WO2015076251A1 - Appareil de sélection et d'élimination de cellules, et procédé de sélection et d'élimination de cellules - Google Patents

Appareil de sélection et d'élimination de cellules, et procédé de sélection et d'élimination de cellules Download PDF

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Publication number
WO2015076251A1
WO2015076251A1 PCT/JP2014/080473 JP2014080473W WO2015076251A1 WO 2015076251 A1 WO2015076251 A1 WO 2015076251A1 JP 2014080473 W JP2014080473 W JP 2014080473W WO 2015076251 A1 WO2015076251 A1 WO 2015076251A1
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cell
cells
channel
predetermined
flow path
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PCT/JP2014/080473
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English (en)
Japanese (ja)
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圭介 合田
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国立大学法人東京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • 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

Definitions

  • the present invention relates to an apparatus for sorting and removing cells and methods thereof.
  • the present invention relates to a cell sorting apparatus, a cell sorting channel device, and a cell sorting method.
  • a cell sorting apparatus that sorts target cells from a cell-containing liquid containing a plurality of types of cells, and a cell sorting channel device used therefor
  • the present invention also relates to a cell sorting method.
  • the specific cell removal apparatus, the specific cell removal flow path, and the specific cell removal method more specifically, autologous hematopoietic stem cell transplantation in which specific cells are removed from a hematopoietic stem cell-containing liquid containing hematopoietic stem cells of a patient and transplanted to the patient.
  • the present invention relates to a specific cell removal apparatus used in the above, a specific cell removal flow path used for removing such specific cells, and a specific cell removal method used for autologous hematopoietic stem cell transplantation.
  • Non-Patent Document 1 an apparatus for sorting cells using fluorescent light emission has been proposed (for example, see Non-Patent Document 1).
  • cell sorting is performed as follows. A fluorescent agent that induces fluorescence by laser irradiation is added to a cell to be sorted into a solution in which a plurality of types of cells are mixed, and the solution is passed through a nozzle through a thin tube that allows one cell to flow down. Dripping. Laser light is irradiated to the solution flowing down in a thin tube to induce fluorescence emission of cells to be sorted.
  • the cells dropped from the nozzle for example, cells that fluoresce in green are charged positively, and cells that fluoresce in red are negatively charged.
  • a cell is dropped between the positively charged electrode and the negatively charged electrode, and the cells that emit green fluorescence are guided to the flow path on the minus electrode side, and the cells that emit fluorescent light in red are positive. Cells that are guided to the electrode-side flow path and do not emit fluorescence are guided to the central flow path to sort the cells.
  • autologous hematopoietic stem cell transplantation was performed by collecting stem cells from the bone marrow into the blood using G-CSF (granulocyte colony-stimulating factor), which is collected from the patient's bone marrow, treated with anticancer drugs, and increases white blood cells.
  • G-CSF granulocyte colony-stimulating factor
  • a hematopoietic stem cell obtained by collection in the same manner as the component blood donation is administered by administering a large amount of an anticancer agent and then transplanting in the same manner as blood transfusion (for example, see Non-patent Document 2).
  • FACS Fluorescence Activated Sorting
  • the number of cells that can be sorted per unit time is small, and it takes time to complete sorting of all cells in the solution. In particular, when cells having a very small content in the solution are selected, a long time is required.
  • fluorescent emission when there are multiple types of cells that emit fluorescence with the same color, it is not possible to sort cells to be selected from the multiple types of cells, and there are multiple types of cells that do not emit fluorescence. Sometimes it is not possible to sort cells to be sorted from these multiple types of cells.
  • the main object of the cell sorting apparatus and cell sorting method of the present invention is to provide an apparatus and method that can sort target cells more rapidly.
  • the main object of the flow channel device for cell sorting used in the cell sorting apparatus of the present invention is to provide a flow channel device for sorting target cells more quickly.
  • hematopoietic stem cell-containing liquid a liquid containing hematopoietic stem cells collected from a patient
  • the tumor cells relapse after transplantation, so the tumor cells are eradicated from the hematopoietic stem cell-containing liquid.
  • the eradication is difficult when the content of tumor cells in the hematopoietic stem cell-containing fluid is very small.
  • the specific cell removal apparatus and the specific cell removal method of the present invention are mainly intended to remove tumor cells that may be mixed in the hematopoietic stem cell-containing liquid.
  • the specific cell removal flow channel of the present invention is intended to provide a flow channel effective for an apparatus or method for removing tumor cells slightly mixed in the hematopoietic stem cell-containing liquid.
  • the cell sorting apparatus, cell sorting channel device and cell sorting method of the present invention employ the following means in order to achieve the above-mentioned main purpose.
  • the cell sorting apparatus of the present invention comprises: A cell sorting device for sorting target cells from a cell-containing liquid containing a plurality of types of cells, An alignment channel that allows cells present at random in the cell-containing liquid to flow in a line, an imaging channel formed at the rear stage of the alignment channel, and a book channel at the rear stage of the imaging channel.
  • a flow path device for cell sorting having a flow path for diversion and a flow path for sorting formed with a branch flow path diverging from the flow path for main flow,
  • a cell imaging means for irradiating the imaging channel with short-wave light at least in a straight line and sequentially illuminating the imaging channel, and forming an image of the cells in the cell-containing liquid based on the reflected light by the irradiation;
  • Determining means for determining whether the cell is the target cell based on the image of the formed cell; Sorting means that sorts the cells determined to be the target cells by at least the determining means by branching from the main flow channel to the branch channel; It is a summary to provide.
  • cells that randomly exist in the cell-containing liquid are lined up by flowing a cell-containing liquid containing a plurality of types of cells through the channel for sorting of the channel sorting channel device. Flowing.
  • short-pulse light is irradiated to the imaging flow channel in which the cells flow in a single row, at least in a straight line, and by a wavelength group with different wavelengths.
  • Form images of cells based on the image of the formed cell, it is determined whether or not the cell is the target cell, and at least the cell determined to be the target cell is branched from the main flow channel to the branch channel. Sort out.
  • the cells basically flow into the main flow channel, but the cells determined to be target cells are branched into the branch flow channel. In this way, since it is not necessary to drop cells one by one from the nozzle, it is possible to sort target cells more quickly by increasing the flow of the cell-containing liquid. In addition, since it is determined by forming an image of a cell, it is not necessary to use fluorescence.
  • the CCD As compared with the case of forming an image using an image sensor (Charge Coupled Device image sensor), it is possible to form an image quickly and with high sensitivity. As a result, the target cells can be quickly selected even if the cells having a very small content in the cell-containing liquid are the target cells.
  • “cells” mainly include living cells, for example, red blood cells, platelets, white blood cells, etc.
  • the “cell-containing liquid” include body fluids such as blood and solutions obtained by diluting them with a solvent such as physiological saline.
  • the “wavelength-specific light group” is not limited to those in which the wavelengths are sequentially different on a straight line, that is, those in which the wavelength is sequentially different for each position on the straight line, and may be those in which the wavelength order is different for each position on the plane.
  • the sorting means is a means for branching the cells determined to be the target cells by cavitation caused by pulse laser irradiation from the main flow channel to the branch channel.
  • Cavitation can be generated up to about 20,000 cells / second using a pulse laser, and therefore target cells can be selected from other cells up to about 20,000 cells / second.
  • the alignment channel has a predetermined width within a range of 100 to 500 micrometers and a predetermined depth within a range of 10 to 100 micrometers and a range of 10 to 20 times. It may be a wavy flow path that meanders at a predetermined number of repetitions. In this way, the cells in the cell-containing liquid can be easily flowed in a line using inertia.
  • the alignment channel includes at least one channel having a predetermined width within a range of 100 to 500 micrometers and a predetermined depth within a range of 10 to 100 micrometers.
  • the surface may be a straight flow path in which irregularities are formed a predetermined number of repetitions within a range of 20 to 50 times. If irregularities are formed on the surface forming the flow path, a wide area and a narrow area of the cross section of the flow path are generated, an inertial force acts on the cells, and the cells gradually flow in a row.
  • the surface on which the unevenness is formed may be any of the top surface, the bottom surface, and the side surface, and the top surface and the bottom surface are particularly preferable.
  • the depth of the flow path changes due to the unevenness, but the cells circulate at locations where the convex portions are formed (locations where the depth is shallow). It is only necessary to have a depth enough to be able to do so, and the depth should be designed according to the size of the cells to be selected.
  • the cell imaging means spectrally divides the short pulse light from the short pulse light source and the short pulse light source that repeatedly outputs short pulse light at intervals of femtoseconds to nanoseconds.
  • An optical unit a wavelength selecting optical fiber that inputs the reflected pulsed light and sequentially outputs light having different wavelengths as time elapses, and an intensity of light for each wavelength output from the wavelength selecting optical fiber.
  • a cell image forming unit that forms an image of the cell based on the above.
  • the flow path device for cell sorting of the present invention comprises: A cell sorting channel device used in a cell sorting apparatus for sorting target cells from a cell-containing liquid containing a plurality of types of cells, An alignment channel that allows cells present at random in the cell-containing liquid to flow in a row; A shooting channel formed in a subsequent stage of the alignment channel; A sorting flow path in which a main flow path and a branch flow path branched from the main flow path are formed at a subsequent stage of the photographing flow path; It is a summary to provide.
  • the cell sorting channel device of the present invention by flowing a cell-containing liquid containing a plurality of types of cells in the sorting channel of the cell sorting channel device, cells that are randomly present in the cell-containing liquid are It flows in a line. Accordingly, since the cells flow in a row in the imaging channel, the cells in the cell-containing liquid can be sequentially imaged, and it is determined whether or not the cells are target cells based on the captured cell images. be able to.
  • the cells determined to be the target cells can be selected from other cells by branching from the main flow channel to the branch channel.
  • the cells in the cell-containing liquid are photographed in a single line, and whether or not the target cell is determined is determined by branching into a branch channel to select the necessary channel with a single channel device. Can do.
  • the cell sorting apparatus can be downsized.
  • the sorting channel has a predetermined width within a range of 100 to 500 micrometers and a predetermined depth within a range of 10 to 100 micrometers and a range of 10 to 20 times.
  • a wavy channel that meanders at a predetermined number of repetitions the imaging channel is a linear channel with the predetermined width and the predetermined depth, and the main channel is less than or equal to the predetermined width
  • the branch channel is a channel that is arranged in a straight line from the photographing channel at the predetermined depth with a width of 10 mm from the main flow channel at a predetermined width with a width smaller than the predetermined width. It may be a flow path that branches at a predetermined angle within a range of 60 degrees to 60 degrees. In this way, the length of the cell sorting channel device can be shortened.
  • the alignment channel forms a channel with a predetermined width within a range of 100 to 500 micrometers and a predetermined depth within a range of 10 to 100 micrometers.
