WO2019049204A1 - Dispositif pour fluide et son utilisation - Google Patents

Dispositif pour fluide et son utilisation Download PDF

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Publication number
WO2019049204A1
WO2019049204A1 PCT/JP2017/031936 JP2017031936W WO2019049204A1 WO 2019049204 A1 WO2019049204 A1 WO 2019049204A1 JP 2017031936 W JP2017031936 W JP 2017031936W WO 2019049204 A1 WO2019049204 A1 WO 2019049204A1
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WIPO (PCT)
Prior art keywords
fluid
flow path
cells
blood cells
flow
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PCT/JP2017/031936
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English (en)
Japanese (ja)
Inventor
百合香 越智
哲臣 高崎
慶治 三井
Original Assignee
株式会社ニコン
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Application filed by 株式会社ニコン filed Critical 株式会社ニコン
Priority to JP2019540155A priority Critical patent/JP7020488B2/ja
Priority to PCT/JP2017/031936 priority patent/WO2019049204A1/fr
Publication of WO2019049204A1 publication Critical patent/WO2019049204A1/fr

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    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/33—Disintegrators
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters

Definitions

  • the present invention relates to fluidic devices and their use. More specifically, the present invention relates to a fluidic device, a method of disrupting cells, and a method of analyzing the number of red blood cells and white blood cells in a blood-derived sample.
  • cells may be broken. Physical methods, chemical methods, and the like exist as methods for disrupting cells.
  • a device or apparatus such as a microbead, a shaker, a centrifuge, or an ultrasonic crusher is used.
  • a worker may come in contact with a sample to be infected with a pathogen or a virus, or may contaminate the environment with an aerosol generated from the sample.
  • Patent No. 3571950 gazette
  • a fluid device includes a circulation channel, a flow channel for flowing a fluid containing cells, and a pump unit provided in the circulation channel and controlling the flow of the fluid or the pressure of the fluid. And an aperture-shaped cell analysis unit provided in the flow path.
  • a method of disrupting cells comprises a flow channel, a pump portion provided in the flow channel, and a compartmental valve for defining a region including the pump portion of the flow channel. Changing the pressure of the fluid by flowing the fluid containing the cells in the flow passage, closing the dividing valve to define a region including the pump portion of the flow passage, and operating the pump portion And c) disrupting the cells.
  • a method of analyzing the number of red blood cells and white blood cells in a blood-derived sample includes a flow path, a pump portion provided in the flow path, and an aperture-shaped cell analysis portion provided in the flow path Flowing the blood-derived sample through the flow channel of the fluidic device, measuring the number of red blood cells and white blood cells in the cell analysis unit, and operating the pump unit to measure the pressure of the blood-derived sample
  • the method comprises the steps of: selectively disrupting the red blood cells by changing; and, after breaking the red blood cells, measuring the number of white blood cells in the cell analysis unit.
  • the present invention comprises a flow path for flowing a fluid containing cells, which comprises a circulation flow path, and a pump unit provided in the circulation flow path for controlling the flow of the fluid or the pressure of the fluid And an aperture-shaped cell analysis unit provided in the flow path.
  • FIG. 1 is a schematic view illustrating the structure of a fluid device 100 according to an embodiment.
  • the fluid device 100 includes a flow channel 110 including a circulation flow channel, a pump unit 130 provided in the circulation flow channel, and an aperture-shaped cell analysis unit 120 provided in the flow channel 110 including the circulation flow channel.
  • the cell analysis unit 120 may be present inside the circulation flow channel, or may be present in a region other than the circulation flow channel in the flow channel 110.
  • the circulation channel means a channel through which the fluid inside can circulate.
  • the circulation flow path may form a closed circuit, or may be configured to be able to form a closed circuit by opening and closing a partition valve 140 described later.
  • the channel 110 is for flowing a fluid L containing cells.
  • the fluid L is not particularly limited, but may be, for example, a blood-derived sample.
  • examples of cells contained in the fluid L include red blood cells, white blood cells and the like.
