WO2015076251A1 - Apparatus for selecting and removing cell, and method for selecting and removing cell - Google Patents

Apparatus for selecting and removing cell, and method for selecting and removing cell Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
channel
predetermined
flow path
Prior art date
Application number
PCT/JP2014/080473
Other languages
French (fr)
Japanese (ja)
Inventor
圭介 合田
Original Assignee
国立大学法人東京大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人東京大学 filed Critical 国立大学法人東京大学
Publication of WO2015076251A1 publication Critical patent/WO2015076251A1/en

Links

Images

Classifications

    • 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—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Optical investigation techniques, e.g. flow cytometry 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—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry 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
    • 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—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Dispersion Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Fluid Mechanics (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

　A cell-containing liquid is poured into a cell selection flow channel device (30) comprising an aligning flow channel (32), an imaging flow channel (34), and a selecting flow channel (36), and after a single-file flow of cells has formed, the cells are imaged by a STEAM camera (40) for forming an image of the cells on the basis of reflected light obtained by shining light having different wavelengths according to a position on a straight line, and when an imaged cell is determined by image matching to be a selection target cell, the selection target cell is selected by causing the selection target cell flowing in a mainstream flow channel (36a) to be pushed out and flow to a branching flow channel (36b) using cavitation. A selection target cell can thereby be selected more rapidly than by dropping one cell at a time from a nozzle. As a result, a selection target cell can be rapidly selected even when the selection target cell is a cell having an extremely small content ratio in the cell-containing liquid.

Description

細胞を選別したり除去する装置およびこれらの方法Apparatus and method for sorting and removing cells
　本発明は、細胞を選別したり除去する装置およびこれらの方法に関する。特に、細胞選別装置、細胞選別用流路デバイス並びに細胞選別方法に関し、詳しくは、複数種類の細胞を含有する細胞含有液体から対象細胞を選別する細胞選別装置およびこれに用いる細胞選別用流路デバイス並びに細胞選別方法に関する。また、特定細胞除去装置および特定細胞除去用流路並びに特定細胞除去方法に関し、詳しくは、患者の造血幹細胞を含有する造血幹細胞含有液体から特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去装置およびこうした特定細胞の除去に用いられる特定細胞除去用流路並びに自家造血幹細胞移植に用いられる特定細胞除去方法に関する。 The present invention relates to an apparatus for sorting and removing cells and methods thereof. In particular, the present invention relates to a cell sorting apparatus, a cell sorting channel device, and a cell sorting method. Specifically, 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. In addition, 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.
　従来、この種の細胞選別装置としては、蛍光発光を利用して細胞選別するものが提案されている（例えば、非特許文献１参照）。この装置では、以下のように細胞選別を行なう。選別対象の細胞に対してレーザー照射により蛍光発光が誘起される蛍光剤を複数種の細胞が混在する溶液に添加しておき、その溶液を１細胞が流下できる程度の細い管を介してノズルから滴下する。細い管で流下する溶液にレーザー光を照射し、選別対象の細胞の蛍光発光を誘起する。ノズルから滴下する細胞のうち例えば緑色に蛍光発光した細胞についてはプラスに帯電させ、赤色に蛍光発光した細胞についてはマイナスに帯電させる。そして、プラスに帯電させた電極とマイナスに帯電させた電極の間に細胞を滴下させ、緑色に蛍光発光した細胞についてはマイナス電極側の流路に誘導し、赤色に蛍光発光した細胞についてはプラス電極側の流路に誘導し、蛍光発光しない細胞についてはその中央の流路に誘導することにより、細胞を選別する。 Conventionally, as this type of cell sorting apparatus, an apparatus for sorting cells using fluorescent light emission has been proposed (for example, see Non-Patent Document 1). In this apparatus, 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. Among 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. Then, 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.
　また、自家造血幹細胞移植は、患者の骨髄からの採取や、抗がん剤治療のあとや白血球を増やすＧ－ＣＳＦ（顆粒球コロニー刺激因子）を使って骨髄から幹細胞を血液中に流れ出させた際の成分献血と同様の方法での採取などにより得られた造血幹細胞を、大量の抗ガン剤を投与した後に、輸血と同様に移植することにより行なわれる（例えば、非特許文献２参照）。大量の抗ガン剤を投与すると、腫瘍細胞の破壊だけでなく、造血機能も損傷するため、その後に造血幹細胞を移植することによって損なわれた造血機能を再生する。造血幹細胞移植は、拒絶反応を考慮すると非血縁家族からの移植は非常に困難であり、可能であれば患者本人から採取した造血幹細胞を移植するのが好ましい。 In addition, 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. 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). When a large amount of an anticancer agent is administered, not only the destruction of tumor cells but also the hematopoietic function is damaged, so that the hematopoietic function impaired by transplanting hematopoietic stem cells is then regenerated. Hematopoietic stem cell transplantation is very difficult to transplant from an unrelated family in consideration of rejection, and it is preferable to transplant hematopoietic stem cells collected from the patient if possible.
　しかしながら、上述の細胞選別装置では、ノズルから１細胞ずつ滴下させる必要があるため、単位時間当たりに選別できる細胞数が少なく、溶液中の全ての細胞の選別を完了するのに時間を要する。特に、溶液中の含有率が極めて小さい細胞を選別する場合には、長時間を要してしまう。また、蛍光発光を用いるから、同色に蛍光発光する複数種の細胞が存在するときにこの複数種の細胞から選別対象の細胞を選別することはできず、蛍光発光しない複数種の細胞が存在するときにこの複数種の細胞から選別対象の細胞を選別することはできない。 However, in the above cell sorting apparatus, since it is necessary to drop cells one by one from the nozzle, 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. In addition, since fluorescent emission is used, 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.
　本発明の細胞選別装置および細胞選別方法は、より迅速に対象細胞を選別することができる装置および方法を提供することを主目的とする。本発明の細胞選別装置に用いる細胞選別用流路デバイスは、より迅速に対象細胞を選別するための流路デバイスを提供することを主目的とする。 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.
　また、患者から採取した造血幹細胞を含む液体（造血幹細胞含有液体）に、僅かでも腫瘍細胞が混在すると、移植後に混在した腫瘍細胞により再発するため、造血幹細胞含有液体から腫瘍細胞を根絶しておく必要があるが、造血幹細胞含有液体中の腫瘍細胞の含有率が非常に小さい場合、その根絶は困難なものとなる。 In addition, if a small amount of tumor cells are mixed in a liquid containing hematopoietic stem cells collected from a patient (hematopoietic stem cell-containing liquid), the tumor cells relapse after transplantation, so the tumor cells are eradicated from the hematopoietic stem cell-containing liquid. Although it is necessary, the eradication is difficult when the content of tumor cells in the hematopoietic stem cell-containing fluid is very small.
　本発明の特定細胞除去装置や特定細胞除去方法は、造血幹細胞含有液体に混在し得る腫瘍細胞を除去することを主目的とする。また、本発明の特定細胞除去用流路は、造血幹細胞含有液体に僅かに混在する腫瘍細胞を除去する装置や方法に有効な流路を提供することを主目的とする。 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. In addition, 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.
　本発明の細胞選別装置、細胞選別用流路デバイス並びに細胞選別方法は、上述の主目的を達成するために以下の手段を採った。 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.
　本発明の細胞選別装置は、
　複数種類の細胞を含有する細胞含有液体から対象細胞を選別する細胞選別装置であって、
　前記細胞含有液体中にランダムに存在する細胞が一列に流れるようにする整列用流路と、前記整列用流路の後段に形成された撮影用流路と、前記撮影用流路の後段に本流用流路と該本流用流路から分岐する分岐流路とが形成された選別用流路と、を有する細胞選別用流路デバイスと、
　短パルス光を少なくとも直線上に波長が順次異なる波長別光群を前記撮影用流路に照射し、該照射による反射光に基づいて前記細胞含有液体中の細胞の画像を形成する細胞撮影手段と、
　前記形成された細胞の画像に基づいて該細胞が前記対象細胞であるか否かを判定する判定手段と、
　少なくとも前記判定手段により前記対象細胞であると判定された細胞については、前記本流用流路から前記分岐流路に分岐させることにより選別する選別手段と、
　を備えることを要旨とする。 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.
　この本発明の細胞選別装置では、細胞選別用流路デバイスの整列用流路に複数種類の細胞を含有する細胞含有液体を流すことにより、細胞含有液体中にランダムに存在する細胞は一列になって流れる。細胞が一列になって流れるようになった撮影用流路に短パルス光を少なくとも直線上に波長が順次異なる波長別光群を照射し、この照射によって反射する反射光に基づいて細胞含有液体中の細胞の画像を形成する。そして、形成した細胞の画像に基づいてその細胞が対象細胞であるか否かを判定し、少なくとも対象細胞であると判定された細胞については、本流用流路から分岐流路に分岐させることにより選別する。即ち、基本的には細胞は本流用流路に流れるが、対象細胞であると判定された細胞については分岐流路に分岐させるのである。このように、ノズルから１細胞ずつ滴下する必要がないから、細胞含有液体の流れを速くすることにより、より迅速に対象細胞を選別することができる。また、細胞の画像を形成して判定するから、蛍光発光を用いる必要がない。しかも、短パルス光を少なくとも直線上に波長が順次異なる波長別光群として細胞含有液体に照射し、この照射によって反射する反射光に基づいて細胞含有液体中の細胞の画像を形成するから、ＣＣＤイメージセンサ（Charge Coupled Device image sensor）を用いて画像形成する場合に比して、迅速に高感度に画像形成することができる。これらの結果、細胞含有液体における含有率が極めて小さい細胞を対象細胞としても対象細胞を迅速に選別することができる。ここで、「細胞」としては、主として生体細胞が含まれ、例えば、血液中の赤血球や血小板、白血球などが含まれる他、記憶Ｂ細胞やナチュラルキラーＴ細胞、Antigen-Specific T Cells、Hematopoietic Stem Cells、Circulating Endothelial Cells (CECs)、Circulating Endothelial progenitor Cells、Circulating Tumor Cells (CTCs)、Circulating Cancer Stem Cellsなどの極めて含有率が低い希少細胞などが含まれる。「細胞含有液体」としては、例えば血液などの体液やこれらを生理的食塩水などの溶媒により薄めた溶液などが含まれる。「波長別光群」としては、直線上に波長が順次異なるもの、即ち、直線上の位置毎に波長が順次異なるものだけでなく、平面上の位置毎に波長順次が異なるものとしてもよい。 In the cell sorting apparatus of the present invention, 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. In the cell-containing liquid, based on the reflected light reflected by this irradiation, 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. Then, 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. That is, 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. Moreover, since the cell-containing liquid is irradiated with a short pulse light as a group of light of different wavelengths at least on a straight line, and an image of the cells in the cell-containing liquid is formed based on the reflected light reflected by this irradiation, 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. Here, “cells” mainly include living cells, for example, red blood cells, platelets, white blood cells, etc. in blood, memory B cells, natural killer T cells, Antigen-Specific T cells, Hematopoietic Stems cells , Circulating Endothelial Cells (CECs), Circulating Endothelial progenitor Cells, Circulating Tumor Cells (CTCs), Circulating Cancer Stem Cells, and the like. 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 “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.
　こうした本発明の細胞選別装置において、前記選別手段は、パルスレーザーの照射によって生じるキャビテーションにより前記対象細胞であると判定された細胞については前記本流用流路から前記分岐流路に分岐させる手段である、ものとすることもできる。キャビテーションは、パルスレーザーを用いて２万個／秒程度まで生じさせることができるから、２万個／秒程度まで対象細胞を他の細胞から選別することができる。 In such a cell sorting apparatus of the present invention, 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. Can also be. 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.
　また、本発明の細胞選別装置において、前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路である、ものとすることもできる。こうすれば、慣性を用いて容易に細胞含有液体中の細胞を一列にして流すことができる。 In the cell sorting apparatus according to the present invention, 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.
　あるいは、本発明の細胞選別装置において、前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで流路を形成する少なくとも１つの面には２０～５０回の範囲内の所定繰り返し回数の凹凸が形成された直線状の流路である、ものとすることもできる。流路を形成する面に凹凸が形成されていると、流路の断面の広いところと狭いところとが生じ、細胞に慣性力が作用し、次第に細胞が一列になって流れるようになる。ここで、凹凸が形成される面としては、流路の断面が矩形流路の場合、頂面や底面、側面のいずれかでよく、頂面や底面が特に好ましい。なお、頂面や側面に凹凸を形成する場合、凹凸により流路の深さが変化することになるが、凸部が形成されている箇所（深さが浅くなっている箇所）で細胞が流通することができる程度の深さになっていればよく、選別する細胞の大きさによって深さあを設計すればよい。 Alternatively, in the cell sorting apparatus according to the present invention, 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. Here, when the cross section of the channel is a rectangular channel, 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. In addition, when unevenness is formed on the top surface or the side surface, 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.
　さらに、本発明の細胞選別装置において、前記細胞撮影手段は、フェムト秒ないしナノ秒オーダーの間隔で繰り返し短パルス光を出力する短パルス光源と、前記短パルス光源からの短パルス光を分光して前記波長別光群とする分光部と、前記波長別光群を前記撮影用流路に照射する照射部と、前記照射により反射した反射波長別光群を集光して反射パルス光とする集光部と、前記反射パルス光を入力して時間の経過に伴って順次波長の異なる光を出力する波長選別用光ファイバと、前記波長選別用光ファイバから出力される波長毎の光の強度に基づいて細胞の画像を形成する細胞画像形成部と、を有する手段である、ものとすることもできる。 Further, in the cell sorting apparatus of the present invention, 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. A spectroscopic unit for the wavelength-specific light group, an irradiation unit for irradiating the imaging channel with the wavelength-specific light group, and a collection of reflected wavelength-specific light groups reflected by the irradiation to be reflected pulsed light. 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. And a cell image forming unit that forms an image of the cell based on the above.
　本発明の細胞選別用流路デバイスは、
　複数種類の細胞を含有する細胞含有液体から対象細胞を選別する細胞選別装置に用いられる細胞選別用流路デバイスであって、
　前記細胞含有液体中にランダムに存在する細胞が一列に流れるようにする整列用流路と、
　前記整列用流路の後段に形成された撮影用流路と、
　前記撮影用流路の後段に本流用流路と該本流用流路から分岐する分岐流路とが形成された選別用流路と、
　を備えることを要旨とする。 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.
　この本発明の細胞選別用流路デバイスでは、細胞選別用流路デバイスの整列用流路に複数種類の細胞を含有する細胞含有液体を流すことにより、細胞含有液体中にランダムに存在する細胞は一列に流れる。したがって、撮影用流路では、細胞が一列になって流れるから、細胞含有液体中の細胞を順次撮影することができ、撮影した細胞の画像に基づいてその細胞が対象細胞か否かを判定することができる。そして、対象細胞であると判定された細胞については本流用流路から分岐流路に分岐させることにより、対象細胞を他の細胞から選別することができる。したがって、細胞含有液体中の細胞を一列に流して撮影し、対象細胞か否かを判定して分岐流路に分岐させて選別する際に必要な流路を単一の流路デバイスで賄うことができる。この結果、細胞選別装置の小型化を図ることができる。 In 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. Therefore, 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. As a result, the cell sorting apparatus can be downsized.
　こうした本発明の細胞選別用流路デバイスにおいて、前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路であり、前記撮影用流路は、前記所定幅で前記所定深さの直線状の流路であり、前記本流用流路は、前記所定幅以下の幅で前記所定深さで前記撮影用流路から直線上に配置された流路であり、前記分岐流路は、前記所定幅より小さな幅で前記所定深さで前記本流用流路から１０度～６０度の範囲内の所定角度で分岐する流路である、ものとすることもできる。こうすれば、細胞選別用流路デバイスを長さを短くすることができる。 In such a cell sorting channel device of the present invention, 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.
　また、本発明の細胞選別用流路デバイスにおいて、前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで流路を形成する少なくとも１つの面には２０～５０回の範囲内の所定繰り返し回数の凹凸が形成された直線状の流路であり、前記撮影用流路は、前記所定幅で前記所定深さの直線状の流路であり、前記本流用流路は、前記所定幅以下の幅で前記所定深さで前記撮影用流路から直線上に配置された流路であり、前記分岐流路は、前記所定幅より小さな幅で前記所定深さで前記本流用流路から１０度～６０度の範囲内の所定角度で分岐する流路である、ものとすることもできる。こうすれば、細胞選別用流路デバイスを長さを短くすることができる。ここで、凹凸が形成される面としては、流路の断面が矩形流路の場合、頂面や底面、側面のいずれかでよく、頂面や底面が特に好ましい。なお、頂面や側面に凹凸を形成する場合、凹凸により流路の深さが変化することになるが、凸部が形成されている箇所（深さが浅くなっている箇所）で細胞が流通することができる程度の深さになっていればよく、選別する細胞の大きさによって深さあを設計すればよい。 In the cell sorting channel device of the present invention, 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. A linear channel having at least one surface formed with irregularities of a predetermined number of repetitions within a range of 20 to 50 times, and the imaging channel is a linear channel having the predetermined width and the predetermined depth. 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. In this way, the length of the cell sorting channel device can be shortened. Here, when the cross section of the channel is a rectangular channel, 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. In addition, when unevenness is formed on the top surface or the side surface, 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.