  • the main flow path is a flow path that is arranged in a straight line from the photographing flow path at the predetermined depth with a width equal to or less than the predetermined width, and the branch flow path has the predetermined width.
  • the flow path may be a flow path that branches at a predetermined angle within a range of 10 degrees to 60 degrees from the main flow path with the predetermined depth and a smaller width.
  • the length of the cell sorting channel device can be shortened.
  • the surface on which the unevenness is formed may be any of the top surface, the bottom surface, and the side surface, and the top surface and the bottom surface are particularly preferable.
  • the depth of the flow path changes due to the unevenness, but the cells circulate at locations where the convex portions are formed (locations where the depth is shallow). It is only necessary to have a depth enough to be able to do so, and the depth should be designed according to the size of the cells to be selected.
  • the sorting channel branches the target cell into the branch channel on the opposite side of the branch channel immediately upstream of the branch point to the branch channel. It is also possible to form a cavitation generation region for generating cavitation for generating the cavitation. If it carries out like this, a target cell can be branched to a branch flow path using the force in the case of expansion by cavitation. Cavitation can be generated up to about 20,000 cells / second using a pulse laser, and therefore target cells can be selected from other cells up to about 20,000 cells / second.
  • the cell sorting method of the present invention comprises: A cell sorting method for sorting target cells from a cell-containing liquid containing a plurality of types of cells, (A) flushing cells randomly present in the cell-containing liquid in a line; (B) irradiating the cell-containing liquid in which the cells flow in a row with a short pulsed light as a group of light beams having different wavelengths at least on a straight line and, based on the reflected light by the irradiation, the cells in the cell-containing liquid Form an image, (C) determining whether the cell is the target cell based on the formed cell image; (D) selecting at least cells determined to be the target cells by branching from the flow of the cell-containing liquid; It is characterized by that.
  • cells randomly present in the cell-containing liquid are made to flow in a row, and short pulse light is sent to the imaging flow path at least in a straight line and with different wavelengths. Irradiation is performed, and an image of the cells in the cell-containing liquid is formed based on the reflected light from the irradiation. Then, it is determined whether or not the cell is a target cell based on the formed image of the cell, and at least the cell determined to be the target cell is selected by branching from the flow of the cell-containing liquid. Since it is not necessary to drop cells one by one from the nozzle, it is possible to sort target cells more quickly by increasing the flow rate of the cell-containing liquid.
  • the CCD As compared with the case of forming an image using an image sensor (Charge Coupled Device image sensor), it is possible to form an image quickly and with high sensitivity.
  • the step (d) may be a step of branching a cell determined to be the target cell using cavitation. Cavitation can be generated up to about 20,000 cells / second using a pulse laser, and therefore target cells can be selected from other cells up to about 20,000 cells / second.
  • the step (a) may be performed by adding the cell-containing liquid at a predetermined width within a range of 100 to 500 micrometers and a predetermined depth within a range of 10 to 100 micrometers. It can also be a step in which cells are arranged to flow in a line by flowing in a wavy flow path that meanders at a predetermined number of repetitions within a range of times. In this way, the cells in the cell-containing liquid can be easily flowed in a line using inertia.
  • the step (b) is performed by spectroscopically irradiating the short pulse light repeatedly output at an interval of femtosecond or nanosecond order and irradiating it as the light group by wavelength, and reflecting by the irradiation.
  • the reflected short-pulse light obtained by condensing the reflected light groups according to the reflected wavelengths is converted into light having different wavelengths with time, and an image of the cell is formed based on the intensity of the light for each wavelength. It can also be.
  • the specific cell removal apparatus the specific cell removal flow path, and the specific cell removal method of the present invention employ the following means in order to achieve the above-described main object.
  • the specific cell removing apparatus of the present invention is A specific cell removal apparatus used for autologous hematopoietic stem cell transplantation, which removes at least specific cells from a hematopoietic stem cell-containing liquid containing hematopoietic stem cells collected from a patient and transplants the same into the patient,
  • An alignment channel that allows cells existing in the hematopoietic stem cell-containing liquid to flow in a row, and a second channel formed downstream of the alignment channel to destroy specific cells in the hematopoietic stem cell-containing liquid.
  • a first flow path having a first treatment flow path and a first separation flow path formed at a subsequent stage of the first treatment flow path in order to separate specific cells destroyed from the hematopoietic stem cell-containing liquid.
  • the device A predetermined cell as a range of cells not containing the hematopoietic stem cell but containing the specific cell and including the cell with the possibility of the specific cell is detected from the hematopoietic stem cell-containing liquid flowing in the first processing channel.
  • Predetermined cell detection means Laser irradiating means capable of irradiating the first processing channel with destructive laser light for cell destruction;
  • the laser irradiation unit is configured to irradiate the detected predetermined cell with the destructive laser beam.
  • Control means for controlling It is a summary to provide.
  • the cells existing in the hematopoietic stem cell-containing liquid are caused to flow in a line by flowing the hematopoietic stem cell-containing liquid through the alignment channel of the first channel device.
  • the predetermined cells in the hematopoietic stem cell-containing liquid in which the cells flow in a line in the first processing flow path are detected, and the predetermined cells are destroyed by irradiating the detected predetermined cells with a destruction laser beam. Then, the predetermined cells broken from the hematopoietic stem cell-containing liquid are separated by the first separation channel of the first channel device.
  • the predetermined cells are cells in a range that does not include hematopoietic stem cells but includes specific cells and includes cells in which the possibility of specific cells is recognized. Therefore, specific cells that do not contain hematopoietic stem cells are detected and destroyed with a disruptive laser beam, and the destroyed cells are separated from the hematopoietic stem cell-containing liquid. Cells can be removed. When tumor cells are considered as specific cells, tumor cells can be removed from the hematopoietic stem cell-containing liquid while leaving the hematopoietic stem cells. Therefore, by performing autologous hematopoietic stem cell transplantation using the hematopoietic stem cell-containing liquid processed by the specific cell removing device, it is possible to effectively suppress recurrence as well as suppress rejection.
  • the laser irradiation means is means capable of irradiating the first processing flow path with the probe laser light for cell detection in addition to the destructive laser light, and the predetermined cell detection
  • the means is means for detecting the predetermined cell using a first marker that expresses fluorescence upon irradiation with the probe laser light
  • the control means is configured to irradiate the first processing flow path with the probe laser light.
  • the marker is CD34 or CD38
  • the predetermined cell detection means detects cells in the hematopoietic stem cell-containing liquid based on scattered light with respect to irradiation with the probe laser light.
  • CD34 is the 34th CD (cluster of differentiation: differentiation antigen group) in the human cell differentiation antigen (HCDM) workshop, and functions as a hematopoietic stem cell marker that is positively expressed in hematopoietic stem cells.
  • CD38 is the 38th CD in the HCDM workshop, is a membrane protein that activates the proliferation of lymphocytes, and is negatively expressed in hematopoietic stem cells. Therefore, by destroying CD34-negative cells as predetermined cells among the detected cells, tumor cells and the like can be destroyed while the hematopoietic stem cells remain. In addition, by destroying CD38 positive cells among the detected cells as predetermined cells, tumor cells and the like can be destroyed while the hematopoietic stem cells remain.
  • the laser device that emits the probe laser light may be configured as a device different from the laser device that emits the destructive laser light, or the laser light may be switched by a single laser device.
  • the predetermined cell detection unit irradiates the first processing flow channel with a short pulse light as a group of light beams having different wavelengths sequentially on at least a straight line, and reflects by the irradiation.
  • Means for detecting the predetermined cell by forming an image of the cell in the hematopoietic stem cell-containing liquid based on light and determining whether the cell is the predetermined cell based on the image of the formed cell It can also be.
  • the predetermined cell detection means includes: a short pulse light source that repeatedly outputs short pulse light at an interval of femtoseconds to nanoseconds; A spectroscopic unit, an irradiating unit that irradiates the imaging light channel with the wavelength-specific light group, a condensing unit that collects the reflected wavelength-specific light group reflected by the irradiation to be reflected pulsed light, and the reflection
  • An optical fiber for wavelength selection that outputs pulsed light and sequentially outputs light of different wavelengths as time passes, and an image of a cell based on the intensity of light for each wavelength output from the optical fiber for wavelength selection It may be a means having a cell image forming part to be formed and a determination part for determining whether or not the cell is the predetermined cell based on the image of the formed cell.
  • images of cells in the hematopoietic stem cell-containing liquid are formed based on irradiation of reflected light of different wavelengths at least on a straight line and the reflected light, so that the amount of photons (light intensity)
  • CCD image sensor Charge-Coupled Device-image image sensor
  • cells can be imaged quickly and with high sensitivity. Can be determined.
  • the specific cells can be rapidly removed even when the content of the specific cells in the hematopoietic stem cell-containing liquid is extremely small.
  • the predetermined cell that is disposed in the subsequent stage of the first flow channel device and remains in the hematopoietic stem cell-containing liquid after separating the predetermined cells destroyed using the first flow channel device.
  • the predetermined cell detecting means detects the predetermined cell in the hematopoietic stem cell-containing liquid flowing in the first processing channel, and the second processing channel.
  • the laser irradiation means is capable of irradiating the first processing flow path with the destructive laser light, in addition to the predetermined cell in the hematopoietic stem cell-containing liquid flowing through the second processing flow path.
  • the control means destroys the predetermined cells detected when the predetermined cells are detected in the hematopoietic stem cell-containing liquid flowing in the first processing flow path by the predetermined cell detection means.
  • the predetermined cell detection means detects the predetermined cell when it is detected in the hematopoietic stem cell-containing liquid flowing in the second processing channel.
  • the laser irradiation unit may be controlled to irradiate the predetermined cell with the destructive laser beam. In this way, since the predetermined cell is detected and destroyed twice, the specific cell in the hematopoietic stem cell-containing liquid can be more reliably removed.
  • the laser irradiation means detects cells in the first processing flow path and the second processing flow path in addition to the destructive laser light.
  • the predetermined cell detecting means is a means capable of irradiating a probe laser light for use, and the predetermined cell detecting means uses the first marker that is fluorescently expressed by the irradiation of the probe laser light, and the hematopoietic stem cell-containing liquid that flows into the first processing channel The hematopoietic stem cell-containing liquid that flows into the second processing channel using first detection means for detecting predetermined cells therein and a second marker different from the first marker that expresses fluorescence when irradiated with the probe laser light Second detecting means for detecting predetermined cells therein, wherein the control means is configured to irradiate the laser beam to the first processing flow path with the probe laser light.
  • the laser irradiation means is controlled so as to irradiate the processing flow path, and when the predetermined cell is detected by the second detection means, the laser irradiation means is applied to irradiate the detected predetermined cell with the destructive laser light. It can also be a means to control.