  • the pump unit 130 By operating the pump unit 130, the flow of the fluid L can be controlled. Also, by operating the pump unit 130, the pressure of the fluid L can be adjusted. For example, by operating the pump unit 130, it is possible to cause the fluid L in a stationary state to flow or to increase the flow rate of the fluid L.
  • the pressure of the fluid L can also be said to be the pressure received by the cells contained in the fluid L. For example, by increasing the flow rate of the fluid L, the pressure of the fluid L can be increased. Alternatively, the pressure of the fluid L can be reduced by reducing the flow rate of the fluid L.
  • Cells can be disrupted using the fluid device of this embodiment. Specifically, cells can be disrupted by changing the pressure of the fluid L flowing through the flow path 110 by the pump unit 130.
  • the cell membrane of a cell can be ruptured by generating a pressure change such as releasing pressure after applying predetermined pressure to the fluid L.
  • a pressure change such as releasing pressure after applying predetermined pressure to the fluid L.
  • only specific cells in the cell mixture can be disrupted. This is considered to be due to the fact that the cell membrane structure is different depending on the cell type, and the resistance to pressure is different.
  • the fluid L contains red blood cells and white blood cells, only red blood cells can be selectively crushed.
  • the fluid device of the present embodiment it is possible to control the scattering of the sample to the outside of the fluid device in order to break up the cells in the fluid device. Therefore, it is possible to reduce the risk of workers being infected with pathogens and viruses and contaminating the environment.
  • the fluidic device of the present embodiment may be a cartridge. That is, the fluid device of the present embodiment is a component that can be freely attached and detached, and may be attached to and detached from the analyzer. Since the fluid device of the present embodiment is a cartridge, it is possible to perform cell disruption and analysis in a closed space within the cartridge, thereby further reducing the risk of infection and contamination.
  • the fluid L inside the circulation flow path provided in the flow path 110 is circulated by operating the pump unit 130 in a state where the partition valves 140a and 140b are closed and the flow path 110 forms a circuit. Can. This makes it possible to disrupt the cells more efficiently.
  • the fluid L flows inside the circulation flow path of the defined volume.
  • “defined” means to distinguish from other areas of the flow path.
  • a part of the flow path 110 may form an introduction flow path 110 a for introducing the fluid L in the area 115 including the pump portion 130, or for discharge for discharging the fluid from the area 115 You may form the flow path 110b.
  • the operation of introducing the fluid L into the region 115 or discharging the fluid L from the region 115 can be easily performed.
  • the introduction channel 110a includes a dividing valve 140a that controls the flow of the fluid L
  • the discharging channel 110b includes a dividing valve 140b that controls the flow of the fluid L.
  • the region 115 including the pump portion 130 is defined from the introduction channel 110a by closing the partition valve 140a and from the discharge channel 110b by closing the partition valve 140b.
  • the fluid device 100 may also include a waste tank 150. By storing the waste liquid in the waste liquid tank 150, the risk of infection or contamination with a germ or virus can be further reduced.
  • the pump unit 130 may be, for example, a valve.
  • the above-mentioned valve is a diaphragm valve provided with a diaphragm member, and the diaphragm member may be formed of an elastomeric material. Then, the flow of the fluid L in the flow path 110 may be controlled by the deformation of the diaphragm member, and the volume of the region 115 including the pump unit 130 may be changed.
  • the deformation direction of the diaphragm member is not particularly limited as long as the flow of the fluid L can be controlled, and may be, for example, a direction perpendicular to the axis of the flow path 110.
  • FIGS. 2A and 2B are cross-sectional views for explaining an example of the structure of the diaphragm valve.
  • 2A shows the diaphragm valve 200 in the open state
  • FIG. 2B shows the diaphragm valve 200 in the closed state.
  • the diaphragm valve 200 includes a first substrate 210, a diaphragm member 230 made of an elastomeric material, and a second substrate 220.
  • the second substrate 220 and the diaphragm member 230 are adhered in close contact with each other. Further, the space between the first substrate 210 and the diaphragm member 230 forms a flow path 110 through which the fluid flows.
  • a through hole 240 is provided in part of the second substrate 220. Further, in the through hole 240, the diaphragm member 230 is exposed.
