EP2027929A2 - Cell separation device and cell separation method - Google Patents
Cell separation device and cell separation method Download PDFInfo
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- 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
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- electrodes
- electric field
- cells
- cell separation
- flow path
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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]
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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
- B03C5/005—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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Abstract
Description
- The present invention relates to a cell separation device and a cell separation method.
- This application is based on Japanese Patent Application Nos.
2007-213922 2008-197043 - Heretofore, techniques for separating particles using differences in their dielectrophoretic properties have been known (for example, see PCT International Publications Nos.
WO1997/34689 WO2001/5512 W02001/5513 WO2001/5514 - According to a technique disclosed in PCT International Publication No.
WO1997/34689 - According to a technique disclosed in PCT International Publication No.
WO2001/5512 - According to a technique disclosed in PCT International Publication No.
W02001/5513 - According to a technique disclosed in PCT International Publication No.
W02001/5514 - According to the technique disclosed in PCT International Publication No.
WO1997/3468 - In addition, according to the technique disclosed in PCT International Publication No.
WO 2001/5512 - In addition, the technique disclosed in PCT International Publication No.
WO 2001/5513 - Furthermore, the technique disclosed in PCT International Publication No.
WO 2001/5514 - 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.
- In order to accomplish the above object, the present invention provides the following solutions.
- According to a first aspect of the present invention, there is provided 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.
- According to the first aspect of the present invention, when an alternating voltage having a direct current component is applied across the electrodes by operation of the power source, 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. On the other hand, cells having negative dielectrophoretic properties contained in the cell suspension receive a dielectrophoretic force in the direction along which the electric field strength decreases, and cells having positive dielectrophoretic properties receive a dielectrophoretic force in the direction along which the electric field strength increases. In addition, a dielectrophoretic force is not applied to cells having no dielectrophoretic properties, such as dead cells, but only the electrophoretic force is applied thereto.
- Accordingly, 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.
- That is, for example, when the direction in which a negative dielectrophoretic force acts is set opposite to the direction in which an electrophoretic force acts, a relatively small force obtained by counteraction between the dielectrophoretic force and the electrophoretic force can be applied to cells having negative dielectrophoretic properties, and on the other hand, a relatively large force formed of only the electrophoretic force can be applied to cells having no dielectrophoretic properties. Hence, cells having no dielectrophoretic properties are attracted by the electrophoretic force, and cells having negative dielectrophoretic properties are made to flow along the flow direction, so that the cell separation can be performed.
- In the above first aspect of the present invention, the electric field gradient forming portion may be an insulating member which has at least one opening and which is disposed between the electrodes.
- With the above structure, 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.
- In addition, in the above first aspect of the present invention, the electrodes may be parallel plate electrodes which form an electric field having an approximately uniform electric field strength.
- With the above structure, according to the combination between the electrodes having a simple structure and the insulating plate, an electric field having an electric field strength gradient can be more easily formed between the electrodes.
- In addition, in the above first aspect of the present invention, the electric field gradient forming portion may be constructed by forming one of the electrodes smaller than that of the other electrode.
- With the above structure, by changing the density of the electric lines of force from the large electrode to the small electrode, an electric field having an electric field strength gradient can be easily formed.
- In addition, according to a second aspect of the present invention, there is provided 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.
- According to the above second aspect of the present invention, when an alternating voltage including a direct current component is 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. On the other hand, cells having negative dielectrophoretic properties contained in the cell suspension receive a dielectrophoretic force in the direction along which the electric field strength decreases, and cells having positive dielectrophoretic properties receive a dielectrophoretic force in the direction along which the electric field strength increases. In addition, to cells having no dielectrophoretic properties, such as dead cells, a dielectrophoretic force is not applied, but only the electrophoretic force is applied.
- Accordingly, 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.