  • the first marker is CD34
  • the second marker is CD38
  • the first detection means detects cells in the hematopoietic stem cell-containing liquid based on scattered light in response to irradiation with the probe laser light.
  • a means for detecting, as the predetermined cell, a cell that does not cause fluorescence expression by irradiation of the probe laser light and the first marker among the detected cells and the second detection means is configured to detect the probe laser light.
  • a cell in the hematopoietic stem cell-containing liquid is detected based on the scattered light with respect to the irradiation, and a cell that produces fluorescence expression by the irradiation of the probe laser light and the second marker is detected as the predetermined cell among the detected cells. It can also be a means.
  • the apparatus for irradiating the second processing flow path with the probe laser light or the destructive laser light is configured as an apparatus different from the apparatus for irradiating the first processing flow path with the probe laser light or the destructive laser light.
  • the irradiation position of the probe laser beam or the destruction laser beam may be switched by a single device.
  • the alignment channel has a predetermined width within a range of 100 to 500 micrometers and a predetermined depth within a range of 10 to 100 micrometers and a predetermined depth within a range of 10 to 20 times. It can also be a wave-like channel that meanders with the number of repetitions. In this way, the cells in the hematopoietic stem cell-containing liquid can be easily flowed in a line using inertia.
  • the alignment channel has at least one surface that forms a channel with a predetermined width within a range of 10 to 100 micrometers and a predetermined width within a range of 100 to 500 micrometers. May be a straight flow path in which irregularities are formed a predetermined number of times within a range of 20 to 50 times. If irregularities are formed on the surface forming the flow path, a wide area and a narrow area of the cross section of the flow path are generated, an inertial force acts on the cells, and the cells gradually flow in a row.
  • the surface on which the unevenness is formed may be any of the top surface, the bottom surface, and the side surface, and the top surface and the bottom surface are particularly preferable.
  • the depth of the flow path changes due to the unevenness, but the cells circulate at locations where the convex portions are formed (locations where the depth is shallow). It is only necessary to have a depth that can be performed, and the depth may be designed according to the size of cells to be selected.
  • the first separation channel is formed with a post array in which a plurality of cylindrical posts are aligned so that the minimum interval is smaller than three times the diameter of the hematopoietic stem cells. It can also be a channel.
  • the diameter of an object (for example, a cell or a broken cell piece) contained in the fluid is more than 1/3 of the interval between adjacent posts. Small objects flow in the main flow of the fluid, and objects whose diameter is larger than 1/3 of the interval between adjacent posts flow away from the main flow of the fluid.
  • the post array should be arranged so that the diameter of the hematopoietic stem cells is larger than 1/3 of the interval between adjacent posts and the diameter of the debris of a predetermined cell is smaller than 1/3 of the interval between adjacent posts. If formed and arranged at a slight angle with respect to the flow of the hematopoietic stem cell-containing liquid, hematopoietic stem cells can be separated from the main flow while containing the cell fragments of the predetermined cells broken in the main flow of the liquid.
  • the predetermined broken cells are separated from the hematopoietic stem cell-containing liquid.
  • the specific cell removal flow path of the present invention comprises: A flow path for removing specific cells used for autologous hematopoietic stem cell transplantation, in which at least specific cells are removed from a hematopoietic stem cell-containing liquid containing hematopoietic stem cells collected from a patient and transplanted to the patient, An alignment channel that allows cells existing in the hematopoietic stem cell-containing liquid to flow in a row, and a second channel formed downstream of the alignment channel to destroy specific cells in the hematopoietic stem cell-containing liquid.
  • a first separation channel formed integrally with a first treatment channel and a first separation channel formed after the first treatment channel in order to separate specific cells destroyed from the hematopoietic stem cell-containing liquid; Comprising one channel device, This is the gist.
  • the cells present in the hematopoietic stem cell-containing liquid flow in a line in the alignment flow channel of the first flow channel device, and the first flow of the first flow channel device is processed.
  • the specific cells in the hematopoietic stem cell-containing liquid in which the cells have flowed in a row are destroyed, and the broken specific cells are separated from the hematopoietic stem cell-containing liquid in the first separation channel of the first channel device.
  • the destruction process includes a process of detecting cells in a range not including hematopoietic stem cells but including specific cells, and irradiating the detected cells with a destruction laser beam to destroy them.
  • the specific cells are removed from the hematopoietic stem cell-containing liquid while leaving the hematopoietic stem cells. can do.
  • the alignment channel, the first processing channel, and the first separation channel that perform such processing are integrally formed, the specific cell removal channel can be made compact.
  • a second cell for destroying the specific cells remaining in the hematopoietic stem cell-containing liquid after the specific cells destroyed in the first separation channel are separated.
  • the destruction process is a process different from the destruction process in the first processing channel, and detects cells in a range not including hematopoietic stem cells but including specific cells, and irradiating the detected cells with a destruction laser beam. Includes destruction. Therefore, since the specific cells are destroyed and separated by different destruction processes, the specific cells can be more reliably removed from the hematopoietic stem cell-containing liquid while leaving the hematopoietic stem cells.
  • the first flow channel device and the second flow channel device may be integrally formed. In this way, the specific cell removal flow path that can remove specific cells more reliably can be made compact.
  • the alignment channel has a predetermined width within a range of 100 to 500 micrometers and a predetermined depth within a range of 10 to 100 micrometers, and is 10 to 20 times. It may be a wavy flow path that meanders at a predetermined number of repetitions within the range. In this way, the cells in the hematopoietic stem cell-containing liquid can be easily flowed in a line using inertia.
  • the alignment flow channel forms a flow channel with a predetermined width within a range of 100 to 500 micrometers and a predetermined depth within a range of 10 to 100 micrometers. It may be a straight flow path in which at least one surface has irregularities formed a predetermined number of repetitions within a range of 20 to 50 times. If irregularities are formed on the surface forming the flow path, a wide area and a narrow area of the cross section of the flow path are generated, an inertial force acts on the cells, and the cells gradually flow in a row.
  • the surface on which the unevenness is formed may be any of the top surface, the bottom surface, and the side surface, and the top surface and the bottom surface are particularly preferable.
  • the depth of the flow path changes due to the unevenness, but the cells circulate at locations where the convex portions are formed (locations where the depth is shallow). It is only necessary to have a depth that can be performed, and the depth may be designed according to the size of cells to be selected.
  • the first separation flow channel is formed with a post array in which a plurality of cylindrical posts are aligned so that the minimum interval is smaller than three times the diameter of the hematopoietic stem cells. It is also possible to be a flow path.
  • the diameter of an object (for example, a cell or a broken cell piece) contained in the fluid is more than 1/3 of the interval between adjacent posts. Small objects flow in the main flow of the fluid, and objects whose diameter is larger than 1/3 of the interval between adjacent posts flow away from the main flow of the fluid.
  • the post array should be arranged so that the diameter of the hematopoietic stem cells is larger than 1/3 of the interval between adjacent posts and the diameter of the debris of a predetermined cell is smaller than 1/3 of the interval between adjacent posts. If formed and arranged at a slight angle with respect to the flow of the hematopoietic stem cell-containing liquid, hematopoietic stem cells can be separated from the main flow while containing the cell fragments of the predetermined cells broken in the main flow of the liquid.
  • the predetermined broken cells are separated from the hematopoietic stem cell-containing liquid.
  • the first specific cell removal method of the present invention comprises: A method for removing specific cells used for autologous hematopoietic stem cell transplantation, wherein at least specific cells are removed from a hematopoietic stem cell-containing liquid containing hematopoietic stem cells collected from a patient, and transplanted to the patient, (A) flowing the hematopoietic stem cell-containing liquid so that the cells present in the hematopoietic stem cell-containing liquid are in a line, (B) Using the hematopoietic stem cell-containing liquid in which the cells flow in a line, irradiation of the probe laser light for cell detection and fluorescence expression by the first marker, the hematopoietic stem cell is not included but the specific cell is included A cell in which the possibility of the specific cell is recognized, detecting a first predetermined cell as a range of cells, (C) destroying the first predetermined cells by irradiating the detected first predetermined cells with a destruction laser beam for cell
  • a second predetermined cell as a range of cells that is not included, but includes the specific cell and a cell in which the possibility of the specific cell is recognized, (F) irradiating the detected second predetermined cells with a destruction laser beam for cell destruction to destroy the second predetermined cells; (G) separating the second predetermined cells destroyed from the hematopoietic stem cell-containing liquid, It is characterized by that.
  • the hematopoietic stem cell-containing liquid is caused to flow so that the cells existing in the hematopoietic stem cell-containing liquid are in a line, and the cells are supplied to the hematopoietic stem cell-containing liquid that flows in a line.
  • the first predetermined cells as a range of cells that do not include hematopoietic stem cells but include specific cells but include cells that can be identified as specific cells are detected using the probe laser light irradiation and fluorescence expression by the first marker.
  • the detected first predetermined cells are destroyed by irradiating the detected first predetermined cells with a destruction laser beam for cell destruction, and the first predetermined cells are separated from the hematopoietic stem cell-containing liquid. Then, the hematopoietic stem cell-containing liquid after separating the destroyed first predetermined cells is irradiated with the probe laser light and the fluorescence expression by the second marker, so that hematopoietic stem cells are not included but specific cells are included and possible
  • the cells having sex are detected as second predetermined cells as a range of cells, and the detected second predetermined cells are destroyed by irradiating with a destruction laser beam for cell destruction, and the second predetermined cells are destroyed from the hematopoietic stem cell-containing liquid.
  • the step (b) uses CD34 as the first marker, and the cells in the hematopoietic stem cell-containing liquid are determined based on scattered light in response to irradiation with the probe laser light.
  • the step (e) includes the step (e) CD38 is used as two markers, and the cells in the hematopoietic stem cell-containing liquid are detected based on the scattered light with respect to the probe laser light irradiation, and among the detected cells, the probe laser light irradiation and the second marker It may be a step of detecting a cell that produces fluorescence expression as the second predetermined cell. That. CD34 and CD38 have been described above.
  • cells negative for CD34 are destroyed and separated as the first predetermined cells, and among the cells in the hematopoietic stem cell-containing liquid after the first predetermined cells are broken and separated, they are positive for CD38.
  • By destroying and separating these cells as second predetermined cells it is possible to more reliably destroy and separate tumor cells and the like while the hematopoietic stem cells remain.