  • the diaphragm valve 200 is disposed in the flow passage 110 and controls the flow of the fluid L or the pressure of the fluid L inside the flow passage 110.
  • the fluid L can flow inside the flow path 110.
  • FIG. 2B when a fluid for valve control is supplied from the through hole 240 of the diaphragm valve 200 and the inside of the through hole 240 is pressurized, the diaphragm member 230 is deformed and the deformed diaphragm member Part of the substrate 230 is in close contact with the first substrate 210.
  • This state is a closed state of the diaphragm valve 200. As a result, the flow of the fluid L inside the flow path 110 is shut off.
  • a convex portion is formed in the region of the first substrate 210 facing the through hole 240. 211 may be formed.
  • Examples of the fluid for valve control include gases such as N 2 gas and air, and liquids such as water and oil.
  • the fluid for valve control can be supplied, for example, by a tube or the like connected to the through hole 240. Alternatively, opening and closing of the valve may be controlled by mechanical force or electromagnetic force.
  • the elastomer material forming the diaphragm member 230 is not particularly limited as long as it is a material that can be deformed in the axial direction of the through hole 240 according to the pressure change inside the through hole 240, for example, polydimethylsiloxane (PDMS) And silicone-based elastomers such as polymethylphenylsiloxane and polydiphenylsiloxane.
  • PDMS polydimethylsiloxane
  • silicone-based elastomers such as polymethylphenylsiloxane and polydiphenylsiloxane.
  • the structure of the diaphragm valve is not limited to the one described above.
  • the diaphragm valve 300 shown in FIGS. 3A and 3B can also be used.
  • 3A shows the valve 300 in the open state
  • FIG. 3B shows the valve 300 in the closed state.
  • the diaphragm member 230 is not disposed on the entire surface of the second substrate 220, and only around the valve 300.
  • the points that are locally arranged are mainly different.
  • the diaphragm member 230 of the valve 300 is provided with the anchor portion 231, so that even when the diaphragm member 230 is deformed by pressure, it is suppressed that the diaphragm member 230 is damaged and peeled off from the second substrate 220. It is done.
  • pump portion 130 may be comprised of three or more valves.
  • the flow control of the fluid L by the pump unit 130 can be more efficiently performed by three or more valves, that is, by arranging three or more valves.
  • FIGS. 4A to 4D are cross-sectional views for explaining the operation of an example of a pump provided with three diaphragm valves 200a, 200b and 200c.
  • valve 200a is controlled to be closed, and the valves 200b and 200c are kept open. As a result, the flow of the fluid L inside the flow path 110 is blocked by the valve 200a.
  • valve 200a and the valve 200b are controlled to be closed, and the valve 200c is kept open.
  • the fluid L existing around the valve 200 b is pushed away by the deformation of the diaphragm member 230.
  • the valve 200a is in the closed state, the displaced fluid moves in the direction shown by the arrow in FIG. 4C, that is, to the right in FIG. 4C.
  • a flow in the direction indicated by the arrow occurs in the fluid L.
  • the fluid L is pressurized.
  • valves 200b and 200c are controlled to be closed.
  • the fluid L existing around the valve 200 c is pushed away by the deformation of the diaphragm member 230.
  • the valve 200a since the valve 200a is in the closed state, the displaced fluid L moves in the direction shown by the arrow in FIG. 4 (d), that is, to the right in FIG. 4 (d).
  • the valve 200a may be controlled to be open as shown in FIG. 4 (d) or may be kept closed.
  • valves 200a, 200b and 200c are all controlled to be in the open state.
  • the pressurization of the fluid L is released.
  • the fluid L inside the flow path 110 may continue to move to the right toward FIG. 4A due to inertia.
  • the flow of the fluid L inside the flow path 110 or the pressure of the fluid L can be controlled by repeating the above steps.
  • the above process is an example of a control method of a pump provided with three valves, and a control method of a pump provided with three valves is not limited to this.
  • the flow of the fluid L inside the flow passage 110 can be controlled in the reverse direction to that described above by reversing the timing of opening and closing the valve 200a and the valve 200c.
