WO2015053393A1 - Trieur de cellules par imagerie - Google Patents

Trieur de cellules par imagerie Download PDF

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WO2015053393A1
WO2015053393A1 PCT/JP2014/077198 JP2014077198W WO2015053393A1 WO 2015053393 A1 WO2015053393 A1 WO 2015053393A1 JP 2014077198 W JP2014077198 W JP 2014077198W WO 2015053393 A1 WO2015053393 A1 WO 2015053393A1
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Prior art keywords
cell
cells
image
cluster
chip
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PCT/JP2014/077198
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English (en)
Japanese (ja)
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安田　賢二
賢徹 金
服部　明弘
英之 寺薗
Original Assignee
公益財団法人神奈川科学技術アカデミー
一般社団法人オンチップ・セロミクス・コンソーシアム
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Priority to JP2015541651A priority Critical patent/JPWO2015053393A1/ja
Publication of WO2015053393A1 publication Critical patent/WO2015053393A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
    • G01N15/0227—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging using imaging, e.g. a projected image of suspension; using holography
    • G01N15/1433—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1484—Electro-optical investigation, e.g. flow cytometers microstructural devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G01N15/149—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1493—Particle size
    • G01N2015/1495—Deformation of particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1497—Particle shape

Definitions

  • the present invention relates to a cell recovery apparatus.
  • cell differentiation 1) Visual morphological cell classification: For example, examination of bladder cancer and urethral cancer by examination of atypical cells appearing in urine, classification of atypical cells in blood, cancer examination by cytology in tissues, etc. Can give. 2) Cell classification by cell surface antigen (marker) staining by the fluorescent antibody method: Cell surface antigens generally called CD markers are stained with a specific fluorescently labeled antibody. Cell sorting by cell sorters, flow cytometers and tissues Used for cancer screening by staining. Of course, these are widely used not only for medical purposes but also for cell physiology research and industrial cell utilization.
  • stem cells containing stem cells are roughly separated using a fluorescent dye incorporated into the cells as a reporter, and then the target stem cells are separated by actually culturing. This is because an effective marker for stem cells has not yet been established, and the target cells are substantially separated by using only those actually cultured and differentiated.
  • Such separation and recovery of specific cells in the culture medium is an important technique in biological and medical analysis.
  • the cells When the cells are separated based on the difference in specific gravity of the cells, they can be separated by a velocity sedimentation method. However, if there is almost no difference in specific gravity between cells that distinguishes unsensitized cells from sensitized cells, the cells are separated one by one based on information stained with fluorescent antibodies or visual information. There is a need to.
  • the cell sorter isolates and drops cells after fluorescence staining in a charged droplet in units of one cell, and based on the presence or absence of fluorescence in the droplet and the amount of light scattering, In the process of dropping, a high electric field is applied in any direction in the normal direction to the direction of drop, and the drop direction of the drop is controlled and fractionated into multiple containers placed underneath.
  • Non-patent document 1 Kamarck, ME, Methods Enzymol. Vol. 151, p150-165 1987 (1987)).
  • the cell sorter created using this microfabrication technique has a slow response speed of sample separation to the observation means, and in order to put it to practical use, a separation processing method that does not damage the sample and has a faster response is required. there were.
  • the separation efficiency of the device cannot be sufficiently increased even at a dilute cell concentration. If the sample is concentrated in a separate device, it is not only difficult to recover the concentrated solution without loss, but it is also desirable for regenerative medicine where cells are contaminated in a complicated pretreatment stage. The problem was that no problems occurred.
  • Patent Document 1 JP 2003-107099
  • Patent Document 2 JP 2004-85323
  • Patent Document 3 WO 2004 / 101731
  • These are well-practical cell sorters at the laboratory level, but for general use for regenerative medicine, new technological development is required for pretreatment such as liquid transport, recovery, and sample preparation. is there.
  • CTCs peripheral blood circulating cancer cells
  • anticancer agents for specific targets have been developed one after another, and if the type of malignant tumor in the blood can be identified, it has become possible to select an anticancer agent that effectively destroys the cells. If technology to monitor CTCs flowing in the blood is realized, the presence of malignant tumor cells that cause metastatic cancer flowing in the blood can be quantitatively measured, thereby quantifying the effect of the administered anticancer drug. This is the realization of the world's first method that can be continuously evaluated and can not only prevent the administration of unnecessary and excessive anticancer drugs, but also detect the presence or absence of recurrence.
  • PCR polymerase chain reaction
  • DNA templates such as complementary DNA reverse transcribed from genomic DNA or messenger RNA in a mixture of various types of nucleic acids, two or more primers, a thermostable enzyme, a salt such as magnesium, and four types
  • dATP, dCTP, dGTP, dTTP deoxyribonucleoside triphosphates
  • the step of separating the nucleic acid into single strands the step of binding the primer to the separated nucleic acid, and the primer bound by a thermostable enzyme
  • a specific nucleic acid sequence can be amplified by repeating the hybridization step using a nucleic acid as a template at least once.
  • a thermal cycle is used by raising and lowering the temperature of a reaction vessel used for a DNA amplification reaction.
  • temperature change mechanisms used there, for example, a mechanism for changing the temperature of the reaction vessel containing the sample by a heater or Peltier element, heat exchange using hot air, and a heater block with different temperatures for the reaction vessel.
  • a mechanism for changing the temperature by alternately contacting the liquid bath and a mechanism for changing the temperature by flowing a sample in a flow path having regions of different temperatures.
  • a light cycler Light Cycler manufactured by Roche Corporation is one of the fastest commercially available devices.
  • the light cycler introduces a sample, a DNA polymerase, a DNA piece serving as a primer, and a fluorescent labeling dye for measurement into each of a plurality of glass capillary tubes, and the temperature of a minute droplet in the capillary tube is set to 55 degrees and 95 degrees, for example. These two temperatures are changed by blowing warm air at the same temperature as the droplet to be changed, and at the same time, this glass capillary tube is irradiated with excitation light of a fluorescent dye, and the obtained fluorescence intensity is measured. It has a mechanism that makes it possible.
  • the current analysis means using cells has a problem that it does not have a means for analyzing whether the target cell is in a state of apoptosis or the like at the time of cell recovery.
  • the narrowing of the sample aqueous solution using the side sheath liquid has a risk of causing dilution of the sample aqueous solution.
  • JP2011-257241-A as a means for detecting the presence or absence of cancer cells in blood, fluorescent dyes are used for antibodies that selectively bind to molecules (cancer markers) present only on the surface of cancer cells.
  • a technique for detecting a cancer cell by causing a cancer cell to emit fluorescence when a cancer cell is present in the blood by reacting with a blood cell is shown.
  • the means and device configuration after irradiating the blood containing cancer cells flowing through the microchip with fluorescence excitation light, the fluorescence emitted from the cancer cells is reflected only by light of a specific wavelength and other wavelengths are reflected.
  • the light passes through a mirror (dichroic mirror) that passes through multiple times, extracts fluorescence with a specific wavelength width at each step, and measures the amount of fluorescence with a photodetector for each fluorescence of that wavelength width.
  • a mirror dichroic mirror
  • the amount of fluorescence from cancer cells that should be detected when passing through the dichroic mirror also attenuates according to the number of passes through the dichroic mirror
  • the fluorescence intensity measured at each wavelength width is caused by the passage through the dichroic mirror. In particular, it was difficult to detect faint fluorescence at a later stage wavelength, and it was difficult to quantitatively detect multiple stained cancer marker molecules.
  • the fluorescence wavelength and the excitation wavelength overlap. Since the light must be decomposed for each wavelength by passing through a plurality of dichroic mirrors, the device configuration becomes complicated, and each wavelength band needs to be narrowed, not only the signal fluorescence is further attenuated, but also the wavelength In principle, it was difficult to separate light with close bands.
  • the present inventors provide a cell analyzer capable of rapidly identifying the type and state of cancer cells flowing in the blood with metastatic ability and the number (concentration). That is, the present invention provides the following apparatuses, systems, and methods.
  • A a first device for concentrating, staining, and washing a cell sample solution from a subject;
  • B a second device for concentrating, separating and purifying the stained cell sample solution from the first device;
  • C a third device for performing gene analysis / expression analysis of purified cells in the cell sample solution from the second device;
  • D a fourth device for continuously feeding the cell sample solution over the first to third devices;
  • E A cell analyzer system comprising a control / analyzer that controls the operation of each of the devices and analyzes the cell sample,
  • the first device is A chamber with a filter for concentrating, staining, and washing cells in the cell sample solution; Containers for storing the cell sample solution, staining solution and washing solution, respectively;
  • the second device is A cell sorter chip having a flow path for flowing a cell sample solution containing cells including target cells, wherein the flow path includes a first flow path in which the cells are concentrated, and detection
  • a cell sorter chip including a second flow path branched from the first flow path, wherein the target cells are selected, and An external force is applied to the cells flowing through the channels so that the cells flowing through the channels are concentrated in the first channel and converged in a desired direction in the second channel.
  • a mechanism to give An optical system including light irradiation means for irradiating the cells flowing in the second flow path, and a high-speed camera for acquiring an image of the cells at an image capture rate of at least 200 frames / second;
  • the third device is A reaction vessel to which a sample solution is added and reacted;
  • a heat exchange tank for exchanging heat with the reaction tank;
  • a temperature control mechanism for controlling the temperature of the heat exchange tank;
  • a cell analyzer system comprising: (2) Contents of the cells conveyed by the fourth device for feeding the cell sample solution before the third device for performing gene analysis / expression analysis of purified cells in the cell sample solution A cell disruption mechanism that elutes the sample solution by cell disruption,
  • the control / analyzer transports the cell sample solution from the second device to the cell disruption mechanism by the fourth device for feeding the cell sample solution, and the sample solution is disrupted by the cell disruption mechanism.
  • the cell disruption mechanism is A container for containing the cell sample; A crushing rotating body for crushing cells in the container, An abrasive for crushing cells in the container, The cell sample and the abrasive are added to the inside of the container, and the cell sample is crushed by the movement of the crushing rotating body in which rotation and revolution are strictly controlled.
  • Cell analyzer system (4)
  • the cell disruption mechanism further includes a rotating shaft, When the crushing rotating body is pressed from above by the rotating shaft, the crushing rotating body rotates inside the container, and the frictional force and slippage between the crushing rotating body and the rotating shaft are different from those of the crushing rotating body.
  • the cell analyzer system according to (3) which is controlled by the pressure between the rotating shafts.
  • the cell crushing mechanism can generate a force for pressing the crushing rotating body in a direction perpendicular to the side surface of the container by shifting the rotating shaft of the crushing rotating body and the rotating shaft of the rotating shaft.
  • the cell analyzer system according to (4) above comprising a mechanism.
  • the cell crushing mechanism is a mechanism that allows the crushing rotating body to be lifted from the inside of the container and removed by the magnetic force of the rotating shaft, electrostatic force, or suction force due to gas pressure difference.
  • the cell analyzer system according to (4) comprising: (7) In the cell disruption mechanism described above, contamination between different cell samples can be eliminated by providing a drive mechanism equipped with a plurality of containers capable of automatic replacement of the containers.
  • the cell analyzer system according to any one of (6).
  • the crushing rotator is sealed and sealed inside the unused container by a confidential seal, and the container and the crushing rotator are placed at the time of crushing the cell sample.
  • a cell sorter chip including a flow path for flowing a cell sample solution containing cells including target cells, wherein the flow path includes a first flow path in which the cells are concentrated, and the concentration A cell sorter chip including a second flow path branched from the first flow path, wherein the detection of the cells and the selection of the target cells are performed, (ii) The cells flowing through each of the channels are concentrated in the first channel and converged in a desired direction in the second channel.
  • a mechanism for applying external force to the (iii) an optical system including a light irradiation means for irradiating the cells flowing in the second flow path, and a high-speed camera for acquiring an image of the cells at an image capture rate of at least 200 frames / second; (iv) a control / analysis unit that controls the operation of each unit and analyzes an image of each cell captured by the optical system;
  • An image detection type single cell separation / purification apparatus comprising: (10) The apparatus according to (9), wherein the external force is ultrasonic radiation pressure, gravity, electrostatic force, or dielectric electrophoresis force. (11) The device according to (9) or (10), wherein the cell sample containing the target cell is derived from blood.
  • the device according to any one of (9) to (11), wherein the target cell includes a cancer cell.
  • the control / analysis unit binarizes the cell image obtained from the optical system, and is selected from the group consisting of the luminance centroid, area, perimeter, major axis, and minor axis of the binarized image.
  • the device according to any one of (9) to (12) above, wherein each of the cells is detected and identified at a single cell level by at least one index.
  • the cells in the cell sample solution are fluorescently labeled, the optical system further includes fluorescence detection means, and information on the fluorescence image of the cells is used as an additional index by the control / analysis unit.
  • the apparatus according to (13) above.
  • the present invention provides the following on-chip cell sorter and on-chip cell sorter system.
  • One sample channel having the same length and cross-sectional area and two buffer liquid channels disposed symmetrically on both sides of the sample channel are arranged to merge, and after the merge, the center of the same length and cross-sectional area is again downstream.
  • the ratio of the cross-sectional area of the sheath liquid reservoir that is distributed to the recovery flow path and the two waste liquid flow paths on both sides and covers the inlets of the three upstream flow paths and the sample liquid reservoir that fills the sample therein
  • the ratio of the number of channels is 2: 1, so that even if the liquid flows, the liquid level of both is the same.
  • the electric field is only applied to the cells that are placed in the I applied it was characterized by the on-chip cell sorter.
  • a stopper disposed on the top surface of the sheath liquid reservoir, means for applying compressed air through the stopper, means for continuously supplying liquid to the sheath liquid reservoir and the sample reservoir,
  • the cell sorter according to (15) above, further comprising an electric sensor capable of measuring the liquid level height in both the sheath liquid reservoir and the reagent reservoir.
  • Flash time Pixel size / flow velocity
  • An on-chip cell sorter system characterized by deciding on the relationship of (20)
  • An on-chip cell sorter system characterized by using an optical system that can maintain the depth of field and the depth of field up to the height of the microchannel by combining an objective lens having a numerical aperture of 0.3 or less and a zoom lens. .
  • (21) An on-chip cell sorter system characterized in that the sample liquid is arranged so as to flow vertically from above to below.
  • An on-chip cell sorter system characterized in that the inner wall surface of the microchannel facing the surface on which the electrode for generating repulsive force is arranged on the sample fine particles has a convex shape.
  • a pair of gel electrodes having a structure in which struts are repeatedly arranged at a certain distance that can prevent the leakage of the liquid into the flow path due to the surface tension of the liquid when the gel is in a sol state.
  • An on-chip cell sorter system characterized by being arranged.
  • An on-chip cell sorter system From the shape of the cells obtained by image recognition, , An on-chip cell sorter system characterized by purifying cells with R less than 1.1 as cardiomyocytes.
  • An on-chip cell sorter system characterized in that oil having a specific gravity lighter than water and not mixed with water is used for the side sheath liquid.
  • An on-chip cell sorter system characterized by using a solution in which the conductivity of the sample aqueous solution is 10 2 ⁇ S / cm or less when collecting the sample fine particles in the aqueous solution.
  • a cell sorter chip including a first flow path for flowing a sample liquid containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells.
  • a cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
  • An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image
  • a target cell flowing through the first flow path or a cell other than the target cell in a second region substantially coincident with the first region upstream from the branch point
  • An external force is applied to the cell to shift the traveling direction of the cell by applying an external force to the target cell recovery channel, and an external force applying mechanism for guiding cells other than the target cell to the waste liquid recovery channel;
  • An on-chip cell sorter system comprising: a control unit that controls operations of the
  • a cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells.
  • a cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
  • An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image
  • a target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point.
  • an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel.
  • An external force application mechanism for A control unit for controlling the operation of the optical system and the external force application mechanism An on-chip cell sorter system, wherein the optical system includes a microscope having an objective lens having a numerical aperture of 0.3 or less and a zoom lens optically coupled to the objective lens.
  • the optical system includes a microscope having an objective lens having a numerical aperture of 0.3 or less and a zoom lens optically coupled to the objective lens.
  • a cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells.
  • a cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
  • An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image
  • a target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point.
  • an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel.