  • the second specific cell removal method of the present invention comprises: A method for removing specific cells used for autologous hematopoietic stem cell transplantation, wherein at least specific cells are removed from a hematopoietic stem cell-containing liquid containing hematopoietic stem cells collected from a patient, and transplanted to the patient, (A) flowing cells present in the hematopoietic stem cell-containing liquid in a line; (B) irradiating the hematopoietic stem cell-containing liquid, in which cells flow in a row, with a short pulse light at least in a straight line as a group of light having different wavelengths, and in the hematopoietic stem cell-containing liquid based on reflected light by the irradiation Forming an image of the cell, and detecting the specific cell by determining whether the cell is the specific cell based on the image of the formed cell, (C) destroying the specific cells by irradiating the detected specific cells with a destruction laser beam for cell destruction;
  • the hematopoietic stem cell-containing liquid is caused to flow so that cells existing in the hematopoietic stem cell-containing liquid are in a line, and the short pulse light is applied to the hematopoietic stem cell-containing liquid that flows in a line.
  • the short pulse light is applied to the hematopoietic stem cell-containing liquid that flows in a line.
  • At least on a straight line as a group of light of different wavelengths and forms an image of cells in the hematopoietic stem cell-containing liquid based on the reflected light from the irradiation, and the cells are identified based on the formed cell image.
  • a specific cell is detected by determining whether it is a cell.
  • the detected specific cells are destroyed by irradiating with a destruction laser beam for cell destruction, and the destroyed specific cells are separated from the hematopoietic stem cell-containing liquid.
  • images of cells in the hematopoietic stem cell-containing liquid are formed based on irradiation of reflected light of different wavelengths at least on a straight line and the reflected light, so that the amount of photons (light intensity) Compared with CCD image sensor (Charge Coupled Device image sensor) that depends on, quickly and sensitively image cells to determine whether the imaged cells are specific fibrillation Can do.
  • CCD image sensor Charge Coupled Device image sensor
  • the specific cells in the hematopoietic stem cell-containing liquid can be more reliably and rapidly removed. Therefore, by performing autologous hematopoietic stem cell transplantation using the hematopoietic stem cell-containing liquid treated by this specific cell removal method, it is possible not only to suppress rejection but also to effectively suppress recurrence.
  • FIG. 2 is a schematic configuration diagram showing an outline of a configuration of a STEAM camera 40.
  • FIG. It is a schematic block diagram which shows the outline of a structure of the cell sorting apparatus 120 of the modification.
  • FIG. 6 is an explanatory view showing a portion of an alignment channel 232 in the A1-A2 cross section in FIG. 4 of a cell sorting channel device 230 of a modified example.
  • FIG. 10 It is a block diagram which shows the outline of a structure of the cell removal apparatus 1020 for 1st hematopoietic stem cells as one Example of this invention.
  • 5 is a flowchart showing an example of CD34 positive test laser irradiation control executed by a control device 1080.
  • 5 is a flowchart showing an example of CD38 negative test laser irradiation control executed by a control device 1080.
  • 1 is a configuration diagram showing an outline of a configuration of a STEAM camera device 1150.
  • FIG. 10 is a flowchart showing an example of destructive laser irradiation control executed by a control device 1180. It is a schematic block diagram which shows the outline of a structure of the flow-path device 1230 for cell destruction removal of a modification.
  • FIG. 16 is an explanatory diagram showing a part of an alignment channel 1232 in the A1-A2 cross section in FIG. 12 of a cell destruction removal channel device 1230 of a modified example.
  • FIG. 1 is a schematic configuration diagram showing an outline of the configuration of the cell sorting apparatus 20 of the embodiment.
  • a cell sorting apparatus 20 according to the embodiment includes a cell sorting channel device 30 for flowing a liquid (cell-containing liquid) containing a plurality of types of cells, and a STEAM camera 40 for photographing cells in the cell-containing liquid.
  • cells mainly include living cells, for example, red blood cells, platelets, white blood cells, etc. in blood, as well as memory B cells, natural killer T cells, antigen-specific T cells, hematopoietic stem cells.
  • CECs Circulating Endothelial Cells
  • CTCs Circulating Tumor Cells
  • Cancer Stem Cells These rare cells will be described later.
  • Examples of the “cell-containing liquid” include body fluids such as blood and solutions obtained by diluting them with a solvent such as physiological saline.
  • the cell sorting channel device 30 allows cells in the cell-containing liquid to flow in a line from the upstream side by a material that is not eroded by the cell-containing liquid such as polydimethylsiloxane (PDMS) or epoxy resin.
  • the cell sorting channel device 30 is preferably formed of polydimethylsiloxane when the flow rate of the cell-containing liquid is less than 1.5 m / s, and the flow rate of the cell-containing liquid is 1.5 m / s or more. In some cases, it is preferably formed of an epoxy resin.
  • the alignment channel 32 is formed as a wavy channel that meanders with a width of 100 to 500 ⁇ m, a depth of 10 to 100 ⁇ m, and a repetition count of 10 to 20 times.
  • the number of repetitions of the sheath meander is appropriately determined depending on the size of the cells contained in the cell-containing liquid.
  • the channels meandering repeatedly, so that cells randomly present in the cell-containing liquid are gradually aligned in a line due to inertia during the meandering.
  • the imaging channel 34 is a channel having the same width and the same depth as the alignment channel 32 and is basically formed in a straight line.
  • the sorting channel 36 includes a main channel 36a continuous from the imaging channel 34, a branch channel 36b branched from the main channel 36a, and a branch channel 36b immediately upstream of the branch point.
  • a cavitation generation region 36c that generates cavitation for branching cells into the branch channel 36b is formed on the side.
  • the main flow channel 36 a is arranged on a straight line of the imaging channel 34 so that the cell-containing liquid from the imaging channel 34 flows smoothly, and the width of the channel is equal to the width of the imaging channel 34.
  • the depth is formed so as to be the same as or slightly smaller than the depth of the photographing channel 34.
  • the branch flow path 36b is a flow path that branches at an angle within a range of 10 to 60 degrees with respect to the flow of the main flow path 36a so that the cells to be sorted branch smoothly from the main flow path 36a.
  • the depth is formed to be the same as the main flow path 36a so that the width is narrower than the main flow path 36a.
  • the cavitation generation region 36c includes a cavitation channel 36d disposed on the opposite side of the main flow channel 36a from the branch flow channel 36b, and the main flow channel 36a and the cavitation channel immediately upstream of the branch point of the branch flow channel 36b.
  • the communication passage 36e communicates with the flow path 36d.
  • the cavitation channel 36d is formed so that the width is the same as or slightly larger than the width of the imaging channel 34, and the depth is the same as that of the imaging channel 34.
  • Inactive liquid for example, physiological saline or cell-containing liquid solvent
  • the communication passage 36e is formed to have a width of 50 to 200 ⁇ m and a depth that is the same as that of other flow paths.
  • the force at the time of expansion due to the cavitation acts on the cell flowing down near the connection point of the communication path 36e of the main flow path 36a via the communication path 36e, and the cell is pushed out to the branch flow path 36b side. As a result, it flows into the branch flow path 36b. Therefore, the selection target cell can be branched to the branch flow path 36b by irradiating the pulse laser at the timing when the selection target cell reaches the vicinity of the connection point of the communication path 36e of the main flow path 36a.
  • the STEAM camera 40 includes a short pulse light source 41 that repeatedly outputs short pulse light at intervals of femtoseconds to nanoseconds, light from the short pulse light source 41 is transmitted, and light from the opposite side is reflected.
  • a half-mirror 42 that splits the light from the short pulse light source 41, and a spectroscope 43 that condenses (synthesizes) the split light from the opposite side, and a lens 44 that collimates the split light.
  • a wavelength selecting optical fiber 45 that sequentially changes the pulsed light reflected from the half mirror 42 with the passage of time, a light intensity detector 46 that detects the intensity of the light, and the light for each wavelength.
  • an image formation processing unit 47 that forms an image based on the intensity.
  • the spectroscope 43 is adjusted so as to perform spectral separation so that the wavelength is sequentially different for each position on the straight line. For this reason, the light split by the spectroscope 43 and converted into parallel light by the lens 44 becomes a group of parallel wavelength-specific light (wavelength-specific light group) having different wavelengths sequentially for each position on the straight line.
  • the road 34 is irradiated. That is, the imaging flow path 34 is irradiated with light having different wavelengths depending on the position on the straight line in the width direction.
  • the reflected light of each wavelength-specific light group (reflected wavelength-specific light group) is collected by the lens 44 with information on the straight line in the width direction of the object flowing through the imaging channel 34 as the light intensity, and collected by the spectroscope 43. Light (combined) is used as pulsed light (reflected pulsed light).
  • the wavelength selecting optical fiber 45 includes a polarization maintaining optical fiber 45a and an optical pump 45b that inputs light in the reverse direction to amplify the intensity of light from the optical fiber 45a. Since the polarization maintaining optical fiber 45a has a property that the output time varies depending on the wavelength of the input light, when the reflected pulse light is input, the light is output in order from the light having the longest wavelength. Therefore, the light intensity of each wavelength can be detected by detecting the light intensity of the light output from the wavelength selecting optical fiber 45 with the light intensity detector 46 over time. As described above, the intensity of light for each wavelength is information on an object on a straight line in the width direction of the imaging flow path 34. Therefore, the image forming processing unit 47 uses the intensity of light for each wavelength. An image of an object flowing in the path 34 can be formed.
  • the image formation processing unit 47 can be configured by a general-purpose computer in which a processing program for forming an image as information on an object on a straight line in the width direction of the imaging channel 34 is installed.
  • the STEAM camera 40 spectrally divides short pulse light, irradiates light having different wavelengths sequentially according to the position on the straight line in the width direction of the imaging channel 34, and condenses (synthesizes) the reflected light.
  • the wavelength selecting optical fiber 45 sequentially outputs light having a longer wavelength, and forms an image of an object (cell) flowing in the imaging channel 34 based on the intensity of light for each wavelength. Therefore, a CCD image sensor (Charge Coupled) is used. As compared with the case where an image is formed using a device (image sensor), an image can be formed quickly and with high sensitivity.
  • the image determination unit 50 is configured by a general-purpose computer in which a processing program for matching an image as to whether or not a cell flowing in the imaging channel 34 imaged by the STEAM camera 40 matches the target cell is installed. Can do. For image matching, it is possible to apply, for example, a method for determining whether or not a cell image input within a certain error range matches a previously registered cell image.
  • the image determination unit 50 outputs an irradiation control signal to the pulse laser 60 at an appropriate timing when the cell images match.
  • the "appropriate timing" is that when the pulse laser 60 irradiates the irradiation spot 36f of the cavitation flow path 36d with the pulse laser to cause cavitation, the expansion force coincides through the communication passage 36e. It is the timing of acting on the cells, and can be determined by experiments or the like based on the flow rate of the cell-containing liquid flowing in the imaging channel 34, the distance from the imaging position of the imaging channel 34 to the communication passage 36e, and the like.