　また、本発明の細胞選別用流路デバイスにおいて、前記選別用流路は、前記分岐流路への分岐点の直近上流の前記分岐流路の反対側に前記対象細胞を前記分岐流路に分岐させるためのキャビテーションを生じさせるキャビテーション発生領域が形成されている、ものとすることもできる。こうすれば、キャビテーションによる膨張の際の力を用いて対象細胞を分岐流路に分岐させることができる。キャビテーションは、パルスレーザーを用いて２万個／秒程度まで生じさせることができるから、２万個／秒程度まで対象細胞を他の細胞から選別することができる。 Further, in the cell sorting channel device of the present invention, 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.
　本発明の細胞選別方法は、
　複数種類の細胞を含有する細胞含有液体から対象細胞を選別する細胞選別方法であって、
（ａ）前記細胞含有液体中にランダムに存在する細胞を一列にして流し、
（ｂ）細胞が一列になって流れる細胞含有液体に短パルス光を少なくとも直線上に波長が順次異なる波長別光群として照射すると共に該照射による反射光に基づいて前記細胞含有液体中の細胞の画像を形成し、
（ｃ）前記形成した細胞の画像に基づいて該細胞が前記対象細胞であるか否かを判定し、
（ｄ）少なくとも前記対象細胞であると判定された細胞を前記細胞含有液体の流れから分岐させることにより選別する、
　ことを特徴とする。 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.
　この本発明の細胞選別方法では、細胞含有液体中にランダムに存在する細胞を一列にして流し、これに短パルス光を少なくとも直線上に波長が順次異なる波長別光群を前記撮影用流路に照射し、照射による反射光に基づいて前記細胞含有液体中の細胞の画像を形成する。そして、形成した細胞の画像に基づいてその細胞が対象細胞であるか否かを判定し、少なくとも対象細胞であると判定された細胞を細胞含有液体の流れから分岐させることにより選別する。ノズルから１細胞ずつ滴下する必要がないから、細胞含有液体の流れを速くすることにより、より迅速に対象細胞を選別することができる。また、細胞の画像を形成して判定するから、蛍光発光を用いる必要がない。しかも、短パルス光を少なくとも直線上に波長が順次異なる波長別光群として細胞含有液体に照射し、この照射によって反射する反射光に基づいて細胞含有液体中の細胞の画像を形成するから、ＣＣＤイメージセンサ（Charge Coupled Device image sensor）を用いて画像形成する場合に比して、迅速に高感度に画像形成することができる。 In this cell sorting method of the present invention, 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. In addition, since it is determined by forming an image of a cell, it is not necessary to use fluorescence. Moreover, since the cell-containing liquid is irradiated with a short pulse light as a group of light of different wavelengths at least on a straight line, and an image of the cells in the cell-containing liquid is formed based on the reflected light reflected by this irradiation, 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.
　こうした本発明の細胞選別方法において、前記ステップ（ｄ）は、キャビテーションを用いて前記対象細胞であると判定された細胞を分岐させるステップである、ものとすることもできる。キャビテーションは、パルスレーザーを用いて２万個／秒程度まで生じさせることができるから、２万個／秒程度まで対象細胞を他の細胞から選別することができる。 In such a cell sorting method of the present invention, 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.
　また、本発明の細胞選別方法において、前記ステップ（ａ）は、前記細胞含有液体を１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路に流すことによって細胞が一列になって流れるように整列するステップである、ものとすることもできる。こうすれば、慣性を用いて容易に細胞含有液体中の細胞を一列にして流すことができる。 In the cell sorting method of the present invention, 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.
　さらに、本発明の細胞選別方法において、前記ステップ（ｂ）は、フェムト秒ないしナノ秒オーダーの間隔で繰り返し出力される短パルス光を分光して前記波長別光群として照射し、該照射により反射した反射波長別光群を集光してなる反射短パルス光を時間の経過に伴って順次波長の異なる光とし、波長毎の光の強度に基づいて細胞の画像を形成するステップである、ものとすることもできる。 Further, in the cell sorting method of the present invention, 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. In this step, 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.
　また、本発明の特定細胞除去装置および特定細胞除去用流路並びに特定細胞除去方法は、上述の主目的を達成するために以下の手段を採った。 In addition, 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.
　本発明の特定細胞除去装置は、
　患者から採取した造血幹細胞を含有する造血幹細胞含有液体から少なくとも特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去装置であって、
　前記造血幹細胞含有液体中に存在する細胞が一列に流れるようにする整列用流路と、前記造血幹細胞含有液体中の特定細胞を破壊処理するために前記整列用流路の後段に形成された第１処理用流路と、前記造血幹細胞含有液体から破壊された特定細胞を分離するために前記第１処理用流路の後段に形成された第１分離用流路と、を有する第１流路デバイスと、
　前記造血幹細胞は含まないが前記特定細胞は含み且つ前記特定細胞の可能性が認められる細胞は含む範囲の細胞としての所定細胞を前記第１処理用流路に流れる前記造血幹細胞含有液体から検出する所定細胞検出手段と、
　前記第１処理用流路に細胞破壊用の破壊レーザ光を照射可能なレーザ照射手段と、
　前記所定細胞検出手段により前記第１処理用流路に流れる前記造血幹細胞含有液体中に前記所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する制御手段と、
　を備えることを要旨とする。 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;
When the predetermined cell is detected in the hematopoietic stem cell-containing liquid flowing in the first processing channel by the predetermined cell detection unit, 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.
　この本発明の特定細胞除去装置では、第１流路デバイスの整列用流路に造血幹細胞含有液体を流すことによって造血幹細胞含有液体中に存在する細胞が一列に流れるようにし、第１流路デバイスの第１処理用流路で細胞が一列に流れるようになった造血幹細胞含有液体中の所定細胞を検出し、検出した所定細胞に破壊レーザ光を照射することによって所定細胞を破壊する。そして、第１流路デバイスの第１分離用流路で造血幹細胞含有液体から破壊された所定細胞を分離する。ここで、所定細胞は、造血幹細胞は含まないが特定細胞は含み且つ特定細胞の可能性が認められる細胞は含む範囲の細胞である。したがって、造血幹細胞は含まないが特定細胞は含む所定細胞を検出して破壊レーザ光により破壊し、破壊した細胞を造血幹細胞含有液体から分離するから、造血幹細胞含有液体から造血幹細胞を残したまま特定細胞を除去することができる。特定細胞として腫瘍細胞を考慮すれば、造血幹細胞含有液体から造血幹細胞を残したまま腫瘍細胞を除去することができる。したがって、この特定細胞除去装置により処理した造血幹細胞含有液体を用いて自家造血幹細胞移植を行なうことにより、拒絶反応を抑制することはもとより、再発を有効に抑制することができる。 In the specific cell removing apparatus of the present invention, 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. Here, 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.
　こうした本発明の特定細胞除去装置において、前記レーザ照射手段は、前記破壊レーザ光の他に前記第１処理用流路に細胞検出用のプローブレーザ光を照射可能な手段であり、前記所定細胞検出手段は、前記プローブレーザ光の照射により蛍光発現する第１マーカーを用いて前記所定細胞を検出する手段であり、前記制御手段は、前記プローブレーザ光を前記第１処理用流路に照射するよう前記レーザ照射手段を制御すると共に前記所定細胞検出手段により前記所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する手段である、ものとすることもできる。この場合、前記マーカーはＣＤ３４またはＣＤ３８であり、前記所定細胞検出手段は、前記マーカーがＣＤ３４のときには、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第１マーカーとによって蛍光発現を生じない細胞を前記所定細胞として検出する手段であり、前記マーカーがＣＤ３８のときには、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第２マーカーとによって蛍光発現を生じる細胞を前記所定細胞として検出する手段である、ものとすることもできる。ここで、ＣＤ３４は、ヒト細胞分化抗原（human cell differentiation molecules (HCDM)）ワークショップで３４番目のＣＤ（cluster of differentiation：分化抗原群）とされ、造血幹細胞に陽性発現する造血幹細胞マーカーとしての機能を有するものである。ＣＤ３８は、ＨＣＤＭワークショップで３８番目のＣＤとされ、リンパ球の増殖を活性化する膜タンパク質であり、造血幹細胞に陰性発現するものである。したがって、検出された細胞のうちＣＤ３４に陰性の細胞を所定細胞として破壊することにより、造血幹細胞を残存したまま腫瘍細胞等を破壊することができる。また、検出された細胞のうちＣＤ３８に陽性の細胞を所定細胞として破壊することにより、造血幹細胞を残存したまま腫瘍細胞等を破壊することができる。なお、プローブレーザ光を照射するレーザ装置は、破壊レーザ光を照射するレーザ装置とは別の装置として構成してもよいし、単一のレーザ装置によりレーザ光を切り替えるものとしてもよい。 In such a specific cell removing apparatus of the present invention, 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, and the control means is configured to irradiate the first processing flow path with the probe laser light. A means for controlling the laser irradiation means to control the laser irradiation means to irradiate the destructive laser light to the predetermined cells detected when the predetermined cells are detected by the predetermined cell detection means; It can also be. In this case, the marker is CD34 or CD38, and when the marker is CD34, 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. A means for detecting, as the predetermined cell, a cell that does not cause fluorescence expression by irradiation with the probe laser light and the first marker among the detected cells, and when the marker is CD38, Means for detecting cells in the hematopoietic stem cell-containing liquid based on scattered light and detecting, as the predetermined cells, cells that generate fluorescence expression by irradiation with the probe laser light and the second marker among the detected cells. There can be. Here, 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. It is what has. 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. Note that 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.
　また、本発明の特定細胞除去装置において、前記所定細胞検出手段は、短パルス光を少なくとも直線上に波長が順次異なる波長別光群として前記第１処理用流路に照射し、該照射による反射光に基づいて前記造血幹細胞含有液体中の細胞の画像を形成し、該形成した細胞の画像に基づいて該細胞が前記所定細胞であるか否かを判定することにより前記所定細胞を検出する手段である、ものとすることもできる。この場合、前記所定細胞検出手段は、フェムト秒ないしナノ秒オーダーの間隔で繰り返し短パルス光を出力する短パルス光源と、前記短パルス光源からの短パルス光を分光して前記波長別光群とする分光部と、前記波長別光群を前記撮影用流路に照射する照射部と、前記照射により反射した反射波長別光群を集光して反射パルス光とする集光部と、前記反射パルス光を入力して時間の経過に伴って順次波長の異なる光を出力する波長選別用光ファイバと、前記波長選別用光ファイバから出力される波長毎の光の強度に基づいて細胞の画像を形成する細胞画像形成部と、前記形成された細胞の画像に基づいて該細胞が前記所定細胞であるか否かを判定する判定部と、を有する手段である、ものとすることもできる。このように、短パルス光を少なくとも直線上に波長が順次異なる波長別光群の照射とその反射光に基づいて造血幹細胞含有液体中の細胞の画像を形成するから、フォトンの量（光強度）に依存するＣＣＤイメージセンサ（Charge Coupled Device image sensor）を用いる場合に比して、迅速に且つ高感度に細胞を画像形成することができ、画像形成された細胞が所定細動であるか否かを判定することができる。この結果、特定細胞の造血幹細胞含有液体における含有率が極めて小さい場合でも特定細胞を迅速に除去することができる。 In the specific cell removal apparatus of the present invention, 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. In this case, 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. In this way, 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 to the case of using a CCD image sensor (Charge-Coupled Device-image image sensor) that depends on the image, cells can be imaged quickly and with high sensitivity. Can be determined. As a result, 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.
　本発明の特定細胞除去装置において、前記第１流路デバイスの後段に配置され、前記第１流路デバイスを用いて破壊された所定細胞を分離した後の前記造血幹細胞含有液体中に残存する所定細胞を破壊処理するための第２処理用流路と、前記造血幹細胞含有液体から破壊された所定細胞を分離するために前記第２処理用流路の後段に形成された第２分離用流路と、を有する第２流路デバイスを備え、前記所定細胞検出手段は、前記第１処理用流路に流れる前記造血幹細胞含有液体中の前記所定細胞を検出する他に前記第２処理用流路に流れる前記造血幹細胞含有液体中の前記所定細胞も検出する手段であり、前記レーザ照射手段は、前記第１処理用流路に前記破壊レーザ光を照射可能な他に前記第２処理用流路に前記破壊レーザ光を照射可能な手段であり、前記制御手段は、前記所定細胞検出手段により前記第１処理用流路に流れる前記造血幹細胞含有液体中に前記所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する他に、前記所定細胞検出手段により前記第２処理用流路に流れる前記造血幹細胞含有液体中に前記所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する手段である、ものとすることもできる。こうすれば、２度に亘って所定細胞を検出して破壊するから、造血幹細胞含有液体中の特定細胞をより確実に除去することができる。 In the specific cell removing apparatus of the present invention, 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. A second processing channel for destroying cells, and a second separation channel formed after the second processing channel to separate predetermined cells destroyed from the hematopoietic stem cell-containing liquid And 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. And 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. Can be irradiated with the destructive laser light And 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. In addition to controlling the laser irradiation means to irradiate laser light, 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. In addition, 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.
　こうした第２流路デバイスを備える態様の本発明の特定細胞除去装置において、前記レーザ照射手段は、前記破壊レーザ光の他に前記第１処理用流路および前記第２処理用流路に細胞検出用のプローブレーザ光を照射可能な手段であり、前記所定細胞検出手段は、前記プローブレーザ光の照射により蛍光発現する第１マーカーを用いて前記第１処理用流路に流れる前記造血幹細胞含有液体中の所定細胞を検出する第１検出手段と、前記プローブレーザ光の照射により蛍光発現する前記第１マーカーとは異なる第２マーカーを用いて前記第２処理用流路に流れる前記造血幹細胞含有液体中の所定細胞を検出する第２検出手段と、を有する手段であり、前記制御手段は、前記プローブレーザ光を前記第１処理用流路に照射するよう前記レーザ照射手段を制御すると共に前記第１検出手段により所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御し、前記プローブレーザ光を前記第２処理用流路に照射するよう前記レーザ照射手段を制御すると共に前記第２検出手段により所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する手段である、ものとすることもできる。この場合、前記第１マーカーはＣＤ３４であり、前記第２マーカーはＣＤ３８であり、前記第１検出手段は、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第１マーカーとによって蛍光発現を生じない細胞を前記所定細胞として検出する手段であり、前記第２検出手段は、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第２マーカーとによって蛍光発現を生じる細胞を前記所定細胞として検出する手段である、ものとすることもできる。こうすれば、検出された細胞のうちＣＤ３４に陰性の細胞を所定細胞として破壊して分離し、更に、検出された細胞のうちＣＤ３８に陽性の細胞を所定細胞として破壊して分離することにより、より確実に造血幹細胞を残存したまま腫瘍細胞等を破壊することができる。ここで、第２処理用流路にプローブレーザ光や破壊レーザ光を照射する装置は、第１処理用流路にプローブレーザ光や破壊レーザ光を照射する装置とは別の装置として構成してもよいし、単一の装置によりプローブレーザ光や破壊レーザ光の照射位置を切り替えて照射するものとしてもよい。 In the specific cell removal apparatus of the present invention having such a second flow path device, 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. And controlling the laser irradiating means to irradiate the detected predetermined cells with the destructive laser light when a predetermined cell is detected by the first detecting means, and supplying the probe laser light to the second 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. In this case, the first marker is CD34, the second marker is CD38, and 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. And 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. In this manner, among the detected cells, cells that are negative for CD34 are destroyed and separated as predetermined cells, and further, among the detected cells, cells that are positive for CD38 are destroyed and separated as predetermined cells, Tumor cells and the like can be destroyed more reliably while hematopoietic stem cells remain. Here, 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. Alternatively, the irradiation position of the probe laser beam or the destruction laser beam may be switched by a single device.
　本発明の特定細胞除去装置において、前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路である、ものとすることもできる。こうすれば、慣性を用いて容易に造血幹細胞含有液体中の細胞を一列にして流すことができる。 In the specific cell removing apparatus of the present invention, 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.
　本発明の特定細胞除去装置において、前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで流路を形成する少なくとも１つの面には２０～５０回の範囲内の所定繰り返し回数の凹凸が形成された直線状の流路である、ものとすることもできる。流路を形成する面に凹凸が形成されていると、流路の断面の広いところと狭いところとが生じ、細胞に慣性力が作用し、次第に細胞が一列になって流れるようになる。ここで、凹凸が形成される面としては、流路の断面が矩形流路の場合、頂面や底面、側面のいずれかでよく、頂面や底面が特に好ましい。なお、頂面や側面に凹凸を形成する場合、凹凸により流路の深さが変化することになるが、凸部が形成されている箇所（深さが浅くなっている箇所）で細胞が流通することができる程度の深さになっていればよく、選別する細胞の大きさによって深さを設計すればよい。 In the specific cell removing apparatus of the present invention, 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. Here, when the cross section of the channel is a rectangular channel, 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. In addition, when unevenness is formed on the top surface or the side surface, 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.