  • An external force application mechanism A control unit for controlling the operation of the optical system and the external force application mechanism,
  • the cell sorter chip is arranged so that the first flow path is substantially parallel to the direction of gravity so that the sample liquid flows substantially vertically downward from the upstream side to the downstream side of the first flow path.
  • An on-chip cell sorter system An on-chip cell sorter system. [5] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells.
  • a cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
  • An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image
  • a target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point.
  • an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel.
  • An external force application mechanism A control unit for controlling the operation of the optical system and the external force application mechanism,
  • the external force applying mechanism includes a gel electrode or a metal electrode for applying an electric force to fine particles including cells flowing through the first flow path, and the conductivity of the sample solution is 10 2 ⁇ S / cm or less.
  • Chip cell sorter system An external force for aligning cells in the sample solution flowing in the first channel in the third region upstream of the first region upstream of the first channel is applied to the cells.
  • the on-chip cell sorter system according to any one of the above [1] to [5], further comprising a further external force applying mechanism applied to the.
  • Chip cell sorter system [8] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells.
  • a cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
  • An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image
  • a target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point.
  • an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel.
  • An external force application mechanism A control unit for controlling the operation of the optical system and the external force application mechanism, further, A reservoir fluidly connected to the upstream side of the first flow path and storing a buffer solution for sheath liquid; A sample solution introduction channel fluidly connected to the upstream side of the channel for introducing a sample solution containing cells into the first channel, An on-chip-chip, wherein a tip end portion of the sample solution introduction channel on the side fluidly connected to the first channel extends to a downstream side of the introduction portion of the buffer solution to the first channel.
  • Cell sorter system [9] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells.
  • a cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
  • a first external force applying mechanism for applying to the cells an external force for aligning the cells in the sample solution flowing through the first flow path in a preliminary region upstream from the branch point;
  • a digital image of the cells in the sample solution flowing in the first flow path in a first region upstream of the branch point and downstream of the preliminary region is obtained, and the digital analysis of the image
  • An optical system for identifying a target cell A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point.
  • an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel.
  • a second external force application mechanism A controller that controls the operation of the optical system and the first and second external force application mechanisms,
  • the first external force applying mechanism is a comb-shaped electrode for generating repulsive force on microparticles containing cells in a sample solution arranged along one surface of the first flow path, and the first flow path
  • An on-chip cell sorter system in which the cross section perpendicular to the flow path is tapered or convex toward the center of the surface opposite to the surface on which the electrodes are arranged in order to promote alignment of the fine particles .
  • a cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells.
  • a cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
  • a first external force applying mechanism for applying to the cells an external force for aligning the cells in the sample solution flowing through the first flow path in a preliminary region upstream from the branch point;
  • a digital image of the cells in the sample solution flowing in the first flow path in a first region upstream of the branch point and downstream of the preliminary region is obtained, and the digital analysis of the image
  • An optical system for identifying a target cell A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point.
  • an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel.
  • a second external force application mechanism A controller that controls the operation of the optical system and the first and second external force application mechanisms,
  • the second external force applying mechanism is a gel electrode disposed so as to come into contact with the sample liquid on both side surfaces of the first flow path, and when the gel is in a sol state, the sol liquid is In order not to leak into the flow path, the sol liquid is provided on both side surfaces along the first flow path at regular intervals so that the surface tension of the sol liquid can prevent the sol liquid from leaking into the flow path.
  • An on-chip cell sorter system configured to contact the sample solution through an array of slits.
  • a cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells.
  • a cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
  • a first external force applying mechanism for applying to the cells an external force for aligning the cells in the sample solution flowing through the first flow path in a preliminary region upstream from the branch point;
  • a digital image of the cells in the sample solution flowing in the first flow path in a first region upstream of the branch point and downstream of the preliminary region is obtained, and the digital analysis of the image
  • An optical system for identifying a target cell A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point.
  • an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel.
  • a second external force application mechanism A controller that controls the operation of the optical system and the first and second external force application mechanisms,
  • the first external force application mechanism includes a pair of flow paths for flowing a side sheath liquid for generating a side sheath flow that is fluidly connected to the upstream side of the first flow path.
  • An on-chip cell sorter system that is light in specific gravity and does not mix with water.
  • the external force applying mechanism that guides the cell to each recovery flow path includes a gel electrode or a metal electrode for applying an electric force to the cell.
  • the target cell is a cardiomyocyte
  • the on-chip cell sorter system according to any one of [1] to [13] above, wherein a cell having R of less than 1.1 is identified as a cardiomyocyte by [15]
  • the present invention provides the following on-chip cell sorter system, a method for identifying candidate cells of blood cancer cells from a cell sample solution derived from a subject using the system, and an optical module.
  • a cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject;
  • An optical system including a bright-field light source and a fluorescent light source for irradiating the cells;
  • a detection system for simultaneously acquiring a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells;
  • Control / analyzing means for identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image;
  • An on-chip cell sorter system comprising: means for selectively recovering the identified multinucleated cells and / or cell clusters.
  • the control / analysis means is i) one or more data selected from the group consisting of the size (area) of the cell, the perimeter, and the value of R indicating the degree of unevenness of the surface of the cell obtained by the area and the perimeter, and ii) 1 selected from the group consisting of the fluorescence wavelength and intensity spectrum of the labeling substance bound to the cell, and the barycentric coordinates and area of each of one or more regions fluorescently stained in the cell.
  • the on-chip cell sorter system according to the above ⁇ 1>, wherein two or more data are acquired, and multinucleated cells and / or cell clusters flowing in the flow path are identified based on the data.
  • ⁇ 3> The on-chip cell sorter system according to the above ⁇ 1> or ⁇ 2>, further comprising means for measuring a nucleic acid sequence of a gene derived from the selectively collected multinucleated cells and / or cell clusters.
  • ⁇ 4> Any one of the above ⁇ 1> to ⁇ 3>, comprising an image dividing mechanism having a function of dividing and displaying the bright field image and the fluorescent image on the light receiving surface of one high-speed camera at the same time.
  • ⁇ 5> The on-chip cell sorter system according to ⁇ 4>, further including a mechanism that adjusts the magnification of the bright field image and the fluorescent image so that the magnification of the image differs.
  • ⁇ 6> The on-chip cell sorter system according to any one of the above ⁇ 1> to ⁇ 5>, which is used for identifying candidate cells of blood cancer cells.
  • ⁇ 7> A method for identifying candidate cancer cells in blood from a cell sample solution derived from a subject using the on-chip cell sorter system according to any one of ⁇ 1> to ⁇ 6> above.
  • the method comprising the step of identifying and selectively recovering from cancer cells by analysis combined with the presence of fluorescence intensity of a fluorescent antibody against one or more biomarkers of cancer cells.
  • a first dichroic mirror with an angle adjustment function capable of adjusting the direction in which light is reflected in three dimensions A filter system in which light including image data reflected by the dichroic mirror is introduced;
  • An image size adjustment system comprising a movable shielding plate for adjusting the image size, into which light is introduced through the filter system, Light introduced through the image size adjustment system is introduced, a second dichroic mirror with an angle adjustment function capable of three-dimensionally adjusting the direction in which the light is reflected, and light introduced through the second dichroic mirror
  • An optical module for use in an optical bright field / fluorescence microscope system comprising an optical lens system for correcting differences in imaging position, An optical module configured such that the image can be enlarged and reduced by the optical lens system, and an image having a different magnification rate can be generated between a bright-field image and a fluorescent image.
  • the present invention provides the following on-chip cell sorter system.
  • a cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject;
  • An optical system including a bright-field light source for irradiating the cells, one or more fluorescent light sources, an optical fiber that conducts light of each wavelength, and a condensing lens that focuses the light on the observation target at the irradiation site;
  • a detection system that simultaneously acquires the fluorescence intensity of the fluorescent labeling substance bound to the cells, comprising a bandpass filter and a fluorescence detector that pass the fluorescence wavelength of A detection system for simultaneously acquiring a bright field image of the cell and a fluorescence image of the cell; Control /
  • the present invention provides the following on-chip cell sorter system.
  • the present invention provides the following on-chip cell sorter system.
  • the ratio R of the perimeter derived from the perimeter of the cell (cluster) and the area as a circular approximation (1 / R) is 0.9 or more
  • the cell (cluster) is an isolated single cell, (1 / R ) Is less than 0.9
  • the on-chip cell sorter system is characterized in that a cell (cluster) is composed of a cluster of two or more cells as a criterion for determining the number of cells in the cell cluster.
  • optical branching module of one unit which is the minimum structural unit, a pair of optical path systems for input / output of image light on the both bottom surfaces of the container and two pairs on the side surface for branching a two-dimensionally developed optical path system
  • Four optical path holes for introducing mirror-reflected light, and six optical path covers that can be freely attached to and detached from each other are arranged, and light is introduced into the holes of the optical path for introducing two-to-four mirror-reflected light on these side surfaces.
  • each of these mirrors can finely adjust the traveling direction of the reflected light, and this makes it possible to connect with the camera.
  • the image position can be freely moved, and the mirror holder can be equipped with a total reflection mirror, high-pass filter, low-pass filter, etc.
  • Two removable adjustable adjustments A removable optical path window is arranged between the mirror holders with a function, the sectional area of the transmitted light can be adjusted, and a removable filter 4304 is arranged.
  • incident light introduced so as to have an area equal to or smaller than (total area of light receiving surface of image acquisition camera system to be measured / parallel light introducing module)
  • a parallel light introducing module for introducing an image optical image in which an optical window filter that cuts out a cross-sectional area of In the first-stage optical branching module connected to the parallel light introducing module, the (wavelength) high-pass filter or the (wavelength) low-pass filter b is introduced into the mirror holder,
  • the transmitted light of the incident light branched into two wavelengths is reflected by the total reflection mirror, and is the same as the first filter of the optical branching module of the second stage (first stage).
  • the light reflected by the filter of the first-stage optical branching module is introduced into the first filter of the second-stage optical branching module, and similarly up to the n-th optical branching module.
  • wavelength band splitting is performed, and for each branch wavelength, positioning to adjust the direction of the optical path so that the images in each wavelength band do not overlap with the light receiving surface of the camera can be performed with each mirror holder with detachable movable adjustment function
  • a total reflection mirror is used for the first filter
  • the first filter in the previous-stage optical branching module is used
  • the high-pass filter or low-pass filter is arranged in the order of the monotonically increasing or monotonically decreasing wavelength for the branch wavelength, and the same filter is used for the first filter in the previous stage and the second filter in the next stage.
  • the image branching display device is characterized in that a total reflection mirror is used for the first-stage second filter and the last-stage first filter.
  • An imaging cell sorter that observes and separates cells in water droplets that are generated in the air and dropped on the water-repellent substrate.
  • a mechanism for producing and dropping water droplets of an optimal size by discharging the sample solution at a constant pressure from the thin tube at the tip of the water droplet forming module with a cell reservoir, in the sample solution, Water droplets are desired by charging the electrostatic field coil that covers from the area where the water just before the water drops are linked to the reservoir to the area where the water drops are just formed, to the opposite charge to the water charge.
  • a mechanism that can be charged with an electric charge A mechanism in which the formed water droplets are dropped onto an optically transparent water-repellent insulating substrate having a Teflon (registered trademark) resin processed on the surface of glass or the like, and rolled down in the direction of inclination of the substrate, , A mechanism in which a high-speed camera capable of measuring a bright field image and a fluorescent image on the back surface of the substrate on the path where the water drops fall and an optical measurement module capable of measuring scattered light intensity, fluorescent intensity, etc.
  • Teflon registered trademark
  • a mechanism to determine whether the target cell is in a water drop After identifying the type of the target cell according to the determination, the charge opposite to the water droplet is transferred to a specific one of the one or more water droplet movement direction control electrostatic field guides for changing the position from the direction in which the water droplet falls.
  • a mechanism to change the drop direction of the water droplets, and to guide to the fractional water droplet reservoir in the subsequent stage A mechanism that can apply the electrostatic field guide for each water droplet movement direction control to the electrode in accordance with the analysis result of the analysis control module, and each electrode is a static for the water droplet movement direction control when not controlled by this mechanism.
  • the electric field guide has a mechanism that does not affect the movement of water drops by grounding
  • a similar electrostatic field guide for controlling the water droplet movement direction is arranged, or a pair of rail-shaped three-dimensional structures sandwiched between the bottom surfaces of water repellent surfaces such as Teflon coat are added.
  • the observation area and the water droplet fractionation start area have means that can arrange water drops closely at equal distances in the falling direction by making the inclination horizontal (perpendicular to gravity) so that the water drops move at a constant speed.
  • a mechanism for controlling the temperature of water droplets that have made minimal contact in a water-repellent state by adjusting the temperature of the substrate In order to selectively collect target cells in the water droplet according to the reaction result, the reaction liquid reservoir water droplet formation mechanism is used to collide the reaction liquid with the sample water droplet and observe and measure the mixed liquid droplet at a certain post-reaction time.
  • a cell sorter that creates reaction droplets with weakly applied reverse charges to the sample water droplets, and can guide the reaction solution to the sample water droplets by the same electrostatic field guide for controlling the direction of water droplet movement.
  • the present invention also includes a process of collecting blood and a cell cluster (cluster) having an area of about 250 ⁇ m 2 or more in the blood, which is collected and cultured, or subjected to genetic mutation test or expression analysis test.
  • the progression of the cancer is presumed to be early in the beginning of metastasis from the primary cancer, and there are many different mutation points while the history of mutations in each cluster is the same.
  • a procedure for recovering cells that are larger in size than normal, especially for phagocytic leukocytes such as macrophages, and examining and identifying genes of heterologous cells such as bacteria in the cells, and B cells From the procedure to diagnose what the immune system is reacting by selectively recovering those whose internal shape has become complicated due to activation of, and those whose size has increased and clarifying the antigen of the B cell A cell cluster diagnostic technique is provided.
  • the present invention also allows the patient's blood to be refluxed using a cell cluster (cluster) removal mechanism such as a membrane filter that removes a cell cluster (cluster) having a cross-sectional area of approximately 250 ⁇ m 2 or more that does not exist in healthy blood.
  • a cell cluster (cluster) removal mechanism such as a membrane filter that removes a cell cluster (cluster) having a cross-sectional area of approximately 250 ⁇ m 2 or more that does not exist in healthy blood.
  • a metastatic cancer treatment device that suppresses the onset and progression of metastatic cancer by effectively removing metastatic cancer cells in blood by a physical technique.
  • the present invention relates to the following on-chip cell sorter system, a method for identifying cancer cell candidate cells in a subject-derived cell sample solution, an optical branching module, an imaging cell sorter, and a cell cluster in blood.
  • a medical treatment apparatus for preventing metastatic cancer progression by physical removal and a method for diagnosing metastatic cancer, organ abnormality, or infection using detection of a cell mass in blood.
  • An on-chip cell sorter system used for identifying cancer cell candidate cells, A cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject; An optical system including a bright-field light source and a fluorescent light source for irradiating the cells; A detection system for simultaneously acquiring a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells; Control / analyzing means for identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image; Means for selectively recovering the identified multinucleated cells and / or cell clusters,
  • the above control / analysis means is provided by the following (i) to (iii): (I) The area of the nucleus of the lump or cluster obtained from the fluorescence image of the nucleus
  • the area of the lump or cluster obtained from a bright field image of a lump of cells or clusters is 250 ⁇ m 2 or more, or (iii) the number of nuclei in the lump of cells or clusters is 3 or more If one condition is met, or (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii) are met , Judge that cancer cells are likely to be present in the cell sample solution, On-chip cell sorter system.
  • the control / analysis means is About the perimeter ratio R derived as a circular approximation from the perimeter and area of cells (clusters), (I) When (1 / R) is 0.9 or more, the cell (cluster) is an isolated cell, or (ii) When (1 / R) is less than 0.9, the cell (cluster) is 2 cells. Consisting of a cluster of cells, As described above, the on-chip cell sorter system according to the above [1], wherein the value of (1 / R) is used as a criterion for determining the presence or absence of clusters in the cell sample solution.