  • the pulse laser 60 for example, the trade name FPL-04CTF (CALMAR LASER) can be used, and as the pulse laser for irradiating the irradiation spot 36f, an irradiation time of about 1 picosecond and an intensity of about 1 nJ are suitable. is there.
  • the irradiation spot 36f is irradiated with the pulse laser, as described above, the pressure of the irradiation spot 36f decreases and cavitation occurs in a radius range of about 100 ⁇ m.
  • this pulse laser 60 it is possible to generate about 20 thousand cavities / second effective for cell sorting.
  • the cell-containing liquid is allowed to flow so that the particle Reynolds number is about 1 or less when the number of cells is one particle from the inlet of the alignment channel 32 of the cell sorting channel device 30.
  • the cells in the cell-containing liquid flow in a line as they flow down the alignment channel 32.
  • the photographing channel 34 cells flowing in a row in the photographing channel 34 are photographed by the STEAM camera 40 to form an image, and the formed image is output to the image determination unit 50.
  • the image determination unit 50 matches the image of the cell to be selected with the image of the cell input from the STEAM camera 40. An irradiation control signal is output.
  • the cell-containing liquid is caused to flow through the cell sorting channel device 30 including the sorting channel 32, the imaging channel 34, and the sorting channel 36.
  • the cells are photographed by the STEAM camera 40 that forms an image of the cells based on the reflected light obtained by irradiating light having different wavelengths depending on the position on the straight line.
  • the cells to be sorted are sorted by pushing the cells to be sorted flowing in the main flow channel 36a into the branch channel 36b by using cavitation, The cells to be sorted can be quickly selected as compared with the case of dropping one cell at a time from the nozzle.
  • the STEAM camera 40 since it is determined by forming an image of a cell, it is not necessary to use fluorescence. In addition, since the STEAM camera 40 is used, it is possible to form an image quickly and with high sensitivity compared with the case where an image is formed using a CCD image sensor (Charge Coupled Device image sensor). Can be sorted out. As a result, the cells to be sorted can be quickly sorted even if the cells having a very small content in the cell-containing liquid are selected.
  • CCD image sensor Charge Coupled Device image sensor
  • the cell sorting flow path device 30 used in the cell sorting apparatus 20 of the embodiment by having the alignment flow path 32, cells that are randomly present in the cell-containing liquid can be lined up only by flowing the cell-containing liquid. It can be made to flow. Further, by having the imaging channel 34, the cells in the cell-containing liquid in which the cells flow in a row can be easily imaged, and whether the cells are the cells to be selected based on the captured cell image. It can be determined whether or not.
  • the cells determined to be the cells to be sorted by having the sorting channel 36 are branched from the main flow channel 36a to the branch channel 36b, so that the cells to be sorted are separated from other cells. Can be sorted.
  • the alignment channel 32, the imaging channel 34, and the sorting channel 36 are provided, the cells in the cell-containing liquid are photographed in a row, and it is determined whether or not the cells are to be sorted.
  • a single flow path device can cover the flow path required for sorting by branching to the branch flow path 36b. As a result, the cell sorting device 20 can be downsized.
  • a cavitation channel 36d is formed as the cavitation generation region 36c of the cell sorting channel device 30, and the cavitation channel 36d is a liquid that does not act on cells in the cell-containing liquid.
  • the cavitation flow path 36d may be configured as a closed region. In this case, the closed region may be filled with physiological saline or a cell-containing liquid solvent.
  • the cavitation flow path 36d and the communication path 36e are formed as the cavitation generation region 36c of the cell sorting flow path device 30, but the cell sorting apparatus 120 of the modified example of FIG.
  • a dent protruding outward
  • the center of the dent is an irradiation spot 136f of the pulse laser.
  • the alignment channel 32, the imaging channel 34, the STEAM camera 40, the image determination unit 50, and the pulse laser 60 in the cell sorting channel device 130 of the cell sorting device 120 of the modified example are the same as those in the above-described embodiment. It is.
  • cavitation is generated by irradiating the irradiation spots 36f and 136f with the pulse laser 60 to cause the cavitation to branch the cells to be sorted into the branch channel 36b.
  • the method of branching the cells to be sorted into the branch flow path 36b may be a method other than using cavitation.
  • a cell determined to be a selection target cell is charged positively or negatively, a negative or positive electrode is arranged on the branch flow path 36b side, and the cell is migrated to the branch flow path 36b side to branch.
  • a technique may be used.
  • the alignment channel 32 of the cell sorting channel devices 30 and 130 has a width of 100 to 500 ⁇ m, a depth of 10 to 100 ⁇ m, and a repetition count of 10 to 20. It is formed as a meandering wavy flow path, but as shown in the alignment flow path 232 of the cell sorting flow path device 230 of the modification of FIGS. 4 and 5, the width is 100 to 500 ⁇ m and the depth is 10 Formed on the top surface forming a rectangular flow path of ⁇ 100 ⁇ m as a straight flow path in which irregularities consisting of a convex part 232a convex downward and a concave part 232b concave downward are formed 20 to 50 times.
  • the width and depth of the alignment channel 232 and the number of irregularities are appropriately determined depending on the size of the cells contained in the cell-containing liquid. For example, when the cells are about 10 ⁇ m in diameter, the alignment channel 232 has an appropriate width of about 100 ⁇ m, an appropriate depth of about 40 ⁇ m, and the height and length of the convex portion 232a is 20 ⁇ m. About 10 ⁇ m and about 10 ⁇ m are appropriate, the length of the recess 232 b is about 100 ⁇ m, and the number of irregularities is about 30 times.
  • the imaging channel 234 and the sorting channel 236 in the cell sorting channel device 230 of the modification are the imaging channel 23 and the sorting channel in the cell sorting channel device 30 of the embodiment illustrated in FIG. 36.
  • the cell sorting channel device 230 of this modification it is preferable to flow the cell-containing liquid through the alignment channel 232 of the cell sorting channel device 230 of this modification so that the Reynolds number is about 80 or less.
  • the alignment flow path 232 of the cell sorting flow path device 230 of the modified example since the top surface is uneven, a wide area and a narrow area of the cross section of the flow path are generated, and the cells in the cell-containing liquid are Inertial forces act and the cells gradually align in a row. Therefore, the cell sorting channel device 230 of the modified example can achieve the same effect as the cell sorting channel device 30 of the embodiment.
  • the top surface of the flow path is uneven, but the bottom surface of the flow path is uneven, What is necessary is just to form an unevenness
  • the applicant refers to the cell sorting channel device 230 of the modified example as the zigzag type because the alignment channel 32 of the cell sorting channel devices 30 and 130 of the example and the modification meanders zigzag.
  • the alignment channel 232 is referred to as a pocket type with the recess 232b regarded as a pocket.
  • the maternal blood contains a few fetal cells (nucleated blood cells and erythroblasts). For this reason, if cell sorting is performed using fetal cells as cells to be sorted using the cell sorting apparatuses 20 and 120 according to the embodiments and the modified examples, genetic DNA is examined by examining the DNA of the sorted fetal cells. Congenital diseases can be diagnosed early after the sixth week of pregnancy.
  • Memory B cells have important effects in various scenes such as infection protection, allergies, and autoimmune diseases, but many details are not yet known.
  • the memory B cells are selected as cells to be sorted and the cells are sorted using the cell sorters 20 and 120 according to the embodiment or the modified example, human immunodeficiency virus using the sorted memory B cells ( It can contribute to creation of vaccines against HIV (Human Immunodeficiency Virus) and other diseases.
  • Natural killer T cells have the properties of both NK cells (natural killer cells, natural killer cells) and T cells (T cells, T lymphocytes). It is responsible for bridging. Therefore, if natural killer T cells are selected as cells to be sorted using the cell sorters 20 and 120 of the examples and modifications, autoimmune disease onset control, allergy regulation, antitumor action, It can contribute to miscarriage.
  • Antigen-specific T cells are rare immune cells that are expected to act as therapeutic agents for various types of cancer treatments and viral infections. Therefore, if antigen-specific T cells are selected as cells to be sorted and cell sorting is performed using the cell sorters 20 and 120 of the examples and modifications, development of therapeutic agents for cancer treatment and viral infections Can help.
  • Hematopoietic Stem Cells are stem cells that differentiate into other blood cells. For this reason, if Hematopoietic Stem Cells are selected as cells to be sorted and the cells are sorted using the cell sorters 20 and 120 of the examples and modifications, it can contribute to leukemia treatment.
  • Circulating Endothelial Cells (CECs) and Circulating Endothelial progenitor cells are cells on the inner wall of the defect and cells before differentiation, and increase during cardiovascular disease and cancer.
  • Circulating Endothelial Cells CECs
  • Circulating Endothelial progenitor Cells CTCs
  • cardiovascular diseases and cancers can be obtained. It can contribute to early detection.
  • Circulating Tumor Cells CTCs
  • Circulating Tumor Cells are cells that cause cancer metastasis and increase as cancer progresses. For this reason, if the cells are selected using the cell sorters 20 and 120 according to the embodiments and the modified examples using Circulating Tumor Cells (CTCs) as cells to be sorted, it is possible to determine the possibility of cancer metastasis and the progression of cancer. Can contribute.
  • Circulating Cancer Stem Cells are cancer stem cells that flow in the blood, are part of circulating tumor cells in the blood, and are responsible for actual cancer metastasis. Therefore, if Circulating Cancer Stem Cells are selected as cells to be selected and cell sorting is performed using the cell sorters 20 and 120 of the examples and modifications, it is possible to determine the possibility of cancer metastasis and the progression of cancer. Can contribute.
  • FIG. 6 is a configuration diagram showing an outline of the configuration of the first hematopoietic stem cell removal device 1020.
  • the first hematopoietic stem cell removal device 1020 of the embodiment includes a first cell destruction / removal channel device 1030 integrally formed to flow a liquid containing hematopoietic stem cells (hematopoietic stem cell-containing liquid).
  • hematopoietic stem cell-containing fluid refers to a fluid collected from the bone marrow of a patient, or stem cells from the bone marrow using G-CSF (granulocyte colony-stimulating factor) that increases leukocytes after the patient is treated with an anticancer agent.
  • G-CSF granulocyte colony-stimulating factor
  • solutions obtained by diluting these collected liquids with a solvent such as physiological saline are also included.
  • the first cell destruction / removal channel device 1030 is made of a material not eroded by the hematopoietic stem cell-containing liquid such as polydimethylsiloxane (PDMS) or epoxy resin, and the cells in the hematopoietic stem cell-containing liquid are lined up from the upstream side.
  • the first separation channel 1036 that separates from the first and the second processing channel 1044 that detects cells in the hematopoietic stem cell-containing liquid as in the first processing channel 1034 and destroys the cells with a destruction laser beam.