　本発明の特定細胞除去装置において、前記第１分離用流路は、最小間隔が造血幹細胞の直径の３倍より小さくなるように複数の円柱形状のポストを整列してなるポストアレイが形成された流路である、ものとすることもできる。円柱形状のポストを整列したポストアレイに流体を流すと、流体力学によると、流体に含有される物体（例えば細胞や破壊された細胞片）のうち直径が隣接するポストの間隔の１／３より小さい物体は流体の主流に含まれて流れ、直径が隣接するポストの間隔の１／３より大きな物体は流体の主流から外れるようにして流れる。したがって、流体の主流に対して対称の位置からポストアレイを若干傾ければ、傾けた方向に直径が隣接するポストの間隔の１／３より大きな物体を流して主流から分離することができる。したがって、造血幹細胞の直径が隣接するポストの間隔の１／３より大きくなるように且つ破壊された所定細胞の細胞片の直径が隣接するポストの間隔の１／３より小さくなるようにポストアレイを形成し、造血幹細胞含有液体の流れに対して若干の角度をもって配置すれば、液体の主流の流れに破壊された所定細胞の細胞片を含ませたまま造血幹細胞を主流から分離することができる。なお、分離される造血幹細胞が含まれる流れの液体を造血幹細胞含有液体と考えれば、破壊された所定細胞を造血幹細胞含有液体から分離したことになる。 In the specific cell removal apparatus of the present invention, 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. When fluid flows through a post array in which cylindrical posts are arranged, according to hydrodynamics, 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. Therefore, if the post array is slightly tilted from a position symmetric with respect to the main flow of the fluid, an object having a diameter larger than 1/3 of the interval between adjacent posts in the tilted direction can be separated from the main flow. Therefore, 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. In addition, if the liquid of the flow containing the hematopoietic stem cell to be separated is considered as the hematopoietic stem cell-containing liquid, the predetermined broken cells are separated from the hematopoietic stem cell-containing liquid.
　本発明の特定細胞除去用流路は、
　患者から採取した造血幹細胞を含有する造血幹細胞含有液体から少なくとも特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去用流路であって、
　前記造血幹細胞含有液体中に存在する細胞が一列に流れるようにする整列用流路と、前記造血幹細胞含有液体中の特定細胞を破壊処理するために前記整列用流路の後段に形成された第１処理用流路と、前記造血幹細胞含有液体から破壊された特定細胞を分離するために前記第１処理用流路の後段に形成された第１分離用流路と、が一体形成された第１流路デバイスを備える、
　ことを要旨とする。 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.
　この本発明の特定細胞除去用流路では、第１流路デバイスの整列用流路では造血幹細胞含有液体中に存在する細胞が一列に流れるようにし、第１流路デバイスの第１処理用流路では細胞が一列に流れるようになった造血幹細胞含有液体中の特定細胞を破壊処理し、第１流路デバイスの第１分離用流路では造血幹細胞含有液体から破壊された特定細胞を分離する。破壊処理としては、造血幹細胞は含まないが特定細胞は含む範囲の細胞を検出し、検出した細胞に破壊レーザ光を照射して破壊する処理が含まれる。したがって、造血幹細胞は含まないが特定細胞は含む範囲の細胞を検出して破壊し、破壊した細胞を造血幹細胞含有液体から分離するから、造血幹細胞含有液体から造血幹細胞を残したまま特定細胞を除去することができる。しかも、こうした処理を行なう整列用流路と第１処理用流路と第１分離用流路とが一体形成されているから、特定細胞除去用流路をコンパクトなものとすることができる。 In the specific cell removal flow channel of the present invention, 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. In the path, 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. Therefore, because cells that do not contain hematopoietic stem cells but contain specific cells are detected and destroyed, and the broken cells are separated from the hematopoietic stem cell-containing liquid, the specific cells are removed from the hematopoietic stem cell-containing liquid while leaving the hematopoietic stem cells. can do. In addition, since 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.
　こうした本発明の特定細胞除去用流路において、前記第１分離用流路で破壊された特定細胞が分離された後の前記造血幹細胞含有液体中に残存する特定細胞を破壊処理するための第２処理用流路と、前記造血幹細胞含有液体から破壊された特定細胞を分離するために前記第２処理用流路の後段に形成された第２分離用流路と、が一体形成された第２流路デバイスを備える、ものとすることもできる。第２流路デバイスの第２処理用流路では第１流路デバイスで特定細胞が破壊・分離された後の造血幹細胞含有液体中に残存する特定細胞を破壊処理し、第２流路デバイスの第２分離用流路では造血幹細胞含有液体から破壊された特定細胞を分離する。破壊処理としては、第１処理用流路における破壊処理とは異なる処理であって、造血幹細胞は含まないが特定細胞は含む範囲の細胞を検出し、検出した細胞に破壊レーザ光を照射して破壊する処理が含まれる。したがって、異なる破壊処理により特定細胞を破壊して分離するから、より確実に造血幹細胞含有液体から造血幹細胞を残したまま特定細胞を除去することができる。この場合、前記第１流路デバイスと前記第２流路デバイスとが一体形成されているものとすることもできる。こうすれば、より確実に特定細胞を除去することができる特定細胞除去用流路をコンパクトなものとすることができる。 In such a specific cell removal channel of the present invention, 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. A second channel in which a processing channel and a second separation channel formed at a subsequent stage of the second processing channel to separate specific cells destroyed from the hematopoietic stem cell-containing liquid are integrally formed. It can also comprise a flow channel device. In the second processing channel of the second channel device, the specific cells remaining in the hematopoietic stem cell-containing liquid after the specific cells are destroyed and separated by the first channel device are destroyed, In the second separation channel, the broken specific cells are separated from the hematopoietic stem cell-containing liquid. 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. In this case, 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.
　また、本発明の特定細胞除去用流路において、前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路である、ものとすることもできる。こうすれば、慣性を用いて容易に造血幹細胞含有液体中の細胞を一列にして流すことができる。 In the specific cell removal channel of the present invention, 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.
　さらに、本発明の特定細胞除去用流路において、前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで流路を形成する少なくとも１つの面には２０～５０回の範囲内の所定繰り返し回数の凹凸が形成された直線状の流路である、ものとすることもできる。流路を形成する面に凹凸が形成されていると、流路の断面の広いところと狭いところとが生じ、細胞に慣性力が作用し、次第に細胞が一列になって流れるようになる。ここで、凹凸が形成される面としては、流路の断面が矩形流路の場合、頂面や底面、側面のいずれかでよく、頂面や底面が特に好ましい。なお、頂面や側面に凹凸を形成する場合、凹凸により流路の深さが変化することになるが、凸部が形成されている箇所（深さが浅くなっている箇所）で細胞が流通することができる程度の深さになっていればよく、選別する細胞の大きさによって深さを設計すればよい。 Furthermore, in the specific cell removal flow channel of the present invention, 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. Here, when the cross section of the channel is a rectangular channel, 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. In addition, when unevenness is formed on the top surface or the side surface, 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.
　本発明の特定細胞除去用流路において、前記第１分離用流路は、最小間隔が造血幹細胞の直径の３倍より小さくなるように複数の円柱形状のポストを整列してなるポストアレイが形成された流路である、ものとすることもできる。円柱形状のポストを整列したポストアレイに流体を流すと、流体力学によると、流体に含有される物体（例えば細胞や破壊された細胞片）のうち直径が隣接するポストの間隔の１／３より小さい物体は流体の主流に含まれて流れ、直径が隣接するポストの間隔の１／３より大きな物体は流体の主流から外れるようにして流れる。したがって、流体の主流に対して対称の位置からポストアレイを若干傾ければ、傾けた方向に直径が隣接するポストの間隔の１／３より大きな物体を流して主流から分離することができる。したがって、造血幹細胞の直径が隣接するポストの間隔の１／３より大きくなるように且つ破壊された所定細胞の細胞片の直径が隣接するポストの間隔の１／３より小さくなるようにポストアレイを形成し、造血幹細胞含有液体の流れに対して若干の角度をもって配置すれば、液体の主流の流れに破壊された所定細胞の細胞片を含ませたまま造血幹細胞を主流から分離することができる。なお、分離される造血幹細胞が含まれる流れの液体を造血幹細胞含有液体と考えれば、破壊された所定細胞を造血幹細胞含有液体から分離したことになる。 In the specific cell removal flow channel of the present invention, 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. When fluid flows through a post array in which cylindrical posts are arranged, according to hydrodynamics, 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. Therefore, if the post array is slightly tilted from a position symmetric with respect to the main flow of the fluid, an object having a diameter larger than 1/3 of the interval between adjacent posts in the tilted direction can be separated from the main flow. Therefore, 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. In addition, if the liquid of the flow containing the hematopoietic stem cell to be separated is considered as the hematopoietic stem cell-containing liquid, the predetermined broken cells are separated from the hematopoietic stem cell-containing liquid.
　本発明の第１の特定細胞除去方法は、
　患者から採取した造血幹細胞を含有する造血幹細胞含有液体から少なくとも特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去方法であって、
（ａ）前記造血幹細胞含有液体中に存在する細胞が一列となるように前記造血幹細胞含有液体を流し、
（ｂ）細胞が一列になって流れる前記造血幹細胞含有液体に細胞検出用のプローブレーザ光の照射と第１マーカーとによる蛍光発現を用いて、前記造血幹細胞は含まないが前記特定細胞は含み且つ前記特定細胞の可能性が認められる細胞は含む範囲の細胞としての第１所定細胞を検出し、
（ｃ）該検出した第１所定細胞に細胞破壊用の破壊レーザ光を照射して前記第１所定細胞を破壊し、
（ｄ）前記造血幹細胞含有液体から破壊された第１所定細胞を分離し、
（ｅ）破壊された第１所定細胞を分離した後の前記造血幹細胞含有液体に前記プローブレーザ光の照射と前記第１マーカーとは異なる第２マーカーによる蛍光発現を用いて、前記造血幹細胞は含まないが前記特定細胞は含み且つ前記特定細胞の可能性が認められる細胞は含む範囲の細胞としての第２所定細胞を検出し、
（ｆ）該検出した第２所定細胞に細胞破壊用の破壊レーザ光を照射して前記第２所定細胞を破壊し、
（ｇ）前記造血幹細胞含有液体から破壊された第２所定細胞を分離する、
　ことを特徴とする。 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 destruction;
(D) separating the first predetermined cells destroyed from the hematopoietic stem cell-containing liquid,
(E) The hematopoietic stem cell is contained in the hematopoietic stem cell-containing liquid after separating the destroyed first predetermined cells using irradiation of the probe laser light and fluorescence expression by a second marker different from the first marker. 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.
　この本発明の第１の特定細胞除去方法では、造血幹細胞含有液体中に存在する細胞が一列となるように造血幹細胞含有液体を流し、細胞が一列になって流れる造血幹細胞含有液体に細胞検出用のプローブレーザ光の照射と第１マーカーとによる蛍光発現を用いて、造血幹細胞は含まないが特定細胞は含み且つ特定細胞の可能性が認められる細胞は含む範囲の細胞としての第１所定細胞を検出し、検出した第１所定細胞に細胞破壊用の破壊レーザ光を照射して破壊し、造血幹細胞含有液体から破壊された第１所定細胞を分離する。そして、破壊された第１所定細胞を分離した後の造血幹細胞含有液体にプローブレーザ光の照射と第２マーカーによる蛍光発現を用いて、造血幹細胞は含まないが特定細胞は含み且つ特定細胞の可能性が認められる細胞は含む範囲の細胞としての第２所定細胞を検出し、検出した第２所定細胞に細胞破壊用の破壊レーザ光を照射して破壊し、造血幹細胞含有液体から破壊された第２所定細胞を分離する。このように、２回に亘って造血幹細胞は含まないが特定細胞は含み且つ特定細胞の可能性が認められる細胞は含む範囲の細胞を検出し、検出した細胞を破壊して分離するから、より確実に造血幹細胞含有液体から特定細胞を除去することができる。特定細胞として腫瘍細胞を考慮すれば、造血幹細胞含有液体から造血幹細胞を残したまま腫瘍細胞を除去することができる。したがって、この特定細胞除去方法により処理した造血幹細胞含有液体を用いて自家造血幹細胞移植を行なうことにより、拒絶反応を抑制することはもとより、再発を有効に抑制することができる。 In the first specific cell removal method of the present invention, 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. 2 Isolate predetermined cells. In this way, since cells that do not include hematopoietic stem cells but include specific cells and cells that are considered to be specific cells are detected twice, the detected cells are destroyed and separated. Certain cells can be reliably removed from the hematopoietic stem cell-containing liquid. 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 treated by this specific cell removal method, it is possible not only to suppress rejection but also to effectively suppress recurrence.
　こうした本発明の第１の特定細胞除去方法において、前記ステップ（ｂ）は、前記第１マーカーとしてＣＤ３４を用い、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第１マーカーとによって蛍光発現を生じない細胞を前記第１所定細胞として検出するステップであり、前記ステップ（ｅ）は、前記第２マーカーとしてＣＤ３８を用い、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第２マーカーとによって蛍光発現を生じる細胞を前記第２所定細胞として検出するステップである、ものとすることもできる。ＣＤ３４とＣＤ３８については上述した。したがって、造血幹細胞含有液体中の細胞のうちＣＤ３４に陰性の細胞を第１所定細胞として破壊して分離し、第１所定細胞を破壊分離した後の造血幹細胞含有液体中の細胞のうちＣＤ３８に陽性の細胞を第２所定細胞として破壊して分離することにより、より確実に造血幹細胞を残存したまま腫瘍細胞等を破壊して分離することができる。 In such a first specific cell removal method of the present invention, 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. Detecting and detecting, as the first predetermined cell, a cell that does not cause fluorescence expression by the irradiation of the probe laser light and the first marker among the detected cells, and 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. Therefore, among the cells in the hematopoietic stem cell-containing liquid, 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.
　本発明の第２の特定細胞除去方法は、
　患者から採取した造血幹細胞を含有する造血幹細胞含有液体から少なくとも特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去方法であって、
（ａ）前記造血幹細胞含有液体中に存在する細胞を一列に流し、
（ｂ）細胞が一列になって流れる前記造血幹細胞含有液体に短パルス光を少なくとも直線上に波長が順次異なる波長別光群として照射すると共に該照射による反射光に基づいて前記造血幹細胞含有液体中の細胞の画像を形成し、前記形成した細胞の画像に基づいて該細胞が前記特定細胞であるか否かを判定することによって特定細胞を検出し、
（ｃ）該検出した特定細胞に細胞破壊用の破壊レーザ光を照射して前記特定細胞を破壊し、
（ｄ）前記造血幹細胞含有液体から破壊された特定細胞を分離する、
　ことを特徴とする。 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;
(D) separating the broken specific cells from the hematopoietic stem cell-containing liquid,
It is characterized by that.
　この本発明の第２の特定細胞除去方法では、造血幹細胞含有液体中に存在する細胞が一列となるように造血幹細胞含有液体を流し、細胞が一列になって流れる造血幹細胞含有液体に短パルス光を少なくとも直線上に波長が順次異なる波長別光群として照射すると共に照射による反射光に基づいて造血幹細胞含有液体中の細胞の画像を形成し、形成した細胞の画像に基づいて該細胞が前記特定細胞であるか否かを判定することによって特定細胞を検出する。そして、検出した特定細胞に細胞破壊用の破壊レーザ光を照射して破壊し、造血幹細胞含有液体から破壊された特定細胞を分離する。このように、短パルス光を少なくとも直線上に波長が順次異なる波長別光群の照射とその反射光に基づいて造血幹細胞含有液体中の細胞の画像を形成するから、フォトンの量（光強度）に依存するＣＣＤイメージセンサ（Charge Coupled Device image sensor）を用いる場合に比して、迅速に且つ高感度に細胞を画像形成して画像形成した細胞が特定細動であるか否かを判定することができる。この結果、特定細胞の造血幹細胞含有液体における含有率が極めて小さい場合でもより確実に且つ迅速に特定細胞を除去することができる。したがって、この特定細胞除去方法により処理した造血幹細胞含有液体を用いて自家造血幹細胞移植を行なうことにより、拒絶反応を抑制することはもとより、再発を有効に抑制することができる。 In the second specific cell removal method of the present invention, 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. 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. Then, 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. In this way, 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. As a result, even when the content rate of the specific cells in the hematopoietic stem cell-containing liquid is extremely small, the specific cells 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.
本発明の一実施例としての細胞選別装置２０の構成の概略を示す概略構成図である。It is a schematic block diagram which shows the outline of a structure of the cell selection apparatus 20 as one Example of this invention. ＳＴＥＡＭカメラ４０の構成の概略を示す概略構成図である。2 is a schematic configuration diagram showing an outline of a configuration of a STEAM camera 40. FIG. 変形例の細胞選別装置１２０の構成の概略を示す概略構成図である。It is a schematic block diagram which shows the outline of a structure of the cell sorting apparatus 120 of the modification. 変形例の細胞選別用流路デバイス２３０の構成の概略を示す概略構成図である。It is a schematic block diagram which shows the outline of a structure of the flow-path device 230 for cell sorting of the modification. 変形例の細胞選別用流路デバイス２３０の図４におけるＡ１－Ａ２断面における整列用流路２３２の部分を示す説明図である。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. 本発明の一実施例としての第１造血幹細胞用細胞除去装置１０２０の構成の概略を示す構成図である。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. 制御装置１０８０により実行されるＣＤ３４陽性試験用レーザ照射制御の一例を示すフローチャートである。5 is a flowchart showing an example of CD34 positive test laser irradiation control executed by a control device 1080. 制御装置１０８０により実行されるＣＤ３８陰性試験用レーザ照射制御の一例を示すフローチャートである。5 is a flowchart showing an example of CD38 negative test laser irradiation control executed by a control device 1080. 本発明の一実施例としての第２造血幹細胞用細胞除去装置１１２０の構成の概略を示す構成図である。It is a block diagram which shows the outline of a structure of the 2nd hematopoietic stem cell removal apparatus 1120 as one Example of this invention. ＳＴＥＡＭカメラ装置１１５０の構成の概略を示す構成図である。1 is a configuration diagram showing an outline of a configuration of a STEAM camera device 1150. FIG. 制御装置１１８０により実行される破壊レーザ照射制御の一例を示すフローチャートである。10 is a flowchart showing an example of destructive laser irradiation control executed by a control device 1180. 変形例の細胞破壊除去用流路デバイス１２３０の構成の概略を示す概略構成図である。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. 変形例の細胞破壊除去用流路デバイス１２３０の図１２におけるＡ１－Ａ２断面における整列用流路１２３２の部分を示す説明図である。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.
