EP2027929A2 - Dispositif de séparation cellulaire et procédé de séparation cellulaire - Google Patents

Dispositif de séparation cellulaire et procédé de séparation cellulaire Download PDF

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Publication number
EP2027929A2
EP2027929A2 EP08014447A EP08014447A EP2027929A2 EP 2027929 A2 EP2027929 A2 EP 2027929A2 EP 08014447 A EP08014447 A EP 08014447A EP 08014447 A EP08014447 A EP 08014447A EP 2027929 A2 EP2027929 A2 EP 2027929A2
Authority
EP
European Patent Office
Prior art keywords
electrodes
electric field
cells
cell separation
flow path
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP08014447A
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German (de)
English (en)
Other versions
EP2027929A3 (fr
Inventor
Masaru Hakoda
Satoshi Yoshida
Hiroki Hibino
Hiroshi Fukuda
Yoshiaki Shiba
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Olympus Corp
Original Assignee
Olympus Corp
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
Priority claimed from JP2008197043A external-priority patent/JP2009065967A/ja
Application filed by Olympus Corp filed Critical Olympus Corp
Publication of EP2027929A2 publication Critical patent/EP2027929A2/fr
Publication of EP2027929A3 publication Critical patent/EP2027929A3/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/005—Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength

Definitions

  • the present invention relates to a cell separation device and a cell separation method.
  • a plurality of traps formed of flat electrodes is disposed in a flow path, beads covered with antibodies which particularly adsorb certain bacteria are supported on the electrodes, and an effluent to be analyzed is made to flow through the flow path, so that bacteria approaching the vicinities of the electrodes are collected by the beads using dielectrophoretic properties.
  • a flow path is formed in which a plurality of wave-shaped electrodes is arranged, and plural types of particles are made to flow in the electrode arrangement direction, so that the particles are separated using the difference in dielectrophoretic properties.
  • particles are separated by applying an electric field having at least two wavelengths so that a plurality of dielectrophoretic forces acts on the particles.
  • bacteria are quantitatively measured by the steps of preparing an analytical chamber at a downstream side in a flow path, measuring dielectrophoretic properties of beads before the bacteria are collected, separating beads that collect certain bacteria from the electrodes, and sending the beads thus separated into an analytical chamber for measurement to determine the difference in properties from beads that collect no bacteria; hence the process of this technique is disadvantageously complicated.
  • the present invention has been conceived in consideration of the above-described situation, and an object of the present invention is to provide a cell separation device and a cell separation method that can efficiently separate plural types of cells having different dielectrophoretic properties using a simple structure.
  • the present invention provides the following solutions.
  • a cell separation device comprising: a flow path through which a cell suspension flows, the cell suspension containing plural types of cells which have different dielectrophoretic properties; electrodes disposed to face each other in a direction intersecting a flow direction of the cell suspension flowing in the flow path; an electric field gradient forming portion which generates an electric field strength gradient between the electrodes; and a power supply applying an alternating voltage having a direct current component across the electrodes.
  • an electric field having an electric field strength gradient is formed between the electrode by the operation of the electric field gradient forming portion, and in addition, charges are unevenly distributed at one electrode side due to the direct current component included in the alternating voltage. That is, the plural types of cells contained in the cell suspension all receive an electrophoretic force caused by the uneven distribution of charges.
  • cells having negative dielectrophoretic properties contained in the cell suspension receive a dielectrophoretic force in the direction along which the electric field strength decreases
  • cells having positive dielectrophoretic properties receive a dielectrophoretic force in the direction along which the electric field strength increases.
  • a dielectrophoretic force is not applied to cells having no dielectrophoretic properties, such as dead cells, but only the electrophoretic force is applied thereto.