  • An on-chip cell sorter system used for identifying cancer cell candidate cells, A cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject; An optical system including a bright-field light source and a fluorescent light source for irradiating the cells; A detection system for simultaneously acquiring a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells; Control / analyzing means for identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image; Means for selectively recovering the identified multinucleated cells and / or cell clusters, The control / analysis means is About the perimeter ratio R derived as a circular approximation from the perimeter and area of cells (clusters), (I) When (1 / R) is 0.9 or more,
  • an on-chip cell sorter system in which the value of (1 / R) is used as a criterion for determining the presence or absence of clusters in the cell sample solution.
  • the on-chip cell sorter system according to any one of the above [1] to [3], further comprising means for measuring a nucleic acid sequence of a gene derived from the selectively recovered multinucleated cells and / or cell clusters.
  • the on-chip cell sorter system according to item.
  • [8] A method for identifying candidate cells of cancer cells in a cell sample solution derived from a subject using the on-chip cell sorter system according to any one of [1] to [7], Flowing a sample solution containing fluorescently-stained cells from a subject through the flow path of the cell sorter chip; Irradiating the cells with light from a bright-field light source and a fluorescent light source, Obtaining a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells; Based on the bright field image, the fluorescence intensity, and the fluorescence image, a step of identifying a multinucleated cell and / or cell cluster flowing through the flow path, and selectively identifying the identified multinucleated cell and / or cell cluster Including the step of collecting, In identifying the multinucleated cells and / or cell clusters, (I) The area of the nucle
  • the area of the lump or cluster obtained from a bright field image of a lump of cells or clusters is 250 ⁇ m 2 or more, or (iii) the number of nuclei in the lump of cells or clusters is 3 or more If one condition is met, or (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii) are met.
  • An optical branching module A substantially rectangular parallelepiped housing; An optical path system including a pair of mirrors for input / output of a pair of image light provided symmetrically on the bottom surface in the housing; Two-to-four openings for introducing mirror reflected light provided on the side surface of the casing, two on the side in the longitudinal direction and one on the side in the short side An aperture formed, 6 optical path covers that can be freely attached to and detached from each of the 2 to 4 openings, A pair of removable movable mirror holders capable of adjusting the mirror so that light is introduced into each of the two to four openings; A detachable optical path window arranged between the pair of mirrors and capable of adjusting a cross-sectional area of the transmitted light, A detachable filter disposed between the pair of mirrors and capable of adjusting a wavelength bandwidth of light; An optical branching module.
  • the bright field image of the cell in the sample solution containing the fluorescence-stained cell, the fluorescence intensity of the fluorescent labeling substance bound to the cell, and the fluorescence image of the cell are used to simultaneously acquire the above [ The optical branching module according to any one of 9] to [12].
  • a water droplet formation module comprising a sample solution reservoir for holding a sample solution containing cells and a thin tube connected to the reservoir; An electrostatic field coil for charging water droplets formed by the water droplet forming module; A substrate having a water-repellent surface for dropping the formed water droplets, the substrate being optically transmissive, A substrate tilt control mechanism for adjusting the tilt angle of the substrate surface; An optical measurement module including a camera capable of measuring a bright-field image and a fluorescent image, disposed on the opposite side of the substrate surface on which the water droplets are dropped, and the water droplets formed on the water-repellent surface can be controlled thereon; An electrostatic field guide for controlling the direction of movement of water droplets, which is one or a plurality of guides for movement, and is charged with a charge opposite to that of the water droplets; A fractionated water droplet reservoir disposed in the subsequent stage of each of the plurality of water droplet movement direction control electrostatic field guides; An electric field switching mechanism for electrostatic field guide for controlling the direction of movement of water droplets
  • a plurality of electrostatic field guides for controlling the direction of movement of the water droplets are provided, at least one of which is for improving the initial water droplet positioning accuracy, and the other guides include water droplets containing target cells and water droplets not containing them.
  • the guide for improving the initial water droplet positioning accuracy is located upstream of the movement of the water droplet, and the guide for selecting the water droplet containing the target cell and the water droplet not containing the target cell is located downstream thereof.
  • the imaging cell sorter described in the above.
  • a metastasis cancer treatment apparatus having a cell mass (cluster) removal mechanism including a membrane filter that removes a cell mass (cluster) having a cross-sectional area of about 250 ⁇ m 2 or more that is not present in healthy blood, the blood of a patient
  • a metastatic cancer treatment device that suppresses the onset and progression of metastatic cancer by effectively removing the metastatic cancer cells in the blood by a physical technique.
  • An on-chip cell sorter system according to any one of the above [1] to [7] used in a diagnostic method for The above diagnostic method is This cell cluster is identified from the selected cell mass by culture, gene mutation test, or expression analysis test, When the identified cell is a metastatic cancer cell in the blood, the gene mutation is confirmed in each cluster unit, and when each cluster is the same mutant, the degree of progression of cancer is If the metastasis from primary cancer is presumed to be early, or if the history of mutations in each cluster is the same but there are many different mutation points, the location of the metastatic cancer Is estimated to be in many areas, If the cell cluster that flows in the blood can be identified as an organ tissue fragment, organ disease is presumed, or for phagocytic leukocytes including macrophages
  • a cell cluster diagnostic method for metastatic cancer, organ abnormality, or infectious disease using detection of a cell mass in blood In a blood sample derived from a subject, a cell cluster (cluster) having an area of 250 ⁇ m 2 or more is selected, This cell cluster is identified from the selected cell mass by culture, gene mutation test, or expression analysis test, When the identified cell is a metastatic cancer cell in the blood, the gene mutation is confirmed in each cluster unit, and when each cluster is the same mutant, the degree of progression of cancer is If the metastasis from primary cancer is presumed to be early, or if the history of mutations in each cluster is the same but there are many different mutation points, the location of the metastatic cancer Is estimated to be in many areas, If the cell cluster that flows in the blood can be identified as an organ tissue fragment, organ disease is presumed, or for phagocytic leukocytes including macrophages, cells larger in size than normal are collected, and By examining and identifying genes of heterologous cells such as bacteria, selectively recovering those whose
  • a method for confirming the presence of abnormal cells in blood Measuring a cell size in a blood sample from a subject containing blood cells excluding red blood cells, Based on the result of the measurement step, a step of obtaining a cell size distribution spectrum of the blood sample, and whether the cell size distribution spectrum has one peak or two or more peaks in the cell size distribution spectrum.
  • a method comprising the step of identifying.
  • a step of selectively recovering cells larger than the threshold from the blood sample with the peak on the large cell side as a threshold when it is identified that there are two or more peaks as a result of the identification step The method according to [22], further comprising: [24] The above [22] or [23], wherein in the step of measuring the cell size and the step of collecting the cells, an on-chip cell sorter chip having a flow path for flowing the blood sample is used. The method described in 1. [25] The method according to [24] above, wherein the on-chip cell sorter chip is part of the on-chip cell sorter system according to any one of [1] to [7].
  • a water droplet formation module comprising a sample solution reservoir for holding a sample solution containing cells and a thin tube connected to the reservoir; A substrate having a water-repellent surface formed by joining regions having two or more different inclination angles for dropping the formed water droplets; A substrate tilt control mechanism for adjusting a tilt angle of a surface having two or more different tilt angles of the substrate; A measurement module for measuring the state of the water droplet and the state inside the water droplet; Means for separating water droplets in a plurality of traveling directions based on the measurement results; An imaging cell sorter for observing and separating cells in water droplets.
  • the means for separating the water droplets in a plurality of traveling directions includes one or more guides for controllably moving the water droplets formed on the water repellent surface,
  • the present invention it is possible to purify a minute amount of target cells in blood in units of one cell, and to analyze accurate gene information and expression information of the target cells.
  • the present invention it is possible to identify whether or not the cells to be examined are clustered (whether or not they are isolated single cells).
  • only the target cells can be separated and purified and collected in real time.
  • the present invention it is possible to measure the intracellular state of only the collected cells at the single cell level, and perform genome analysis and expression analysis at the single cell level.
  • the collected cells can be re-cultured.
  • cell information such as a difference in cell size, a size ratio between the nucleus inside the cell and the cytoplasm, etc. can be acquired, and the cell can be purified by discrimination based on the result.
  • the present invention by collecting cells that are dividing in the blood, it is possible to collect cells having a dividing ability such as blood cancer cells and stem cells.
  • the present invention makes it possible to effectively recover multinucleated cells and cell clusters that are candidates for cancer cells circulating in the blood. According to the present invention, it becomes possible to simultaneously excite cells labeled with fluorescent antibodies of multiple wavelengths with excitation light of multiple wavelengths and simultaneously detect the emitted multiple fluorescences, and effectively recover target cells. It becomes possible. According to the present invention, cancer cells and diseased organ tissue sections can be identified and recovered quantitatively from image data from blood cells. According to the present invention, metastatic cancer cell clusters can be removed from blood cells to prevent the progression of metastatic cancer.
  • FIG. 1 It is a schematic diagram which shows notionally an example of the means in the apparatus corresponding to the whole process of the cell analysis performed using the cell analyzer apparatus of this invention, and each process. It is a figure which shows typically one example of the whole structure of the cell analyzer system of this invention in FIG. It is a figure which shows typically an example of a structure of the cell concentration / staining / decolorization module in FIG. It is a figure which shows typically an example of a structure of the image detection type 1-cell separation and refinement
  • FIG. 7 It is a schematic diagram which shows notionally an example of the means in the apparatus corresponding to the whole process in the case of including the cell destruction process in the cell analysis performed using the cell analyzer apparatus of this invention, and each process. It is a figure which shows typically an example of the crushing mechanism comprised in the cell destruction process in the process shown in FIG. 7 comprised from a container, a rotary body, and a rotating shaft. It is a figure which shows typically the various variations of the basic cell crushing mechanism shown in FIG. The mechanism which ensures the adhesiveness of a container and a rotary body is illustrated. It is a schematic diagram which shows the example of the various shapes of the rotary body of a cell crushing mechanism used in this invention, and a rotating shaft.
  • FIG. 16 is a diagram schematically showing an example of a cell purification process in the image detection type 1-cell separation / purification (cell sorter) module of FIG. 15.
  • FIG. 19 shows typically the image recognition of the disappearance process of the nucleus at the time of the cell division which is one of the identification indexes of the cell purification in the image detection type 1-cell separation / purification (cell sorter) module of FIG. It is a figure which shows typically an example of the light emission timing of the high-speed flash light source for preventing the image blurring in an image detection type 1 cell separation and refinement
  • FIG. 19B shows typically an example of a structure of the optical system for preventing the image blurring in an image detection type
  • FIG. 19A shows typically an example of a structure of the optical system for preventing the image blurring in an image detection type
  • FIG. 19A shows typically an example of a structure of the optical system for preventing the image blurring in an image detection type
  • FIG. 19A shows typically an example of a structure of the optical system for preventing the image blurring in an image detection type
  • FIG. 19A shows typically an example of a structure of the optical system for preventing the image blurring in an image detection type
  • FIG. 19 shows a comparison between an image of fine particles observed with a conventional optical system (FIG. 19B) and an example of an image of fine particles observed with an optical system of the present invention (FIG. 19C).
  • FIG. 19B shows typically an example of the structure which has arrange
  • FIG. 19C shows typically an example of the structure which has arrange
  • FIG. 19 shows typically an example of a structure of the part where the sample solution and buffer solution of a cell sorter chip merge.
  • FIG. 1 It is a figure which shows typically an example of a structure of the chip
  • FIG. 4 is a schematic diagram showing an example of a process for identifying cardiomyocytes and fibroblasts to be separated by an image in an image recognition type cell sorter system. It is a schematic diagram which shows an example of a structure of the cell sorter system which combined water and oil. It is a schematic diagram which shows an example of a structure of the joint area
  • FIG. 41A is a distribution diagram of the area of cell nuclei when blood cancer cells are measured by the apparatus of the present invention, and a bright field photograph and a fluorescence photograph of cells in each distribution region.
  • FIG. 41B is a graph showing the ratio of the peripheral length of cells (clusters) measured by the apparatus of the present invention and the peripheral length derived by circular approximation from the area of the cells (clusters) when blood cancer cells are measured. .
  • FIG. 43B and 43C and FIG. 43C are examples showing an example of image acquisition of a plurality of images captured by the camera shown in FIG. 43B.
  • FIG. 44A is a diagram schematically illustrating an example of a configuration of an imaging cell sorter that observes and separates cells in water droplets. 44B is a diagram of the configuration of the chip illustrated in FIG. 44A as viewed from above.
  • Figure (A) schematically showing an example of a process for detecting and analyzing a cell cluster in human blood, and an example of an integrated measurement analysis system for realizing the process shown in Figure 45A It is the figure (B and C) shown typically.
  • Figure (A) schematically showing an example of a process for detecting and analyzing a cell cluster in human blood, and an example of an integrated measurement analysis system for realizing the process shown in Figure 45A It is the figure (B and C) shown typically. It is the figure which showed typically an example of the method of removing the cell lump (cluster) in the blood beyond a fixed size as a metastatic cancer treatment technique.
  • the distribution spectrum of blood cell size is determined for each of normal blood (FIG.
  • FIG. 47A schematically shows a method using a substrate having an acceleration region and a constant velocity moving region by having two inclination angles in the means for moving the water droplet dropped on the water repellent substrate described above with reference to FIG. FIG.
  • the cell analyzer of the present invention generally comprises: (1) Cell concentration / staining / decolorization part that continuously performs processes including cell concentration, fluorescent antibody labeling (or staining and washing with reversible fluorescent labeling markers such as aptamers if necessary) , (2) Obtain image data of about 10,000 images of cells per second from cells flowing through the microchannel formed on the chip substrate, and purify 10,000 cells per second in real time based on the analysis result of the image information An image detection type 1 cell separation / purification (cell sorter) unit, (3) a 1-cell genome analysis / expression analysis unit that measures the intracellular state at the 1-cell level; (4) a liquid feeding part for carrying the sample liquid between the above parts, (5) A control analysis unit for controlling the operation of each unit and performing the analysis.
  • a typical embodiment of the cell analyzer of the present invention is characterized in that the three modules (1) to (3) are continuously combined in the order described above, and the cells are continuously connected by the flow path. Therefore, a small amount of cells can be eliminated by contamination or manipulation.
  • the presence or absence of fluorescent labeling of the cells is detected and confirmed at the single cell level, and the fluorescence-labeled cells are confirmed to be isolated single cells that are not clustered. In addition, it can be determined whether apoptosis is occurring in the cells. Therefore, according to the cell analyzer of the present invention, cells can be separated and purified based on an index that cannot be identified by the conventional scattered light detection type cell sorter technique.
  • cells that are stained accurately in units of 1 cell are selectively collected, and cell states such as apoptosis of the cells to be collected are confirmed, and fluorescence information and cell state information of each cell are confirmed.
  • cell genetic information and expression information can be analyzed.
  • the cell concentration / staining / decoloring part (1) above a small amount of cells contained in the reaction solution continuously sent from the previous module by the non-contact force is continuously captured and concentrated, and a certain number of cells is obtained.
  • the cell label staining solution is introduced and the cells are stained, the unbound reagent is washed away, and then the cells are delivered to the next module at a constant concentration.
  • cell capture / concentration using the feature that cells gather by “dielectrophoretic force” created by an alternating electric field by a metal electrode created in a microchannel as a non-contact force
  • the means for performing separation / purification in units of one cell based on the result of the image detection of (2) above details such as the difference in cell size, the ratio of the nucleus inside the cell to the cytoplasm, etc.
  • Cell information is acquired as image information, and the cell is purified based on the result.
  • a high-speed camera is used, the light emission of the light source is adjusted in accordance with the shutter cycle of the high-speed camera, and light from the light source is emitted for a certain period of time during which each shutter is released.
  • the shutter speed is 1 / 10,000th of a second
  • illuminate the target cells with a light source that can be controlled at high speed, such as an LED light source or a pulsed laser light source, for a period of 1 / 10th of the shutter speed.
  • a light source that can be controlled at high speed, such as an LED light source or a pulsed laser light source, for a period of 1 / 10th of the shutter speed.
  • the present invention makes it possible to completely prevent cross-contamination of the device by making the main part of the cell sorter into a chip, and to separate cells without cross-contamination essential in the medical field, particularly in the field of regenerative medicine. Provide a system.
  • the cells assumed as detection targets in the present invention are bacteria for small ones and animal cells (for example, cancer cells) for large ones. Therefore, the cell size typically ranges from about 0.5 ⁇ m to about 30 ⁇ m.