  • a second separation channel 1046 that separates the broken cells from the hematopoietic stem cell-containing liquid in the same manner as the first separation channel 1036. It is.
  • the first cell destruction / removal channel device 1030 is preferably formed of polydimethylsiloxane when the flow rate of the hematopoietic stem cell-containing liquid is less than 1.5 m / s, and the flow rate of the hematopoietic stem cell-containing liquid is 1. When it is 5 m / s or more, it is preferably formed of an epoxy resin.
  • the alignment channel 1032 is a wavy channel having a meandering width of 100 to 500 ⁇ m, a depth of 10 to 100 ⁇ m and a number of repetitions of 10 to 20 times.
  • the width is 330 ⁇ m and the depth is 50 ⁇ m and the number of repetitions is 10 to 100 ⁇ m. It is formed as a wavy flow path that fluctuates 15 times.
  • the width and depth of the alignment channel 1032 and the number of times of meandering may be appropriately determined depending on the size of cells contained in the hematopoietic stem cell-containing liquid.
  • the aligning flow path 1032 the flow path repeatedly meanders, so that cells randomly present in the hematopoietic stem cell-containing liquid gradually align in a line due to inertia during the meandering.
  • the first processing flow path 1034 is formed at the subsequent stage of the alignment flow path 1032 so as to be basically linear with a flow path having the same width and the same depth as the alignment flow path 1032. Yes.
  • the first separation channel 1036 is formed at the subsequent stage of the first processing channel 1034, and the sheath flow channels 1036a and 1036b for joining the same sheath flow as the solvent of the hematopoietic stem cell-containing liquid from both sides to the main flow.
  • a post-array flow path 1036d in which a plurality of cylindrical posts 1036c are regularly formed to distinguish hematopoietic stem cells from broken cells, and hematopoietic stem cells containing hematopoietic stem cells separated from broken cells
  • the communication channel 1036e is configured to flow the liquid to the second processing channel 1044, and the separation channel 1036f is configured to flow the broken cells.
  • the sheath flow channels 1036a and 1036b are about half the width of the first processing channel 1034 and are formed at the same depth as the first processing channel 1034.
  • the flow from the first processing channel 1034 is as follows.
  • the solvent is flowed at a slightly high pressure to form a sheath flow.
  • the hematopoietic stem cell-containing liquid can be flowed to the center of the flow path.
  • the plurality of posts 1036c are formed as cylinders having a radius of 60 ⁇ m, and are arranged so that the minimum width between adjacent posts 1036c is 30 ⁇ m.
  • the post array has a slight angle with respect to the flow path direction (for example, about 5 to 6 degrees), that is, the straight line of the posts 1036c arranged in the flow direction has a slight angle with respect to the flow path.
  • a plurality of posts 1036c are arranged so that When a fluid is caused to flow through the post array channel 1036d, according to fluid dynamics, particles smaller than 1/3 of the minimum width of the adjacent post 1036c (broken cells) flow in the same manner as the fluid flow. Particles that are larger than 1/3 of the minimum width of adjacent posts 1036c (unbroken cells) flow away from the fluid flow to the left and right.
  • the post array is formed at a slight angle with respect to the direction of the flow path, particles (unbroken cells) whose diameter is larger than 1/3 of the minimum width of the adjacent post 1036c are not collected. It can be set apart from the flow in a certain direction.
  • the post array is slightly rotated counterclockwise to have an angle, so that large particles (unbroken cells) flow away in the right direction. Therefore, large particles (unbroken cells) are close to the communication channel 1036e near the outlet of the post-array channel 1036d, and therefore flow into the communication channel 1036e together with the solvent of the sheath flow.
  • the connecting channel 36e is formed to have the same width and the same depth as the first processing channel 1034.
  • the second processing channel 1044 is a channel having the same width and the same depth as the communication channel 1036e and basically a straight line, similarly to the first processing channel 1034, downstream of the communication channel 1036e. It forms so that it may become a shape.
  • the second separation channel 1046 has a configuration that is the same as that of the first separation channel 1036, that is, a sheath flow channel 1046a, 1046b, a post-array channel 1046d, in the subsequent stage of the second processing channel 1044.
  • An output channel 1046e corresponding to the communication channel 1036e and a separation channel 1046f are configured.
  • the post-array flow path 1036d of the second separation flow path 1046 is also rotated to the left to give an angle so that large particles ( Non-destructed cells) flow away to the right and flow into the output channel 1046e, and small particles (broken cells) flow near the center of the post-array channel 1046d and flow into the separation channel 1046f. .
  • the laser 1060 for example, a source-integrated high-power CW green laser manufactured by Spectra-Physics Co., Ltd. can be used, and the output can be changed or separated by a beam splitter. Thus, a probe laser beam for cell detection and a destructive laser beam for cell destruction can be output.
  • Laser light from the laser 1060 is separated into a mirror 1062 and a movable mirror 1066 by a beam splitter 1061.
  • the laser light applied to the mirror 1062 is applied to the first processing channel 1034 via the movable mirror 1063 and the half mirror 1064, and the scattered light and reflected light are light transmitted via the half mirror 1064 and the mirror 1065. Input to detector 1070.
  • the laser light applied to the movable mirror 1066 is applied to the second processing flow path 1044 via the half mirror 1067, and the scattered light and reflected light are detected by the photodetector via the half mirror 1067 and the mirror 1065. 1070 is input.
  • the movable mirrors 1063 and 1066 are provided with actuators 1063a and 1066a for changing the rotation angle of the mirror.
  • the rotation angle may be changed by an angle corresponding to the distance that the cell moves (flows) in the first processing channel 1034 or the second processing channel 1044 in the time from the irradiation of the laser beam to the irradiation of the destructive laser beam. It can be done. This rotation angle is determined by the flow rate of the hematopoietic stem cell-containing liquid in the first processing channel 1034 and the second processing channel 1044, the movable mirrors 1063 and 1066, the first processing channel 1034 and the second processing channel 1044. And the time required from the irradiation of the probe laser beam to the irradiation of the destructive laser beam.
  • the photodetector 1070 detects the light in the hematopoietic stem cell-containing liquid flowing in the first processing channel 1034 and the second processing channel 1044 by detecting the light intensity of the scattered light with respect to the probe laser light irradiated.
  • a cell expresses fluorescence by using a marker, it is composed of a plurality of optical sensors (for example, photodiodes) that detect the light intensity due to the fluorescence expression.
  • control device 1080 is configured as a microcomputer centered on a CPU, and includes a ROM, a RAM, an input port, an output port and the like in addition to the CPU.
  • a detection signal from the photodetector 1070 is input to the control device 1080 via an input port, and a drive control signal to the laser 1060 and the movable mirrors 1063 and 1066 is output from the output port from the control device 1080. It is output.
  • the first cell destruction / removal channel device 1030 has a hematopoietic stem cell so that the particle Reynolds number is about 1 or less when the cell is one particle from the entrance of the alignment channel 1032. It is assumed that the contained liquid is flowing. As described above, when the hematopoietic stem cell-containing liquid is caused to flow through the alignment channel 1032 so that the particle Reynolds number is about 1 or less, the cells in the hematopoietic stem cell-containing liquid are lined up while flowing down the alignment channel 1032. And become flowing.
  • CD34 is added to the hematopoietic stem cell-containing liquid as a marker before reaching the first processing channel 1034.
  • CD34 is the 34th CD (cluster of differentiation: differentiation antigen group) in the human cell differentiation antigen (HCDM) workshop and has a function as a hematopoietic stem cell marker that is positively expressed in hematopoietic stem cells. It is.
  • CD38 is added to the hematopoietic stem cell-containing liquid as a marker before reaching the second processing channel 1044.
  • CD38 is the 38th CD in the HCDM workshop, is a membrane protein that activates the proliferation of lymphocytes, and is negatively expressed in hematopoietic stem cells.
  • the communication channel 1036e is configured as a channel that is long enough for the CD 34 to lose its function.
  • FIG. 7 is a flowchart showing an example of CD34 positive test laser irradiation control executed by the control device 1080 on the first processing flow path 1034.
  • an irradiation control signal is output to the laser 1060 so that the laser beam 1060 is irradiated with the probe laser light to the first processing flow path 1034 (step S1100).
  • a light detection signal based on scattered light or reflected light by irradiation of the probe laser light is input from the light detector 1070 (step S1110), and it is determined whether or not a cell is detected by the scattered light (step S1120). Then, it is determined whether CD34 is positively expressed (step S1130). If the cell is not detected, it is determined that it is not necessary to irradiate the destruction laser beam, and this routine is finished.
  • the cell is positively expressed even if the cell is detected, the cell is a hematopoietic stem cell. It is determined that there is a high possibility, and this routine is terminated without irradiating the destructive laser beam.
  • the cell is a first predetermined cell (a cell belonging to a range including not a hematopoietic stem cell and a tumor cell or a cell that may be a tumor cell). Is output, an irradiation control signal is output to the laser 1060 so as to irradiate the first processing flow path 1034 with the destructive laser beam for destroying the cell (step S1140), and this routine ends.
  • the laser 1060 destroys the cell. Since the first predetermined cells are destroyed by irradiation with laser light, the first predetermined cells including tumor cells (specific cells) can be destroyed while the hematopoietic stem cells in the hematopoietic stem cell-containing liquid remain.
  • the hematopoietic stem cell-containing liquid thus processed in the first processing flow path 1034 flows into the first separation flow path 1036, but the post-array flow path 1036d of the first separation flow path 1036 passes through the adjacent post 1036c.
  • the hematopoietic stem cells in the hematopoietic stem cell-containing liquid flow into the communication channel 1036e by forming so that 1/3 of the width is smaller than the diameter of the hematopoietic stem cells and larger than the diameter of the cell pieces of the destroyed cells.
  • the broken cell pieces in the hematopoietic stem cell-containing liquid flow into the separation channel 1036f.
  • the post 1036c is formed as a cylinder having a radius of 60 ⁇ m so that the hematopoietic stem cells and the broken cell pieces are separated in this way, and the minimum width between adjacent posts 1036c is 30 ⁇ m.
  • the post array channel 1036d was formed so that the post array had a slight angle (for example, about 5 to 6 degrees) with respect to the direction of the channel.
  • the cell fragments of the cells destroyed by the first separation flow path 1036 are separated and the hematopoietic stem cell-containing liquid flowing in the communication flow path 1036e is obtained.
  • hematopoietic stem cells in the hematopoietic stem cell-containing liquid are maintained as they are in the range containing tumor cells (specific cells) or cells that may be tumor cells (specific cells) (second cell).
  • the predetermined cells are subjected to destruction processing (hereinafter referred to as “treatment by CD38 negative test”).
  • step S1200 an irradiation control signal is output to the laser 1060 so that the laser beam 1060 irradiates the second processing flow path 1044 with the probe laser light.