  • the flow of the fluid L or the fluid L can also be adjusted by adjusting the cycle of repeating the operation of FIGS.
  • the pressure of the can be controlled.
  • Division valve Fluid device 100 may further include compartment valve 140.
  • the pressure of the fluid L can be controlled more efficiently by closing the dividing valve 140 (140a, 140b) to define the region 115 including the pump portion 130 of the flow path 110 and operating the pump portion 130.
  • closing the partition valve 140 results in the area 115 including the pump portion 130 being defined. That is, in the fluid device 100, by closing the dividing valve 140 (140a, 140b), the area 115 including the pump portion 130 of the flow path 110 becomes a circulation flow path (loop flow path) and becomes independent from other parts. it can.
  • the flow velocity of the fluid temporarily decreases or stops. Thereafter, when the pump unit 130 is operated, the flow velocity in the region 115 is increased, and the pressure applied to the fluid L is increased.
  • the pump portion 130 is a diaphragm valve
  • closing the diaphragm valve deforms the diaphragm member toward the flow path 110, and the volume in the region 115 is reduced.
  • the pressure of the fluid L in the area 115 is increased.
  • opening the diaphragm valve the diaphragm member is deformed in the direction opposite to the flow path side, and the volume in the region 115 is increased.
  • the pressure of the fluid L decreases.
  • the pressure of the fluid L can be adjusted by changing the volume in the region 115.
  • the dividing valve 140 may be a diaphragm valve including a diaphragm member, and may be similar to the valve constituting the pump unit described above.
  • Compartment valve 140 may be a normally closed valve or a normally open valve.
  • the normally closed valve is a valve that is closed in a steady state and is opened by operating the valve.
  • the normally open valve is a valve that is open in a steady state and is closed by operating the valve.
  • the partition valve 140 When the partition valve 140 is a normally closed valve, at steady state, the partition valve 140 is closed, and by operating the valve, the region 115 defined by the partition valve 140 is released.
  • the dividing valve 140 when the dividing valve 140 is a normally open valve, the dividing valve 140 is open in the steady state and is closed by operating the valve, and a part of the flow path 110 is the dividing valve 140. It becomes an area defined by
  • the fluid device 100 may include one compartment valve 140 or two or more compartment valves. When there are two or more partition valves 140, closing each partition valve 140 defines a region 115 including the pump portion, which can be a temporarily independent space.
  • the cell analysis unit 120 is an area for analyzing cells flowing in the flow channel 110.
  • cell analysis means analyzing predetermined parameters of fluid containing cells.
  • the parameters are not particularly limited, and include, for example, the number of cells in the fluid containing the cells, the size of the cells, and the like.
  • the cell analysis unit has, for example, an aperture shape.
  • the aperture shape is a shape in which the cross-sectional area of the flow channel cross section orthogonal to the flow direction of the fluid in the cell analysis unit 120 is smaller than the cross-sectional area of the flow channel cross section orthogonal to the flow direction of the fluid.
  • the cross-sectional area of the cell analysis unit is smaller than the cross-sectional area of the flow channels of the flow channels 110 before and after the cell analysis unit 120.
  • the diameter of the cell analysis unit 120 is smaller than the diameter of the other region of the flow channel 110. That is, the flow channel cross section of the cell analysis unit having an aperture shape may have a size suitable for analysis of an analyte, or may be a size in which cells to be analyzed pass one by one. Thereby, at least in the cell analysis unit 120, it becomes possible to pass cells one by one. As a result, since cells can be detected for each cell, analysis of cells is facilitated.
  • Cell analysis may be performed, for example, optically or electrically.
  • the analysis items of cells can be set as appropriate. For example, the number of cells may be measured, or the size of cells may be measured.
  • the cross-sectional shape of the flow path and the aperture shape is not limited to a rectangular shape, and may be, for example, a circular shape, or any polygonal shape.
  • the cross section of the flow channel 110 may be a rectangle having a width of 1.5 mm and a height of 0.3 mm.