-
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Fig. 1 is an overall schematic structural view showing a cell separation device according to one embodiment of the present invention. -
Fig. 2 is a view showing an alternating voltage waveform generated by a power source of the cell separation device shown inFig. 1 . -
Fig. 3A is a view showing electric lines of force formed between electrodes of the cell separation device shown inFig. 1 and forces applied to cells, the view showing the state before separation. -
Fig. 3B is a view showing electric lines of force formed between the electrodes of the cell separation device shown inFig. 1 and forces applied to the cells, the view showing the state after separation. -
Fig. 4 is a view illustrating an operation for separating cells having different dielectrophoretic properties with the cell separation device shown inFig. 1 . -
Fig. 5 is a partial enlarged vertical cross-sectional view showing a modification of the cell separation device shown inFig. 1 . -
Fig. 6 is a graph showing the change in frequency of an alternating voltage generated by using the cell separation device shown inFig. 5 . -
Fig. 7 is a graph showing frequency properties at a cell retention boundary. -
Fig. 8 is a graph showing retention boundaries and retention areas of living cells and dead cells. -
Fig. 9 is an overall schematic structural view showing a cell separation device according to one Example of the present invention. -
Fig. 10 is a schematic view of a porous insulating plate of the cell separation device according to the above Example of the present invention. -
Fig. 11 is a view showing retention rates of living cells and dead cells. - A
cell separation device 1 according to one embodiment of the present invention will be described with reference toFigs. 1 to 4 . - The
cell separation device 1 according to this embodiment includes, as shown inFigs. 1 to 4 , aflow 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 flow path 2; an insulating plate (insulating member: electric field gradient forming member) 4 disposed between theelectrodes power source 5 applying a voltage across theelectrodes - The
flow path 2 is divided by theelectrically insulating plate 4 into afirst flow path 2a and asecond flow path 2b, both of which extend in a flowing direction. - The
electrodes flow path 2 to face each other in the direction perpendicular to a flow direction L. - In addition, the insulating
plate 4 is formed to have a flat plate shape, is disposed at a central position of a space formed between theelectrodes - Hence, the
first flow path 2a and thesecond flow path 2b communicate with each other via the through-hole 4a formed in the insulatingplate 4. - Accordingly, since electric lines of force C generated from one side to the other side between the
electrodes power source 5 are blocked by the insulatingplate 4, the electric lines of force C pass only through the through-hole 4a formed in the insulatingplate 4, as shown inFigs. 3A and 3B . That is, since the electric lines of force C are bundled by the through-hole 4a, and the density thereof is increased thereby, in an electric field formed between theelectrodes hole 4a and gradually decreases along the directions toward theelectrodes - The
power source 5 is, as shown inFig. 2 , designed to apply a sine-wave alternating voltage having a direct current component offset in one direction (such as plus (+) direction) across theelectrodes - A cell separation method using the
cell separation device 1 according to this embodiment, having the structure as described above, will now be described. - In order to separate the plural types of cells A and B having different dielectrophoretic properties using the
cell separation device 1 according to this embodiment, an alternating voltage is applied across theelectrodes power source 5 to form the electric lines of force C, as shown inFigs. 3A and 3B , between theelectrodes - In the state described above, 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. In this case, for example, the cell-suspension liquid is made to flow through thefirst flow path 2a between the minus (-)side electrode 3a and the insulatingplate 4, and a medium containing no cells A and B is made to flow through thesecond flow path 2b between the plus (+)side electrode 3b and the insulatingplate 4. The flow velocities of the cell-suspension liquid and the medium are set sufficiently slow. - Once the cells A having negative dielectrophoretic properties reach an area between the
electrodes electrodes Figs. 3A and 3B . In addition, since the alternating voltage supplied from thepower 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 inFigs. 3A and 3B . Hence, when the dielectrophoretic force f1 and the electrophoretic force f2, which are applied to the cells A, are balanced, the cells A having negative dielectrophoretic properties move smoothly along the flow of the cell-suspension liquid flowing in thefirst flow path 2a without being attracted by theelectrodes - On the other hand, since the dielectrophoretic force f1 is not applied to the cells B having no dielectrophoretic properties, the electrophoretic force f2 is only applied to the cells B. As a result, due to the electrophoretic force f2, the cells B are attracted by the plus (+)
side electrode 3b and flow into thesecond flow path 2b through the through-hole 4a formed in the insulatingplate 4. - That is, by operating the
power source 5, the amplitude and frequency of the alternating voltage applied across theelectrodes first flow path 2a without being interrupted and a flow of the cells B introduced into thesecond flow path 2b by being attracted by theelectrode 3b, as shown inFigs. 4 ; hence, the two types of cells can be effectively separated. - As described above, according to the
cell separation device 1 and the cell separation method of this embodiment, by adopting the structure in which only theparallel plate electrodes plate 4 are disposed in theflow 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. - In this embodiment, although at least one through-
hole 4a is provided in the insulatingplate 4 which is disposed between theelectrodes holes 4a is not particularly limited. In addition, 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. - In addition, although the insulating
plate 4 is disposed at the central position of the space formed between theparallel plate electrodes Fig. 5 , the insulatingplate 4 may be disposed in close contact with the surface of oneelectrode 3a of the aboveparallel plate electrodes electrode 3a covered with the insulatingplate 4, 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 theother 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 theflow path 2. - Hereinafter, an Example using the
cell separation device 1 shown inFig. 5 will be described with reference toFig. 6 . - As the
electrodes electrodes electrodes plate 4, a Kapton film having a thickness of 50 µm was used. In addition, as the through-hole 4a formed in the insulatingplate 4, a hole having a diameter of approximately 100 µm was formed. - In addition, instead of the cells A and B having different dielectrophoretic properties, particles D made of polystyrene beads having a diameter of approximately 10 µm were used.
- As the alternating voltage, 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.