  • dielectrophoretic forces having different directions and an electrophoretic force can be applied to cells having different dielectrophoretic properties, and by maintaining an appropriate balance therebetween, the cells having different dielectrophoretic properties can be effectively separated from each other.
  • the electric field gradient forming portion may be an insulating member which has at least one opening and which is disposed between the electrodes.
  • the electric lines of force formed between the electrodes can be squeezed by the insulating member so as to pass through the opening, and an electric field having an electric field strength gradient can be easily formed between the electrodes.
  • the electrodes may be parallel plate electrodes which form an electric field having an approximately uniform electric field strength.
  • an electric field having an electric field strength gradient can be more easily formed between the electrodes.
  • the electric field gradient forming portion may be constructed by forming one of the electrodes smaller than that of the other electrode.
  • a cell separation method comprising the steps of: making a cell suspension containing plural types of cells having different dielectrophoretic properties flow in a flow path; and applying an alternating voltage including a direct current component across electrodes which faces each other in a direction intersecting a flow direction of the cell suspension flowing in the flow path to form an electric field having an electric field strength gradient between the electrodes.
  • an alternating voltage including a direct current component when applied across the electrodes, an electric field having an electric field strength gradient is formed between the electrodes, and at the same time, charges are unevenly distributed at one electrode side due to the direct current component included in the alternating voltage. That is, all types of cells contained in the cell suspension receive an electrophoretic force caused by the uneven distribution of charges.
  • cells having negative dielectrophoretic properties contained in the cell suspension receive a dielectrophoretic force in the direction along which the electric field strength decreases
  • cells having positive dielectrophoretic properties receive a dielectrophoretic force in the direction along which the electric field strength increases.
  • a dielectrophoretic force is not applied, but only the electrophoretic force is applied.
  • dielectrophoretic forces having different directions and an electrophoretic force can be applied to cells having different dielectrophoretic properties, and by maintaining an appropriate balance therebetween, the cells having different dielectrophoretic properties can be effectively separated from each other.
  • the present invention provides an advantage in that plural types of cells having different dielectrophoretic properties can be effectively separated with a simple structure.
  • a cell separation device 1 according to one embodiment of the present invention will be described with reference to Figs. 1 to 4 .
  • the cell separation device 1 includes, as shown in Figs. 1 to 4 , a flow path 2 through which a cell-suspension liquid containing plural types of cells A and B, such as living cells A and dead cells B, flows in one direction; electrodes 3a and 3b disposed in the flow path 2; an insulating plate (insulating member: electric field gradient forming member) 4 disposed between the electrodes 3a and 3b; and a power source 5 applying a voltage across the electrodes 3a and 3b.
  • a flow path 2 through which a cell-suspension liquid containing plural types of cells A and B, such as living cells A and dead cells B, flows in one direction
  • electrodes 3a and 3b disposed in the flow path 2
  • an insulating plate (insulating member: electric field gradient forming member) 4 disposed between the electrodes 3a and 3b
  • a power source 5 applying a voltage across the electrodes 3a and 3b.
  • the flow path 2 is divided by the electrically insulating plate 4 into a first flow path 2a and a second flow path 2b, both of which extend in a flowing direction.
  • the electrodes 3a and 3b are, for example, parallel plate electrodes and are disposed on facing wall surfaces of the flow path 2 to face each other in the direction perpendicular to a flow direction L.
  • the insulating plate 4 is formed to have a flat plate shape, is disposed at a central position of a space formed between the electrodes 3a and 3b, and has at least one through-hole (opening) 4a.
  • the first flow path 2a and the second flow path 2b communicate with each other via the through-hole 4a formed in the insulating plate 4.
  • the power source 5 is, as shown in Fig. 2 , designed to apply a sine-wave alternating voltage having a direct current component offset in one direction (such as plus (+) direction) across the electrodes 3a and 3b.