  • the cross section of the cell analysis unit having an aperture shape may be a rectangle having a width of 30 ⁇ m and a height of 30 ⁇ m. With such a shape and size, for example, white blood cells can pass through a single cell analysis unit that is in the form of flow paths and apertures.
  • the cell analysis unit 120 is present inside the circulation flow channel provided in the flow channel 110. That is, the cell analysis unit 120 is present in the circulation channel.
  • the presence of the cell analysis unit 120 inside the circulation flow channel allows the state of the cells inside the circulation flow channel to be analyzed in real time while the pump unit 130 is operated to break up the cells. As a result, for example, it becomes possible to stop the pump unit 130 when the cell crushing rate reaches an arbitrary value, to continue the cell crushing operation until the cells are completely broken, and the like.
  • FIG. 5 is a schematic view illustrating the structure of the fluid device 500 according to the second embodiment.
  • the fluid device 500 according to the second embodiment is different from the fluid device 500 in that the cell analysis unit 120 exists outside the circuit when the partition valves 140a and 140b are closed to form a circuit in which the circulation channel of the channel 110 is closed.
  • This differs mainly from the fluidic device 100 according to one embodiment. That is, in the fluid device 500, the cell analysis unit 120 is present in a flow channel other than the circulation flow channel.
  • the flow of the fluid L inside the flow path 110 is not influenced by the cell analysis unit 120 because the cell analysis unit 120 is present outside the circuit. Therefore, the degree of freedom in control of the flow of the fluid L by the operation of the pump unit 130 is higher than that of the fluid device 100 according to the first embodiment.
  • the cell analysis unit 120 analyzes the cells flowing in the flow channel 110 before starting the cell disruption or after completing the cell disruption.
  • the cell analysis unit 120 is present in the discharge channel 110b.
  • cells can be analyzed by flowing the fluid L in one passage in the order of the introduction channel 110a, the circulation channel, and the discharge channel 110b.
  • the position of the cell analysis unit 120 is not limited to this, and the cell analysis unit 120 may be present in the introduction channel 110a.
  • cells can be analyzed by flowing the fluid L in the opposite direction again to the introduction channel 110a.
  • FIG. 6 is a schematic view for explaining a modification of the fluid device.
  • the fluid device 600 is mainly different from the fluid device 100 according to the first embodiment in that the circulation channel provided in the flow channel 110 forms two or more closed circuits by controlling the opening and closing of the dividing valve 140. .
  • the flow of the fluid L inside the flow path 110 can be controlled to analyze cells.
  • the cell analysis unit 120 is present inside the circuit.
  • the position of the cell analysis unit 120 is not limited to this, and may be configured to exist outside the circuit.
  • the fluid device 600 includes a first circulation channel and a second circulation channel, and the circuit in which the first circulation channel and the second circulation channel are closed by closing the dividing valve 140. May be formed.
  • the first circulation channel and the second circulation channel in the fluid device 600 share at least a part of the channel, and include a pump unit in the shared channel, and the first circulation
  • the cell analysis unit 120 may be provided in the channel or the second circulation channel.
  • the first circulation channel and the second circulation channel are not shared in the vicinity of both ends of the channel shared by the first circulation channel and the second circulation channel.
  • Each of the flow paths may have a valve.
  • the present invention is a method of disrupting cells, comprising: a flow path, a pump portion provided in the flow path, and a compartment valve for defining a region including the pump portion of the flow path. And allowing the fluid containing the cells to flow in the flow path of the fluidic device, closing the compartment valve to define a region including the pump portion of the flow path, and operating the pump portion Breaking the cells by changing the pressure of the fluid.
  • FIG. 1 is a schematic view illustrating the structure of a fluid device 100 according to an embodiment.
  • the fluid device 100 includes a flow channel 110 including a circulation flow channel, a pump unit 130 provided in the circulation flow channel, and an aperture-shaped cell analysis unit 120 provided in the flow channel 110 including the circulation flow channel. There is.
  • the pump unit 130 may include three or more valves. As described above, control of the flow of the fluid L by the pump unit 130 can be performed more efficiently by using three or more valves, that is, arranging three or more valves.