Ａ．細胞選別関係
　次に、本発明の一実施例としての細胞選別装置２０について説明する。図１は、実施例の細胞選別装置２０の構成の概略を示す概略構成図である。図示するように、実施例の細胞選別装置２０は、複数種の細胞を含有する液体（細胞含有液体）を流す細胞選別用流路デバイス３０と、細胞含有液体中の細胞を撮影するＳＴＥＡＭカメラ４０（STEAM：Serial Time-Encoded Amplified Imaging / Microscopy）と、撮影された細胞の画像に基づいてその細胞が選別対象細胞であるか否かを判定する画像判定部５０と、選別対象細胞であると判定された細胞を細胞含有液体の流れから分岐させるためのキャビテーションを発生させるパルスレーザー６０と、を備える。ここで、「細胞」としては、主として生体細胞が含まれ、例えば、血液中の赤血球や血小板、白血球などが含まれる他、記憶Ｂ細胞やナチュラルキラーＴ細胞、Antigen-Specific T Cells、Hematopoietic Stem Cells、Circulating Endothelial Cells (CECs)、Circulating Endothelial progenitor Cells、Circulating Tumor Cells (CTCs)、Circulating Cancer Stem Cellsなどの極めて含有率が低い希少細胞などが含まれる。これらの希少細胞については後述する。「細胞含有液体」としては、例えば血液などの体液やこれらを生理的食塩水などの溶媒により薄めた溶液などが含まれる。 A. Cell Sorting Relationship Next, a cell sorting apparatus 20 as one embodiment of the present invention will be described. FIG. 1 is a schematic configuration diagram showing an outline of the configuration of the cell sorting apparatus 20 of the embodiment. As shown in the figure, 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. (STEAM: Serial Time-Encoded Amplified Imaging / Microscopy), an image determination unit 50 that determines whether or not the cell is a selection target cell based on the captured image of the cell, and a determination that the cell is a selection target cell And a pulsed laser 60 for generating cavitation for branching the formed cells from the flow of the cell-containing liquid. Here, “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. And rare cells such as Circulating Endothelial Cells (CECs), Circulating Endothelial progenitor Cells, Circulating Tumor Cells (CTCs), and Circulating 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.
　細胞選別用流路デバイス３０は、ポリジメチルシロキサン（PDMS：polydimethylsiloxane）やエポキシ樹脂などの細胞含有液体に浸食されない材料により、上流側から、細胞含有液体中の細胞が一列になって流れるようにする整列用流路３２と、細胞含有液体中の細胞をＳＴＥＡＭカメラ４０で撮影するための撮影用流路３４と、選別対象細胞を細胞含有液体の流れから分岐させて選別する選別用流路３６と、からなるように形成されている。なお、細胞選別用流路デバイス３０は、細胞含有液体の流速が１．５ｍ／ｓ未満であるときにはポリジメチルシロキサンにより形成されるのが好ましく、細胞含有液体の流速が１．５ｍ／ｓ以上であるときにはエポキシ樹脂により形成されるのが好ましい。 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. An alignment channel 32, an imaging channel 34 for imaging cells in the cell-containing liquid with the STEAM camera 40, and a sorting channel 36 for sorting and sorting the cells to be sorted from the flow of the cell-containing liquid. It is formed to consist of. 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.
　整列用流路３２は、幅が１００～５００μｍで深さが１０～１００μｍで繰り返し回数が１０～２０回の蛇行する波状の流路として形成されているが、整列用流路３２の幅や深さや蛇行の繰り返し回数は、細胞含有液体に含まれる細胞の大きさなどによって適宜定められるものである。整列用流路３２では、流路が繰り返し蛇行していることにより、細胞含有液体中にランダムに存する細胞は、蛇行の際の慣性により徐々に一列に整列するようになる。 The alignment channel 32 is formed as a wavy channel that meanders with a width of 100 to 500 μm, a depth of 10 to 100 μm, 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. In the alignment channel 32, 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.
　撮影用流路３４は、整列用流路３２と同一の幅で同一の深さの流路で基本的には直線状となるように形成されている。 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.
　選別用流路３６には、撮影用流路３４から連続する本流用流路３６ａと、この本流用流路３６ａから分岐する分岐流路３６ｂと、分岐点の直近上流の分岐流路３６ｂの反対側に分岐流路３６ｂに細胞を分岐させるためのキャビテーションを生じさせるキャビテーション発生領域３６ｃと、が形成されている。本流用流路３６ａは、撮影用流路３４からの細胞含有液体がスムースに流れるように撮影用流路３４の直線上に配置されており、流路の幅は撮影用流路３４の幅と同一か若干小さくなるように、深さは撮影用流路３４の深さと同一となるように形成されている。分岐流路３６ｂは、選別対象の細胞が本流用流路３６ａからスムースに分岐するように本流用流路３６ａの流れに対して１０度～６０度の範囲内の角度で分岐する流路として、幅は本流用流路３６ａより狭くなるように、深さは本流用流路３６ａと同一になるように形成されている。キャビテーション発生領域３６ｃは、本流用流路３６ａの分岐流路３６ｂとは反対側に配置されたキャビテーション用流路３６ｄと、分岐流路３６ｂの分岐点の直近上流で本流用流路３６ａとキャビテーション用流路３６ｄとを連絡する連絡通路３６ｅと、により構成されている。キャビテーション用流路３６ｄは、幅は撮影用流路３４の幅と同一か若干大きくなるように、深さは撮影用流路３４と同一となるように形成されており、細胞含有液体中の細胞に対して無作用の液体（例えば、生理的食塩水や細胞含有液体の溶媒など）が流されている。連絡通路３６ｅは、幅は５０～２００μｍで深さは他の流路と同一となるように形成されている。キャビテーション用流路３６ｄの連絡通路３６ｅの近傍の照射スポット３６ｆにパルスレーザーを照射すると、照射スポット３６ｆの圧力が低下してキャビテーションが生じる。このキャビテーションによる膨張する際の力は、連絡通路３６ｅを介して本流用流路３６ａの連絡通路３６ｅの連絡点近傍を流下している細胞に作用するから、その細胞は分岐流路３６ｂ側に押し出され、分岐流路３６ｂに流れるようになる。したがって、選別対象細胞が本流用流路３６ａの連絡通路３６ｅの連絡点近傍に至ったタイミングでパルスレーザーを照射することにより、選別対象細胞を分岐流路３６ｂに分岐させることができる。 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) is flowed. The communication passage 36e is formed to have a width of 50 to 200 μm and a depth that is the same as that of other flow paths. When the irradiation spot 36f in the vicinity of the communication passage 36e of the cavitation flow path 36d is irradiated with a pulse laser, the pressure of the irradiation spot 36f is reduced to cause cavitation. 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.
　ＳＴＥＡＭカメラ４０の構成の概略を図２に示す。図示するように、ＳＴＥＡＭカメラ４０は、フェムト秒ないしナノ秒オーダーの間隔で繰り返し短パルス光を出力する短パルス光源４１と、短パルス光源４１からの光は透過すると共に反対側からの光は反射するハーフミラー４２と、短パルス光源４１からの光については分光すると共に反対側からの分光された光については集光（合成）する分光器４３と、分光した光を平行光とするレンズ４４と、ハーフミラー４２から反射されたパルス光を時間の経過に伴って順次波長の異なる光とする波長選別用光ファイバ４５と、光の強度を検出する光強度検出器４６と、波長毎の光の強度に基づいて画像形成する画像形成処理部４７と、を備える。 The outline of the configuration of the STEAM camera 40 is shown in FIG. As shown in the figure, 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. And an image formation processing unit 47 that forms an image based on the intensity.
　分光器４３は、直線上の位置毎に波長が順次異なるように分光するよう調整されている。このため、分光器４３によって分光されレンズ４４によって平行光とされた光は、直線上の位置毎に順次波長が異なる平行な波長別の光の群（波長別光群）となって撮影用流路３４に照射されることになる。即ち、撮影用流路３４には、幅方向の直線上の位置によって異なる波長の光が照射されるのである。この波長別光群の反射光（反射波長別光群）は、撮影用流路３４に流れる物体の幅方向の直線上の情報を光強度として持ってレンズ４４によって集められ、分光器４３によって集光（合成）されてパルス光（反射パルス光）とされる。 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).
　波長選別用光ファイバ４５は、偏波保持型の光ファイバ４５ａと、光ファイバ４５ａからの光の強度を増幅するために逆方向に光を入力する光ポンプ４５ｂとにより構成されている。偏波保持型の光ファイバ４５ａは、入力した光の波長の長短により出力する時間が変化する性質を有するため、反射パルス光を入力すると、波長の長い光から順に出力する。したがって、波長選別用光ファイバ４５から出力される光に対して時間の経過に伴って光強度検出器４６により光強度を検出することにより、波長毎の光の強度を検出ことができる。上述したように波長毎の光の強度は、撮影用流路３４の幅方向の直線上の物体の情報となるから、この波長毎の光の強度を用いて画像形成処理部４７により撮影用流路３４に流れる物体の画像を形成することができる。画像形成処理部４７は、光の波長と強度とを撮影用流路３４の幅方向の直線上の物体の情報として画像形成する処理プログラムがインストールされた汎用のコンピュータにより構成することができる。 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.
　ＳＴＥＡＭカメラ４０は、上述したように、短パルス光を分光して撮影用流路３４の幅方向の直線上の位置によって波長が順次異なる光を照射し、その反射光を集光（合成）して波長選別用光ファイバ４５により波長の長い光から順に出力させ、波長毎の光の強度に基づいて撮影用流路３４に流れる物体（細胞）の画像形成を行なうから、ＣＣＤイメージセンサ（Charge Coupled Device image sensor）を用いて画像形成する場合に比して、迅速に高感度に画像形成することができる。 As described above, 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.
　画像判定部５０は、ＳＴＥＡＭカメラ４０によって画像形成された撮影用流路３４に流れる細胞が対象細胞に一致するか否かの画像のマッチングを行なう処理プログラムがインストールされた汎用のコンピュータにより構成することができる。画像のマッチングは、予め登録した選別対象の細胞の画像に対して一定の誤差の範囲内で入力した細胞の画像が一致するか否かを判定するものなどを適用することができる。画像判定部５０では、細胞の画像が一致したときに、適当なタイミングでパルスレーザー６０に照射制御信号を出力する。「適当なタイミング」は、パルスレーザー６０がキャビテーション用流路３６ｄの照射スポット３６ｆにパルスレーザーを照射してキャビテーションを生じさせたときに、その膨張する際の力が連絡通路３６ｅを介して一致した細胞に作用するタイミングであり、撮影用流路３４に流れる細胞含有液体の流速や撮影用流路３４の撮影位置から連絡通路３６ｅまでの距離などに基づいて実験などにより定めることができる。 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.
　パルスレーザー６０は、例えば、商品名FPL-04CTF(CALMAR LASER)を用いることができ、照射スポット３６ｆに照射するパルスレーザーとしては、照射時間が１ピコ秒程度で強度が１ｎＪ程度のものが好適である。パルスレーザーが照射スポット３６ｆに照射されると、上述したように、照射スポット３６ｆの圧力が低下して半径１００μｍ程度の範囲でキャビテーションが生じる。このパルスレーザー６０を用いると、細胞選別に有効なキャビテーションを２ 万個／秒程度発生させることができる。 As 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. When 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 μm. By using this pulse laser 60, it is possible to generate about 20 thousand cavities / second effective for cell sorting.
　次に、こうして構成された実施例の細胞選別装置２０による細胞選別の様子について説明する。細胞選別用流路デバイス３０の整列用流路３２の入口から細胞を１粒子としたときに粒子レイノルズ数（particle Reynolds number）が値１程度以下となるように細胞含有液体を流す。細胞含有液体中の細胞は、整列用流路３２を流下するうちに一列になって流れるようになる。撮影用流路３４では、ＳＴＥＡＭカメラ４０により撮影用流路３４を一列になって流れる細胞が撮影されて画像形成され、形成された画像が画像判定部５０に出力される。画像判定部５０では、選別対象の細胞の画像とＳＴＥＡＭカメラ４０から入力した細胞の画像のマッチングが行なわれ、一定の誤差の範囲内で一致したと判定されると適当なタイミングでパルスレーザー６０に照射制御信号が出力される。パルスレーザー６０から照射スポット３６ｆにパルスレーザーが照射されると、照射スポット３６ｆの圧力の低下によりキャビテーションが生じ、その膨張する際の力が連絡通路３６ｅを介して本流用流路３６ａに伝達され、本流用流路３６ａの連絡通路３６ｅの近傍の選別対象の細胞に作用し、その選別対象の細胞は分岐流路３６ｂ側に押し出され、分岐流路３６ｂに流れる。一方、パルスレーザー６０からのパルスレーザーの照射がないときには、キャビテーションは生じないから、本流用流路３６ａを流れる細胞は分岐流路３６ｂに押し出されることなく、本流用流路３６ａを流下する。 Next, the state of cell sorting by the cell sorting apparatus 20 of the embodiment configured as described above will be described. 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. In 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. When the pulse laser 60 irradiates the irradiation spot 36f from the pulse laser 60, cavitation occurs due to a decrease in the pressure of the irradiation spot 36f, and the expansion force is transmitted to the main flow path 36a through the communication path 36e. It acts on the cells to be sorted in the vicinity of the communication passage 36e of the main flow channel 36a, and the cells to be sorted are pushed out to the branch channel 36b and flow to the branch channel 36b. On the other hand, when there is no pulse laser irradiation from the pulse laser 60, cavitation does not occur. Therefore, the cells flowing through the main flow path 36a flow down the main flow path 36a without being pushed out to the branch flow path 36b.
　以上説明した実施例の細胞選別装置２０によれば、整列用流路３２と撮影用流路３４と選別用流路３６とからなる細胞選別用流路デバイス３０に細胞含有液体を流し、細胞が一列になって流れるようになった後に、直線上の位置によって波長が異なる光を照射して得られる反射光に基づいて細胞の画像を形成するＳＴＥＡＭカメラ４０により細胞を撮影し、画像のマッチングにより撮影した細胞が選別対象の細胞であると判定したときにはキャビテーションを用いて本流用流路３６ａに流れる選別対象の細胞を分岐流路３６ｂに押し出して流すことによって選別対象の細胞を選別することにより、ノズルから１細胞ずつ滴下するものに比して、迅速に選別対象の細胞を選別することができる。また、細胞の画像を形成して判定するから、蛍光発光を用いる必要がない。しかも、ＳＴＥＡＭカメラ４０を用いるから、ＣＣＤイメージセンサ（Charge Coupled Device image sensor）を用いて画像形成する場合に比して、迅速に高感度に画像形成することができ、極めて迅速に選別対象の細胞を選別することができる。これらの結果、細胞含有液体における含有率が極めて小さい細胞を選別対象の細胞としても選別対象の細胞を迅速に選別することができる。 According to the cell sorting apparatus 20 of the embodiment described above, 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. After starting to flow in a row, 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. When it is determined that the photographed cells are the cells to be sorted, 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. In addition, 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.
　実施例の細胞選別装置２０に用いる細胞選別用流路デバイス３０によれば、整列用流路３２を有することにより、細胞含有液体を流すだけで、細胞含有液体中にランダムに存在する細胞を一列にして流すことができる。また、撮影用流路３４を有することにより、細胞が一列になって流れる細胞含有液体中の細胞を容易に撮影することができ、撮影した細胞の画像に基づいてその細胞が選別対象の細胞か否かを判定することができる。そして、選別用流路３６を有することにより、選別対象の細胞であると判定された細胞については本流用流路３６ａから分岐流路３６ｂに分岐させることにより、選別対象の細胞を他の細胞から選別することができる。このように整列用流路３２と撮影用流路３４と選別用流路３６とを備えるから、細胞含有液体中の細胞を一列に流して撮影し、選別対象の細胞か否かを判定して分岐流路３６ｂに分岐させて選別する際に必要な流路を単一の流路デバイスで賄うことができる。この結果、細胞選別装置２０の小型化を図ることができる。 According to 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. As described above, since 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.
　実施例の細胞選別装置２０では、細胞選別用流路デバイス３０のキャビテーション発生領域３６ｃとしてキャビテーション用流路３６ｄを形成し、キャビテーション用流路３６ｄに細胞含有液体中の細胞に対して無作用の液体が流されているものとしたが、キャビテーション用流路３６ｄを閉鎖領域として構成するものとしてもよい。この場合、閉鎖領域には生理的食塩水や細胞含有液体の溶媒が充填されるようにすればよい。 In the cell sorting apparatus 20 of the embodiment, 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. However, 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.
　実施例の細胞選別装置２０では、細胞選別用流路デバイス３０のキャビテーション発生領域３６ｃとしてキャビテーション用流路３６ｄと連絡通路３６ｅとを形成するものとしたが、図３の変形例の細胞選別装置１２０に示すように、細胞選別用流路デバイス１３０のキャビテーション発生領域１３６ｃとして本流用流路１３６ａから見ると分岐流路１３６ｂの反対側の凹み（外側に凸）を形成するものとしてもよい。この場合、その凹みの中央がパルスレーザーの照射スポット１３６ｆとなる。なお、変形例の細胞選別装置１２０の細胞選別用流路デバイス１３０における整列用流路３２や撮影用流路３４、ＳＴＥＡＭカメラ４０、画像判定部５０、パルスレーザー６０は、上述した実施例と同一である。 In the cell sorting apparatus 20 of the embodiment, 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. As shown in the figure, a dent (protruding outward) on the opposite side of the branch flow path 136b when viewed from the main flow path 136a may be formed as the cavitation generation region 136c of the cell sorting flow path device 130. In this case, the center of the dent is an irradiation spot 136f of the pulse laser. Note that 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.
