each of the divided multilayer fluids S1 is further rotated by 90 degrees with respect to the flow direction, so that the cross section is rotated by 90 degrees. Therefore, the cross section of the multilayer fluid S2 is 180 ° compared to the multilayer fluid S1.
  • the stacking order of the fluid composing each layer is reversed and the number of stacks is doubled.
  • the multilayer fluid S1 is divided in the cross-sectional direction by passing through the micromixer 21 shown in FIG. 2, and the cross-section itself is rotated 180 degrees.
  • each layer is more mixed and uniform than the multilayer fluid S 1. Therefore, the mixing and stirring of the magnetic beads and cells contained in each layer is promoted, and the cells adhere to the magnetic beads with high efficiency.
  • an antigen as a surface marker is attached to the surface of the cell, and an antibody that causes an antigen-antibody reaction with the antigen is attached to the surface of the magnetic bead.
  • the cells can be efficiently and firmly attached to the magnetic beads through the antigen-antibody reaction.
  • FIG. 5 is an enlarged view showing a peripheral portion including the separator 31 in the cell separation device 10 shown in FIG.
  • the separator 31 includes a pair of magnets 311 and 312 arranged to face each other with the flow path 40 interposed therebetween.
  • the magnets 311 and 312 need not be arranged so as to sandwich the flow path 40, but may be disposed close to the flow path 40.
  • arrows indicate the direction of fluid flow
  • black circles indicate magnetic beads
  • scaly members indicate cells.
  • the separator unit 30 When the fluid including the magnetic beads and cells that have passed through the micromixer unit 20 reaches the separator unit 30, it is affected by the magnetic field B between the magnets 311 and 312. At this time, the magnetic beads and the cells attached to the magnetic beads are attracted to the right by the magnetic field B and separated and removed from the rest of the fluid.
  • the separated magnetic beads and adherent cells reach the valve 13 and are taken out through the outlet 15.
  • the remaining portion of the fluid reaches the valve 14 and is taken out through the outlet 16. Therefore, through the above steps, only desired cells are attached to the magnetic beads, and the cells can be easily and reliably separated.
  • a pure water separation device can be provided on the downstream side of the separator portion 30.
  • Figure 6 shows the cell separation device shown in Figure 1.
  • 3 is an enlarged view of a peripheral portion including a pure water separator 51 in the apparatus 10;
  • the purification / separation device 51 includes a pair of magnets 511 and 512 arranged to face each other with the flow path 40 interposed therebetween.
  • arrows indicate the direction of fluid flow
  • black circles indicate magnetic beads
  • scaly members indicate cells.
  • the magnetic beads and the adherent cells that have been separated and removed by passing through the separator 30 are introduced into the pure water separator 51 together with the buffer solution. Since the buffer solution has an action of separating the cells from the magnetic beads, when the mixed solution is affected by the magnetic field B between the magnets 511 and 512, the magnetic beads are independent of whether or not the cells are separated. It is drawn to the right by the influence of magnetic field B, and is taken out through the nozzle 13 and the outlet 15. On the other hand, the cells separated from the magnetic beads are taken out through the valve 14 and the outlet 16 without being affected by the magnetic field B.
  • the magnetic beads can be easily recovered and only the cells to be separated can be easily extracted.
  • FIG. 7 is a configuration diagram showing a modification of the cell separation device shown in FIG. Components that are the same as or similar to the components shown in FIG. 1 are denoted by the same reference numerals.
  • a bypass channel 60 is provided between the inlets 11 and 12 and the micromixer unit 20 and between the separator unit 30 and the valves 13 and 14, and is separated and removed by the separator unit 30.
  • the fluid containing the magnetic beads is circulated by the pump 61 to the upstream side of the micromixer section 20. According to such a configuration, the magnetic beads to be adsorbed with cells are always circulated and reused, so that the total amount of magnetic beads used for cell separation can be reduced.
  • the corners can be chamfered in at least one of the portion 231 and the third opening 241 provided in the third plate-like member 24. If there are sharp corners in these openings, the flow velocity decreases in the corners as the multilayer fluid S1 flows through those openings. As a result, the multilayer fluid SI may not be sufficiently mixed. Therefore, in order to suppress the occurrence of these problems, it is preferable to chamfer the corners of the opening as described above.
  • Each plate-like member can be formed of any material. However, as long as the above-described fluid mixing method of the present invention can be realized, a force such as resin, metal, glass, etc. can be formed. . Therefore, preparation of each plate-like member and processing for each plate-like member are facilitated, and the formation of the above-described opening, that is, the formation of the micromixer itself can be easily performed.
  • the present invention is not limited to the case where the magnetic beads and the like constitute a multilayer fluid. Even when it is a fluid of a layer, it can be preferably used.
  • the micromixer unit 20 is composed of a plurality of micromixers 21 and the separator unit 30 is composed of a plurality of separators 31. A single separator can also be constructed.
  • the multilayer fluid S1 is caused to flow backward from the fourth opening 222 to the first opening 221 through the second opening 231 and the third opening 241. You can also.
  • the multilayer fluid S1 divides the multilayer fluid S1 upward at the second opening 231, divides the multilayer fluid S1 downward at the third opening 241 and upwards at the first opening 221. It is also possible to join the divided multilayer fluid S 1 and the multilayer fluid S 1 divided below. Even in this case, the multilayer fluid S1 can be sufficiently mixed in the manner shown in FIGS. 3 and 4 to obtain the multilayer fluid S2.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Il est prévu un fluide contenant des cordons magnétiques et des cellules spécifiées réparti vers la droite et vers la gauche à l’aide d’un premier moyen diviseur de fluide prévu dans un micromélangeur, et le fluide réparti vers la droite et vers la gauche est ensuite réparti vers le haut à l’aide d’un second moyen diviseur de fluide prévu dans le micromélangeur. Ensuite, le fluide réparti vers la droite et vers la gauche est réparti vers le bas à l’aide d’un troisième moyen diviseur de fluide prévu dans le micromélangeur. Le fluide réparti vers le haut et le fluide réparti vers le bas sont unis à l’aide d’un moyen de jonction de fluide prévu dans le micromélangeur. Ensuite, les cordons magnétiques dans le fluide après avoir traversé le micromélangeur et les cellules collant aux cordons magnétiques sont séparés du reste du fluide à l’aide d’un séparateur.
PCT/JP2005/010876 2004-06-24 2005-06-14 Séparateur de cellule et procédé de séparation de cellule WO2006001196A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004186321A JP2006006166A (ja) 2004-06-24 2004-06-24 細胞分離装置、及び細胞分離方法
JP2004-186321 2004-06-24