  • step S1200 a light detection signal based on scattered light or reflected light by irradiation of probe laser light is input from the light detector 1070 (step S1210), and it is determined whether or not a cell is detected by the scattered light (step S1220). Then, it is determined whether or not positive expression is caused by CD38 (step S1230).
  • the cell is a second predetermined cell (not a hematopoietic stem cell and a cell belonging to a range including tumor cells or cells that may be tumor cells).
  • the irradiation control signal is output to the laser 1060 so as to irradiate the second processing flow path 1044 with the destructive laser beam for destroying the cell (step S1240), and this routine is terminated.
  • a cell is detected and it is determined whether or not the cell is the second predetermined cell.
  • the laser 1060 destroys the cell. Since the second predetermined cells are destroyed by irradiating the laser beam, the second predetermined cells including tumor cells (specific cells) can be destroyed while the hematopoietic stem cells in the hematopoietic stem cell-containing liquid remain.
  • the hematopoietic stem cell-containing liquid thus processed in the second processing flow path 1044 flows into the second separation flow path 1046, and the post-array flow path 1046d of the second separation flow path 1046 passes through the first separation flow path 1036.
  • the post 1036c is formed as a cylinder having a radius of 60 ⁇ m so that hematopoietic stem cells and broken cell pieces are separated, and the minimum width between adjacent posts 1036c is 30 ⁇ m.
  • the post array by forming the post array so as to have a slight angle (for example, about 5 to 6 degrees) with respect to the direction of the flow path, the hematopoietic stem cells in the hematopoietic stem cell-containing liquid can be output flow path 1046e.
  • the broken cell pieces in the hematopoietic stem cell-containing liquid flow into the separation channel 1046f.
  • the hematopoietic stem cells remain by the above-described treatment by the CD34 positive test and the treatment by the CD38 negative test. Since the cells (either the first predetermined cells or the second predetermined cells) belonging to the range including cells that may be specific cells) are destroyed, the hematopoietic stem cells in the hematopoietic stem cell-containing liquid remain more reliably. Tumor cells (specific cells) can be destroyed as they are. In general, it is said that the treatment by the CD34 positive test can remove the tumor cells with a probability of 99.99%, and the treatment by the CD38 negative test can remove the tumor cells with a probability of 99.99%. Therefore, in the first hematopoietic stem cell removal apparatus 1020 of the embodiment, by using both of them, tumor cells can be more reliably removed from the hematopoietic stem cell-containing liquid.
  • the cells in the hematopoietic stem cell-containing liquid flow in a line in the alignment flow path 1032 of the first cell disruption removal flow path device 1030.
  • the first processing channel 1034 is irradiated with the probe laser light and the CD34 positive test using the CD 34 is performed to detect the first predetermined cells including the tumor cells and destroy them with the destruction laser light.
  • the disrupted cells in the hematopoietic stem cell-containing liquid are separated and removed by the separation channel 1036, and further, the processing by the CD38 negative test using the probe laser light and the CD 38 is performed in the second processing channel 1044.
  • the second predetermined cells including tumor cells are detected and destroyed by the destruction laser beam, and the broken cells in the hematopoietic stem cell-containing liquid are separated and removed by the second separation channel 1046. And it makes it possible to reliably remove tumor cells from leaving the hematopoietic stem cells into hematopoietic stem cells containing liquid. Therefore, by performing autologous hematopoietic stem cell transplantation using the hematopoietic stem cell-containing liquid processed by the cell device for hematopoietic stem cells 20, it is possible to effectively suppress recurrence as well as suppress rejection.
  • the hematopoietic stem cell-containing liquid can be obtained simply by flowing the hematopoietic stem cell-containing liquid by having the alignment channel 1032. Cells randomly present in the liquid can flow in a row. Further, by having the first processing flow path 1034 and the second processing flow path 1044, the first predetermined cells containing tumor cells that are not hematopoietic stem cells by performing the processing by the CD34 positive test or the processing by the CD38 negative test, The second predetermined cell can be destroyed.
  • the first separation channel 1036 and the second separation channel 1046 it is possible to separate the cell fragments of the cells and hematopoietic stem cells from the hematopoietic stem cell-containing liquid.
  • the alignment channel 1032, the first processing channel 1034, the first separation channel 1036, the second processing channel 1044, and the second separation channel 1046 are provided, the hematopoietic stem cell-containing liquid is provided.
  • the first predetermined cells and the second predetermined cells including tumor cells are irradiated with a destruction laser beam to be destroyed and separated.
  • a single flow path device can provide the necessary flow path. As a result, the size of the first hematopoietic stem cell removal device 1020 can be reduced.
  • the alignment channel 1032, the first processing channel 1034, the first separation channel 1036, the second processing channel 1044, and the second separation channel are used.
  • the first cell destruction / removal flow channel device 1030 integrally formed with 1046 is used, but the alignment flow channel, the first processing flow channel, and the first separation flow channel are integrally formed. It may be composed of a flow path device and a second flow path device in which the second processing flow path and the second separation flow path are integrally formed. In this case, the hematopoietic stem cell-containing liquid from the connecting flow path of the first separation flow path in the first flow path device may be flowed to the second processing flow path in the second flow path device.
  • the alignment channel device in which only the alignment channel 1032 is formed, the first processing separation channel device in which the first processing channel 1034 and the first separation channel 1036 are integrally formed, and the second The processing flow channel 1044 and the second separation flow channel 1046 may be formed of a second processing separation flow channel device integrally formed.
  • the first processing separation channel device and the second processing separation channel device may be a common member.
  • the process by the CD38 negative test is performed in the second processing flow path 1044.
  • the processing by the CD34 positive test may be executed in the second processing flow path 1044.
  • the process by the CD38 negative test is performed in the second processing flow path 1044.
  • the flow path device may include the alignment flow path 1032, the first processing flow path 1034, and the first separation flow path 1036.
  • the execution of the processing by the CD34 positive test in the first processing flow path 1034 and the execution of the processing by the CD38 negative test in the second processing flow path 1044 are a single laser. 1060, but includes a first laser for performing processing by the CD34 positive test in the first processing flow path 1034, and executes processing by the CD38 negative test in the second processing flow path 1044.
  • a second laser may be provided.
  • irradiation with the probe laser light and irradiation with the destruction laser light are performed by the single laser 1060.
  • the detection laser for irradiation with the probe laser light is provided and the destruction for irradiation with the destruction laser light is performed.
  • a laser for use. That is, a configuration including only a single laser 1060 as in the embodiment, a first laser that irradiates a probe laser beam and a destructive laser beam for executing processing by the CD34 positive test in the first processing channel 1034
  • a probe array for executing processing by the CD34 positive test in the first processing flow path 1034 Sum of two lasers respectively irradiating light and destruction laser light, and two lasers each irradiating probe laser light and destruction laser light for executing processing by the CD38 negative test in the second processing flow path 1044 Any
  • a process of destroying the first predetermined cells is performed in the first processing flow path 1034 using CD34, and the second processing flow path 1044 is used using CD38.
  • the processing for destroying the second predetermined cells was performed.
  • a marker other than CD34 is replaced with CD34.
  • a marker other than CD38 may be used instead of CD38.
  • FIG. 9 is a configuration diagram showing an outline of the configuration of the second hematopoietic stem cell removal device 1120 of the example.
  • the second hematopoietic stem cell removal device 1120 of the example includes a second cell destruction / removal channel device 1130 integrally formed to flow a hematopoietic stem cell-containing liquid containing hematopoietic stem cells, and a hematopoietic stem cell.
  • a STEAM camera device 1150 that detects tumor cells (specific cells) and cells that may be tumor cells (third predetermined cells), a laser 1160 that emits destructive laser light that destroys the cells, and a STEAM camera And a control device 1180 that controls the laser 1160 based on a control signal from the device 1150.
  • the second cell destruction / removal channel device 1130 is similar to the alignment channel 1032, the first processing channel 1034, and the first separation channel 1036 of the first cell destruction / removal channel device 1030 described above.
  • the alignment channel 1132, the first processing channel 1136, and the first separation channel 1136 are formed integrally. Therefore, in the first separation channel 1136, as in the first separation channel 1036, the sheath flow channels 1136 a and 1136 b, the post-array channel 1136 d, and the output channel 1136 e corresponding to the communication channel 1136 e. And a separation channel 1136f.
  • the STEAM camera device 1150 includes a short pulse light source 1151 that repeatedly outputs short pulse light at intervals of femtoseconds to nanoseconds, and light from the short pulse light source 1151 transmits and light from the opposite side.
  • a spectroscope 1153 that splits the light from the reflecting half mirror 1152, the light from the short pulse light source 1151, and condenses (combines) the split light from the opposite side, and a lens 1154 that collimates the split light.
  • a wavelength selecting optical fiber 1155 that sequentially changes the pulsed light reflected from the half mirror 1152 over time, a light intensity detector 1156 that detects the light intensity, and light for each wavelength.
  • An image formation processing unit 1157 that forms an image based on the intensity of the image, and the image-formed cells are third predetermined cells including tumor cells Includes either a determination unit 1158 whether or not a determination, the.
  • the spectroscope 1153 is adjusted so that the wavelength is sequentially different for each position on the straight line. For this reason, the light split by the spectroscope 1153 and converted into parallel light by the lens 1154 becomes a group of light by wavelength (wavelength-specific light group) having different wavelengths sequentially for each position on the straight line.
  • the application channel 1134 is irradiated. That is, the first processing channel 1134 is irradiated with light having different wavelengths depending on the position on the straight line in the width direction.
  • the reflected light of each wavelength-specific light group (reflected wavelength-specific light group) is collected by the lens 1154 with information on the straight line in the width direction of the object flowing through the first processing flow path 1134 as the light intensity, and the spectroscope 1153.
  • the light is condensed (combined) by the light to be pulsed light (reflected pulsed light).
  • the wavelength selecting optical fiber 1155 includes a polarization maintaining optical fiber 1155a and an optical pump 1155b that inputs light in the reverse direction in order to amplify the intensity of light from the optical fiber 1155a. Since the polarization maintaining optical fiber 1155a has a property that the output time varies depending on the wavelength of the input light, when the reflected pulse light is input, the light is output in order from the light having the longest wavelength. Therefore, by detecting the light intensity of the light output from the wavelength selecting optical fiber 1155 by the light intensity detector 1156 over time, the light intensity for each wavelength can be detected. As described above, the light intensity for each wavelength is information on the object on the straight line in the width direction of the first processing flow channel 1134. Therefore, the image forming processing unit 1157 uses the light intensity for each wavelength to generate the first information. It is possible to form an image of an object flowing in the one processing flow path 1134.