  • the first problem is the channel width (cross-sectional shape).
  • the channel is formed in a substantially two-dimensional plane using a space of about 10 to about 100 ⁇ m in the thickness direction of the substrate on one of the substrate surfaces. In terms of cell size, the most typical size is about 5 to about 10 ⁇ m in the thickness direction for bacteria, and about 10 to about 50 ⁇ m in the thickness direction for animal cells.
  • the cell analyzer of the present invention typically comprises a cell concentrating unit having a function of concentrating cells, a cell arrangement unit having a function of separating and purifying cells, and a cell separation / purification in the same chip. And an optical analysis unit for identifying and judging cells to be separated and purified.
  • a sample solution that has not been subjected to concentration treatment is introduced into the cell concentration section from one inlet, and the sample solution is discharged from a discharge section disposed downstream of the cell concentration section.
  • ultrasonic radiation pressure, gravity, electrostatic force, dielectric electrophoretic force and the like can be used, but are not limited thereto.
  • an arrangement is used in which these external forces can be applied in a direction perpendicular to the flow of the sample solution in the concentration section and in the direction of the concentrated cell recovery port.
  • all cells should flow in one of the two channels that are branched into two downstreams by applying external force to the cells so that the cells are arranged in the center of the channel where the cells are flowing. Subsequently, by applying an external force to only the cells to be collected out of the arranged cells and moving the flow position of the cells, the external force is applied to the flow path branched into the above two. Only when the cells are introduced into another channel.
  • the external force means for arranging cells into nodes of standing waves by ultrasonic radiation pressure can be used.
  • a means for arranging cells at the position of the apex of the wedge can be used by combining wedge-shaped electrode arrays.
  • the cell detection function of the cell analyzer of the present invention resides in the image detection type 1-cell separation / purification part (2) above.
  • a part to be observed by a CCD camera is provided upstream of the flow path branching part, and a cell separation region is provided downstream of the part if necessary.
  • the cells passing through the flow path are irradiated with a laser or the like, and when the cells cross and the scattered light or the cells are modified with fluorescence, the fluorescence can be detected with a photodetector.
  • a separation channel point that becomes a cell separation region is installed downstream of the detection unit.
  • the sorting unit which is a cell separation region
  • a pair of comb-shaped electrodes is used as a means for moving the cells by applying external force to the cells from the outside, for example, when using a dielectrophoretic force.
  • a pair of comb-shaped electrodes is used. Install and provide a flow path that can separate and drain cells.
  • electrostatic force a voltage is applied to the electrode to change the position of the cell in the flow path. At this time, since the cell is generally charged negatively, it moves toward the positive electrode.
  • the waste liquid outlet (outlet 213) of the cell concentrating unit 215 and the purified cell of the cell sorting unit 217 It is desirable that the pressure at the outlet (cell recovery unit 224) and the outlet of the waste liquid of the cell sorting unit (waste liquid recovery unit 223) be substantially the same (see FIG. 4B).
  • a flow path resistance adjusting portion for pressure adjustment is arranged immediately before each outlet such as a thin flow path or a long S-shaped flow path.
  • the cell recognition and separation algorithm has the following characteristics.
  • the cell image is first binarized and its center of gravity is obtained.
  • the luminance center of gravity, area, perimeter length, major axis, minor axis of the binarized cells are obtained, and each cell is numbered using these parameters. It is possible to automatically save each cell image as an image at this point because it is beneficial to the user.
  • the separation index may be information such as the luminance center of gravity, area, circumference length, major axis, minor axis, or the like, or information using fluorescence may be obtained by using fluorescence detection separately from the image. In any case, the cells obtained by the detection unit are separated according to the numbering.
  • the movement speed (V) of the numbered cells is calculated from the image captured every predetermined time, and the distance from the detection unit to the selection unit with respect to the cell movement speed (V) (L),
  • the application timing from (L / V) to (L / V + T) depending on the application time (T) the cells are electrically distributed and separated when the cells of the target number come between the electrodes.
  • the means for high-speed single-cell genome analysis / expression analysis of (3) used in the present invention for example, the reaction control device used, For a plurality of temperatures to be changed, means for changing a plurality of liquids having different heat capacities at a high speed at high speed using a liquid having a large heat capacity maintained at each temperature as a heat exchange medium, and a liquid having a large heat capacity And a microreaction tank in which heat exchange with the sample liquid is performed quickly.
  • the reaction control device used in the present invention includes a reaction vessel that has a structure and material suitable for heat exchange, and a reaction that circulates a liquid having a temperature suitable for each reaction outside the reaction vessel.
  • Liquid is transferred from any liquid reservoir tank to the outside of the reaction tank to rapidly change the temperature of the tank heat exchange tank, heat sources that maintain the temperature of the liquid with high accuracy, and the temperature of the micro reaction tank And a mechanism for preventing the mixing of liquids at different temperatures when the valve system is switched.
  • Advantages of controlling the temperature of the reaction vessel with the refluxing liquid include the following points. First, the temperature overshoot problem can be solved. Since the temperature of the liquid that is constantly refluxing is constant, the temperature of the reaction vessel surface and the temperature of the liquid are instantaneously equilibrated. Since the heat capacity of the reaction vessel and the sample is insignificant compared to the liquid being refluxed, the liquid flows continuously even if heat is locally deprived from the liquid. Basically does not occur. Of course, the temperature of the reaction vessel does not exceed the temperature of the liquid. It is possible to change the temperature by 30 degrees or more within 0.5 seconds by sequentially pouring liquids of different temperatures into the reaction tank heat exchange tank.
  • FIG. 1 illustrates an example of a procedure from collection of a blood sample to analysis performed using the cell analyzer of the present invention.
  • the blood sample collected from the patient is directly introduced into the cell concentration / staining part.
  • a fluorescent labeling agent such as a fluorescent cancer marker
  • the excess fluorescent labeling agent that did not react is washed away.
  • the cells are introduced into the image detection type 1-cell separation and purification unit in a form adjusted to the optimum cell concentration and solution for the next image detection type 1-cell separation and purification unit.
  • the primary detection the presence or absence of fluorescence emission based on the fluorescent label at the 1-cell level is confirmed. Thereby, it can be confirmed by a conventional labeling technique whether the cell is a target cell.
  • the cells emitting fluorescence are isolated cells or become cell clusters with other cells 2) Determine whether the cells emitting fluorescence are in a healthy state or in a state such as apoptosis in which the cell nucleus and cell shape are deformed, and depending on the purpose, Recover healthy cells or recover cells that are undergoing apoptosis, and perform gene analysis and expression analysis on the next stage so that gene analysis and expression analysis can be performed separately for each cell morphology. Can be introduced into the department. In particular, in the case of a cell mass, since cells other than the target cell are mixed, collection is not performed even when there are cells that emit fluorescence.
  • the cells identified and purified at this stage can be re-cultured in a contamination-free manner in purified cells, in addition to being introduced into the gene analysis / expression analysis section.
  • the gene analysis / expression analysis unit converts the introduced cells into a small amount of cells, as one cell that is identified as the same cell based on information from the image detection type 1 cell separation and purification unit, or a group of the same cells Gene identification or expression identification is performed in units.
  • FIG. 2 shows an example of an overall image of the cell analyzer system 1 that realizes the procedure shown in FIG.
  • the apparatus system 1 includes a concentration / staining / decoloring module 10 that introduces a blood sample and pretreats cells, an image detection type 1-cell separation / purification module 20 that identifies and purifies cells in units of cells, and is purified.
  • 1-cell genome analysis / expression analysis module 30 for performing gene analysis and expression analysis of the collected cells
  • liquid feeding module 40 for transporting samples between modules, and controlling the operation of the entire system and analyzing the analysis results
  • a control / analysis module (computer) 50 is provided.
  • 3 to 6 show an example of the configuration of each module in the example shown in FIG.
  • FIG. 3 shows an example of the configuration of the cell concentration / staining / decoloring module 10 that introduces a blood sample derived from a subject (eg, cancer patient) and pretreats the cells.
  • the cell concentration / staining / decoloring module 10 is integrally disposed on the chassis 114, and each solution of the sample cell sample, the staining agent, and the cleaning agent is held in the module.
  • a sample cell sample such as blood is introduced into the concentration chamber 108, and the liquid component is discharged through the filter to the waste liquid collection tube 110 by the pressure pump 109, thereby concentrating the cells.
  • the dyeing solution is introduced using the dispensing head 104 and reacted for a predetermined time, and then the dyeing solution is discharged again by the pressure pump 109.
  • a decolorizing agent into the concentration chamber 108, excess dyeing agent is washed and discharged.
  • a diluent also serving as a cleaning agent is introduced to dilute the cells to a desired concentration, and the cells are introduced into the collection tube 112 through the collection head 111 having the collection tip 113 at the tip. Yes.
  • FIG. 4 shows an example of the configuration of the image detection type 1-cell separation / purification module 20 for identifying and purifying cells in units of one cell.
  • the image detection type 1-cell separation / purification module 20 includes a light source 201, a mirror 202, a condensing lens 203, a dichroic mirror 204, a filter 205, a light detection element 206 for fluorescence detection, a high-speed camera 207, And a cell sorter chip 209 for introducing a cell sample.
  • a light source 201 such as a pulse laser or a high-intensity LED light source
  • a light detection element 208 such as a photodiode that detects passage of cells with scattered light, and fluorescence are detected for cells passing through the cell sorter chip 209.
  • a plurality of pieces of information can be detected simultaneously by a high-sensitivity light detection element 206 such as a photomultiplier, a high-speed camera 207, or the like.
  • the light emitted from the light source continuous light may be emitted, but in order to increase the spatial resolution of the image without blurring, pulse light is generated in synchronization with the shutter cycle of the high-speed camera 207.
  • the processing using an image and the processing using fluorescence or scattered light may be used in combination.
  • the image data obtained by the high-speed camera 207 can be displayed on the monitor of the computer 50 for use by the user.
  • the filter 205 is appropriately adjusted to transmit a plurality of excitation lights, and a wavelength that does not overlap with the fluorescence wavelength for fluorescence detection in the lower stage is selected to light the cells.
  • a plurality of devices to which devices from the dichroic mirror 204 to the filter 205 and the fluorescence detector 206 are added in accordance with the type of fluorescence to be observed.
  • the cell recognition and separation algorithm has the following characteristics.
  • the cell image is first binarized and its center of gravity is obtained.
  • the luminance center of gravity, area, perimeter length, major axis, minor axis of the binarized cells are obtained, and each cell is numbered using these parameters. It is possible to automatically save each cell image as an image at this point because it is beneficial to the user.
  • the separation index may be information such as the luminance center of gravity, area, circumference length, major axis, minor axis, or the like, or information using fluorescence may be obtained by using fluorescence detection separately from the image. In any case, the cells obtained by the detection unit are separated according to the numbering.
  • the movement speed (V) of the numbered cells is calculated from the image captured every predetermined time, and the distance from the detection unit to the selection unit with respect to the cell movement speed (V) (L),
  • the application timing from (L / V) to (L / V + T) depending on the application time (T) the cells are electrically distributed and separated when the cells of the target number come between the electrodes.
  • An example of the configuration for cell separation and purification is as follows. A series of microfabricated channels arranged in a two-dimensional manner on a planar chip, from concentration to arrangement and purification of cells in the sample solution, and force applied to the cells incorporated in the chip Consists of means.
  • FIG. 4B schematically shows an example of a cell sorter chip 209 configured on such a chip.
  • a microchannel 211 is provided inside the chip substrate 210, and an opening communicating with the channel is provided on the upper surface to serve as a supply port for a sample and a necessary buffer solution (medium).
  • the flow path can be created by so-called injection molding in which a plastic such as PMMA is poured into a mold, or can be created by bonding a plurality of glass substrates.
  • the size of the chip is, for example, 50 ⁇ 70 ⁇ 1 mm (t), but is not limited thereto.
  • the cells envisaged in the present invention are bacteria at a small size, and animal cells at a large size, such as cancer cells. Therefore, the cell size is typically in the range of about 0.5 ⁇ m to 30 ⁇ m, but is not strictly limited to this range, and cells of any size can be used as long as the present invention is effectively used. Can be done.
  • the first problem is the flow channel width (cross-sectional shape).
  • the channel 211 is formed on one of the substrate surfaces in a substantially two-dimensional plane in a space typically 10 to 100 ⁇ m in the thickness direction of the substrate. The appropriate size is 5 to 10 ⁇ m in the thickness direction for bacteria and 10 to 50 ⁇ m in the thickness direction for animal cells based on the cell size.
  • the sample solution is introduced from the inlet 212 into the microchannel 211 by a syringe pump or cell introduction means that does not generate a pulsating flow such as air pressure.
  • the sample liquid containing the cells introduced into the microchannel 211 flows along the streamline of the particle flow 218 before application toward the outlet 213 downstream, and is discharged.
  • means for continuously applying an external force to the cells is introduced so that the cells are concentrated toward the cell concentrate inlet 214 arranged in a part of the side wall of the microchannel 211.
  • the cells are concentrated along the cell flow 219 after application, and a cell concentrate having a concentration 100 times or more the cell concentration introduced at the inlet 212 is introduced into the cell concentrate inlet 214.
  • ultrasonic radiation pressure gravity, electrostatic force, dielectric electrophoretic force
  • a traveling wave of ultrasonic waves is generated in the direction of the cell concentrate inlet 214 and orthogonal to the flow of the sample liquid, and is applied by the ultrasonic radiation pressure after application.
  • a cell stream 219 can be generated.
  • a PZT-based piezoelectric element may be adhered to the surface of the chip 209, or a comb-shaped electrode array is disposed on the surface of the piezoelectric element so that surface acoustic waves are generated in the cell concentration unit 215 This may be applied to the surface of the cell concentrating unit 215, and the fact that the soaked ultrasonic waves are introduced into the cell concentrating unit 215 may be used.
  • the spatial arrangement of the chip 209 may be adjusted so that the direction of the concentrated liquid inlet 214 is perpendicular to the flow of the sample liquid and the direction of the concentrated liquid inlet 214 is the direction of gravity, or
  • the chip 209 may be arranged on a disc that can rotate, perpendicular to the flow of the sample solution, and the cell concentrate in the same direction as the radial direction of the disc.
  • electrostatic force an electrode is arranged on the side wall of the microchannel 211 so that the cell receives an external force toward the side wall. In that case, the cell of the target cell It may be determined which charge is applied depending on whether the surface potential is positive or negative.
  • the flow path distance of the micro flow path 211 must be flexibly adjusted according to the type and strength of the external force applied to the cell, for example, sufficiently long in the case of electrostatic force It must be a thing.
  • the dielectrophoretic force is used as an external force, the dielectrophoretic force is applied in the cell concentration unit 215 so as to be orthogonal to the flow of the sample liquid and in the direction of the concentrate inlet 214.
  • An electrode may be disposed.
  • the concentrated cell liquid into which the cell concentrated liquid has been introduced into the concentrated liquid inlet 214 is arranged in a line along the flow in the solution at the converging unit 216. Specifically, it has means for generating an external force so that cells are attracted to the central part of the flow path of the converging unit 216 by using a dielectric electrophoretic force or a standing wave mode of ultrasonic radiation pressure. is there.
  • the cells arranged in a straight line in the center in this way are measured in the cell detection region 218 arranged in the preceding stage of the cell sorting unit 217, and after determining the type of each cell, from upstream to downstream.
  • the wedge-shaped electrodes (converging V-shaped comb electrodes) 225 are alternately arranged. And applying an alternating voltage to the converging V-shaped comb electrode contact allows the cell to be directed toward the position of the wedge-shaped apex, thereby applying an external force to the cell. As a result, the position of the wedge-shaped apex Cells can be continuously concentrated.
  • the shape of the electrode arranged in the flow path that the electrode has an angle toward the downstream side, and that this electrode has a sharp tip instead of a straight line, and Because of the comb-shaped electrode array that has the shape of the axis, the cell that receives the dielectric electrophoretic force is affected by the flow regardless of whether it receives a repulsive force or an attractive force.
  • the cells are guided and arranged at the acute electrode tip portion by the resultant force of the force swept downstream and the force applied to the cell toward the sharp tip portion.
  • the cell is forced to flow downstream by the flow and the dielectric electrophoresis directed toward this acute angle tip direction.