  • the region 115 of the flow channel 110 forms a closed circuit.
  • the partition valves 140a and 140b of the fluid device 100 are opened, and the partition valves 140c and 140d are closed.
  • the fluid L containing cells is introduced from the introduction channel 110a.
  • the fluid L containing cells may be, for example, a blood-derived sample containing red blood cells and white blood cells.
  • the inside of the flow path 110 is filled with the fluid L containing cells.
  • the excess fluid L passes through the discharge channel 110 b and is stored in the waste liquid tank 150.
  • the flow of the fluid L containing cells may be generated by operating the pump unit 130.
  • the operating condition of the pump unit 130 may be a condition that does not crush cells.
  • the compartment valves 140a and 140b are closed to open 140c.
  • the region 115 of the flow channel 110 forms a closed circuit.
  • the pump unit 130 is operated under the condition of changing the pressure of the fluid L containing cells.
  • the conditions for changing the pressure of the fluid L the driving time of the pump, the driving pressure, the frequency and the like can be mentioned.
  • the flow velocity in the region 115 is increased, and the pressure applied to the fluid L is increased.
  • the pump portion 130 is a diaphragm valve, the diaphragm member is deformed toward the flow passage 110 by closing the diaphragm valve, and the volume in the region 115 is reduced.
  • the pressure of the fluid L in the area 115 is increased.
  • the diaphragm member is deformed in the direction opposite to the flow path side, and the volume in the region 115 is increased. As a result, the pressure of the fluid L decreases. Such pressure changes disrupt the cells.
  • red blood cells in a blood-derived sample containing, for example, red blood cells and white blood cells by adjusting the conditions under which the pressure is changed.
  • the pump unit 130 by operating the pump unit 130 in a state in which the region 115 of the flow channel 110 is closed, the efficiency of cell disruption can be increased.
  • the present invention is a method of measuring the number of red blood cells and white blood cells in a blood-derived sample, comprising: a flow path; a pump unit provided in the flow path; and an aperture provided in the flow path Flowing the blood-derived sample through the flow channel of the fluidic device having the shape of a cell analysis unit, measuring the number of red blood cells and white blood cells in the cell analysis unit, and operating the pump unit to operate the pump unit.
  • a method is provided comprising the steps of: selectively disrupting the red blood cells by changing the pressure of a blood-derived sample; and measuring the number of white blood cells in the cell analysis unit after breaking the red blood cells. It can be said that the method of the present embodiment is a method of blood cell count (blood calculation).
  • the partition valves 140a and 140b of the fluid device 500 are opened, and the partition valves 140c and 140d are closed.
  • a blood-derived sample is introduced from the introduction channel 110a.
  • the introduction of the blood-derived sample may be performed by pressurization or may be performed by operating the pump unit 130.
  • the operating condition of the pump unit 130 be a condition that does not crush cells.
  • the inside of the flow channel 110 is filled with the blood-derived sample.
  • the excess blood-derived sample passes through the cell analysis unit 120 and the discharge channel 110 b and is stored in the waste liquid tank 150.
  • the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120.
  • red blood cells and white blood cells are not broken. For this reason, the number of cells measured at this stage is the sum of the numbers of red blood cells and white blood cells.
  • the compartment valves 140a and 140b are closed to open 140c.
  • the region 115 of the flow channel 110 forms a closed circuit.
  • the pump unit 130 is operated under conditions to selectively crush the red blood cells in the blood-derived sample to change the pressure of the blood-derived sample, thereby selectively crush the red blood cells.
  • the compartment valve 140b is opened to feed the blood-derived sample. Then, the blood-derived sample in the flow path 110 passes through the cell analysis unit 120 and the discharge flow path 110 b and is stored in the waste liquid tank 150.
  • the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120.
  • the number of cells measured at this stage is the number of white blood cells.
  • the number of white blood cells in the blood-derived sample can be measured. Also, the number of erythrocytes can be determined more accurately by subtracting the number of leukocytes measured later from the total number of erythrocytes and leukocytes measured first. However, since the number of white blood cells is usually very small relative to the number of red blood cells, the total number of red blood cells and white blood cells initially measured may be used as the number of red blood cells.