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WO (1) WO2006001196A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111534412A (zh) * 2020-05-26 2020-08-14 南京智能高端装备产业研究院有限公司 一种用于细胞磁珠标记的器件
JP2021119799A (ja) * 2015-06-05 2021-08-19 ノバルティス アーゲー フロースルー式常磁性粒子をベースにした細胞分離および常磁性粒子除去

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007267635A (ja) * 2006-03-30 2007-10-18 Kitakyushu Foundation For The Advancement Of Industry Science & Technology 細胞分離具及びそれを用いた細胞分離方法
DE102011004806A1 (de) * 2011-02-28 2012-08-30 Siemens Aktiengesellschaft Magnetische Durchflusszytometrie für hohen Probendurchsatz
WO2018169060A1 (fr) 2017-03-16 2018-09-20 富士フイルム株式会社 Procédé de séparation des mégacaryocytes et des plaquettes, et kit de séparation des plaquettes

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WO1999040174A1 (fr) * 1998-02-05 1999-08-12 Aclara Biosciences, Inc. Dispositifs microfluidiques integres
WO2001094635A2 (fr) * 2000-06-05 2001-12-13 California Institute Of Technology Dispositifs et procedes microfluidiques a flux actif integre
WO2002046355A1 (fr) * 2000-12-07 2002-06-13 Effector Cell Institute Unite cupulaire pour detecter la chimiotaxie cellulaire et separer les cellules chimiotactiques

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JP3638151B2 (ja) * 1996-03-28 2005-04-13 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング 少量液体の混合デバイス
JP2001120972A (ja) * 1999-10-21 2001-05-08 Shimadzu Corp 液体混合器
JP2003180336A (ja) * 2000-12-07 2003-07-02 Effector Cell Institute Inc 細胞走化性検出及び走化細胞分離装置のためのウエルユニット
JP2004016870A (ja) * 2002-06-13 2004-01-22 Atec Japan:Kk マイクロリアクター及びそれを用いた化学反応方法

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WO1999040174A1 (fr) * 1998-02-05 1999-08-12 Aclara Biosciences, Inc. Dispositifs microfluidiques integres
WO2001094635A2 (fr) * 2000-06-05 2001-12-13 California Institute Of Technology Dispositifs et procedes microfluidiques a flux actif integre
WO2002046355A1 (fr) * 2000-12-07 2002-06-13 Effector Cell Institute Unite cupulaire pour detecter la chimiotaxie cellulaire et separer les cellules chimiotactiques

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021119799A (ja) * 2015-06-05 2021-08-19 ノバルティス アーゲー フロースルー式常磁性粒子をベースにした細胞分離および常磁性粒子除去
JP7361070B2 (ja) 2015-06-05 2023-10-13 ノバルティス アーゲー フロースルー式常磁性粒子をベースにした細胞分離および常磁性粒子除去
US11912978B2 (en) 2015-06-05 2024-02-27 Novartis Ag Flow-through paramagnetic particle-based cell separation and paramagnetic particle removal
CN111534412A (zh) * 2020-05-26 2020-08-14 南京智能高端装备产业研究院有限公司 一种用于细胞磁珠标记的器件
CN111534412B (zh) * 2020-05-26 2024-02-20 南京智能高端装备产业研究院有限公司 一种用于细胞磁珠标记的器件

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