- The results are shown in
Fig. 6 . -
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 insulatingplate 4 by a predetermined distance and corresponding to the through-hole 4a formed in the insulatingplate 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 theelectrodes - At all amplitudes, since the value of a necessary direct current component was maximized in the vicinity of a frequency of 1 MHz, it was understood that a large electrophoretic force f2 was applied to the particles D. Hence, in the vicinity of a frequency of 1 MHz, it was understood that a large dielectrophoretic force f1 counteracting the large electrophoretic force f2 was exerted.
- As a result, by adjusting the amplitude and the direct current component of the alternating voltage, 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. - In addition, in the above embodiment, by covering the surface of the
electrode 3a with the insulatingplate 4, only the portion exposed through the through-hole 4a was used as theelectrode 3a; however, an electrode (not shown) having a small area similar to that of the through-hole 4a may be employed from the beginning. - Next, another Example using the
cell separation device 1 shown inFig. 1 will be described with reference toFigs. 7 and 8 . - As the device conditions, the distance between the
electrodes plate 4, a plate having a thickness of 0.2 mm was used. The width of theflow path 2 was set to 0.5 mm (the width of theflow path 2a and that of theflow path 2b were each set to 0.5 mm), and as the through-hole 4a formed in the insulatingplate 4, a hole having a diameter of approximately 0.2 mm was used. - As living cells and dead cells, 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 inFig. 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. When the conditions of thecell separation device 1 were set to obtain a separation possible area shown inFig. 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. - In addition, still another Example using a
cell separation device 111 shown inFig. 9 will be described with reference toFigs. 9 to 11 . - As the device conditions, in the
cell separation device 111 having a width of 12 mm, a length of 90 mm, and a height of 30 mm, the distance between theelectrodes 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 insulatingplate 4, a hole having a diameter of approximately 0.2 mm was used. In the arrangement shown inFig. 10 , the distances between holes were set to 0.3 mm and 0.6 mm. - As shown in
Fig. 9 , a cell-suspension liquid was fed into thecell separation device 111 through a pump P and was transferred with a bulk liquid in a flow direction L. When 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 theelectrodes plate 4, and simultaneously, by the direct current component included in the alternating voltage, charges were unevenly distributed at one electrode side. For example, by this uneven distribution of charges, 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. Alternatively, when an electric field was formed so that, by this uneven distribution of charges, the living cells received a dielectrophoretic force in the direction in which the electric field strength increased, a dielectrophoretic force was not applied to the dead cells having no dielectrophoretic properties, but only the electrophoretic force was applied thereto; hence the living cells and the dead cells could be separated at the transmission side and the retention 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.
- As the living cells and the dead cells, 3-2H3 cells on the second day after culture at a concentration of 5.0×105 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.
- As shown in
Fig. 11 , with thecell 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.
Claims (5)
- 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; anda power supply applying an alternating voltage having a direct current component across the electrodes.
- The cell separation device according to Claim 1, wherein the electric field gradient forming portion is an insulating member which has at least one opening and which is disposed between the electrodes.
- The cell separation device according to Claim 2, wherein the electrodes are parallel plate electrodes which form an electric field having an approximately uniform electric field strength.
- The cell separation device according to Claim 1, wherein the electric field gradient forming portion is constructed by forming one of the electrodes is smaller than 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; andapplying an alternating voltage, including a direct current component, across electrodes which face 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.
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JP2008197043A JP2009065967A (en) | 2007-08-20 | 2008-07-30 | Cell separation device and cell separation method |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103194371A (en) * | 2013-03-26 | 2013-07-10 | 上海交通大学 | Separation and collection device for biological compound |
CN103217311A (en) * | 2013-03-26 | 2013-07-24 | 上海交通大学 | Biological composition separation and collection method |
EP2754709A1 (en) * | 2011-09-07 | 2014-07-16 | Panasonic Healthcare Co., Ltd. | Microorganism quantity measurement cell and microorganism quantity measurement device comprising same |
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US6824664B1 (en) * | 1999-11-04 | 2004-11-30 | Princeton University | Electrode-less dielectrophorises for polarizable particles |
DE10311716A1 (en) * | 2003-03-17 | 2004-10-14 | Evotec Oai Ag | Method and device for separating particles in a liquid flow |
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WO1997034689A1 (en) | 1996-03-18 | 1997-09-25 | University College Of North Wales | Apparatus with electrode arrays for carrying out chemical, physical or physico-chemical reactions |
WO2001005512A1 (en) | 1999-07-20 | 2001-01-25 | University Of Wales, Bangor | Dielectrophoretic apparatus & method |
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CN103194371A (en) * | 2013-03-26 | 2013-07-10 | 上海交通大学 | Separation and collection device for biological compound |
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CN103217311B (en) * | 2013-03-26 | 2015-04-01 | 上海交通大学 | Biological composition separation and collection method |
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US20090050482A1 (en) | 2009-02-26 |
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