  • the conditions for selectively breaking up the red blood cells in the blood-derived sample may be, for example, conditions in which the driving pressure of the pump unit having a diaphragm member is more than 0 kPa, such as 50 to 110 kPa, such as 70 to 110 kPa.
  • the drive frequency of the pump unit having the diaphragm member may be more than 0 Hz, for example, 0.5 to 3.3 Hz, for example, 2 to 3.3 Hz.
  • the operating time of the pump unit may be, for example, 1 to 30 minutes, 5 to 20 minutes, or about 10 minutes.
  • the diameter of the above-mentioned diaphragm member may be, for example, 1 to 3 mm, or 2 to 3 mm.
  • the thickness of the diaphragm member may be, for example, 10 to 2000 ⁇ m, and may be, for example, 100 to 1000 ⁇ m. As the thickness of the diaphragm member is thinner, the required displacement can be obtained at a lower driving pressure.
  • the material of the diaphragm member may be a silicone-based elastomer such as polydimethylsiloxane (PDMS), polymethylphenylsiloxane, polydiphenylsiloxane or the like.
  • erythrocytes can be selectively disrupted.
  • the crushing rate of white blood cells under the above conditions is 2.5% or less.
  • FIGS. 7A to 7C are schematic diagrams illustrating a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 600.
  • FIG. 7A to 7C are schematic diagrams illustrating a method of measuring the number of red blood cells and white blood cells in a blood-derived sample using the fluid device 600.
  • the partition valves 140a2, 140b1, 140c1, 140c2 and 140c5 of the fluid device 600 are opened, and the partition valves 140a1, 140b2, 140c3 and 140c4 are closed.
  • a blood-derived sample is introduced from the introduction channel 110a1.
  • the introduction of the blood-derived sample may be performed by pressurization or may be performed by operating the pump unit 130. In this case, it is preferable that the operating condition of the pump unit 130 be a condition that does not crush cells.
  • the blood-derived sample flows inside the flow channel 110, and the inside of the flow channel 110 is filled with the blood-derived sample.
  • the excess blood-derived sample passes through the discharge channel 110 b 1 and is stored in the waste liquid tank 150.
  • the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120.
  • red blood cells and white blood cells are not broken. For this reason, the number of cells measured at this stage is the sum of the numbers of red blood cells and white blood cells.
  • the partition valves 140a1, 140b1, 140c3, 140c5 are closed, and the valves 140a2, 140b2, 140c1, 140c2, 140c4 are closed. Open. As a result, the region 115 of the flow channel 110 forms a closed circuit.
  • the pump unit 130 is operated under conditions to selectively crush the red blood cells in the blood-derived sample to change the pressure of the blood-derived sample, thereby selectively crush the red blood cells.
  • the blood-derived sample circulates inside the closed circuit formed by the flow path 110.
  • the blood origin is present in the area including the cell analysis unit 120 of the channel 110.
  • the sample can be drained.
  • the discharge fluid include N 2 gas, gas such as air, buffer, liquid such as oil, and the like.
  • the blood-derived sample passes through the discharge channel 110 b 2 and is stored in the waste liquid tank 150.
  • the discharge fluid is introduced from the introduction channel 110a1.
  • the introduction of the discharge fluid may be performed by pressurization or may be performed by operating the pump unit 130.
  • the operating condition of the pump unit 130 be a condition that does not crush cells.
  • the blood-derived sample in the flow path 110 passes through the cell analysis unit 120 and the discharge flow path 110b2 and is stored in the waste liquid tank 150.
  • the cell analysis unit 120 measures the number of cells passing through the cell analysis unit 120.
  • the number of cells measured at this stage is the number of white blood cells.
  • the device shown in FIG. 8B (hereinafter referred to as “device B”) was the same as device A except that the diameter of each valve was 2 mm.
  • the devices A and B were manufactured for the purpose of evaluating whether or not cells can be disrupted by the operation of the pump unit, and the cell analysis unit was not included.
  • a sample was introduced into each fluid device.
  • the amount of sample introduced was 62.5 ⁇ L for each device.