　実施例や変形例の細胞選別装置２０，１２０では、パルスレーザー６０により照射スポット３６ｆ，１３６ｆにパルスレーザーを照射することによってキャビテーションを生じさせて選別対象の細胞を分岐流路３６ｂに分岐させるものとしたが、選別対象の細胞を分岐流路３６ｂに分岐させる手法はキャビテーションを用いる以外の手法であってもよい。例えば、選別対象の細胞と判定された細胞を正または負に帯電させ、分岐流路３６ｂ側に負または正の電極を配置して細胞を分岐流路３６ｂ側に泳動させて分岐させるものなどの手法を用いてもよい。 In the cell sorting apparatuses 20 and 120 according to the embodiment and the modification, 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. However, the method of branching the cells to be sorted into the branch flow path 36b may be a method other than using cavitation. For example, 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.
　実施例や変形例の細胞選別装置２０，１２０では、細胞選別用流路デバイス３０，１３０の整列用流路３２として、幅が１００～５００μｍで深さが１０～１００μｍで繰り返し回数が１０～２０回の蛇行する波状の流路として形成したが、図４および図５の変形例の細胞選別用流路デバイス２３０の整列用流路２３２に示すように、幅が１００～５００μｍで深さが１０～１００μｍで矩形流路を形成する頂面には下向きに凸の凸部２３２ａと下向きに凹の凹部２３２ｂとからなる凹凸が２０～５０回に亘って形成された直線状の流路として形成してもよい。整列用流路２３２の幅や深さや凹凸の回数は、細胞含有液体に含まれる細胞の大きさなどによって適宜定められるものである。例えば、細胞が直径１０μｍ程度の大きさの場合、整列用流路２３２は、幅は１００μｍ程度が妥当であり、深さは４０μｍ程度が妥当であり、凸部２３２ａの高さ及び長さは２０μｍ程度および１０μｍ程度が妥当であり、凹部２３２ｂの長さは１００μｍ程度が妥当であり、凹凸の回数は３０回程度が妥当である。変形例の細胞選別用流路デバイス２３０における撮影用流路２３４および選別用流路２３６は、図１に例示した実施例の細胞選別用流路デバイス３０における撮影用流路２３および選別用流路３６と同一である。この変形例の細胞選別用流路デバイス２３０の整列用流路２３２には、レイノルズ数（Reynolds number）が８０程度以下となるように細胞含有液体を流すのが好ましい。変形例の細胞選別用流路デバイス２３０の整列用流路２３２では、頂面に凹凸が形成されているため、流路の断面の広いところと狭いところとが生じ、細胞含有液体中の細胞に慣性力が作用し、次第に細胞が一列に整列するようになる。したがって、変形例の細胞選別用流路デバイス２３０でも実施例の細胞選別用流路デバイス３０と同一の効果を奏することができる。この変形例の細胞選別用流路デバイス２３０の整列用流路２３２では、流路の頂面に凹凸を形成するものとしたが、流路の底面に凹凸を形成するものとしたり、流路の側面の一方に凹凸を形成するものとしたりするなど、流路を形成する少なくとも１つの面に凹凸を形成すれものとすればよい。細胞を一列に整列させる効果を考慮すると、流路の頂面や底面に凹凸を形成するのが好ましい。なお、出願人は、実施例や変形例の細胞選別用流路デバイス３０，１３０の整列用流路３２はジグザクと蛇行しているからジグザグタイプと称し、変形例の細胞選別用流路デバイス２３０の整列用流路２３２は凹部２３２ｂをポケットとみなしてポケットタイプと称している。 In the cell sorting apparatuses 20 and 120 of the embodiments and modifications, the alignment channel 32 of the cell sorting channel devices 30 and 130 has a width of 100 to 500 μm, a depth of 10 to 100 μm, 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 μm and the depth is 10 Formed on the top surface forming a rectangular flow path of ˜100 μm 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. May be. 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 μm in diameter, the alignment channel 232 has an appropriate width of about 100 μm, an appropriate depth of about 40 μm, and the height and length of the convex portion 232a is 20 μm. About 10 μm and about 10 μm are appropriate, the length of the recess 232 b is about 100 μm, 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. 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. In 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. In the alignment flow path 232 of the cell sorting flow path device 230 according to this modified example, 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 | corrugation in at least 1 surface which forms a flow path, such as forming an unevenness | corrugation in 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 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.
　次に、実施例や変形例の細胞選別装置２０，１２０を細胞含有液体における含有率が極めて小さい細胞に適用した場合の事例について説明する。 Next, a case will be described in which the cell sorting apparatuses 20 and 120 of the examples and modifications are applied to cells having a very small content in the cell-containing liquid.
（１）母体血液には胎児細胞（有核血球や赤芽球）が僅かに含まれている。このため、胎児細胞を選別対象の細胞として実施例や変形例の細胞選別装置２０，１２０を用いて細胞選別を行なうものとすれば、選別された胎児細胞のＤＮＡを検査することによって遺伝性の先天性疾患を妊娠６週以降の早期に診断することができる。
（２）記憶Ｂ細胞は感染防御やアレルギー、自己免疫疾患などの様々な場面で重要な影響を与えているが、まだその詳細の多くは知られていない。このため、記憶Ｂ細胞を選別対象の細胞として実施例や変形例の細胞選別装置２０，１２０を用いて細胞選別を行なうものとすれば、選別された記憶Ｂ細胞を用いたヒト免疫不全ウイルス（ＨＩＶ：Human Immunodeficiency Virus）やその他の疾患へのワクチンの創製に資することができる。
（３）ナチュラルキラーＴ細胞（natural killer T cell）は、ＮＫ細胞（ナチュラルキラー細胞，natural killer細胞）とＴ細胞（T cell、T lymphocyte）の両方の性質を持っており、自己免疫と獲得免疫の橋渡しを担っている。このため、ナチュラルキラーＴ細胞を選別対象の細胞として実施例や変形例の細胞選別装置２０，１２０を用いて細胞選別を行なうものとすれば、自己免疫疾患発症制御やアレルギー調節、抗腫瘍作用、流産などに資することができる。
（４）Antigen-Specific T Cellsは、様々なタイプのガン治療やウイルス感染症への治療薬としての働きが期待される希少免疫細胞である。このため、Antigen-Specific T Cellsを選別対象の細胞として実施例や変形例の細胞選別装置２０，１２０を用いて細胞選別を行なうものとすれば、ガン治療やウイルス感染症への治療薬の開発に資することができる。 (1) 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.
(2) Memory B cells have important effects in various scenes such as infection protection, allergies, and autoimmune diseases, but many details are not yet known. For this reason, if 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.
(3) 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.
(4) 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.
（５）Hematopoietic Stem Cellsは、他の血液細胞に分化する幹細胞である。このため、Hematopoietic Stem Cellsを選別対象の細胞として実施例や変形例の細胞選別装置２０，１２０を用いて細胞選別を行なうものとすれば、白血病治療に資することができる。
（６）Circulating Endothelial Cells (CECs)やCirculating Endothelial progenitor Cellsは、欠陥の内側の壁の細胞とその分化前の細胞であり、心血管疾患やガンの際に増加する。このため、Circulating Endothelial Cells (CECs)やCirculating Endothelial progenitor Cellsを選別対象の細胞として実施例や変形例の細胞選別装置２０，１２０を用いて細胞選別を行なうものとすれば、心血管疾患やガンの早期発見に資することができる。
（７）Circulating Tumor Cells (CTCs)は、ガン転移の元となる細胞であり、ガンの進行によって増加する。このため、Circulating Tumor Cells (CTCs)を選別対象の細胞として実施例や変形例の細胞選別装置２０，１２０を用いて細胞選別を行なうものとすれば、ガン転移の可能性やガンの進行の判断などに資することができる。
（８）Circulating Cancer Stem Cellsは、血中を流れるガン幹細胞であり、血中循環腫瘍細胞の一部とされており、実際のガン転移の原因とされている。このため、Circulating Cancer Stem Cellsを選別対象の細胞として実施例や変形例の細胞選別装置２０，１２０を用いて細胞選別を行なうものとすれば、ガン転移の可能性やガンの進行の判断などに資することができる。 (5) 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.
(6) 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. For this reason, if cell sorting is performed using the cell sorting devices 20 and 120 according to the embodiments and the modified examples using Circulating Endothelial Cells (CECs) and Circulating Endothelial progenitor Cells as cells to be sorted, cardiovascular diseases and cancers can be obtained. It can contribute to early detection.
(7) Circulating Tumor Cells (CTCs) 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.
(8) 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.
Ｂ．細胞除去装置関係
　次に、本発明の一実施例としての第１造血幹細胞用細胞除去装置１０２０について説明する。図６は、第１造血幹細胞用細胞除去装置１０２０の構成の概略を示す構成図である。図示するように、実施例の第１造血幹細胞用細胞除去装置１０２０は、造血幹細胞を含有する液体（造血幹細胞含有液体）を流すために一体形成された第１細胞破壊除去用流路デバイス１０３０と、造血幹細胞含有液体中の細胞を検出するプローブレーザ光を出力すると共に細胞を破壊する破壊レーザ光を出力するレーザ１０６０と、プローブレーザ光の散乱光に基づいて細胞を検出すると共に細胞が蛍光発現しているかを検出する光検出器１０７０と、光検出器１０７０からの検出信号に基づいてレーザ１０６０を制御する制御装置１０８０と、を備える。ここで、「造血幹細胞含有液体」としては、患者の骨髄から採取した液体や、患者に抗がん剤治療したあとや白血球を増やすＧ－ＣＳＦ（顆粒球コロニー刺激因子）を使って骨髄から幹細胞を血液中に流れ出させた際に成分献血と同様の方法で採取した液体の他、これらの採取した液体を生理的食塩水などの溶媒により薄めた溶液なども含まれる。 B. Cell Removal Device Next, a first hematopoietic stem cell removal device 1020 as one embodiment of the present invention will be described. FIG. 6 is a configuration diagram showing an outline of the configuration of the first hematopoietic stem cell removal device 1020. As shown in the figure, 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). A laser 1060 for outputting a probe laser beam for detecting cells in a hematopoietic stem cell-containing liquid and outputting a destructive laser beam for destroying the cells, and detecting the cells based on the scattered light of the probe laser beam and expressing the cells with fluorescence. A photodetector 1070 that detects whether the laser beam is detected and a control device 1080 that controls the laser 1060 based on a detection signal from the photodetector 1070. Here, “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. In addition to liquids collected in the same manner as the component blood donation when flowing into the blood, solutions obtained by diluting these collected liquids with a solvent such as physiological saline are also included.
　第１細胞破壊除去用流路デバイス１０３０は、ポリジメチルシロキサン（PDMS：polydimethylsiloxane）やエポキシ樹脂などの造血幹細胞含有液体に浸食されない材料により、上流側から、造血幹細胞含有液体中の細胞が一列になって流れるようにする整列用流路１０３２と、造血幹細胞含有液体中の細胞を検出したり破壊レーザ光により細胞を破壊したりする第１処理用流路１０３４と、破壊した細胞を造血幹細胞含有液体から分離する第１分離用流路１０３６と、第１処理用流路１０３４と同様に造血幹細胞含有液体中の細胞を検出したり破壊レーザ光により細胞を破壊したりする第２処理用流路１０４４と、第１分離用流路１０３６と同様に破壊した細胞を造血幹細胞含有液体から分離する第２分離用流路１０４６と、からなるように一体形成されている。なお、第１細胞破壊除去用流路デバイス１０３０は、造血幹細胞含有液体の流速が１．５ｍ／ｓ未満であるときにはポリジメチルシロキサンにより形成されるのが好ましく、造血幹細胞含有液体の流速が１．５ｍ／ｓ以上であるときにはエポキシ樹脂により形成されるのが好ましい。 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. A flow path for alignment 1032 for allowing the cells to flow in the first flow path 1034 for detecting cells in the hematopoietic stem cell-containing liquid or destroying the cells with a destructive laser beam, and the hematopoietic stem cell-containing liquid for destroying the cells. 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. And 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.
　整列用流路１０３２は、幅が１００～５００μｍで深さが１０～１００μｍで繰り返し回数が１０～２０回の蛇行する波状の流路、実施例では幅が３３０μｍで深さが５０μｍで繰り返し回数が１５回の蛇行する波状の流路として形成されている。整列用流路１０３２の幅や深さや蛇行の繰り返し回数は、造血幹細胞含有液体に含まれる細胞の大きさなどによって適宜定めればよい。整列用流路１０３２では、流路が繰り返し蛇行していることにより、造血幹細胞含有液体中にランダムに存する細胞は、蛇行の際の慣性により徐々に一列に整列するようになる。 The alignment channel 1032 is a wavy channel having a meandering width of 100 to 500 μm, a depth of 10 to 100 μm and a number of repetitions of 10 to 20 times. In the embodiment, the width is 330 μm and the depth is 50 μm and the number of repetitions is 10 to 100 μm. 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. In 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.
　第１処理用流路１０３４は、整列用流路１０３２の後段に、この整列用流路１０３２と同一の幅で同一の深さの流路で基本的には直線状となるように形成されている。 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.
　第１分離用流路１０３６は、第１処理用流路１０３４の後段に形成されており、本流に両サイドから造血幹細胞含有液体の溶媒と同一のシース流を合流させるシース流用流路１０３６ａ，１０３６ｂと、円柱形状の複数のポスト１０３６ｃが規則的に形成されて造血幹細胞と破壊された細胞とを峻別するポストアレイ流路１０３６ｄと、破壊された細胞と分離された造血幹細胞を含有する造血幹細胞含有液体を第２処理用流路１０４４に流す連絡流路１０３６ｅと、破壊された細胞を流す分離流路１０３６ｆと、により構成されている。シース流用流路１０３６ａ，１０３６ｂは、幅が第１処理用流路１０３４の半分程度で第１処理用流路１０３４と同一の深さに形成されており、第１処理用流路１０３４からの流れに対して若干高い圧力によって溶媒を流してシース流とする。こうしたシース流を用いることにより、造血幹細胞含有液体を流路の中央に流すことができる。複数のポスト１０３６ｃは、実施例では、半径６０μｍの円柱として形成されており、隣接するポスト１０３６ｃとの最小幅が３０μｍとなるように配置されている。そして、ポストアレイは流路の方向に対して若干の角度（例えば５度～６度程度）となるように、即ち、流れ方向に並ぶポスト１０３６ｃの並びの直線が流路に対して若干の角度となるように複数のポスト１０３６ｃを配置するのである。このポストアレイ流路１０３６ｄに流体を流すと、流体力学によれば、直径が隣接するポスト１０３６ｃとの最小幅の１／３より小さい粒子（破壊された細胞）は流体の流れと同様に流れ、直径が隣接するポスト１０３６ｃとの最小幅の１／３より大きい粒子（破壊されていない細胞）は流体の流れから左右に離れるように流れる。したがって、ポストアレイを流路の方向に対して若干の角度となるように形成すれば、直径が隣接するポスト１０３６ｃとの最小幅の１／３より大きい粒子（破壊されていない細胞）を流体の流れに対して一定方向に離れるようにすることができる。実施例では、図６中、ポストアレイを若干左回転させて角度をもたせているから、大きい粒子（破壊されていない細胞）は右方向に離れるように流れる。このため、大きい粒子（破壊されていない細胞）は、ポストアレイ流路１０３６ｄの出口付近では、連絡流路１０３６ｅ側に寄っているから、シース流の溶媒と共に連絡流路１０３６ｅに流れ込む。一方、小さい粒子（破壊された細胞）は、ポストアレイ流路１０３６ｄの中央付近を流れるから、分離流路１０３６ｆに流れ込む。なお、実施例では、連結流路３６ｅは第１処理用流路１０３４と同一の幅で同一の深さとなるように形成されている。 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. On the other hand, the solvent is flowed at a slightly high pressure to form a sheath flow. By using such a sheath flow, the hematopoietic stem cell-containing liquid can be flowed to the center of the flow path. In the embodiment, the plurality of posts 1036c are formed as cylinders having a radius of 60 μm, and are arranged so that the minimum width between adjacent posts 1036c is 30 μm. 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. Therefore, if 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. In the embodiment, in FIG. 6, 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. On the other hand, small particles (broken cells) flow near the center of the post-array channel 1036d, and therefore flow into the separation channel 1036f. In the embodiment, the connecting channel 36e is formed to have the same width and the same depth as the first processing channel 1034.
　第２処理用流路１０４４は、連絡流路１０３６ｅの後段に、第１処理用流路１０３４と同様に、連絡流路１０３６ｅと同一の幅で同一の深さの流路で基本的には直線状となるように形成されている。 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.