  • the resultant force and force gather at the acute angle tip.
  • FIG. 5 illustrates an example of the configuration of a one-cell genome analysis / expression analysis module 30 that performs gene analysis and expression analysis of purified cells.
  • the reaction tank 301 is composed of a thin plate of aluminum, nickel, or gold having a plurality of depressions. The thickness of the thin plate in the depression region is about 10 to 30 microns, and the region between adjacent depressions has a thickness of 100 to 500 microns in order to ensure overall strength.
  • the reaction vessel 301 is fixed to the bottom surface of a square or circular reaction vessel frame, and has a structure that can be easily detached from the reaction vessel heat exchange vessel 302. The temperature of the liquid introduced into the reaction tank heat exchange tank 302 is overheated by a heat source disposed inside the liquid reservoir tank 303.
  • An agitation mechanism is provided to quickly remove heat from the surface of the heat source and to make the temperature inside the liquid reservoir tank 303 uniform.
  • the liquid in the liquid reservoir tank is guided inside the flow path by the pump 304.
  • the liquid is led to the reaction tank heat exchange tank 302 by the switching valve 305 or directly returned to the liquid reservoir tank 303 by being led to the bypass flow path. If necessary, the temperature of the liquid is slightly controlled by the auxiliary temperature control mechanism 306 so as to suppress temperature fluctuation in the liquid reservoir tank 303.
  • the basic configuration of the reaction tank heat exchange tank 302 includes an inlet A (307) and an inlet B (308) for introducing liquids having different temperatures.
  • the number corresponding to the plurality of temperatures for which the temperature of the sample solution is to be changed will be prepared as a plurality of two temperatures or more.
  • the number is three. It is not limited to two.
  • a plurality of outlets, outlet A (309) and outlet B (310) are provided in order to return the liquid in the reaction tank heat exchange tank 302 to the liquid reservoir tank 303.
  • the number is not limited to two.
  • Various types of reaction vessels can be used, and reaction vessel A, reaction vessel B, reaction vessel C, and reaction vessel D are shown as an example.
  • water may be used, but a liquid having a large heat capacity and low viscosity may be used.
  • liquid ammonia For example, liquid ammonia.
  • the sample liquid is surely set to 100 degrees, or the liquid having a lower freezing point than water is used. It is also possible to reliably change the temperature up to the freezing point of water while preventing freezing of the circulating liquid.
  • the reaction vessel frame can measure the change in fluorescence intensity of the fluorescent dye in the sample solution, which changes due to the reaction of the sample solution 311 in the reaction vessel 301, for each of the one or more reaction vessels 301.
  • An optical window that transmits the excitation light of the fluorescent dye and the fluorescence is arranged, and the fluorescence detector 312 is arranged to measure the temporal change of the measured fluorescence intensity of each reaction vessel 301. it can.
  • each of the plurality of fluorescence detectors 312 includes an excitation light irradiation mechanism and a fluorescence detection mechanism, and each of a plurality of reaction vessels 301 to which different primers or different sample solutions are dropped.
  • the fluorescence intensity data acquired by the fluorescence detector 312 is recorded by the control analysis unit 313 and has a function of estimating the amount of DNA or mRNA in the sample solution obtained by the PCR reaction. Further, the control analysis unit 313 obtains the switching information of the switching valve 305, thereby estimating whether the temperature change of the sample liquid 311 after the valve switching has reached the target temperature from the change in fluorescence intensity over time, And a mechanism for controlling valve switching based on the result.
  • one detector is arranged in each reaction vessel 301.
  • the fluorescence intensity change in a plurality of reaction vessels can be changed by combining a fluorescent example light source and a camera capable of quantitative fluorescence detection such as a cooled CCD camera. You may measure.
  • the fluorescence intensity of all reaction vessels can be measured by combining a mechanical drive mechanism that can move at high speed on the XY plane with the detectors. You may do it.
  • a freeze-dried reagent it is convenient to freeze and dry the reagents necessary for the reaction. It is possible to prepare a freeze-dried reagent at the bottom of the reaction vessel. Further, if a plug-like freeze-dried reagent is formed inside a dispensing tip used when dispensing a sample, the reagent can be dissolved in the sample by moving the sample up and down. Alternatively, it is also possible to dissolve the freeze-dried reagent by forming a freeze-dried reagent on the surface of the fiber ball on which nylon fibers or the like are bundled, and inserting the sample into a sample inside the reaction vessel and stirring.
  • the reaction vessel frame is preferably formed of a heat-insulating material such as polystyrene, polycarbonate, PEEK, acrylic, and the like, and the reaction area of the reaction vessel 301 can be controlled quickly and with high accuracy. Is desirable.
  • a thread is formed on the surface of the reaction tank frame and the reaction tank frame is screwed. In order to maintain watertightness, it is desirable to attach a seal to the opening.
  • valve switching mechanism There are an inlet valve A and an inlet valve B for introducing a liquid into the reaction tank 301, and an outlet valve A and an outlet valve B for introducing the liquid to the outside.
  • the liquid guided from the inlet valve A returns from the outlet valve A to the liquid reservoir tank, and the liquid guided from the inlet valve A returns from the outlet bawl B to another liquid reservoir tank.
  • the sample in the reaction vessel can be reacted.
  • the inlet valve B and the outlet valve A, or the inlet valve A and the outlet valve B are simultaneously opened for a moment so that liquids of different temperatures are mixed.
  • the conditions for PCR are, for example, reaction buffer 1.0 ⁇ L, 2 mM dNTP (dATP, dCTP, dGTP, dTTP) 1 ⁇ L, 25 mM magnesium sulfate 1.2 ⁇ L, 10% fetal bovine serum 0.125 ⁇ L, SYBR Green I 0.5 ⁇ L, primer Two types can be used: 0.6 ⁇ L each, sterilized water 3.725 ⁇ L, KOD plus polymerase 0.25 ⁇ L, and genomic DNA 1.0 ⁇ L.
  • As the temperature condition first, 95 ° C. for 10 seconds, and then temperature change of 95 ° C. for 1 second and 60 ° C. for 3 seconds can be measured in 40 cycles.
  • FIG. 6 illustrates an example of a configuration of a liquid feeding module 40 that transports a sample between the modules. It has a dispensing head 401 and a dispensing tip 402 for exchanging liquid between each module arranged on the chassis 406, and controls the height direction of the dispensing head in the Z-axis direction.
  • the Z-axis movement guide 403 and the Z-axis movement motor 404 as functions and the arm rotation motor 405 as an arm rotation mechanism have a function of controlling the position of the dispensing head 401 on the XY plane.
  • FIG. 7 shows cell expression of a sample in which a nucleic acid component in a cell is not easily eluted into a sample solution by a shell covering a cell such as an anthrax spore in a cell analysis performed using the cell analyzer of the present invention.
  • An example of a procedure from collection of a sample into which a procedure for crushing a shell covering a cell is introduced before an analysis procedure is shown.
  • the present cell analyzer can analyze cells such as spores of Bacillus anthracis by means similar to the means for analyzing blood cells described above.
  • FIG. 8 schematically shows an example of a basic structure for automatically crushing a shell of a spore or the like covering a small amount of sample cells in order to analyze intracellular genes and expression information for cells having spores such as anthrax.
  • a minute sample 802 is dispensed into the container 801, and a rotating body 803 for crushing is placed inside the container 801.
  • the rotating body 803 is pressed against the container 801 by the rotating shaft 804.
  • the sample 805 in the minute sample is ground by the abrasive 806.
  • the processed sample 805 can be easily recovered by removing the rotating body 803. Since the rotating body 803 and the container 1 have a simple structure, there is no problem even if they are handled as consumables.
  • FIG. 9 schematically shows various variations of the basic cell disruption mechanism shown in FIG.
  • the container 811 including the rotating body 810 is held by a flexible structure 812 such as rubber. Since the tip portion 814 of the shaft 813 is cut obliquely, when the shaft 813 is pressed against the rotating body 810, the rotating body 810 presses the container 811 downward and laterally, and the flexible structure 812 By deforming, pressure is absorbed. As a result, the sample can be crushed while holding the rotating body 810 and the container 811 closely without giving excessive stress to the rotating shaft 813.
  • FIG. 9B as a method of releasing stress, it is possible to incorporate a spring mechanism 815 that deforms vertically and laterally inside the rotating shaft.
  • FIG. 10 shows the possibility of rotating bodies and rotating shafts of various shapes in the cell disruption mechanism used in the present invention.
  • the shaft whose tip is cut obliquely (FIG. 10a)
  • it may be recessed in a gently curved surface (FIG. 10b), bowl-shaped (FIG. 10c), or the like.
  • the rotating body does not have to be a true sphere, and may have a structure in which the shaft and the rotating body are gently engaged with each other (FIG. 10d).
  • the hemisphere may be rotated with a rotating shaft cut diagonally (FIG. 10e).
  • it may have an egg-shaped rotating body (FIG. 10f) or a protruding structure that meshes with the rotating shaft (FIG. 10g). It is also possible to rotate a dish-like rotating body with a shaft (FIG. 10h).
  • FIG. 11 shows an example of the cell disruption step in the present invention.
  • a rotating body 831 and an abrasive 832 are enclosed in the container 830 (FIG. 11a).
  • the seal 833 is broken (FIG. 11b), and a sample 834 containing cells is dispensed into the container 830 (FIG. 11c).
  • the cells in the sample are crushed by the abrasive 832 and the component 836 is eluted (FIG. 11e).
  • the sample can be easily collected by removing the rotating body 831 from the container 830 (FIG. 11f).
  • negative pressure, magnetic force, and electrostatic force can be used, and such a mechanism can be incorporated in the rotating shaft.
  • a special mechanism may be prepared separately.
  • FIG. 12 shows a conceptual diagram of a mechanism that can be used when the cell disruption process in the present invention is automated.
  • a plurality of containers 840 are integrally formed, and a rotating body is sealed in advance in each container.
  • a rotating shaft In order to break the seal, it is possible to directly press and tear the rotating shaft (FIG. 12A), or to break with the opening cutter 841 attached to the rotating shaft (FIG. 12B).
  • the relative position of the shaft and the container can be automatically changed, and a plurality of samples can be crushed one after another.
  • FIG. 13 shows an example of the on-chip cell sorter chip of the present invention that can also be used in the cell analyzer system of the present invention.
  • the cell sorter chip 1301 three axial flow paths are arranged symmetrically on the upstream side (1302, 1304, 1306) and downstream side (1303, 1305, 1307) on the chip substrate.
  • the three flow paths merge while maintaining a laminar flow, and maintain the state as it is and branch to the three downstream flow paths. Therefore, the upstream side 1302 of the central flow path through which the sample flows is changed to the downstream central flow path 1303, and the upstream side flow path 1304 to the downstream side 1305, and the upstream flow path 1306, respectively, for the two side sheath flows.
  • the inlets of the three upstream channels are connected to inlet openings 1308, 1309, and 1310, respectively.
  • the inlet opening 1308 upstream of the flow path through which the sample flows is connected to the sample reservoir 1322, typically (but not limited to) by adding a small annular cap (or stopper).
  • the inlet openings 1309 and 1310 of the flow path for flowing the sheath liquid stored in the sheath liquid reservoir 1311 are separated from each other and are arranged so that the sample solution does not diffuse.
  • the downstream reservoir is also arranged in the same manner as the upstream side, and the waste liquid reservoir 1312 is connected to the outlet openings 1313 and 1314 of the flow path through which the two side sheath liquids flow. Is connected to the outlet opening 1315 of the recovered purified sample, and typically (but not limited to) a small annular cap is added to the outlet opening 1315 to recover the recovered purified sample. Does not diffuse into the waste reservoir.
  • the flow rate is generated using a gravity type that utilizes the difference between the liquid level of the sample reservoir and the sheath liquid reservoir and the liquid level of the waste / recovered liquid reservoir, or pressurized air with a cap attached to the upper surface of the reservoir.
  • a gravity type that utilizes the difference between the liquid level of the sample reservoir and the sheath liquid reservoir and the liquid level of the waste / recovered liquid reservoir, or pressurized air with a cap attached to the upper surface of the reservoir.
  • the ratio of the cross-sectional area of the side sheath flow (or waste liquid) reservoir to the cross-sectional area of the inner sample / recovery sample reservoir is 1 (sample / recovery reservoir): 2 (side sheath liquid reservoir / waste liquid reservoir). It is desirable. This is because if the change in the liquid level of each reservoir is different, the rate of decrease in the liquid level will be different, which will eventually destroy the generation of laminar flow at the confluence. It is. Therefore, since the flow rate of the liquid flow per unit time is 1: 2 in the sheath liquid having two inlets with respect to the sample inlet 1, the cross-sectional area of each reservoir is adjusted so that the liquid level is the same. The ratio was set to 1: 2. To make this universal, it is desirable that the ratio of the total cross-sectional areas of the flow paths coupled to the respective reservoirs matches the ratio of the cross-sectional areas of the respective reservoirs.
  • an electrode is arranged at a point where three laminar flows without walls join, where all six channels join.
  • the electrode is typically composed of a gel electrode.
  • a gel electrode for example, an agarose gel in which NaCl is dissolved so that the electrolyte becomes a current carrier is used.
  • the gel is placed in a Y-shaped channel 1316 for gel filling so that the gel tip can be in contact with the agarose gel in a sol state from the inlet 1317 so that the gel can be directed to the outlet 1318.
  • the gel does not penetrate into the cell sorter flow path and stops at the boundary line due to surface tension.
  • a wire 1319 such as a platinum wire connected to a power source 1320 to apply an electric field to this gel introduction point, at the gel electrode boundary in contact with the flow path, a normal metal electrode Even if the bubbles are raised to a voltage higher than the voltage generated in the flow path, no bubbles are generated and a current can be applied.
  • the on-off of the electric field application can be adjusted using the switch 1321, for example.
  • FIG. 14 schematically shows an example of the cross section of the upstream reservoir, particularly in the AA cross section of FIG.
  • a flow path 1409 is embedded in the cell sorter chip 1401.
  • the upper surface of the outer sheath fluid reservoir 1403 is blocked by the cap 1402, so that air pressure at an appropriate flow rate is supplied from the pressurized air introduction pipe 1405.
  • the flow path 1409 through which the sample flows is connected to the sample reservoir 1404 so that the sample liquid and the sheath liquid are not mixed.
  • the ratio of the cross-sectional area between the sample reservoir and the sheath fluid reservoir is 1: 2 because the ratio of the number of channels is 1: 2, and the ratio of each reservoir connected to each channel is The liquid level is adjusted to be the same.
  • a mechanism for supplying liquid can be added so that a larger amount of sample can be processed.
  • This includes a sample solution introduction tube 1406 or a sheath liquid introduction tube 1407, and a water level measurement sensor 1408 using conductivity measurement on the wall surface of each reservoir.
  • the water level measurement sensor 1408 can be configured by electrodes or electrode pairs disposed at the lower limit of the water level and the upper limit of the water level, which are desired to be set.
  • FIG. 15 shows an example of another configuration for handling a large amount of sample in the cell sorter of the present invention.
  • Three large reservoirs 1502 are arranged on the chip 1501 upstream of each of the three flow paths, and these are distributed more flexibly using the distribution valve 1505 from the air pressure application device 1503 through the pressure sensor 1504. can do.
  • sample collection both the sorted (purified) sample and the waste liquid are collected in the sorted sample collection reservoir 1506 and the waste liquid collection reservoir 1507, respectively, disposed at positions below the chip.
  • FIG. 16 schematically shows a procedure for collecting an actual sample in the chip.
  • the sample solution stream 1601 flowing from the upstream is sandwiched between the two side sheath solution streams 1602 and 1603 and proceeds to the cell monitoring region 1604 while maintaining the arrangement. Therefore, the shape discrimination of each cell, the presence or absence of a fluorescent label, etc. are confirmed, and cell separation is performed downstream based on the results.
  • the cells to be collected flow, they flow as they are to the downstream sorted sample collection flow channel 1606, and when the cells or fine particles to be discarded flow, they are arranged to face each other regardless of whether the charge is positive or negative.
  • the two gel electrodes 1605 by applying a voltage to the two gel electrodes 1605, the gel can move to one of the two side sheath flows 1607 and be eliminated.
  • FIG. 17 is a schematic diagram for explaining one of the indices of cell recovery in the image processing cell sorter.