  • an alternating voltage is applied across the electrodes 3a and 3b by operating the power source 5 to form the electric lines of force C, as shown in Figs. 3A and 3B , between the electrodes 3a and 3b.
  • a cell-suspension liquid containing the plural types of cells A and B having different dielectrophoretic properties is made to flow in the first flow path 2a.
  • the cell-suspension liquid is made to flow through the first flow path 2a between the minus (-) side electrode 3a and the insulating plate 4, and a medium containing no cells A and B is made to flow through the second flow path 2b between the plus (+) side electrode 3b and the insulating plate 4.
  • the flow velocities of the cell-suspension liquid and the medium are set sufficiently slow.
  • the cells A having negative dielectrophoretic properties reach an area between the electrodes 3a and 3b, due to an electric field having an electric field strength gradient formed between the electrodes 3a and 3b, the cells A receive a dielectrophoretic force f1 in the direction along which the electric field strength decreases, as shown in Figs. 3A and 3B .
  • the alternating voltage supplied from the power source 5 includes the direct current component, the cells A and B each receive an electrophoretic force f2 in the direction opposite to that of the dielectrophoretic force f1 in accordance with the charges of the cells A and B, as shown in Figs. 3A and 3B .
  • the cells A having negative dielectrophoretic properties move smoothly along the flow of the cell-suspension liquid flowing in the first flow path 2a without being attracted by the electrodes 3a and 3b.
  • the electrophoretic force f2 is only applied to the cells B.
  • the cells B are attracted by the plus (+) side electrode 3b and flow into the second flow path 2b through the through-hole 4a formed in the insulating plate 4.
  • the amplitude and frequency of the alternating voltage applied across the electrodes 3a and 3b are adjusted so as to adjust the dielectrophoretic force f1 applied to the cells A having dielectrophoretic properties, and the absolute value of the direct current component included in the alternating voltage is adjusted so as to adjust the electrophoretic force f2 applied to the cells A and B.
  • the cell separation device 1 and the cell separation method of this embodiment by adopting the structure in which only the parallel plate electrodes 3a and 3b and the insulating plate 4 are disposed in the flow path 2 through which the cell-suspension liquid flows, it is possible to obtain an advantage in that the plural types of cells A and B having different dielectrophoretic properties can be efficiently separated from each other.
  • the number of the through-holes 4a is not particularly limited.
  • the shape, arrangement, and intervals of the through-holes 4a may be arbitrarily determined as long as a sufficient electric field gradient can be formed.
  • the insulating plate 4 is disposed at the central position of the space formed between the parallel plate electrodes 3a and 3b, instead of this arrangement, as shown in Fig. 5 , the insulating plate 4 may be disposed in close contact with the surface of one electrode 3a of the above parallel plate electrodes 3a and 3b.
  • the electric lines of force C are formed only from the portion exposed through the through-hole 4a, and the electric field is formed such that the electric field strength gradually decreases in the direction toward the other electrode 3b. Accordingly, as in the case described above, the cells A and B having different dielectrophoretic properties can be separated in the direction intersecting the flow direction L in the flow path 2.
  • the electrodes 3a and 3b As the electrodes 3a and 3b, a pair of flat plate-shaped electrodes 3a and 3b made of titanium having a thickness of 1 mm was used. The space between the electrodes 3a and 3b was set to approximately 1 mm.
  • the insulating plate 4 As the insulating plate 4, a Kapton film having a thickness of 50 ⁇ m was used.
  • the through-hole 4a formed in the insulating plate 4 a hole having a diameter of approximately 100 ⁇ m was formed.
  • particles D made of polystyrene beads having a diameter of approximately 10 ⁇ m were used.
  • sine waveform voltages having amplitudes of 1, 1.2, and 1.5 V, a direct current component of 0 to 3 V, and frequencies of 10 kHz, 100 kHz, 500 kHz, 1 MHz, 5 MHz, and 10 MHz were used.
  • Fig. 6 shows the relationships among the frequency, direct current component, and amplitude of the alternating voltage, each relationship indicating conditions under which the particles D were made to stay still at a position away from the insulating plate 4 by a predetermined distance and corresponding to the through-hole 4a formed in the insulating plate 4. That is, when the amplitudes of the alternating voltage were set to 1, 1.2, and 1.5 V, and the frequency was sequentially changed at the above individual amplitudes, the values of the direct current components at which the particles D were not attracted by either of the electrodes 3a and 3b were plotted.