　第２分離用流路１０４６は、第２処理用流路１０４４の後段に、第１分離用流路１０３６と同様の構成、即ち、シース流用流路１０４６ａ，１０４６ｂと、ポストアレイ流路１０４６ｄと、連絡流路１０３６ｅに相当する出力流路１０４６ｅと、分離流路１０４６ｆと、により構成されている。第２分離用流路１０４６のポストアレイ流路１０４６ｄでも、第１分離用流路１０３６のポストアレイ流路１０３６ｄと同様に、ポストアレイを若干左回転させて角度をもたせているから、大きい粒子（破壊されていない細胞）は、右方向に離れるように流れて出力流路１０４６ｅに流れ込み、小さい粒子（破壊された細胞）は、ポストアレイ流路１０４６ｄの中央付近を流れて分離流路１０４６ｆに流れ込む。 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. Similarly to the post-array flow path 1036d of the first separation flow path 1036, 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. .
　レーザ１０６０は、例えば、スペクトラ・フィジックス株式会社（Spectra-Physics）製の源一体型高出力CWグリーンレーザー Millennia eV（商標登録）などを用いることができ、出力を変更したりビームスプリッターで分離したりして細胞検出用のプローブレーザ光と細胞破壊用の破壊レーザ光とを出力することができる。レーザ１０６０からのレーザ光は、ビームスプリッター１０６１によってミラー１０６２と可動ミラー１０６６とに分離される。ミラー１０６２に照射されたレーザ光は、可動ミラー１０６３とハーフミラー１０６４とを介して第１処理用流路１０３４に照射され、その散乱光や反射光は、ハーフミラー１０６４、ミラー１０６５を介して光検出器１０７０に入力される。一方、可動ミラー１０６６に照射されたレーザ光は、ハーフミラー１０６７を介して第２処理用流路１０４４に照射され、その散乱光や反射光は、ハーフミラー１０６７、ミラー１０６５を介して光検出器１０７０に入力される。可動ミラー１０６３，１０６６は、ミラーの回転角を変更するアクチュエータ１０６３ａ，１０６６ａが取り付けられており、プローブレーザ光によって細胞が検出され、その細胞を破壊するために破壊レーザ光を照射するときに、プローブレーザ光の照射から破壊レーザ光の照射までの時間で細胞が第１処理用流路１０３４や第２処理用流路１０４４において移動する（流れる）距離に応じた角度だけ回転角を変更することができるようになっている。この回転角は、第１処理用流路１０３４や第２処理用流路１０４４における造血幹細胞含有液体の流速と、可動ミラー１０６３，１０６６と第１処理用流路１０３４や第２処理用流路１０４４との距離と、プローブレーザ光の照射から破壊レーザ光の照射までに要する時間と、により定めることができる。 As 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. On the other hand, 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. When the cells are detected by the probe laser light and the destruction laser light is irradiated to destroy the cells, the probe is used. 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.
　光検出器１０７０は、プローブレーザ光に照射に対する散乱光の光強度を検出して第１処理用流路１０３４や第２処理用流路１０４４に流れている造血幹細胞含有液体中の細胞を検出したりマーカーを用いることにより細胞が蛍光発現したときにはその蛍光発現による光強度を検出する複数の光センサ（例えば、フォトダイオードなど）により構成されている。 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. When 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.
　制御装置１０８０は、図示しないが、ＣＰＵを中心とするマイクロコンピュータとして構成されており、ＣＰＵの他にＲＯＭやＲＡＭ、入力ポート、出力ポートなどを備える。制御装置１０８０には、光検出器１０７０からの検出信号などが入力ポートを介して入力されており、制御装置１０８０からは、レーザ１０６０や可動ミラー１０６３，１０６６への駆動制御信号などが出力ポートから出力されている。 Although not shown, the 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.
　次に、こうして構成された第１造血幹細胞用細胞除去装置１０２０の動作について説明する。なお、第１細胞破壊除去用流路デバイス１０３０には、整列用流路１０３２の入口から細胞を１粒子としたときに粒子レイノルズ数（particle Reynolds number）が値１程度以下となるように造血幹細胞含有液体が流されているものとする。このように、粒子レイノルズ数が値１程度以下となるように造血幹細胞含有液体を整列用流路１０３２に流すと、造血幹細胞含有液体中の細胞は、整列用流路１０３２を流下するうちに一列になって流れるようになる。また、造血幹細胞含有液体には、第１処理用流路１０３４に至る前にマーカーとしてＣＤ３４が添加されるものとする。ＣＤ３４は、ヒト細胞分化抗原（human cell differentiation molecules (HCDM)）ワークショップで３４番目のＣＤ（cluster of differentiation：分化抗原群）とされ、造血幹細胞に陽性発現する造血幹細胞マーカーとしての機能を有するものである。さらに、造血幹細胞含有液体には、第２処理用流路１０４４に至る前にマーカーとしてＣＤ３８が添加されるものとする。ＣＤ３８は、ＨＣＤＭワークショップで３８番目のＣＤとされ、リンパ球の増殖を活性化する膜タンパク質であり、造血幹細胞に陰性発現するものである。また、連絡流路１０３６ｅは、ＣＤ３４が機能を失う程度に十分に長い流路として構成されているものとする。 Next, the operation of the first hematopoietic stem cell removing apparatus 1020 configured as described above will be described. 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. In addition, 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. Furthermore, 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. Further, it is assumed that the communication channel 1036e is configured as a channel that is long enough for the CD 34 to lose its function.
　第１造血幹細胞用細胞除去装置１０２０の第１処理用流路１０３４では、整列用流路１０３２により細胞が一列になって流れるようになった造血幹細胞含有液体に対して、ＣＤ３４を用いたＣＤ３４陽性試験により造血幹細胞含有液体中の造血幹細胞はそのままに腫瘍細胞（特定細胞）や腫瘍細胞（特定細胞）の可能性のある細胞を含む範囲に属する細胞（第１所定細胞）については破壊する処理（以下、「ＣＤ３４陽性試験による処理」という。）が行なわれる。図７は、第１処理用流路１０３４に対して制御装置１０８０により実行されるＣＤ３４陽性試験用レーザ照射制御の一例を示すフローチャートである。ＣＤ３４陽性試験用レーザ照射制御では、まず、レーザ１０６０から第１処理用流路１０３４にプローブレーザ光が照射されるようレーザ１０６０に照射制御信号が出力される（ステップＳ１１００）。続いて、光検出器１０７０からプローブレーザ光の照射による散乱光や反射光に基づく光検出信号を入力し（ステップＳ１１１０）、散乱光により細胞が検出されたか否かを判定すると共に（ステップＳ１１２０）、ＣＤ３４により陽性発現しているか否かを判定する（ステップＳ１１３０）。細胞が検出されていないときは破壊レーザ光を照射する必要がないと判断して本ルーチンを終了し、細胞が検出されてもその細胞が陽性発現しているときには、その細胞は造血幹細胞である可能性が高いと判断し、破壊レーザ光を照射することなく本ルーチンを終了する。一方、細胞が検出されているがその細胞が陽性発現していないときには、その細胞は第１所定細胞（造血幹細胞ではなく且つ腫瘍細胞か腫瘍細胞の可能性のある細胞を含む範囲に属する細胞）であると判断し、その細胞を破壊するための破壊レーザ光を第１処理用流路１０３４に照射するようレーザ１０６０に照射制御信号を出力して（ステップＳ１１４０）、本ルーチンを終了する。このように、プローブレーザ光とＣＤ３４とを用いることにより、細胞を検出すると共にその細胞が第１所定細胞であるか否かを判断し、第１所定細胞であると判断したときにはレーザ１０６０から破壊レーザ光を照射して第１所定細胞を破壊するから、造血幹細胞含有液体中の造血幹細胞を残存したまま腫瘍細胞（特定細胞）を含む第１所定細胞を破壊することができる。 In the first treatment flow path 1034 of the first hematopoietic stem cell removal apparatus 1020, CD34 positive using CD34 with respect to the hematopoietic stem cell-containing liquid in which the cells flow in a line by the alignment flow path 1032. A treatment for destroying cells (first predetermined cells) belonging to a range including cells that may be tumor cells (specific cells) or tumor cells (specific cells) while maintaining the hematopoietic stem cells in the hematopoietic stem cell-containing liquid by the test ( Hereinafter, “treatment by CD34 positive test” is performed. 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. In the CD34 positive test laser irradiation control, first, 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). Subsequently, 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. If 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. On the other hand, when a cell is detected but the cell is not positively expressed, 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. As described above, by using the probe laser beam and the CD 34, a cell is detected and it is determined whether or not the cell is the first predetermined cell. When it is determined that the cell is the first predetermined cell, 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.
　こうして第１処理用流路１０３４で処理された造血幹細胞含有液体は、第１分離用流路１０３６に流れるが、第１分離用流路１０３６のポストアレイ流路１０３６ｄを隣接するポスト１０３６ｃとの最小幅の１／３が造血幹細胞の直径より小さくなると共に破壊された細胞の細胞片の直径より大きくなるように形成しておくことにより、造血幹細胞含有液体中の造血幹細胞は連絡流路１０３６ｅに流れるようになり、造血幹細胞含有液体中の破壊された細胞の細胞片は分離流路１０３６ｆに流れるようになる。実施例では、このように造血幹細胞と破壊された細胞の細胞片とが分離されるように、ポスト１０３６ｃを半径６０μｍの円柱として形成すると共に隣接するポスト１０３６ｃとの最小幅が３０μｍとなるように、且つ、ポストアレイが流路の方向に対して若干の角度（例えば５度～６度程度）となるように、ポストアレ流路１０３６ｄを形成した。 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. As a result, the broken cell pieces in the hematopoietic stem cell-containing liquid flow into the separation channel 1036f. In the embodiment, the post 1036c is formed as a cylinder having a radius of 60 μm 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 μm. In addition, 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.
　第１造血幹細胞用細胞除去装置１０２０の第２処理用流路１０４４では、第１分離用流路１０３６により破壊された細胞の細胞片とが分離されて連絡流路１０３６ｅを流れる造血幹細胞含有液体に対して、ＣＤ３８を用いたＣＤ３８陰性試験により造血幹細胞含有液体中の造血幹細胞はそのままに腫瘍細胞（特定細胞）や腫瘍細胞（特定細胞）の可能性のある細胞を含む範囲に属する細胞（第２所定細胞）については破壊する処理（以下、「ＣＤ３８陰性試験による処理」という。）が行なわれる。図８は、第２処理用流路１０４４に対して制御装置１０８０により実行されるＣＤ３８陰性試験用レーザ照射制御の一例を示すフローチャートである。ＣＤ３８陰性試験用レーザ照射制御では、まず、レーザ１０６０から第２処理用流路１０４４にプローブレーザ光が照射されるようレーザ１０６０に照射制御信号が出力される（ステップＳ１２００）。続いて、光検出器１０７０からプローブレーザ光の照射による散乱光や反射光に基づく光検出信号を入力し（ステップＳ１２１０）、散乱光により細胞が検出されたか否かを判定すると共に（ステップＳ１２２０）、ＣＤ３８により陽性発現しているか否かを判定する（ステップＳ１２３０）。細胞が検出されていないときは破壊レーザ光を照射する必要がないと判断して本ルーチンを終了し、細胞が検出されているがその細胞が陽性発現していないときには、その細胞は造血幹細胞である可能性が高いと判断し、破壊レーザ光を照射することなく本ルーチンを終了する。一方、細胞が検出されており且つその細胞が陽性発現しているときには、その細胞は第２所定細胞（造血幹細胞ではなく且つ腫瘍細胞か腫瘍細胞の可能性のある細胞を含む範囲に属する細胞）であると判断し、その細胞を破壊するための破壊レーザ光を第２処理用流路１０４４に照射するようレーザ１０６０に照射制御信号を出力して（ステップＳ１２４０）、本ルーチンを終了する。このように、プローブレーザ光とＣＤ３８とを用いることにより、細胞を検出すると共にその細胞が第２所定細胞であるか否かを判断し、第２所定細胞であると判断したときにはレーザ１０６０から破壊レーザ光を照射して第２所定細胞を破壊するから、造血幹細胞含有液体中の造血幹細胞を残存したまま腫瘍細胞（特定細胞）を含む第２所定細胞を破壊することができる。 In the second treatment flow path 1044 of the first hematopoietic stem cell removal apparatus 1020, 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. On the other hand, 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”). FIG. 8 is a flowchart showing an example of CD38 negative test laser irradiation control executed by the control device 1080 on the second processing flow path 1044. In the laser irradiation control for CD38 negative test, first, 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). Subsequently, 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). If no cells are detected, it is determined that irradiation with a destructive laser beam is not necessary, and this routine ends.If cells are detected but the cells are not positively expressed, the cells are hematopoietic stem cells. It is determined that there is a high possibility, and this routine is finished without irradiating the destructive laser beam. On the other hand, when a cell is detected and the cell is positively expressed, 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. As described above, by using the probe laser beam and the CD 38, a cell is detected and it is determined whether or not the cell is the second predetermined cell. When it is determined that 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.
　こうして第２処理用流路１０４４で処理された造血幹細胞含有液体は、第２分離用流路１０４６に流れるが、第２分離用流路１０４６のポストアレイ流路１０４６ｄを第１分離用流路１０３６のポストアレイ流路１０３６ｄと同様に、造血幹細胞と破壊された細胞の細胞片とが分離されるように、ポスト１０３６ｃを半径６０μｍの円柱として形成すると共に隣接するポスト１０３６ｃとの最小幅が３０μｍとなるように、且つ、ポストアレイが流路の方向に対して若干の角度（例えば５度～６度程度）となるように形成することにより、造血幹細胞含有液体中の造血幹細胞は出力流路１０４６ｅに流れ、造血幹細胞含有液体中の破壊された細胞の細胞片は分離流路１０４６ｆに流れる。 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. In the same manner as the post-array flow path 1036d, the post 1036c is formed as a cylinder having a radius of 60 μm so that hematopoietic stem cells and broken cell pieces are separated, and the minimum width between adjacent posts 1036c is 30 μm. In addition, 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.
　出力流路１０４６ｅに流れる造血幹細胞含有液体には、上述したＣＤ３４陽性試験による処理とＣＤ３８陰性試験による処理とにより、造血幹細胞について残存し、造血幹細胞ではなく且つ腫瘍細胞（特定細胞）か腫瘍細胞（特定細胞）の可能性のある細胞を含む範囲に属する細胞（第１所定細胞か第２所定細胞のいずれか）については破壊するから、より確実に、造血幹細胞含有液体中の造血幹細胞を残存したまま腫瘍細胞（特定細胞）を破壊することができる。一般的に、ＣＤ３４陽性試験による処理では９９．９９％の確率で腫瘍細胞を除去することができ、ＣＤ３８陰性試験による処理では９９．９９％の確率で腫瘍細胞を除去することができるといわれているから、実施例の第１造血幹細胞用細胞除去装置１０２０では、その双方を用いることにより、より確実に造血幹細胞含有液体から腫瘍細胞を除去することができる。 In the hematopoietic stem cell-containing liquid flowing in the output channel 1046e, 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.
　以上説明した実施例の第１造血幹細胞用細胞除去装置１０２０によれば、第１細胞破壊除去用流路デバイス１０３０の整列用流路１０３２で造血幹細胞含有液体中の細胞が一列になって流れるようにし、第１処理用流路１０３４でプローブレーザ光の照射とＣＤ３４とを用いたＣＤ３４陽性試験による処理を実行して腫瘍細胞を含む第１所定細胞を検出して破壊レーザ光により破壊し、第１分離用流路１０３６で造血幹細胞含有液体中の破壊された細胞を分離除去し、更に、第２処理用流路１０４４でプローブレーザ光の照射とＣＤ３８とを用いたＣＤ３８陰性試験による処理を実行して腫瘍細胞を含む第２所定細胞を検出して破壊レーザ光により破壊し、第２分離用流路１０４６で造血幹細胞含有液体中の破壊された細胞を分離除去することにより、造血幹細胞含有液体中に造血幹細胞を残したままより確実に腫瘍細胞を除去することができる。したがって、この造血幹細胞用細胞装置２０により処理した造血幹細胞含有液体を用いて自家造血幹細胞移植を行なうことにより、拒絶反応を抑制することはもとより、再発を有効に抑制することができる。 According to the first hematopoietic stem cell removal apparatus 1020 of the embodiment described above, 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. Then, 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.
　実施例の第１造血幹細胞用細胞除去装置１０２０に用いる第１細胞破壊除去用流路デバイス１０３０によれば、整列用流路１０３２を有することにより、造血幹細胞含有液体を流すだけで、造血幹細胞含有液体中にランダムに存在する細胞を一列にして流すことができる。また、第１処理用流路１０３４や第２処理用流路１０４４を有することにより、ＣＤ３４陽性試験による処理やＣＤ３８陰性試験による処理を実行して造血幹細胞ではない腫瘍細胞を含む第１所定細胞や第２所定細胞を破壊することができる。そして、第１分離用流路１０３６や第２分離用流路１０４６を有することにより、造血幹細胞含有液体から破壊した細胞の細胞片と造血幹細胞とを分離することができる。このように、整列用流路１０３２と第１処理用流路１０３４と第１分離用流路１０３６と第２処理用流路１０４４と第２分離用流路１０４６とを備えるから、造血幹細胞含有液体中の細胞を一列に流してＣＤ３４陽性試験による処理やＣＤ３８陰性試験による処理を行なって腫瘍細胞を含む第１所定細胞や第２所定細胞については破壊レーザ光を照射して破壊して分離する際に必要な流路を単一の流路デバイスで賄うことができる。この結果、第１造血幹細胞用細胞除去装置１０２０の小型化を図ることができる。 According to the first cell destruction / removal channel device 1030 used in the first hematopoietic stem cell cell removal device 1020 of the example, 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. Then, by having 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. As described above, since 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. When the cells are flown in a row and processed by CD34 positive test or CD38 negative test, 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.