  • the cell is in the G0 cycle and has a nucleus (FIG. 17A (a)), which is clearly image-recognized as a black sphere inside the cell (FIG. 17B (a)).
  • FIG. 17A (b) since the nucleus of the cell in the division phase has disappeared (FIG. 17A (b)), the nucleus cannot be confirmed even if the cell is image-recognized (FIG. 17B (b)).
  • conventional labeling techniques such as antibody labeling, it is difficult to confirm the state of the cell.
  • the presence or absence of a nucleus in the cell Dividing cells can be recovered.
  • most normal cells flowing in the blood are already terminally differentiated, but by collecting cells that have undergone cell division in the blood according to the present invention, blood cancer cells, stem cells, etc. It becomes possible to collect cells having the ability to divide.
  • FIG. 18 is an example of an operation timing chart when using a flash light source when actually operating the image recognition type cell sorter of the present invention.
  • the pixel size of a 1/2000 second camera is 12 ⁇ m ⁇ 12 ⁇ m
  • the pixel resolution when viewed with a 20 ⁇ objective lens is 0.6 ⁇ m / pixel. If an LED light source that can perform flash firing at a high speed is used, it is possible to actually obtain an image without blurring.
  • the present invention also provides an on-chip cell sorter system that describes exemplary aspects below.
  • control of each part for example, image acquisition and analysis by an optical system, external force addition by an external force application device, etc.
  • a control device including a personal computer or the like as in the above embodiment. be able to.
  • FIG. 19 is a diagram schematically showing an example of the configuration of an optical system for preventing image blur in the image detection type 1-cell separation / purification (cell sorter) module.
  • the magnification ratio of the object image is determined only by the magnification of the objective lens. It depends on the magnification and numerical aperture of the objective lens. The higher the magnification of the objective lens, the shallower the focal depth and depth of field of the optical system.
  • an objective lens and a zoom lens may be combined and this magnified image may be captured by an image acquisition device such as a CCD camera.
  • the image can be obtained up to about 15 ⁇ m without any problem. You can get that.
  • an objective lens with a numerical aperture of 20 times 20 times and an objective lens with a numerical aperture of 0.6 and magnification of 40 times an image with a blur of only about 5 ⁇ m can be acquired.
  • the image is a combination of an objective lens 10 ⁇ and a zoom lens 1 ⁇ combined image, an objective lens 10 ⁇ combined with a zoom lens 2 ⁇ image, and an objective lens 10 ⁇ combined with a zoom lens 4 ⁇ image. It can be seen that when the depth and the depth of field are combined and the image is not blurred, the image can be sufficiently observed even when the magnification of the zoom lens is different up to 25 ⁇ m.
  • This result is obtained by combining a 10 ⁇ objective lens with a 4 ⁇ zoom lens when an image processing cell sorter system obtains an image with the same magnification as that conventionally observed with a 40 ⁇ objective lens.
  • An example of a configuration that can obtain an image that is optimal for cell sorting and that has no blur in the height direction of the flow path is shown.
  • the cell sorter chip 2001 in the cell sorter system is vertically arranged as shown in FIG. 20, and the upstream of the flow is installed upward, and the downstream is installed downward.
  • the buffer introduction device 2003 and the sample solution introduction device 2006 are arranged. The sample solution and the buffer solution whose pressures are controlled by the pressure sensor 2004 are introduced into each solution reservoir 2002 connected to the upper surface of the chip.
  • the flow of each flow path can be finely adjusted by finely adjusting the open / closed state of the distribution valve 2005 according to the pressure and state of each reservoir.
  • the liquids introduced from the reservoir are joined via the sample liquid channel 2007 or the buffer channel 2008, and the quantitative measurement of the amount of fluorescence and the amount of light scattering such as image acquisition of the cell type or labeling measurement such as fluorescence.
  • the cell is moved vertically in the flow direction in the flow path by the sorting external force applying mechanism 2009, and as a result, the cells can be collected by the plurality of downstream reservoirs 20101, 20102, and 20103.
  • FIG. 21 shows an example of another configuration of the cell sorter arranged in the vertical type shown in FIG.
  • the cell sorter chip 2101 in the cell sorter system is arranged vertically as in FIG. 20, and the upstream of the flow is installed upward and the downstream thereof is installed downward.
  • the sample liquid and the buffer solution are introduced into the solution reservoir 2102 connected to the upper surface of the chip.
  • the sample solution introduced after passing through is introduced into the flow path 2107 while being sandwiched between the buffer solutions.
  • the sort of external force application mechanism 2109 sorts the cells in the flow path in accordance with the determination based on the result of quantitative measurement of the amount of fluorescence and light scattering, such as image acquisition of fluorescence or other labeling measurement. As a result, the plurality of downstream reservoirs 21101 and 21102 can be collected.
  • FIG. 22 is a diagram schematically showing an example of the configuration of the portion where the sample solution and the buffer solution of the cell sorter chip shown in FIG. 21 merge.
  • the sample liquid is introduced into the flow path 2107 by the capillary tube 21061
  • the buffer liquid is introduced into the reservoir 2102 connected to the upper surface of the end point of the flow path 2107
  • the buffer liquid is arranged to be introduced into the flow path 2107.
  • the outlet of the capillary tube 21061 is disposed downstream of the reservoir portion, so that the sample solution can be introduced into a desired position in the buffer flow path.
  • the flow of the sample solution is arranged in the buffer channel using the capillary tube.
  • the capillary tube is not particularly used.
  • the same effect can be obtained by combining the flow paths configured by microfabrication.
  • FIG. 23 is a diagram schematically showing an example of a chip configuration incorporating a cell sorting mechanism in the cell sorter system.
  • a sample introduction port 23002 is arranged upstream in the chip 23001.
  • target particles 23003 to be collected and unnecessary particles 23004 to be discarded are mixed.
  • the sample solution flows through the microchannel 23005 and is first introduced into the particle alignment mechanism 23006.
  • a particle alignment external force input (electric force or sheath flow) 23007 is arranged.
  • a gel electrode is introduced on both sides of the flow path 23005 to introduce an electric field.
  • a buffer solution is introduced.
  • the type of particles is identified by the particle detection mechanism 23008, and the particles are separated by the particle purification mechanism 23009 which is the next step.
  • a particle purification external force input (gel or metal) electrode + electric force) 23010 is arranged, and an electric force can be applied to the position of the particle purification mechanism 23009 using a gel electrode or a metal electrode. it can.
  • the particle purification mechanism 23009 the particle is allowed to flow downstream without applying an external force and collected at the collection port 23011.
  • the particles other than the particle to be collected have arrived, By applying an external force, the particles can be guided to unnecessary particle reservoirs 23012 and 23013.
  • FIG. 24 is a diagram showing an example of the arrangement of electrodes for applying an external force in the flow path of the cell sorter system.
  • the metal thin film electrode first layer 24002 and the metal thin film electrode The two comb-shaped electrode portions of the two-layer 24003 are arranged so as to be slightly shifted from each other with the insulating film layer 24004 interposed therebetween, and the second-layer electrode 24003 is in direct contact with the sample channel 24005.
  • FIG. 24 (c) shows a photograph of an example in which the comb-shaped electrode array is actually arranged on the bottom surface of the channel with such a configuration.
  • FIG. 25 is a diagram showing an example of the arrangement of electrode arrays for applying an external force in the flow path of the cell sorter system.
  • FIG. 25 (a) shows a case where the sample particles 25004 introduced into the sample introduction port 25001 flow through the micro flow channel 25002, and the metal thin film stacked parallel type having the configuration as shown in FIG.
  • the sample particles 25004 are directed to the upper surface by the dielectric electrophoretic force.
  • FIG. 25 (a) shows a case where the sample particles 25004 introduced into the sample introduction port 25001 flow through the micro flow channel 25002, and the metal thin film stacked parallel type having the configuration as shown in FIG.
  • FIG. 25 (d) when the cross-sectional shape of the flow path receives repulsive force from the bottom surface and the particles progress to the top surface, the semicircular cross-sectional configuration is such that the particles gather at a specific top surface position. It has become. Although a semicircular shape is used here, a triangular cross-sectional configuration may be used.
  • FIG. 25 (b) uses a metal thin film laminated type V-shaped comb electrode 25005. Similarly, by generating repulsive force from the bottom electrode 25007, particles are induced to the top wall, Depending on the shape, the fine particles can be arranged in a line in the center of the flow path.
  • FIG. 25 (c) shows an arrangement of fine particles by adding a buffer fluid sheath channel 25006 from both sides of the channel 25002.
  • FIG. 26 is a schematic diagram showing an example of a cell purification process in the flow path of the cell sorter system. The following cell purification process after cell identification is described.
  • the target particles 26001, unnecessary particles (negatively charged) 26002, unnecessary particles (positively charged) 26003 are arranged in a line and flow through the microchannel 26004, where the target particles are in the region of the particle purification electrode 26005, Since the electrode is OFF, the target particles are guided to the flow 26006 to the target particle recovery port.
  • FIG. 27 is a schematic diagram showing an example of the arrangement of gel electrodes that give an external electric field in the flow path of the cell sorter system, and a photograph of an example of an actual chip.
  • the electrode gel liquid junction part 27004 is arranged adjacent to the microchannel 27001.
  • the electrode gel liquid junction part 27004 which is a boundary separating the flow path 27001 and the gel, introduces the gel from the electrode gel inlet 27002 to the electrode gel passage 27003 and fills the electrode gel outlet 27005.
  • the gel is prevented from leaking out by surface tension so that the gel does not flow out to the channel 27001.
  • the side surface of the flow path 27001 has a structure in which a large number of columns are arranged instead of walls, and the gel and the liquid flowing through the channel are in contact with each other through the space between the columns.
  • the width is preferably 500 ⁇ m or less.
  • the end point of the gel filled with the electrolyte is connected to the metal wire 27006, and bubbles and the like generated when an electric field is applied to the electrode are generated not at the flow path but at the position of the electrode wire.
  • This electrode is connected to a DC voltage source 27007 and controls ON / OFF based on the observation result of particles flowing by the voltage application switching mechanism 27008.
  • the position of the particle in the flow path can be changed, whereby the fine particles can be purified.
  • the gel electrode is used here, the metal thin film electrode 27010 may be used when the applied voltage does not reach the potential at which bubbles are generated (FIGS. 27B and 27D).
  • FIG. 28 is a schematic diagram showing an example of a process for identifying cardiomyocytes and fibroblasts to be separated according to an image in the image recognition type cell sorter system.
  • the surface is very smooth (smooth surface) as can be seen from the image (upper: original image) taken by the image acquisition mechanism of the cell sorter in FIG. It can be seen that the surface is very uneven (rough surface).
  • This image is binarized like the image in the middle of FIG. 28 (binarized image) to clarify the image of the cell boundary surface.
  • the length l of the boundary line of the boundary is measured from the number of pixels of the boundary, and at the same time, the area S of the filled inner surface is calculated from the number of filled pixels.
  • the surface roughness (R) can be quantitatively quantified by comparing the actual perimeter length with the perimeter length when converted from the area into a circle.
  • the numerical value of R is shown in the lower part of FIG. 28, but by performing more detailed measurement, when the value of R is less than about 1.1, this cell is a cardiomyocyte, and when the value is larger than this, It is known to be another cell. In this way, by using a specific numerical value R for purifying cardiomyocytes by image recognition as an index, it becomes possible to identify cells based on the difference in unevenness of the cell surface.
  • FIG. 29 is a schematic diagram showing an example of the configuration of a cell sorter system in which water and oil are combined.
  • the sheath liquid reservoir 1311 is filled with oil having a specific gravity lower than that of water such as silicon oil, and when a sample aqueous solution is dropped into the sample inlet opening 1308, water containing the sample is obtained only in the channel 1302. Oil flows in the flow paths 1304 and 1306 on both sides.
  • water and oil are not mixed, it is not necessary to isolate the entrance of the sheath liquid and the sample liquid with a cap or the like.
  • FIG. 30 is a schematic diagram illustrating an example of a configuration of a water and oil merging region of a cell sorter system in which water and oil are combined.
  • oil is filled and the sample aqueous solution is introduced into the sample aqueous solution introduction port 3001, and the oil in the reservoir is introduced as it is into the remaining oil introduction ports 3002 and 3003.
  • the introduced sample aqueous solution and oil merge at the merge region 3004.
  • the sample aqueous solution 3005 is arranged as shown in FIG. 30B, the sample aqueous solution 3005 can be narrowed down by the oil 3006 as shown in FIG. .
  • the sample aqueous solution can be easily recovered without diluting.
  • FIG. 31 is a graph showing the relationship between the electrolytic mass (conductivity) in an aqueous solution under various solution conditions and the cell separation rate.
  • a solution composition with an ionic strength such that the conductivity of the aqueous sample solution is 10 2 ⁇ S / cm or less. By doing so, it becomes easy to move the fine particles in the sample liquid by the electric field.
  • a solution composition that maintains osmotic pressure while lowering ionic strength is particularly important when sorting cells alive.
  • molecules that do not directly contribute to the increase in ionic strength such as sugars and polymers, are desirably used as sample solutions during cell purification.
  • FIG. 32 is a diagram schematically showing an example of the configuration of an analysis system that simultaneously performs fluorescence intensity measurement and high-speed bright-field microscopic image acquisition.
  • Monochromatic light for observation emitted from a bright field light source 3200 such as an LED flash light source synchronized with a frame interval of a high-speed camera is collected by a condenser lens 3201, and the flow of target cells in blood as described above flows.
  • the cells in the cell sorting unit 3202 including the cell and the cell sorting chip incorporating the cell sorting mechanism are irradiated.
  • the cells in the channel can be focused with the objective lens 3203.
  • a depth-of-field improving technique incorporating the zoom lens system may be incorporated.
  • Fluorescence can be generated from the formed nucleus.
  • the intensity of the obtained fluorescence can be quantitatively measured by a fluorescence detection system 3205, 3207, 3209 including a fluorescence intensity measurement system such as a photomultiplier tube or a photodiode.
  • a plurality of fluorescences can be excited by a single excitation light, so that a plurality of free combinations are possible.
  • a bright field image of a cell can be simultaneously acquired by the high-speed camera 3210 while performing fluorescence detection of the cell.
  • FIG. 33 is a diagram schematically showing an example of a specific configuration of the analysis system that simultaneously performs the fluorescence intensity measurement and the high-speed bright-field microscopic image acquisition shown in FIG.
  • a high-intensity LED flash light source that emits monochromatic light in the infrared region is used as a light source for a bright field (high-speed camera), and lasers of 375 nm, 488 nm, and 515 nm are used as excitation light sources for fluorescent dyes.
  • the introduction of fluorescence into a fluorescence detector using a dichroic mirror is arranged so that the wavelength increases monotonically from short wavelength light to long wavelength light as shown in FIG.
  • the wavelength region is located in the high speed camera.
  • the fluorescence intensity at various wavelengths and the bright field image of the cells in the cell sorter chip disposed in the microchip holder can be simultaneously measured.
  • FIG. 34 is a diagram schematically showing an example of the configuration of an analysis system that simultaneously performs fluorescence intensity measurement, high-speed bright-field microscopic image acquisition, and high-speed fluorescent microscopic image acquisition in the apparatus system shown in FIG.
  • Monochromatic light for observation emitted from a bright field light source 3400 such as an LED flash light source synchronized with a frame interval of a high-speed camera is collected by a condenser lens 3401, and the flow of target cells in blood as described above flows.
  • the cells in the cell sorting unit 3402 including the path and the cell sorting chip incorporating the cell sorting mechanism are irradiated.
  • the cells in the channel can be focused with the objective lens 3403.
  • a depth-of-field improving technique incorporating the zoom lens system may be incorporated.
  • fluorescent excitation light irradiated to the objective lens from a plurality of fluorescent light sources 3404, 3406, 3408 such as monochromatic lasers, the fluorescent antibody bound to the cells in the flow path, or a nuclear staining fluorescent dye (DAPI, Hoechst33258, etc.)
  • DAPI nuclear staining fluorescent dye
  • Fluorescence can be generated from stained nuclei and the like.
  • the intensity of the obtained fluorescence can be quantitatively measured by a fluorescence detection system 3405, 3407, 3409 including a fluorescence intensity measurement system such as a photomultiplier tube or a photodiode.