  • the particles D are made to migrate toward the electrode 3a by the electrophoretic force f2, and particles D to which the dielectrophoretic force f1 is applied continue to flow in the same way or are made to migrate in the opposite direction, so that separation can be performed.
  • the distance between the electrodes 3a and 3b was set to approximately 1.2 mm, and as the insulating plate 4, a plate having a thickness of 0.2 mm was used.
  • the width of the flow path 2 was set to 0.5 mm (the width of the flow path 2a and that of the flow path 2b were each set to 0.5 mm), and as the through-hole 4a formed in the insulating plate 4, a hole having a diameter of approximately 0.2 mm was used.
  • 3-2H3 cells on the second day of culture were used, and as a bulk liquid, a mixture of an aqueous solution containing 8.5 percent by weight of sucrose and an aqueous solution containing 0.3 percent by weight of glucose was used.
  • Fig. 7 shows frequency properties of a living cell retention boundary when the amplitude of the alternating voltage was set to 80 V, and the frequency was changed to 1, 5, and 10 kHz. As shown in Fig. 7 , it was found that the highest offset voltage was output at a frequency of 5 kHz, and in addition, the retention area was increased.
  • Fig. 8 shows retention boundaries and retention areas of living cells and dead cells when the frequency was set to 5 kHz, and the amplitude of the alternating voltage was changed to 60, 80, 100, and 120 V.
  • the conditions of the cell separation device 1 were set to obtain a separation possible area shown in Fig. 8 , the dead cells were transmitted, and the living cells could be retained; hence, the living cells and the dead cells could be separated from each other.
  • the distance between the electrodes 3a and 3b was set to approximately 1.2 mm, and as the insulating plate 4, a plate having a thickness of 0.2 mm was used.
  • the number of holes was set to approximately 1,000, and as the through-hole 4a formed in the insulating plate 4, a hole having a diameter of approximately 0.2 mm was used.
  • the distances between holes were set to 0.3 mm and 0.6 mm.
  • a cell-suspension liquid was fed into the cell separation device 111 through a pump P and was transferred with a bulk liquid in a flow direction L.
  • the alternating voltage was applied by operation of an arbitrary waveform generator G, an electric field having an electric field strength gradient was formed between the electrodes 3a and 3b by operation of the insulating plate 4, and simultaneously, by the direct current component included in the alternating voltage, charges were unevenly distributed at one electrode side.
  • living cells received a dielectrophoretic force in the direction in which the electric field strength decreased, and to dead cells having no dielectrophoretic properties, the dielectrophoretic force was not applied but only the electrophoretic force was applied; hence the living cells and the dead cells could be separated at the retention side and the transmission side, respectively.
  • Fig. 11 shows retention rates of living cells and dead cells when separation of a mixture containing living and dead cells was performed such that the frequency was set to 5 kHz, the amplitude of the alternating voltage was set to 100 V, and the offset voltage was output in the range of 0 to 1.0 V.
  • the flow rates of the cell-suspension liquid, bulk liquid, retention liquid, and transmission liquid were set to 0.3, 0.3, 0.2, and 0.4 ml/minute, respectively.
  • 3-2H3 cells on the second day after culture at a concentration of 5.0 ⁇ 10 5 cells/ml were each used, and as the bulk liquid, a mixture of an aqueous solution containing 8.5 percent by weight of sucrose and an aqueous solution containing 0.3 percent by weight of glucose was used.
  • the cell separation device 111 since the offset voltage was output, the retention rate of the dead cells decreased, that is, the dead cells were transmitted; hence, the living cells, keeping a high retention rate, could be separated from the dead cells having a decreased retention.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Electrostatic Separation (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP08014447A 2007-08-20 2008-08-13 Dispositif de séparation cellulaire et procédé de séparation cellulaire Withdrawn EP2027929A3 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2007213922 2007-08-20
JP2008197043A JP2009065967A (ja) 2007-08-20 2008-07-30 細胞分離装置および細胞分離方法