　実施例の第１造血幹細胞用細胞除去装置１０２０では、整列用流路１０３２と第１処理用流路１０３４と第１分離用流路１０３６と第２処理用流路１０４４と第２分離用流路１０４６とが一体形成された第１細胞破壊除去用流路デバイス１０３０を用いるものとしたが、整列用流路と第１処理用流路と第１分離用流路とが一体形成された第１流路デバイスと、第２処理用流路と第２分離用流路とが一体形成された第２流路デバイスとからなるものとしてもよい。この場合、第１流路デバイスにおける第１分離用流路の連絡流路からの造血幹細胞含有液体を、第２流路デバイスにおける第２処理用流路に流すようにすればよい。また、整列用流路１０３２だけが形成された整列流路デバイスと、第１処理用流路１０３４と第１分離用流路１０３６とが一体形成された第１処理分離流路デバイスと、第２処理用流路１０４４と第２分離用流路１０４６とが一体形成された第２処理分離流路デバイスとからなるものとしてもよい。この場合、第１処理分離流路デバイスと第２処理分離流路デバイスは、共通部材としてもよい。 In the first hematopoietic stem cell removal apparatus 1020 of the embodiment, 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. In addition, 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. In this case, the first processing separation channel device and the second processing separation channel device may be a common member.
　実施例の第１造血幹細胞用細胞除去装置１０２０では、第１処理用流路１０３４でＣＤ３４陽性試験による処理を実行した後に、第２処理用流路１０４４でＣＤ３８陰性試験による処理を実行するものとしたが、第１処理用流路１０３４でＣＤ３８陰性試験による処理を実行した後で、第２処理用流路１０４４でＣＤ３４陽性試験による処理を実行するものとしてもよい。 In the cell removal apparatus 1020 for the first hematopoietic stem cell of the example, after performing the process by the CD34 positive test in the first processing flow path 1034, the process by the CD38 negative test is performed in the second processing flow path 1044. However, after the processing by the CD38 negative test is executed in the first processing flow path 1034, the processing by the CD34 positive test may be executed in the second processing flow path 1044.
　実施例の第１造血幹細胞用細胞除去装置１０２０では、第１処理用流路１０３４でＣＤ３４陽性試験による処理を実行した後に、第２処理用流路１０４４でＣＤ３８陰性試験による処理を実行するものとしたが、ＣＤ３４陽性試験による処理かＣＤ３８陰性試験による処理の一方だけを実行するものとしてもよい。この場合、流路デバイスとしては、整列用流路１０３２と第１処理用流路１０３４と第１分離用流路１０３６とを備えればよい。 In the cell removal apparatus 1020 for the first hematopoietic stem cell of the example, after performing the process by the CD34 positive test in the first processing flow path 1034, the process by the CD38 negative test is performed in the second processing flow path 1044. However, only one of the processing by the CD34 positive test or the processing by the CD38 negative test may be executed. In this case, the flow path device may include the alignment flow path 1032, the first processing flow path 1034, and the first separation flow path 1036.
　実施例の第１造血幹細胞用細胞除去装置１０２０では、第１処理用流路１０３４におけるＣＤ３４陽性試験による処理の実行も第２処理用流路１０４４におけるＣＤ３８陰性試験による処理の実行も単一のレーザ１０６０により行なわれるものとしたが、第１処理用流路１０３４におけるＣＤ３４陽性試験による処理を実行するための第１レーザを備えると共に、第２処理用流路１０４４におけるＣＤ３８陰性試験による処理を実行するための第２レーザを備えるものとしてもよい。また、プローブレーザ光に照射と破壊レーザ光の照射とを単一のレーザ１０６０により行なうものとしたが、プローブレーザ光を照射するための検出用レーザを備えると共に破壊レーザ光を照射するための破壊用レーザを備えるものとしてもよい。即ち、実施例のように単一のレーザ１０６０だけを備える構成、第１処理用流路１０３４におけるＣＤ３４陽性試験による処理を実行するためのプローブレーザ光と破壊レーザ光とを照射する第１レーザと第２処理用流路１０４４におけるＣＤ３８陰性試験による処理を実行するためのプローブレーザ光と破壊レーザ光とを照射する第２レーザとの２つのレーザを備える構成、第１処理用流路１０３４におけるＣＤ３４陽性試験による処理および第２処理用流路１０４４におけるＣＤ３８陰性試験による処理のためのプローブレーザ光を照射する検出用レーザと両処理のための破壊レーザ光を照射する破壊用レーザとの２つのレーザを備える構成、第１処理用流路１０３４におけるＣＤ３４陽性試験による処理を実行するためのプローブレーザ光と破壊レーザ光とを各々照射する２つのレーザと第２処理用流路１０４４におけるＣＤ３８陰性試験による処理を実行するためのプローブレーザ光と破壊レーザ光とを各々照射する２つのレーザとの合計４つのレーザを備える構成、のうちいずれの構成としてもよいのである。 In the first hematopoietic stem cell removal apparatus 1020 of the embodiment, 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. In addition, irradiation with the probe laser light and irradiation with the destruction laser light are performed by the single laser 1060. However, the detection laser for irradiation with the probe laser light is provided and the destruction for irradiation with the destruction laser light is performed. It may be provided with 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 configuration including two lasers, a probe laser beam and a second laser beam that irradiates a destruction laser beam, for executing processing by the CD38 negative test in the second processing channel 1044, CD34 in the first processing channel 1034 Two lasers: a detection laser that irradiates probe laser light for processing by a positive test and a CD38 negative test in the second processing flow path 1044 and a destructive laser that irradiates destructive laser light for both treatments 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 of the four lasers may be used.
　実施例の第１造血幹細胞用細胞除去装置１０２０では、ＣＤ３４を用いて第１処理用流路１０３４において第１所定細胞を破壊する処理を実行すると共にＣＤ３８を用いて第２処理用流路１０４４において第２所定細胞を破壊する処理を実行するものとしたが、マーカーとして造血幹細胞に対しては陽性であると共に腫瘍細胞に対しては陰性であるものであればＣＤ３４以外のマーカーをＣＤ３４に代えて用いたり、マーカーとして造血幹細胞に対しては陰性であると共に腫瘍細胞に対しては陽性であるものであればＣＤ３８以外のマーカーをＣＤ３８に代えて用いたりしてもよい。 In the first hematopoietic stem cell removal apparatus 1020 of the embodiment, 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. However, if the marker is positive for hematopoietic stem cells and negative for tumor cells, a marker other than CD34 is replaced with CD34. As long as it is negative for hematopoietic stem cells and positive for tumor cells, a marker other than CD38 may be used instead of CD38.
　次に、本発明の一実施例としての第２造血幹細胞用細胞除去装置１１２０について説明する。図９は、実施例の第２造血幹細胞用細胞除去装置１１２０の構成の概略を示す構成図である。図示するように、実施例の第２造血幹細胞用細胞除去装置１１２０は、造血幹細胞を含有する造血幹細胞含有液体を流すために一体形成された第２細胞破壊除去用流路デバイス１１３０と、造血幹細胞含有液体中の腫瘍細胞（特定細胞）および腫瘍細胞の可能性のある細胞（第３所定細胞）を検出するＳＴＥＡＭカメラ装置１１５０と、細胞を破壊する破壊レーザ光を照射するレーザ１１６０と、ＳＴＥＡＭカメラ装置１１５０からの制御信号に基づいてレーザ１１６０を制御する制御装置１１８０と、を備える。 Next, a second hematopoietic stem cell removal device 1120 according to an embodiment of the present invention will be described. FIG. 9 is a configuration diagram showing an outline of the configuration of the second hematopoietic stem cell removal device 1120 of the example. As shown in the figure, 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.
　第２細胞破壊除去用流路デバイス１１３０は、上述の第１細胞破壊除去用流路デバイス１０３０の整列用流路１０３２と第１処理用流路１０３４と第１分離用流路１０３６と同様の、整列用流路１１３２と第１処理用流路１１３６と第１分離用流路１１３６とから構成されており、これらが一体形成されている。したがって、第１分離用流路１１３６には、第１分離用流路１０３６と同様に、シース流用流路１１３６ａ，１１３６ｂと、ポストアレイ流路１１３６ｄと、連絡流路１１３６ｅに相当する出力流路１１３６ｅと、分離流路１１３６ｆと、が形成されている。 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.
　ＳＴＥＡＭカメラ装置１１５０の構成の概略を図１０に示す。図示するように、ＳＴＥＡＭカメラ装置１１５０は、フェムト秒ないしナノ秒オーダーの間隔で繰り返し短パルス光を出力する短パルス光源１１５１と、短パルス光源１１５１からの光は透過すると共に反対側からの光は反射するハーフミラー１１５２と、短パルス光源１１５１からの光については分光すると共に反対側からの分光された光については集光（合成）する分光器１１５３と、分光した光を平行光とするレンズ１１５４と、ハーフミラー１１５２から反射されたパルス光を時間の経過に伴って順次波長の異なる光とする波長選別用光ファイバ１１５５と、光の強度を検出する光強度検出器１１５６と、波長毎の光の強度に基づいて画像形成する画像形成処理部１１５７と、画像形成された細胞が腫瘍細胞を含む第３所定細胞であるか否かの判定を行なう判定部１１５８と、を備える。 A schematic configuration of the STEAM camera device 1150 is shown in FIG. As shown in the figure, 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.
　分光器１１５３は、直線上の位置毎に波長が順次異なるように分光するよう調整されている。このため、分光器１１５３によって分光されレンズ１１５４によって平行光とされた光は、直線上の位置毎に順次波長が異なる平行な波長別の光の群（波長別光群）となって第１処理用流路１１３４に照射されることになる。即ち、第１処理用流路１１３４には、幅方向の直線上の位置によって異なる波長の光が照射されるのである。この波長別光群の反射光（反射波長別光群）は、第１処理用流路１１３４に流れる物体の幅方向の直線上の情報を光強度として持ってレンズ１１５４によって集められ、分光器１１５３によって集光（合成）されてパルス光（反射パルス光）とされる。 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).
　波長選別用光ファイバ１１５５は、偏波保持型の光ファイバ１１５５ａと、光ファイバ１１５５ａからの光の強度を増幅するために逆方向に光を入力する光ポンプ１１５５ｂとにより構成されている。偏波保持型の光ファイバ１１５５ａは、入力した光の波長の長短により出力する時間が変化する性質を有するため、反射パルス光を入力すると、波長の長い光から順に出力する。したがって、波長選別用光ファイバ１１５５から出力される光に対して時間の経過に伴って光強度検出器１１５６により光強度を検出することにより、波長毎の光の強度を検出ことができる。上述したように波長毎の光の強度は、第１処理用流路１１３４の幅方向の直線上の物体の情報となるから、この波長毎の光の強度を用いて画像形成処理部１１５７により第１処理用流路１１３４に流れる物体の画像を形成することができる。 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.
　判定部１１５８は、画像形成された第１処理用流路１１３４に流れる細胞が造血幹細胞ではなく且つ腫瘍細胞（特定細胞）および腫瘍細胞（特定細胞）である可能性のある細胞の範囲である細胞（第３所定細胞）であるか否かの画像のマッチングを行なう。画像のマッチングは、予め登録した腫瘍細胞（特定細胞）の画像に対して一定の誤差の範囲内で入力した細胞の画像が一致するか否かを判定するものなどを適用することができる。この判定部１１５８と上述した画像形成処理部１１５７はそれぞれの処理プログラムがインストールされた汎用のコンピュータにより構成することができる。 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.
　ＳＴＥＡＭカメラ装置１１５０は、上述したように、短パルス光を分光して第１処理用流路１１３４の幅方向の直線上の位置によって波長が順次異なる光を照射し、その反射光を集光（合成）して波長選別用光ファイバ１１５５により波長の長い光から順に出力させ、波長毎の光の強度に基づいて第１処理用流路１１３４に流れる物体（細胞）の画像形成を行なうから、フォトンの量（光強度）に依存するＣＣＤイメージセンサ（Charge Coupled Device image sensor）を用いて画像形成する場合に比して、迅速に高感度に画像形成することができる。 As described above, 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.
　レーザ１１６０は、上述のレーザ１０６０と同様に、例えば、スペクトラ・フィジックス株式会社（Spectra-Physics）製の源一体型高出力CWグリーンレーザー Millennia eV（商標登録）などを用いることができる。実施例では、プローブレーザ光の照射は不要なので、破壊レーザ光のみを照射できるものを用いることもできる。 As 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. In the embodiment, since irradiation with the probe laser beam is unnecessary, it is possible to use one that can irradiate only the destructive laser beam.
　制御装置１１８０は、上述の制御装置１０８０と同様に、汎用のマイクロコンピューターとして構成されており、ＳＴＥＡＭカメラ装置１１５０からの判定結果信号などが入力ポートを介して入力されており、レーザ１１６０への駆動制御信号などが出力ポートから出力されている。なお、制御装置１１８０は、ＳＴＥＡＭカメラ装置１１５０の画像形成処理部１１５７および判定部１１５８と共に単一のコンピューターにより構成されるものとしてもよい。 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.
　次に、こうして構成された第２造血幹細胞用細胞除去装置１１２０の動作について説明する。なお、第２細胞破壊除去用流路デバイス１１３０には、上述の第１細胞破壊除去用流路デバイス１０３０と同様に、整列用流路１０３２の入口から細胞を１粒子としたときに粒子レイノルズ数が値１程度以下となるように造血幹細胞含有液体が流されている。 Next, the operation of the second hematopoietic stem cell removing apparatus 1120 configured as described above will be described. 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.
　第２造血幹細胞用細胞除去装置１１２０の第１処理用流路１１３４では、整列用流路１１３２により細胞が一列になって流れるようになった造血幹細胞含有液体に対して、ＳＴＥＡＭカメラ装置１１５０により一列になって流れる細胞を撮影して細胞が腫瘍細胞あるいは腫瘍細胞の可能性のある細胞としての第３所定細胞であるか否かの判定を行ない、その判定結果に基づいてレーザ１１６０から破壊レーザ光が照射される。図１１は、制御装置１１８０により実行される破壊レーザ照射制御の一例を示すフローチャートである。破壊レーザ照射制御では、まず、ＳＴＥＡＭカメラ装置１１５０から出力される判定結果を入力し（ステップＳ１３００）、判定結果により撮影した細胞に対する画像マッチングによりその細胞が腫瘍細胞（特定細胞）や腫瘍細胞である可能性のある細胞の範囲として第３所定細胞であるか否かを判定する（ステップＳ１３１０）。撮影した細胞が第３所定細胞ではないときには、破壊レーザ光を照射する必要がないと判断して本ルーチンを終了し、撮影した細胞が第３所定細胞であるときには、その細胞を破壊するための破壊レーザ光を第１処理用流路１１３４に照射するようレーザ１１６０に照射制御信号を出力して（ステップＳ１３２０）、本ルーチンを終了する。このように、ＳＴＥＡＭカメラ装置１１５０により造血幹細胞含有液体中の細胞が第３所定細胞であるか否かを判定し、撮影した細胞が第３所定細胞であると判定したときにはレーザ１１６０から破壊レーザ光を照射して第３所定細胞を破壊するから、造血幹細胞含有液体中の造血幹細胞を残存したまま腫瘍細胞（特定細胞）を含む第３所定細胞を破壊することができる。 In the first processing flow path 1134 of the second hematopoietic stem cell removal apparatus 1120, 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. In destructive laser irradiation control, first, 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. As described above, 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. To destroy the third predetermined cells, 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.
　こうして第１処理用流路１１３４で処理された造血幹細胞含有液体は、第１分離用流路１１３６に流れるが、第１分離用流路１１３６のポストアレイ流路１１３６ｄは実施例の第１分離用流路１０３６のポストアレイ流路１０３６ｄと同様に、造血幹細胞と破壊された細胞の細胞片とが分離されるように、ポスト１０３６ｃを半径６０μｍの円柱として形成すると共に隣接するポスト１０３６ｃとの最小幅が３０μｍとなるように、且つ、ポストアレイが流路の方向に対して若干の角度（例えば５度～６度程度）となるように形成されているから、造血幹細胞含有液体中の造血幹細胞は出力流路１１３６ｅに流れ、造血幹細胞含有液体中の破壊された細胞の細胞片は分離流路１１３６ｆに流れる。したがって、出力流路１１３６ｅに流れる造血幹細胞含有液体には、腫瘍細胞（特定細胞）を含む第３所定細胞については破壊されて分離除去されていることになる。 The hematopoietic stem cell-containing liquid thus treated in the first treatment channel 1134 flows into the first separation channel 1136, but the post-array channel 1136d of the first separation channel 1136 is used for the first separation in the embodiment. Similarly to the post-array channel 1036d of the channel 1036, the post 1036c is formed as a cylinder having a radius of 60 μm and the minimum width between adjacent posts 1036c so that hematopoietic stem cells and broken cell fragments are separated. Is 30 μm, 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. Therefore, 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.