  • an image dividing system 3410 for dividing an optical microscopic image into a bright field image and a fluorescent image and simultaneously acquiring a plurality of images with a single high-speed camera light receiving element will be described later with reference to FIG. Then, while detecting the fluorescence intensity of the cells in this way, a bright field image of the cells can be simultaneously acquired by the high speed camera 3411.
  • FIG. 35 is a diagram schematically showing an example of an apparatus configuration that simultaneously acquires high-speed bright-field microscopic image acquisition and high-speed fluorescence microscopic image acquisition with a single high-speed camera light-receiving surface.
  • the image data that has entered from the input optical path 3501 is first introduced into the first image dividing unit 3510.
  • the filter system 3512 includes an ND filter for intensity adjustment for aligning the bright field or fluorescence intensity to some extent on a high-speed camera, or a bandpass filter for obtaining a fluorescent image in a sharper wavelength band.
  • a plurality of divided images are finely adjusted three-dimensionally in the direction of reflection through an image size adjustment system 3513 including a movable shielding plate for reducing the image size so that the light receiving surface of the high-speed camera does not overlap.
  • a dichroic mirror 3514 with an adjustable angle function and an optical lens system 3515 for correcting a difference in image formation position from an image of another path caused by a path difference of an optical system including a wavelength difference of light to be further handled.
  • the second image dividing unit 3520 having the same configuration is introduced. Furthermore, the image 3502 that is introduced into the third image dividing unit 3530 having the same configuration and finally output is such that microscopic images composed of monochromatic light of different wavelengths do not overlap on the light receiving surface of the high-speed camera. They are arranged in a cut-out size.
  • three image dividing units having the same configuration are combined, but two or four or more may be used in combination.
  • the division system is “a dichroic mirror 3511 with an angle adjustment function capable of finely adjusting the reflection direction in three dimensions, a filter system 3512, and a movable shield for cutting and reducing the image size.
  • FIG. 36 is a diagram schematically illustrating an example of an image obtained by simultaneously acquiring a high-speed bright-field microscopic image and one high-speed fluorescence microscopic image with a single high-speed camera light-receiving surface, and an example of analysis information.
  • the image 3601 at the time of input to the divided system is data of the cell 3600 in which information of light of a plurality of wavelengths is superimposed.
  • the bright field image 3610 and The fluorescence image 3620 of the nucleus can be acquired simultaneously on one light receiving surface 3602.
  • the extra area on both sides of the input image is cut out by the image size adjustment system in the division system, so that a plurality of images can be acquired with a size that does not overlap on the light receiving surface.
  • the position of each image on the light-receiving surface of the high-speed camera can be freely adjusted by adjusting the surface position of a plurality of dichroic mirrors with an angle adjustment function that can be finely adjusted in three dimensions.
  • the optical lens system 3515 for enlarging or reducing the image of a bright field image or fluorescent image, it is possible to form images with different magnifications on one high-speed camera light receiving surface 3602.
  • An optical system combining images with different magnifications is a divided optical system shown in FIG. 35, “a dichroic mirror 3511 with an angle adjustment function that can finely adjust the reflecting direction in three dimensions, a filter system 3512, and a small image size.
  • An image size adjustment system 3513 composed of a movable shielding plate, a dichroic mirror 3514 with an angle adjustment function capable of finely adjusting the reflection direction in three dimensions, and an optical lens system 3515 for correcting a difference in imaging position.
  • the combined configuration is modularized and used as a set, it is not limited to the application to an imaging cell sorter, but is a general optical bright field / It can also be incorporated into a fluorescence microscope system.
  • the cell size (area) and the cell perimeter can be obtained from the total number of pixels in the area where the data remains after subtraction or the total number of pixels at the boundary of the area where the data remains. . Furthermore, using these two data, it is possible to obtain the degree R of the irregularities of the cell shown in the above-described equation 1. Here, if R is about 1.3 or more, the cell cluster can be determined only from the cell cluster and the bright field image.
  • a fluorescence image 3621 of the nucleus is obtained from the fluorescence image (nuclear staining) 3620, and the total number of nuclei, the area of the nucleus, and the fluorescence intensity, that is, the integrated value of luminance (comparable to the photomal data) is obtained. Can do. Further, since the bright field image and the fluorescent image are only taken at different wavelengths at the same place, the coordinate axes of both are the same. Therefore, although the shape of a cell cannot be measured with a fluorescent image, the relative position of a stained nucleus in a cell can be estimated using coordinates in a bright field image.
  • the relative coordinate system is similarly combined with a relative coordinate system that takes into account the difference in magnification rate with the same origin (image center) as the center. If used, the same processing can be performed.
  • FIG. 37 is a photograph showing an example of an image obtained by simultaneously obtaining a high-speed bright-field microscopic image and a high-speed fluorescence microscopic image stained with nuclear fluorescence with a single high-speed camera light-receiving surface.
  • the position of the nucleus that can be identified by the fluorescence image can be determined at any part of the cell image or cell cluster image of the bright field image. Whether nuclei are distributed can be collated using relative coordinates of each other. By comparing the relative coordinates, it can be seen that in normal cells, one nucleus shines with fluorescence in a smooth cell surface and a normal size cell.
  • (1) a method for identifying and selectively recovering cell clusters (lumps) that do not exist in healthy blood as blood cancer cell candidates, (2) healthy To identify and selectively collect polynuclear cells that do not exist in healthy blood as cancer cell candidates in blood, and (3) identify and select giant cells that do not exist in healthy blood as cancer cell candidates (4)
  • cancer cell biomarkers for example, EpCam antibody, K-ras antibody, site
  • Blood cancer cells instead of conventional molecular biomarkers by a method that identifies and selectively recovers cancer cells by analysis combined with the presence of fluorescence intensity of fluorescent antibodies against one or more of keratin antibodies) ,"cell It is possible to identify and select and collect a new biomarker such as an image of the shape, grouping state, or internal structure such as multinucleation.
  • blood cancer cell candidates collected by the above method can be combined with the above-described gene analysis means such as the PCR analysis technology for microcells, and finally measured for gene mutations. If it is a cancer cell, it is possible to finally identify the characteristics of the cancer cell.
  • evaluation by R> 1.3 using a bright-field image, or the number and distribution of nuclei by size and fluorescence image by a bright-field image that is, the distance between the centers of gravity of adjacent nuclei images is 3 ⁇ m It can be determined by the fact that they are far apart.
  • R ⁇ 1.3 is determined by the bright field image, and the number and distribution of nuclei (that is, the distance between the centroids of the images of adjacent nuclei is within 3 ⁇ m from each other). can do.
  • a combination of (1) to (3) above can be determined as a cancer cell if there is one or more matching conditions.
  • FIG. 38 shows an example of simultaneously irradiating a plurality of wavelengths of fluorescence excitation light to cells such as blood flowing in a microchip simultaneously using a fiber optic array, It is the figure which showed typically an example of the apparatus structure for acquiring a fluorescence image simultaneously.
  • the apparatus of the embodiment of FIG. 38 includes an excitation light source unit (3801-3807) including a fluorescent excitation light source for generating six different monochromatic excitation lights and a bright-field microscopic image light source.
  • Each excitation light source is connected to a controller 3808-3814 that can individually control the light emission timing, light emission time, and light emission intensity.
  • a light source combining a filter with an ordinary broadband light source such as a xenon lamp or a mercury lamp, or a laser light source such as a semiconductor excitation solid state laser or a He-Ne laser may be used.
  • an LED light source that has a narrow wavelength range of output light, can control the intensity stably, is small, and can easily control pulse emission in less than a millisecond.
  • a controller 3808-3814 connected to the excitation light source can control the intensity and output time of the excitation light source, and can irradiate light from continuous light to pulsed light.
  • the technique described in FIG. 18 can be used.
  • the excitation light source can be used for the purpose of exciting fluorescence, but one or more of them can be used as a bright field light source for acquiring a bright field microscopic image.
  • the sample can be irradiated with only light having a specific wavelength by the excitation light filter 3821.
  • each filter 3821 that optimizes the optical bandwidth of the excitation light of each light source 3801 to 3807 is arranged, and each lens 3822 is arranged at the subsequent stage.
  • Each excitation light generated by each excitation light source is focused and irradiated to the end face of each optical fiber 3824 for each excitation light, and the excitation light is introduced into each independent optical fiber 3824.
  • These optical fibers are bundled, irradiated from the opposite end face, and irradiated through the condensing microlens 3826 to the sample flowing through the microchannel in the microchip 3827.
  • the shape of the flow path of a typical microchip may be the one described in FIG.
  • the diameter of the optical fiber is typically 100 microns, and the width of the microchannel through which the sample cells in the microchip pass is typically 10 to 100 microns.
  • a microlens 3826 for condensing excitation light is disposed immediately above the microchip. By adjusting the focal position of the microlens 3826, it is possible to irradiate only the very narrow region of about 1 micron diameter of the microchannel in the microchip with the excitation light focused, and the irradiation region diameter is 100 microns. It is also possible to irradiate the entire microchannel width as a measure.
  • fluorescence of a specific wavelength is emitted from the sample such as a cancer cell in a spherical wave shape.
  • the fluorescence emitted in one hemispherical direction (in the embodiment of FIG. 38 in the direction of the upper surface of the chip) is guided to the end face where the optical fibers 3825 prepared for the number of fluorescence wavelength bands to be measured are bundled by the microlens 3826. Further, the light is guided to a fluorescence intensity detection unit that performs fluorescence intensity detection via an optical fiber.
  • a fluorescence intensity detection unit comprising fluorescence detectors 3815-3820 for detecting fluorescence intensities in six different fluorescence wavelength regions is mounted in this embodiment, and is emitted from the end face of each optical fiber 3825. Fluorescence can be measured by first guiding the fluorescence to the lens 3822 disposed at the end point of each optical fiber, and then guiding the fluorescence to the filter 3823 of each fluorescence wavelength to be measured and the fluorescence detector 3815-3820.
  • a fluorescence detector it is suitable to use a photomultiplier tube that can detect weak light and easily quantify the amount of received light. However, both the excitation light source and the fluorescence detector are suitable for the measurement target.
  • a fluorescent filter 3823 that can be exchanged according to measurement conditions can be mounted on the detection port of the fluorescence detector.
  • the detected fluorescence amount is analyzed and processed by the fluorescence detection control unit 3832, and a specific cell shape or cell cluster obtained from the specific fluorescence or the high-speed camera 3830 when a specific fluorescence or a combination of fluorescence is detected.
  • a feedback signal pulse voltage
  • a feedback signal is sent to the microchip 3827.
  • a threshold amount set in advance by the fluorescence detection control unit 3832
  • a feedback signal is transmitted to the microchip.
  • a voltage is applied to the electrodes mounted on the microchip to recover the target cancer cells.
  • Fluorescence emitted in the remaining hemispherical direction passes through the objective lens 3828, and then simultaneously acquires a bright-field microscopic image and a fluorescent microscopic image with a single high-speed camera light-receiving surface.
  • a bright-field microscopic image and a plurality of fluorescent microscopic images are obtained by passing through a device 3829 (multi-view system; device details are, for example, the configuration of the embodiment shown in FIG. 35 as an example) and being captured by the high-speed camera 3830. All can be acquired simultaneously.
  • the timing of the imaging of the high-speed camera and the excitation light source pulse irradiation can be synchronized.
  • a clear cell image without shape distortion can be acquired (for example, the configuration of the embodiment shown in FIG. 18 is an example of the details of synchronization).
  • FIG. 39 shows an example of six types of fluorescence excitation light sources and one type of bright-field microscopic image acquisition light source mounted on the apparatus of FIG.
  • an example of 6 wavelengths of fluorescence excitation, 1 wavelength of bright-field microscopic image light source, and 6 wavelengths of fluorescence detection is shown, but the number can be easily increased by increasing the number of light sources, detectors, and optical fibers.
  • the excitation light source central wavelengths are 370, 440, 465, 498, 533, 618 nm
  • the bright field microscopic image light source is 750 nm
  • the fluorescence center wavelengths are 488, 510, 580, 610, 640, and 660 nm.
  • the feature of the apparatus system configuration of the present embodiment is that a group of dichroic mirrors are arranged in the optical path system between the objective lens 3828 and the multi-view system 3829, the wavelength bands are divided, and the excitation light sources 3801-3807 and Instead of arranging the fluorescence detectors 3815-3820, as shown in the embodiment of FIG. 38, an optical fiber array is arranged on the opposite side of the objective lens, and each optical fiber of the optical fiber array has one wavelength band. Excitation light source 3801-3807 and fluorescence detector 3815-3820 are arranged. This avoids the problem that fluorescent light emitted from a sample such as cancer cells has been attenuated when passing through a dichroic mirror multiple times, and also eliminates the need for light separation for each wavelength.
  • FIG. 40 is a diagram schematically showing the configuration of the present embodiment whose example is shown in FIG.
  • Monochromatic light for observation emitted from a bright-field light source 4000 such as an LED flash light source synchronized with the frame interval of the high-speed camera is collected by the lens 4001 and the flow path through which target cells in the blood flow as described above.
  • the cells in the cell sorting unit 4002 including the cell sorting chip incorporating the cell sorting mechanism are irradiated.
  • the cells in the channel can be focused with the objective lens 4003.
  • a depth-of-field improving technique incorporating the zoom lens system may be incorporated.
  • Fluorescent light sources 4004, 4006, 4008 such as monochromatic lasers and the like, and fluorescence excitation light irradiated to the cell sorting unit 4002 from above, fluorescent antibodies bound to cells in the flow path, and nuclear staining fluorescent dyes (DAPI, Hoechst33258, etc.) ) Can generate fluorescence from nuclei stained with).
  • the intensity of the obtained fluorescence can be quantitatively measured by a fluorescence detection system 4005, 4007, 4009 including a fluorescence intensity measurement system such as a photomultiplier tube or a photodiode.
  • a plurality of fluorescences can be excited by a single excitation light, so that a plurality of free combinations are possible.
  • an image division system for dividing an optical microscopic image into a bright-field image and a fluorescent image and acquiring a plurality of images at the same time with a single high-speed camera light-receiving element is described in detail later in FIG. Through 4010, a bright field image of a cell can be simultaneously acquired by the high-speed camera 4011 while detecting the fluorescence intensity of the cell in this way.
  • the description is based on the combination with the image detection unit. However, naturally, it does not include the image detection unit using the high-speed camera, and the simultaneous detection of the multiple excitation light and the multiple fluorescence using only the optical fiber array. It can also be used as a system.
  • FIG. 41A shows an example of a distribution diagram of cell nucleus areas when blood cancer cells are measured by the apparatus of the present invention.
  • the area of each nucleus is compared between an image of cells in blood when cancer tissue is actually transplanted and an image of cells in healthy blood for comparison. From this result, it can be seen that the presence of cancer cells can be clearly identified as cancer cells when the area of the fluorescence image of the nucleus of about 150 ⁇ m 2 or more is observed.
  • (b) to (g) of FIG. 41A show the bright field image and the fluorescence image of typical cells at the respective nucleus sizes in the graph of FIG. (A).
  • the cells are clustered in the region where the area of the fluorescence image of the nucleus of 150 ⁇ m 2 or more is observed.
  • this is when measured by integrating the total area in the image for the nuclei in the cell clusters, in about 150 [mu] m 2 or less of conditions, and also shows that it is impossible to distinguish cancer cells and normal cells,
  • the above about 150 ⁇ m 2 or more is one of the sufficient conditions for the existence of cancer cell clusters. This index is also effective for identifying multinucleated cells that are characteristic of cancer cells.
  • FIG. 41B is a graph showing the ratio of the peripheral length of the blood cancer cells measured by the apparatus of the present invention to the peripheral length derived by circular approximation from the area of the cells (cluster).
  • An example is shown.
  • the isolated one cell has (1 / R)> about 0.9
  • the cluster of two or more cells has (1 / R) ⁇ about 0.9. From the result (1 / R), when this is about 0.9 or more, it can be determined that the cell is an isolated cell, and when it is less than about 0.9, two or more cells are clumped. It can be seen that it is a cell cluster.
  • FIG. 42 shows a table showing the distribution of cell (population) area and number of nuclei when blood cancer cells are measured with the apparatus of the present invention.
  • the bright field is compared.