  • the valve 140 of each device was opened and closed to form a closed circuit of the flow path 110. Thereby, the sample was sealed in the circuit of each device.
  • the pump unit 130 was operated to break up the cells.
  • the operating conditions of the pump unit 130 were set to the conditions shown in Table 1. Moreover, the operating time of the pump part 130 was 10 minutes in all the conditions.
  • Hemolysis rate (%) (absorbance of sample ⁇ absorbance of negative control) / (absorbance of positive control ⁇ absorbance of negative control) ⁇ 100 (1)
  • FIG. 9 is a graph showing the measurement results of the hemolytic rate. As a result, when the valve diameter, the driving pressure and the frequency were increased, the ratio of the erythrocytes to be crushed tended to increase.
  • a sample was introduced into device A.
  • As a sample rabbit blood was used.
  • the amount of sample introduced was 62.5 ⁇ L.
  • the valve 140 of the device A was opened and closed to form a closed circuit of the flow path 110. Thereby, the sample was sealed in the circuit of device A.
  • the pump unit 130 was operated to break up the cells.
  • the operating conditions of the pump unit 130 were set to the conditions shown in Table 2. Moreover, the operating time of the pump part 130 was 10 minutes in all the conditions.
  • the valve 140 of the device A was opened and closed to recover the sample.
  • the hemolysis rate of each collected sample was measured in the same manner as in Experimental Example 2 using a spectrophotometer (UV-1800 / Shimadzu Corporation).
  • the amount of DNA in each sample is subjected to real-time quantitative PCR (Light Cycler 480 System II / Roche Diagnostics). Measured. When white blood cells are destroyed, the amount of DNA in the sample increases. The relative value of the amount of DNA in the recovered sample was calculated when the total amount of DNA contained in the original sample was 100.
  • FIG. 10 is a graph showing the measurement results of the hemolysis rate and the amount of DNA. As a result, it became clear that even if the red blood cells were destroyed by the device A, the white blood cells were hardly destroyed.

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Abstract

L'invention concerne un dispositif pour fluide qui comprend : un trajet d'écoulement à travers lequel s'écoule un fluide contenant des cellules, et qui comprend un trajet d'écoulement de circulation ; une section pompe qui est disposée dans le trajet d'écoulement de circulation, et commande l'écoulement du fluide ou la pression du fluide ; et une section d'analyse de cellule disposée dans le trajet d'écoulement, la surface de section transversale de la section d'analyse de cellule étant plus petite que la surface de section transversale de la section de trajet d'écoulement à l'exclusion de la section d'analyse de cellule.
PCT/JP2017/031936 2017-09-05 2017-09-05 Dispositif pour fluide et son utilisation WO2019049204A1 (fr)

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JP2019540155A JP7020488B2 (ja) 2017-09-05 2017-09-05 流体デバイス及びその使用
PCT/JP2017/031936 WO2019049204A1 (fr) 2017-09-05 2017-09-05 Dispositif pour fluide et son utilisation

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008136475A (ja) * 2006-11-10 2008-06-19 Univ Waseda 細胞捕捉装置及びそれを利用した細胞操作方法
JP2014132860A (ja) * 2013-01-10 2014-07-24 Aquatech Co Ltd マイクロポンプユニット
WO2016013395A1 (fr) * 2014-07-22 2016-01-28 株式会社日立ハイテクノロジーズ Mécanisme de mesure de dispersion cellulaire, et système de culture cellulaire repiquée l'utilisant

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070105206A1 (en) 2005-10-19 2007-05-10 Chang Lu Fluidic device

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008136475A (ja) * 2006-11-10 2008-06-19 Univ Waseda 細胞捕捉装置及びそれを利用した細胞操作方法
JP2014132860A (ja) * 2013-01-10 2014-07-24 Aquatech Co Ltd マイクロポンプユニット
WO2016013395A1 (fr) * 2014-07-22 2016-01-28 株式会社日立ハイテクノロジーズ Mécanisme de mesure de dispersion cellulaire, et système de culture cellulaire repiquée l'utilisant

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