  • the determination unit 1158 is a cell that is not a hematopoietic stem cell and that is a range of cells that may be a tumor cell (specific cell) and a tumor cell (specific cell). Image matching is performed to determine whether or not it is a (third predetermined cell). For image matching, it is possible to apply, for example, a method for determining whether an image of a cell input within a certain error range matches an image of a tumor cell (specific cell) registered in advance.
  • the determination unit 1158 and the image forming processing unit 1157 described above can be configured by a general-purpose computer in which the respective processing programs are installed.
  • the STEAM camera device 1150 divides the short pulse light, irradiates light having different wavelengths sequentially according to the position on the straight line in the width direction of the first processing channel 1134, and condenses the reflected light ( Since the wavelength selection optical fiber 1155 sequentially outputs the light having the longest wavelength and forms an image of the object (cell) flowing in the first processing flow channel 1134 based on the intensity of the light for each wavelength. As compared with the case of forming an image using a CCD image sensor (Charge Coupled Device image sensor) that depends on the amount of light (light intensity), an image can be formed quickly and with high sensitivity.
  • CCD image sensor Charge Coupled Device image sensor
  • the laser 1160 for example, a source-integrated high-power CW green laser Millennia eV (trademark registration) manufactured by Spectra-Physics Co., Ltd. can be used.
  • a source-integrated high-power CW green laser Millennia eV (trademark registration) manufactured by Spectra-Physics Co., Ltd. can be used.
  • irradiation with the probe laser beam is unnecessary, it is possible to use one that can irradiate only the destructive laser beam.
  • the control device 1180 is configured as a general-purpose microcomputer, similarly to the control device 1080 described above, and a determination result signal from the STEAM camera device 1150 is input via the input port, and the laser 1160 is driven. A control signal or the like is output from the output port.
  • the control device 1180 may be configured by a single computer together with the image formation processing unit 1157 and the determination unit 1158 of the STEAM camera device 1150.
  • the second cell destructive removal channel device 1130 has a particle Reynolds number when the cell is defined as one particle from the inlet of the alignment channel 1032 as in the first cell destructive removal channel device 1030 described above.
  • the hematopoietic stem cell-containing liquid is allowed to flow so that the value is about 1 or less.
  • the STEAM camera apparatus 1150 makes a line for the hematopoietic stem cell-containing liquid in which the cells flow in a line by the alignment flow path 1132.
  • the cell flowing in the above state is photographed to determine whether the cell is a third predetermined cell as a tumor cell or a cell that may be a tumor cell, and based on the determination result, the laser 1160 destroys the laser beam. Is irradiated.
  • FIG. 11 is a flowchart illustrating an example of destructive laser irradiation control executed by the control device 1180.
  • a determination result output from the STEAM camera device 1150 is input (step S1300), and the cell is a tumor cell (specific cell) or a tumor cell by image matching with respect to a cell photographed based on the determination result. It is determined whether or not it is the third predetermined cell as a range of possible cells (step S1310). When the photographed cell is not the third predetermined cell, it is determined that it is not necessary to irradiate the destruction laser beam, and this routine is ended. When the photographed cell is the third predetermined cell, the routine is for destroying the cell.
  • An irradiation control signal is output to the laser 1160 so as to irradiate the first laser beam 1134 with the destructive laser beam (step S1320), and this routine ends.
  • the STEAM camera device 1150 determines whether or not the cells in the hematopoietic stem cell-containing liquid are the third predetermined cells, and when it is determined that the photographed cells are the third predetermined cells, the laser 1160 generates a destructive laser beam.
  • the third predetermined cells including tumor cells (specific cells) can be destroyed while the hematopoietic stem cells in the hematopoietic stem cell-containing liquid remain.
  • the post 1036c is formed as a cylinder having a radius of 60 ⁇ m and the minimum width between adjacent posts 1036c so that hematopoietic stem cells and broken cell fragments are separated. Is 30 ⁇ m, and the post array is formed at a slight angle (for example, about 5 to 6 degrees) with respect to the direction of the flow path.
  • the hematopoietic stem cells in the hematopoietic stem cell-containing liquid are Flowing to the output flow path 1136e, the broken cell debris in the hematopoietic stem cell containing liquid flows to the separation flow path 1136f. Therefore, in the hematopoietic stem cell-containing liquid flowing in the output flow channel 1136e, the third predetermined cells including tumor cells (specific cells) are destroyed and separated and removed.
  • the cells in the hematopoietic stem cell-containing liquid flow in a line in the alignment flow path 1132 of the second cell destruction removal flow path device 1130.
  • the STEAM camera device 1150 is used to detect whether or not the cell is a third predetermined cell, and a destruction laser is irradiated to destroy the cell.
  • the STEAM camera device 1150 can be used, the cells in the hematopoietic stem cell-containing liquid can be photographed quickly and with high accuracy compared to the case where a CCD image sensor (Charge Coupled Device image sensor) is used.
  • the flow rate of the hematopoietic stem cell-containing liquid in the one processing flow path 1134 can be increased, and the tumor cells can be more reliably removed more rapidly while leaving the hematopoietic stem cells in the hematopoietic stem cell-containing liquid.
  • the hematopoietic stem cell-containing liquid can be obtained simply by flowing the hematopoietic stem cell-containing liquid. Cells randomly present in the liquid can flow in a row. Further, by having the first processing flow path 1034, it is possible to detect and destroy third predetermined cells including tumor cells using the STEAM camera device 1150 and the laser 1160. Then, by having the first separation channel 1136, it is possible to separate cell fragments and hematopoietic stem cells that have been destroyed from the hematopoietic stem cell-containing liquid.
  • the alignment channel 1132, the first processing channel 1134, and the first separation channel 1136 are provided, the cells in the hematopoietic stem cell-containing liquid are caused to flow in a row, and the STEAM camera device 1150 and the laser 1160 Can be used to detect and destroy third predetermined cells including tumor cells, and to provide a single flow channel device for the flow path required for separating the broken cell fragments from hematopoietic stem cells. As a result, the size of the second hematopoietic stem cell removal device 1120 can be reduced.
  • the third predetermined cells including the cells are detected and destroyed, and the cell pieces of the destroyed cells are separated from the hematopoietic stem cells.
  • a cell destruction removal flow path device having a second processing flow path and a second separation flow path is used.
  • the second processing channel may not include hematopoietic stem cells but may destroy predetermined cells in a range including tumor cells.
  • the third predetermined cells including tumor cells may be detected and destroyed using the STEAM camera device 1150 and the laser 1160 in both the first processing channel 1134 and the second processing channel,
  • One of the first processing channel 1134 and the second processing channel is used to detect and destroy third predetermined cells including tumor cells using the STEAM camera device 1150 and the laser 1160, and the other channel. It is good also as what performs the process by CD34 positive test and the process by CD38 negative test by a flow path.
  • the alignment flow path of the first cell destruction removal flow path device 1030 and the second cell destruction removal flow path device 1130 1032 and 1132 were formed as meandering wavy channels having a width of 100 to 500 ⁇ m, a depth of 10 to 100 ⁇ m, and a repetition number of 10 to 20 times.
  • the top surface forming a rectangular channel having a width of 100 to 500 ⁇ m and a depth of 10 to 100 ⁇ m has a convex portion 1232a that is downwardly convex and a concave portion that is downwardly concave.
  • the alignment channel 1232 has an appropriate width of about 100 ⁇ m, an appropriate depth of about 40 ⁇ m, and the height and length of the convex portion 1232 a are 20 ⁇ m.
  • about 10 ⁇ m and about 10 ⁇ m are appropriate, about 100 ⁇ m is appropriate for the length of the recess 1232 b, and about 30 times is appropriate for the number of irregularities.
  • the first processing channel 1234 and the first separation channel 1236 in the cell destruction / removal channel device 1230 of the modification are the first in the first cell destruction / removal channel device 1030 of the embodiment illustrated in FIG. This is the same as the processing channel 1032 and the first separation channel 1036. It is preferable to flow the cell-containing liquid in the alignment flow path 1232 of the cell destruction removal flow path device 1230 of this modification so that the Reynolds number is about 80 or less. In the alignment flow path 1232 of the modified cell destruction removal flow path device 1230, since the top surface is uneven, a wide area and a narrow area of the cross section of the flow path are generated, and the cells in the cell-containing liquid An inertial force acts on the cells, and the cells gradually align in a line.
  • the cell destruction / removal channel device 1230 of the modification can achieve the same effects as the first cell destruction / removal channel device 1030 and the second cell destruction / removal channel device 1130 of the example.
  • the top surface of the flow path is uneven, but the bottom surface of the flow path is uneven, It is only necessary to form irregularities on at least one surface forming the flow path, such as forming irregularities on one of the side surfaces. Considering the effect of aligning the cells in a row, it is preferable to form irregularities on the top and bottom surfaces of the channel.
  • the applicant referred to the zigzag type because the alignment channel 1032 of the first cell destruction removal channel device 1030 and the second cell destruction removal channel device 1130 of the embodiment meanders zigzag,
  • the alignment flow path 1232 of the cell destruction / removal flow path device 1230 in the example is referred to as a pocket type with the recess 1232b regarded as a pocket.
  • the present invention can be used in the cell sorting device manufacturing industry, the specific cell removal device manufacturing industry used for autologous stem cell transplantation, the flow channel device manufacturing industry used in such a specific cell removal device, and the like.

Abstract

　Selon la présente invention, un liquide contenant des cellules est versé dans un dispositif (30) à canal d'écoulement pour sélection de cellules, comprenant un canal d'écoulement pour alignement (32), un canal d'écoulement pour imagerie (34) et un canal d'écoulement pour sélection (36) et, après qu'un écoulement en file indienne de cellules a été formé, les cellules sont imagées par une caméra STEAM (40) pour former une image des cellules sur la base de la lumière réfléchie obtenue par envoi d'une lumière ayant différentes longueurs d'ondes selon une position sur une ligne droite et, quand une cellule imagée est déterminée par concordance d'image prouvant qu'il s'agit d'une cellule cible de sélection, la cellule cible de sélection est sélectionnée par le fait que la cellule cible de sélection s'écoulant dans un canal d'écoulement principal (36a) va être poussée vers l'extérieur et s'écouler vers un canal d'écoulement en ramification (36b) par utilisation d'une cavitation. Une cellule cible de sélection peut ainsi être sélectionnée plus rapidement que par la chute d'une cellule à la fois à partir d'une buse. En conséquence, une cellule cible de sélection peut être rapidement sélectionnée même quand la cellule cible de sélection est une cellule présente selon une proportion extrêmement petite dans le liquide contenant des cellules.
PCT/JP2014/080473 2013-11-21 2014-11-18 Appareil de sélection et d'élimination de cellules, et procédé de sélection et d'élimination de cellules WO2015076251A1 (fr)

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