  • the presence of cancer cells is clearly identified as a cancer cell cluster if the area of the bright field image of cells of approximately 250 ⁇ m 2 or more. You can see that you can.
  • the area of the nucleus of about 150 ⁇ m 2 or more of the cell (cluster) is measured from the acquired image (2)
  • the area of about 250 ⁇ m 2 or more of the cell (cluster) is measured from the acquired image (3)
  • the cell (cluster) The presence of three or more nuclei in (1) is measured from the acquired image, or a combination of the above three conditions in AND, that is, (1) and (2), or (1) and (3 ), (2) and (3), or (1), (2) and (3) may be used as criteria for determining the presence of blood cancer cells.
  • FIG. 43 schematically shows a more specific configuration for practical application of the device configuration for simultaneously acquiring microscopic images in a plurality of different wavelength bands with one high-speed camera light-receiving surface for the device shown in FIG.
  • FIG. 43A in FIG. 43A is a schematic view of the entire internal configuration of one unit of the optical branching module, which is the minimum configuration unit, as viewed from above.
  • This apparatus is a two-dimensionally developed optical path system viewed from the top, and as shown in the figure, a pair of image light input / output optical path systems symmetrically on both bottom surfaces of a rectangular parallelepiped container, and On the side surface, 2 to 4 holes for introducing optical paths of mirror reflected light are arranged, and six optical path covers 4301 that can be freely attached and detached are arranged on each side. Two detachable movable adjustment function mirror holders 4302 are arranged on these side surfaces so that light is introduced into the holes of the two to four mirror reflected light introduction optical paths. Adjustment enables the traveling direction of the reflected light to be moved minutely, so that the imaging position of the camera can be freely moved.
  • the mirror holder can be provided with a total reflection mirror, a high pass filter, a low pass filter, and the like.
  • a detachable optical path window 4303 is arranged between the two mirror holders 4302 with detachable movable adjustment function, and the cross-sectional area of the transmitted light can be adjusted.
  • a detachable filter 4304 is provided, and the wavelength bandwidth of light can be finely adjusted by a band pass filter or the like.
  • FIG. 43B schematically shows one of the embodiments for observing a combination of images of a plurality of wavelengths by actually connecting the units shown in FIG. 43A.
  • an image optical image to be observed is introduced from the parallel light introduction module 4305 to the first light branching module 4300.
  • the optical path cover 4301 in the optical path introduced here is removed, and is fixed to the parallel light introducing module 4305 in such a manner as to maintain hermeticity by the connecting portion.
  • the parallel light introducing module 4305 incorporates a lens system so that incident light is introduced into the light branching module 4300 as parallel light, and the cross-sectional area of the incident light to be introduced is set at the front stage of the lens system.
  • a small optical window filter may be arranged.
  • the cross-sectional area of the incident light cut out by the optical window is an image acquisition camera that is finally measured in order to project an independent image corresponding to the number of modules to be connected so as not to overlap the light receiving surface on the observation camera.
  • the area is adjusted to be equal to or smaller than (total area of the light receiving surface / parallel light introducing module).
  • the first-stage light branching module 4300 it is introduced into the mirror holder 4302 in which the (wavelength) high-pass filter or the (wavelength) low-pass filter b is incorporated, and the incident light is branched into two wavelengths.
  • the transmitted light is reflected by the total reflection mirror a, and is introduced into the second filter b of the second-stage light branching module 4300 (the same filter as the first filter of the preceding-stage (first-stage) light-branching module).
  • the light reflected by the filter b of the first-stage light branch module 4300 is introduced into the first filter c of the second-stage light branch module.
  • the same wavelength band branching is performed up to the fifth stage optical branching module, and the direction of the optical path is adjusted for each branch wavelength so that the images of the respective wavelength bands do not overlap on the light receiving surface of the camera.
  • Positioning is performed by each detachable movable adjustment function mirror holder 4302.
  • the total reflection mirror a is used for the first filter
  • the first filter e in the preceding-stage (fourth-stage) light branching module 4300 is used for the second filter.
  • b, c, d, and e which are high-pass filters or low-pass filters, are arranged in order of monotonically increasing or monotonically decreasing wavelengths (b ⁇ c ⁇ d ⁇ e or b> c> d> e), the same filter is used for the first filter in the previous stage and the second filter in the next stage, and a total reflection mirror is used in the second filter in the first stage and the first filter in the final stage. Shall be used.
  • FIG. 43C schematically shows how an actually input image is projected on the camera system 4306.
  • the acquired image (left figure) is 1 / of the size of the light receiving surface of the camera system by an optical window filter in the parallel light introducing module 4305 that cuts off the cross-sectional area of the incident light introduced in the previous stage of the lens system.
  • the image is cut into a size that allows an image having an area of 5 or less to pass through and is introduced into the optical branching module 4300 (center view).
  • the image forming positions on the light receiving surface of each light branch module are arranged so that there is no overlapping portion (right diagram).
  • images of each optical wavelength bandwidth created by the five optical branching modules are simultaneously formed on the light receiving surface of one camera, and particularly when a high-speed camera is used, a high-speed image of one image. Only by processing, comparative analysis of images of a plurality of wavelengths can be performed at one time.
  • FIG. 43D shows another application example in which an optical branching module is combined.
  • the second mirror holder of the optical branching module can be removed and another camera system can be connected here, or the light intensity measurement 4307 can be connected to measure the light intensity of a specific wavelength bandwidth. it can.
  • FIG. 44 is a diagram schematically showing an example of the configuration of an imaging cell sorter that observes and separates cells in water droplets.
  • the apparatus shown in FIG. 44A produces and drops water droplets of an optimal size by discharging cells in the sample solution from the thin tube at the tip of the water droplet forming module 4401 with a cell reservoir at a constant pressure. Can do.
  • the electrostatic field coil 4402 covering the region from the region where the water just before the water droplet is formed to the reservoir to the region where the water droplet is just formed to the opposite charge to the water droplet to be charged, Water droplets can be charged with a desired charge. For example, when it is desired to charge a water droplet with a negative charge, the coil may be charged with a positive charge.
  • the formed charged water droplet 4403 is dropped on an optically transparent water-repellent insulating substrate having a Teflon resin processed surface such as glass, and rolls down in the tilt direction of the substrate.
  • a high-speed camera capable of measuring bright field images and fluorescent images and an optical measurement module 4405 capable of measuring scattered light intensity, fluorescent intensity, etc. are arranged on the back of the substrate on the path where the water drops fall, and the acquired information is analyzed. By analyzing with the control module 4410, it can be determined whether the target cell is in a water droplet.
  • a charge opposite to the water droplet is given to one specific path among the plurality of water droplet movement direction control electrostatic field guides 4406 for changing the position from the water droplet falling direction.
  • the electric charge is applied so as to hold, and the falling direction of the water droplet is changed and guided to the fraction water droplet reservoir 4407 at the subsequent stage.
  • a plurality of water droplet movement direction control electrostatic field guides 4406 and a fractional water droplet reservoir 4407 may be arranged in accordance with the types of the targets.
  • the electrodes of the electrostatic field guides for controlling each water droplet movement direction are arranged in the lower stage of the substrate 4404, and each of these electrodes is connected to the electric field switching mechanism 4409 for electrostatic field guides for controlling the water droplet movement direction.
  • a control charge from 4410 can be applied.
  • the analysis control module analyzes the acquired image and calculates the cell (cluster) area and perimeter, the nucleus area and the number in the cell cluster by calculation, and the calculation result obtained (for example, a computer). It can be configured by a mechanism (for example, a computer and a power source) that identifies the types of cells to be collected in combination and applies an electric field to an electrode of a specific guide to give an electric charge.
  • the switching mechanism can be composed of a removable inset groove for fixing the substrate and an array of electrical contacts for applying electric charges from the analysis control module, which are aligned with the position of the electrode line of the substrate guide. .
  • each electrode has a mechanism that does not affect the movement of the water droplet by grounding the electrostatic field guide for controlling the direction of water droplet movement.
  • a similar water droplet movement direction control electrostatic field guide 4406 is disposed, or a pair of rails sandwiched between the lower surfaces of a water repellent surface such as a Teflon coat These three-dimensional structures may be added.
  • any material can be used as long as it can build a physical structure.
  • a material whose deformation and rigidity do not change significantly at a temperature of 150 ° C. or higher. Further, it is desirable that it is optically transparent or constant absorption in the wavelength region used for observation. Specifically, glass, SU8, PDMS, etc. are mentioned.
  • One of the features of this technology is that it has a substrate tilt control mechanism 4408 that can freely control the drop speed of water droplets, that is, the measurement time.
  • a mechanism in which a tilt mechanism is added to a flat plate is shown, but a substrate having a combination of different tilts may be used. For example, by using a steeper slope for the transport and separation of water drops and a gentle slope in the measurement area, it is possible to observe the water drops in a state where the distance between adjacent water drops is short and the moving speed is slow. Can do.
  • the water droplets in the observation region and the water droplet fractionation start region, can be arranged closely at equal distances in the falling direction by making them horizontal (perpendicular to gravity) so that the water droplets move at a constant speed. Further, if the substrate is lengthened under such a particularly horizontal condition, the distance for sequentially observing various measurements under the condition that the water droplet moving speed is the same can be made sufficiently long. Further, by adjusting the temperature of the substrate, it is possible to control the temperature of water droplets that have made minimal contact in a water-repellent state. Further, a temperature / humidity control container 4411 and a temperature / humidity control mechanism 4412 are added to prevent evaporation of water droplets.
  • temperature / humidity control container 4411 and the temperature / humidity control mechanism 4412 for example, an acrylic optically transparent container or a light-shielding metal container may be used, and temperature control by a Peltier element is possible. It can comprise with the air blower etc. to the said container.
  • reaction liquid reservoir water droplet formation mechanism 4413 creates a reaction liquid to which a charge opposite to that of the sample water droplet is weakly applied, and the liquid droplet movement direction control electrostatic field guide 4414 can guide the reaction liquid to the sample water droplet.
  • FIG. 44B is a diagram schematically showing the apparatus from the top in order to explain a mechanism for actually separating and collecting water droplets.
  • the water droplets falling on the straight line are arranged so as to overlap at the end points of the plurality of branching water droplet movement direction control electrostatic field guides 4406.
  • each guide 4406 is arranged so that a water droplet falls obliquely with a resultant force of gravity with an angle in the tilt direction, and when a charge opposite to the water droplet is applied to the guide, Is configured to move.
  • a fractional water droplet reservoir 4407 serving as a U-shaped water droplet receiver for capturing water droplets is disposed below.
  • each guide extends to the end of the substrate, and by arranging there, it is possible to easily apply a charge to each guide. It has a configuration.
  • FIG. 45A is a diagram schematically showing an example of a process for detecting a cell cluster in human blood and analyzing it for diagnosis.
  • 45B and C are diagrams schematically showing an example of an integrated measurement analysis system for realizing the process shown in FIG. 45A.
  • the degree of cancer progression is determined from the primary cancer. If there are many different mutation points while the history of mutations in each cluster is the same, the location of the metastatic cancer is spread over many regions. Can be estimated.
  • the cell cluster that flows in the blood can be identified as a liver tissue fragment, the liver disease can be estimated, and similarly, it can be identified from other organ sections It can be estimated that these are diseases of each organ.
  • FIG. 45B schematically shows the components of the system for enabling the measurement.
  • FIG. 45C shows an example of a configuration created to actually realize the concept of FIG. 45B.
  • the cell pretreatment mechanism is arranged in the upper stage, the imaging cell sorter is arranged in the middle stage, and the droplet type PCR apparatus is arranged in the lower stage.
  • FIG. 46 is a diagram schematically showing an example of a technique for removing cell clusters (clusters) in blood of a certain size or more as a metastatic cancer treatment technique.
  • the cell blood (cluster) removing mechanism 4601 such as a membrane filter that removes a cell mass (cluster) having a cross-sectional area of about 250 ⁇ m 2 or more that does not exist in healthy blood due to the above discovery is used to circulate the patient's blood. Therefore, a technique that suppresses the onset and progression of metastatic cancer by effectively removing metastatic cancer cells in the blood by physical methods without using drugs such as anticancer drugs is schematically shown. It is what.
  • the membrane filter to remove about 250 [mu] m 2 or more of the cross-sectional area of the cell mass (cluster) if a technique for removing about 250 [mu] m 2 or more of the cross-sectional area of the cell mass (cluster), physical A non-contact capture technique such as a ladder array for selective capture or capture by ultrasonic radiation pressure may be used.
  • a technique for removing about 250 [mu] m 2 or more of the cross-sectional area of the cell mass (cluster) physical A non-contact capture technique such as a ladder array for selective capture or capture by ultrasonic radiation pressure may be used.
  • the establishment of implantation / metastasis of cancer cells that have been clustered (clustered) can be performed in the blood with a weakly isolated single cell that easily causes apoptosis. This stems from an exponential increase in potential for flowing cancer cells.
  • FIG. 47 shows the distribution spectrum of blood cell size in the case of measuring the particle size distribution of leukocytes in blood for diagnosis of metastatic cancer in blood, normal blood (FIG. 47A), metastatic cancer cells.
  • FIG. 47B schematically shows each of the blood (FIGS. 47B and 47C) flowing.
  • the presence of abnormal cells such as cancer cells was measured based on the size of each cell relative to the absolute indicator of cell size.
  • the cell size distribution in the blood is plotted on the X axis.
  • the distribution curve (spectrum) is obtained with the quantity (or the ratio of the quantity to the whole) as the Y axis, and when the distribution has a monotonous single peak as shown in FIG. 47A, normal blood It is determined.
  • the value of the abnormal cell size peak larger than the peak of normal cells is used as an index (threshold), and blood is again
  • the cell size spectrum is measured in the first step, and the obtained abnormal cell size peak is obtained in the second step.
  • Accurate cell recovery can be realized in two steps for recovering cells that are equal to or greater than the threshold value (peak value of distribution: see arrows in FIG. 47B, C abnormal cell distribution portion).
  • the peak of abnormal cells corresponds to a giant cell or a cell cluster depending on the case.
  • FIG. 48 shows a method using a substrate having an acceleration region and a constant velocity moving region by having two inclination angles in the means for moving the water droplet dropped on the water repellent substrate described in FIG. 44 on the surface of the substrate.
  • the moving speed of the water droplet is adjusted by adjusting the inclination angle of one flat plate, but only one surface is “acceleration only” or “constant velocity motion only”.
  • the movement speed of the water droplet becomes too high with “acceleration alone”, and higher-speed processing is required for the subsequent measurement and separation.
  • the dropped water droplet 4801 is placed on a water repellent treated substrate whose inclination angle can be adjusted along the water droplet dropping direction 4802. For example, the moving speed is increased by using the inclined surface 4803 having the inclination ⁇ as an “acceleration region”. While rolling down. Then, on the horizontal plane 4804 having the function of adjusting the inclination angle that follows, the water droplet moves at a constant speed as the “constant velocity region”. In addition, after performing processing such as analysis and separation in the constant velocity region, the inclined surface may be newly set as an “acceleration region”.
  • the “constant velocity region” is basically leveled, but in actuality, when decelerating due to minute resistance on the surface of the water-repellent surface, a slight inclination angle is maintained to maintain the constant velocity of water droplets. You may give it.
  • the present invention is useful for purifying a minute amount of target cells in blood in units of one cell, and analyzing accurate gene information and expression information of the target cells.
  • the present invention is useful for purifying a very small amount of target cells having spores such as Bacillus anthracis per cell and analyzing accurate gene information and expression information of the target cells at high speed.
  • the present invention is also useful as a technique for identifying and / or collecting cancer cells circulating in the blood.

Abstract

La présente invention concerne un dispositif de concentration et purification cellulaire ayant : une fonction pour concentrer des cellules en continu ; une fonction pour disposer ensuite en continu les cellules sur une région spécifique d'un trajet ; une fonction pour reconnaître simultanément la forme et l'émission de fluorescence des cellules une par une, à partir d'une image ; et une fonction pour séparer et purifier les cellules en reconnaissant les cellules sur la base des informations de forme et d'émission de fluorescence des cellules.
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CN110047107B (zh) * 2018-01-13 2021-04-13 深圳华大智造科技股份有限公司 相机标定方法及图像配准方法、基因测序仪及系统
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