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EP2027929A2 true EP2027929A2 (fr) 2009-02-25
EP2027929A3 EP2027929A3 (fr) 2013-03-27

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103194371A (zh) * 2013-03-26 2013-07-10 上海交通大学 生物复合物的分离与收集装置
CN103217311A (zh) * 2013-03-26 2013-07-24 上海交通大学 生物复合物的分离与收集方法
EP2754709A1 (fr) * 2011-09-07 2014-07-16 Panasonic Healthcare Co., Ltd. Cellule de mesure de quantité de microorganismes et dispositif de mesure de quantité de microorganismes contenant celle-ci

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034689A1 (fr) 1996-03-18 1997-09-25 University College Of North Wales Appareil a reseau d'electrodes servant a effectuer des reactions chimiques, physiques ou physico-chimiques
WO2001005513A1 (fr) 1999-07-20 2001-01-25 University Of Wales, Bangor Manipulation de particules dans des milieux liquides
WO2001005514A1 (fr) 1999-07-20 2001-01-25 University Of Wales, Bangor Appareil de dielectrophorese a onde progressive et procede correspondant
WO2001005512A1 (fr) 1999-07-20 2001-01-25 University Of Wales, Bangor Appareil pour dielectrophorese et methode correspondante
JP2007213922A (ja) 2006-02-08 2007-08-23 Mitsubishi Electric Corp 高圧放電ランプ点灯装置およびこれを用いた投射型ディスプレイ装置
JP2008197043A (ja) 2007-02-15 2008-08-28 Yokogawa Electric Corp 光信号測定装置

Family Cites Families (5)

* Cited by examiner, † Cited by third party
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US6641708B1 (en) * 1996-01-31 2003-11-04 Board Of Regents, The University Of Texas System Method and apparatus for fractionation using conventional dielectrophoresis and field flow fractionation
JP2002519176A (ja) * 1998-06-26 2002-07-02 エボテック バイオシステムズ アクチェン ゲゼルシャフト マイクロシステムに機能電界障壁を発生させるための電極構造
US6824664B1 (en) * 1999-11-04 2004-11-30 Princeton University Electrode-less dielectrophorises for polarizable particles
DE10311716A1 (de) * 2003-03-17 2004-10-14 Evotec Oai Ag Verfahren und Vorrichtung zur Trennung von Partikeln in einer Flüssigkeitsströmung
KR100624460B1 (ko) * 2005-02-12 2006-09-19 삼성전자주식회사 나노 내지 마이크로 크기의 포어가 형성되어 있는 막을 포함하는 미세유동장치 및 그를 이용하여 분극성 물질을 분리하는 방법

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997034689A1 (fr) 1996-03-18 1997-09-25 University College Of North Wales Appareil a reseau d'electrodes servant a effectuer des reactions chimiques, physiques ou physico-chimiques
WO2001005513A1 (fr) 1999-07-20 2001-01-25 University Of Wales, Bangor Manipulation de particules dans des milieux liquides
WO2001005514A1 (fr) 1999-07-20 2001-01-25 University Of Wales, Bangor Appareil de dielectrophorese a onde progressive et procede correspondant
WO2001005512A1 (fr) 1999-07-20 2001-01-25 University Of Wales, Bangor Appareil pour dielectrophorese et methode correspondante
JP2007213922A (ja) 2006-02-08 2007-08-23 Mitsubishi Electric Corp 高圧放電ランプ点灯装置およびこれを用いた投射型ディスプレイ装置
JP2008197043A (ja) 2007-02-15 2008-08-28 Yokogawa Electric Corp 光信号測定装置

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2754709A1 (fr) * 2011-09-07 2014-07-16 Panasonic Healthcare Co., Ltd. Cellule de mesure de quantité de microorganismes et dispositif de mesure de quantité de microorganismes contenant celle-ci
EP2754709A4 (fr) * 2011-09-07 2015-01-21 Panasonic Healthcare Co Ltd Cellule de mesure de quantité de microorganismes et dispositif de mesure de quantité de microorganismes contenant celle-ci
CN103194371A (zh) * 2013-03-26 2013-07-10 上海交通大学 生物复合物的分离与收集装置
CN103217311A (zh) * 2013-03-26 2013-07-24 上海交通大学 生物复合物的分离与收集方法
CN103217311B (zh) * 2013-03-26 2015-04-01 上海交通大学 生物复合物的分离与收集方法

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EP2027929A3 (fr) 2013-03-27
US20090050482A1 (en) 2009-02-26

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