　以上説明した実施例の第２造血幹細胞用細胞除去装置１１２０によれば、第２細胞破壊除去用流路デバイス１１３０の整列用流路１１３２で造血幹細胞含有液体中の細胞が一列になって流れるようにし、第１処理用流路１１３４でＳＴＥＡＭカメラ装置１１５０を用いて細胞が第３所定細胞であるか否かを検出して破壊レーザを照射して破壊し、第１分離用流路１１３６で造血幹細胞含有液体中の破壊された細胞を分離除去することにより、造血幹細胞含有液体中に造血幹細胞を残したままより確実に腫瘍細胞を除去することができる。しかも、ＳＴＥＡＭカメラ装置１１５０を用いることにより、ＣＣＤイメージセンサ（Charge Coupled Device image sensor）を用いる場合に比して、迅速に高精度に造血幹細胞含有液体中の細胞を撮影することができるから、第１処理用流路１１３４の造血幹細胞含有液体の流速を速くすることができ、より迅速に高精度に造血幹細胞含有液体中に造血幹細胞を残したままより確実に腫瘍細胞を除去することができる。 According to the second hematopoietic stem cell removal apparatus 1120 of the embodiment described above, 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. In the first processing channel 1134, 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. By separating and removing the destroyed cells in the stem cell-containing liquid, tumor cells can be more reliably removed while leaving the hematopoietic stem cells in the hematopoietic stem cell-containing liquid. Moreover, since 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.
　実施例の第２造血幹細胞用細胞除去装置１１２０に用いる第２細胞破壊除去用流路デバイス１１３０によれば、整列用流路１１３２を有することにより、造血幹細胞含有液体を流すだけで、造血幹細胞含有液体中にランダムに存在する細胞を一列にして流すことができる。また、第１処理用流路１０３４を有することにより、ＳＴＥＡＭカメラ装置１１５０とレーザ１１６０と用いて腫瘍細胞を含む第３所定細胞を検出して破壊することができる。そして、第１分離用流路１１３６を有することにより、造血幹細胞含有液体から破壊した細胞の細胞片と造血幹細胞とを分離することができる。このように、整列用流路１１３２と第１処理用流路１１３４と第１分離用流路１１３６とを備えるから、造血幹細胞含有液体中の細胞を一列に流してＳＴＥＡＭカメラ装置１１５０とレーザ１１６０とを用いて腫瘍細胞を含む第３所定細胞を検出して破壊し、破壊した細胞の細胞片を造血幹細胞から分離する際に必要な流路を単一の流路デバイスで賄うことができる。この結果、第２造血幹細胞用細胞除去装置１１２０の小型化を図ることができる。 According to the second cell disruption / removal channel device 1130 used in the second hematopoietic stem cell cell removal device 1120 of the embodiment, by having the alignment channel 1132, 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. As described above, since 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.
　実施例の第２造血幹細胞用細胞除去装置１１２０では、第２処理用流路や第２分離用流路を有しない第２細胞破壊除去用流路デバイス１１３０を用いて造血幹細胞含有液体中の腫瘍細胞を含む第３所定細胞を検出して破壊し、破壊した細胞の細胞片を造血幹細胞から分離するものとしたが、実施例の第１細胞破壊除去用流路デバイス１０３０と同様に、整列用流路１１３２や第１処理用流路１１３４，第１分離用流路１１３６の他に第２処理用流路と第２分離用流路とを有する細胞破壊除去用流路デバイスを用い、第１処理用流路１１３４の他に第２処理用流路で造血幹細胞は含まないが腫瘍細胞を含む範囲の所定細胞を破壊するものとしてもよい。この場合、第１処理用流路１１３４と第２処理用流路の双方でＳＴＥＡＭカメラ装置１１５０とレーザ１１６０と用いて腫瘍細胞を含む第３所定細胞を検出して破壊するものとしてもよいし、第１処理用流路１１３４と第２処理用流路とのうちの一方の流路でＳＴＥＡＭカメラ装置１１５０とレーザ１１６０と用いて腫瘍細胞を含む第３所定細胞を検出して破壊し、他方の流路でＣＤ３４陽性試験による処理やＣＤ３８陰性試験による処理を行なうものとしてもよい。 In the second hematopoietic stem cell removal device 1120 of the example, the tumor in the hematopoietic stem cell-containing liquid using the second cell destruction removal flow channel device 1130 that does not have the second processing flow channel or the second separation flow channel. 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. In addition to the flow path 1132, the first processing flow path 1134, and the first separation flow path 1136, a cell destruction removal flow path device having a second processing flow path and a second separation flow path is used. In addition to the processing channel 1134, the second processing channel may not include hematopoietic stem cells but may destroy predetermined cells in a range including tumor cells. In this case, 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.
　実施例の第１造血幹細胞用細胞除去装置１０２０や第２造血幹細胞用細胞除去装置１１２０では、第１細胞破壊除去用流路デバイス１０３０や第２細胞破壊除去用流路デバイス１１３０の整列用流路１０３２，１１３２として、幅が１００～５００μｍで深さが１０～１００μｍで繰り返し回数が１０～２０回の蛇行する波状の流路として形成したが、図１２および図１３の変形例の細胞破壊除去用流路デバイス１２３０の整列用流路１２３２に示すように、幅が１００～５００μｍで深さが１０～１００μｍで矩形流路を形成する頂面には下向きに凸の凸部１２３２ａと下向きに凹の凹部１２３２ｂとからなる凹凸が２０～５０回に亘って形成された直線状の流路として形成してもよい。整列用流路１２３２の幅や深さや凹凸の回数は、細胞含有液体に含まれる細胞の大きさなどによって適宜定められるものである。例えば、細胞が直径１０μｍ程度の大きさの場合、整列用流路１２３２は、幅は１００μｍ程度が妥当であり、深さは４０μｍ程度が妥当であり、凸部１２３２ａの高さ及び長さは２０μｍ程度および１０μｍ程度が妥当であり、凹部１２３２ｂの長さは１００μｍ程度が妥当であり、凹凸の回数は３０回程度が妥当である。変形例の細胞破壊除去用流路デバイス１２３０における第１処理用流路１２３４および第１分離用流路１２３６は、図６に例示した実施例の第１細胞破壊除去用流路デバイス１０３０における第１処理用流路１０３２および第１分離用流路１０３６と同一である。この変形例の細胞破壊除去用流路デバイス１２３０の整列用流路１２３２には、レイノルズ数（Reynolds number）が８０程度以下となるように細胞含有液体を流すのが好ましい。変形例の細胞破壊除去用流路デバイス１２３０の整列用流路１２３２では、頂面に凹凸が形成されているため、流路の断面の広いところと狭いところとが生じ、細胞含有液体中の細胞に慣性力が作用し、次第に細胞が一列に整列するようになる。したがって、変形例の細胞破壊除去用流路デバイス１２３０でも実施例の第１細胞破壊除去用流路デバイス１０３０や第２細胞破壊除去用流路デバイス１１３０と同一の効果を奏することができる。この変形例の細胞破壊除去用流路デバイス１２３０の整列用流路１２３２では、流路の頂面に凹凸を形成するものとしたが、流路の底面に凹凸を形成するものとしたり、流路の側面の一方に凹凸を形成するものとしたりするなど、流路を形成する少なくとも１つの面に凹凸を形成すれものとすればよい。細胞を一列に整列させる効果を考慮すると、流路の頂面や底面に凹凸を形成するのが好ましい。なお、出願人は、実施例の第１細胞破壊除去用流路デバイス１０３０や第２細胞破壊除去用流路デバイス１１３０の整列用流路１０３２はジグザクと蛇行しているからジグザグタイプと称し、変形例の細胞破壊除去用流路デバイス１２３０の整列用流路１２３２は凹部１２３２ｂをポケットとみなしてポケットタイプと称している。 In the first hematopoietic stem cell removal apparatus 1020 and the second hematopoietic stem cell removal apparatus 1120 of the embodiment, 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 μm, a depth of 10 to 100 μm, and a repetition number of 10 to 20 times. As shown in the alignment channel 1232 of the channel device 1230, the top surface forming a rectangular channel having a width of 100 to 500 μm and a depth of 10 to 100 μm has a convex portion 1232a that is downwardly convex and a concave portion that is downwardly concave. It may be formed as a linear flow path in which the unevenness comprising the recess 1232b is formed 20 to 50 times. The width and depth of the alignment channel 1232 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 have a diameter of about 10 μm, the alignment channel 1232 has an appropriate width of about 100 μm, an appropriate depth of about 40 μm, and the height and length of the convex portion 1232 a are 20 μm. About 10 μm and about 10 μm are appropriate, about 100 μm 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. Therefore, 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. In the alignment flow path 1232 of the cell destruction removal flow path device 1230 of this modified 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.
　以上、本発明を実施例を用いて説明したが、本発明はこうした実施例に何等限定されるものではなく、本発明の要旨を逸脱しない範囲内において、種々なる形態で実施し得ることは勿論である。 The present invention has been described with reference to the embodiments. However, the present invention is not limited to these embodiments, and can of course be implemented in various forms without departing from the gist of the present invention. It is.
　本発明は、細胞選別装置の製造産業や、家造血幹細胞移植に用いられる特定細胞除去装置の製造産業、こうした特定細胞除去装置に用いられる流路デバイスの製造産業などに利用可能である。 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.

Claims (30)

  1. 　複数種類の細胞を含有する細胞含有液体から対象細胞を選別する細胞選別装置であって、
    　前記細胞含有液体中にランダムに存在する細胞が一列に流れるようにする整列用流路と、前記整列用流路の後段に形成された撮影用流路と、前記撮影用流路の後段に本流用流路と該本流用流路から分岐する分岐流路とが形成された選別用流路と、を有する細胞選別用流路デバイスと、
    　短パルス光を少なくとも直線上に波長が順次異なる波長別光群を前記撮影用流路に照射し、該照射による反射光に基づいて前記細胞含有液体中の細胞の画像を形成する細胞撮影手段と、
    　前記形成された細胞の画像に基づいて該細胞が前記対象細胞であるか否かを判定する判定手段と、
    　少なくとも前記判定手段により前記対象細胞であると判定された細胞については、前記本流用流路から前記分岐流路に分岐させることにより選別する選別手段と、
    　を備える細胞選別装置。     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;
    A cell sorting apparatus comprising:
  2. 　請求項１記載の細胞選別装置であって、
    　前記選別手段は、パルスレーザーの照射によって生じるキャビテーションにより前記対象細胞であると判定された細胞を前記本流用流路から前記分岐流路に分岐させる手段である、
    　細胞選別装置。     The cell sorting apparatus according to claim 1, wherein
    The sorting means is 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 flow channel.
    Cell sorter.
  3. 　請求項１記載の細胞選別装置であって、
    　前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路である、
    　細胞選別装置。     The cell sorting apparatus according to claim 1, wherein
    The alignment channel is a wavy channel that meanders at a predetermined width within a range of 10 to 100 micrometers and a predetermined depth within a range of 10 to 20 times with a predetermined width within a range of 100 to 500 micrometers. Is,
    Cell sorter.
  4. 　請求項１記載の細胞選別装置であって、
    　前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで流路を形成する少なくとも１つの面には２０～５０回の範囲内の所定繰り返し回数の凹凸が形成された直線状の流路である、
    　細胞選別装置。     The cell sorting apparatus according to claim 1, wherein
    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 at least one surface forming the flow channel has a range of 20 to 50 times. Is a linear flow path in which irregularities of a predetermined number of repetitions are formed,
    Cell sorter.
  5. 　請求項１記載の細胞選別装置であって、
    　前記細胞撮影手段は、フェムト秒ないしナノ秒オーダーの間隔で繰り返し短パルス光を出力する短パルス光源と、前記短パルス光源からの短パルス光を分光して前記波長別光群とする分光部と、前記波長別光群を前記撮影用流路に照射する照射部と、前記照射により反射した反射波長別光群を集光して反射パルス光とする集光部と、前記反射パルス光を入力して時間の経過に伴って順次波長の異なる光を出力する波長選別用光ファイバと、前記波長選別用光ファイバから出力される波長毎の光の強度に基づいて細胞の画像を形成する細胞画像形成部と、を有する手段である、
    　細胞選別装置。     The cell sorting apparatus according to claim 1, wherein
    The cell imaging means includes a short pulse light source that repeatedly outputs short pulse light at an interval of femtoseconds to nanoseconds, and a spectroscopic unit that splits short pulse light from the short pulse light source into the wavelength-specific light group, , An irradiation unit for irradiating the imaging light channel with the wavelength-specific light group, a condensing unit for collecting the reflected wavelength-specific light group reflected by the irradiation to be reflected pulsed light, and inputting the reflected pulse light Then, a wavelength selecting optical fiber that sequentially outputs light having different wavelengths as time passes, and a cell image that forms an image of a cell based on the intensity of light for each wavelength output from the wavelength selecting optical fiber Forming means,
    Cell sorter.
  6. 　複数種類の細胞を含有する細胞含有液体から対象細胞を選別する細胞選別装置に用いられる細胞選別用流路デバイスであって、
    　前記細胞含有液体中にランダムに存在する細胞が一列に流れるようにする整列用流路と、
    　前記整列用流路の後段に形成された撮影用流路と、
    　前記撮影用流路の後段に本流用流路と該本流用流路から分岐する分岐流路とが形成された選別用流路と、
    　を備える細胞選別用流路デバイス。     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;
    A cell sorting channel device comprising:
  7. 　請求項６記載の細胞選別用流路デバイスであって、
    　前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路であり、
    　前記撮影用流路は、前記所定幅で前記所定深さの直線状の流路であり、
    　前記本流用流路は、前記所定幅以下の幅で前記所定深さで前記撮影用流路から直線上に配置された流路であり、
    　前記分岐流路は、前記所定幅より小さな幅で前記所定深さで前記本流用流路から１０度～６０度の範囲内の所定角度で分岐する流路である、
    　細胞選別用流路デバイス。     The cell sorting channel device according to claim 6,
    The alignment channel is a wavy channel that meanders at a predetermined width within a range of 10 to 100 micrometers and a predetermined depth within a range of 10 to 20 times with a predetermined width within a range of 100 to 500 micrometers. And
    The photographing channel is a linear channel having the predetermined width and the predetermined depth,
    The main flow channel is a channel arranged in a straight line from the photographing channel at the predetermined depth with a width equal to or less than the predetermined width,
    The branch flow path is a flow path that branches at a predetermined angle within a range of 10 degrees to 60 degrees from the main flow path at a predetermined depth with a width smaller than the predetermined width.
    Channel device for cell sorting.
  8. 　請求項６記載の細胞選別用流路デバイスであって、
    　前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで流路を形成する少なくとも１つの面には２０～５０回の範囲内の所定繰り返し回数の凹凸が形成された直線状の流路であり、
    　前記撮影用流路は、前記所定幅で前記所定深さの直線状の流路であり、
    　前記本流用流路は、前記所定幅以下の幅で前記所定深さで前記撮影用流路から直線上に配置された流路であり、
    　前記分岐流路は、前記所定幅より小さな幅で前記所定深さで前記本流用流路から１０度～６０度の範囲内の所定角度で分岐する流路である、
    　細胞選別用流路デバイス。     The cell sorting channel device according to claim 6,
    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 at least one surface forming the flow channel has a range of 20 to 50 times. Is a linear flow path in which irregularities of a predetermined number of repetitions are formed,
    The photographing channel is a linear channel having the predetermined width and the predetermined depth,
    The main flow channel is a channel arranged in a straight line from the photographing channel at the predetermined depth with a width equal to or less than the predetermined width,
    The branch flow path is a flow path that branches at a predetermined angle within a range of 10 degrees to 60 degrees from the main flow path at a predetermined depth with a width smaller than the predetermined width.
    Channel device for cell sorting.
  9. 　請求項６記載の細胞選別用流路デバイスであって、
    　前記選別用流路は、前記分岐流路への分岐点の直近上流の前記分岐流路の反対側に前記対象細胞を前記分岐流路に分岐させるためのキャビテーションを生じさせるキャビテーション発生領域が形成されている、
    　細胞選別用流路デバイス。     The cell sorting channel device according to claim 6,
    In the sorting channel, a cavitation generation region for generating cavitation for branching the target cell into the branch channel is formed on the opposite side of the branch channel immediately upstream of the branch point to the branch channel. ing,
    Channel device for cell sorting.
  10. 　複数種類の細胞を含有する細胞含有液体から対象細胞を選別する細胞選別方法であって、
    （ａ）前記細胞含有液体中にランダムに存在する細胞を一列にして流し、
    （ｂ）細胞が一列になって流れる細胞含有液体に短パルス光を少なくとも直線上に波長が順次異なる波長別光群として照射すると共に該照射による反射光に基づいて前記細胞含有液体中の細胞の画像を形成し、
    （ｃ）前記形成した細胞の画像に基づいて該細胞が前記対象細胞であるか否かを判定し、
    （ｄ）少なくとも前記対象細胞であると判定された細胞を前記細胞含有液体の流れから分岐させることにより選別する、
    　ことを特徴とする細胞選別方法。     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;
    A cell sorting method characterized by the above.
  11. 　患者から採取した造血幹細胞を含有する造血幹細胞含有液体から少なくとも特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去装置であって、
    　前記造血幹細胞含有液体中に存在する細胞が一列に流れるようにする整列用流路と、前記造血幹細胞含有液体中の特定細胞を破壊処理するために前記整列用流路の後段に形成された第１処理用流路と、前記造血幹細胞含有液体から破壊された特定細胞を分離するために前記第１処理用流路の後段に形成された第１分離用流路と、を有する第１流路デバイスと、
    　前記造血幹細胞は含まないが前記特定細胞は含み且つ前記特定細胞の可能性が認められる細胞は含む範囲の細胞としての所定細胞を前記第１処理用流路に流れる前記造血幹細胞含有液体から検出する所定細胞検出手段と、
    　前記第１処理用流路に細胞破壊用の破壊レーザ光を照射可能なレーザ照射手段と、
    　前記所定細胞検出手段により前記第１処理用流路に流れる前記造血幹細胞含有液体中に前記所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する制御手段と、
    　を備える特定細胞除去装置。     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;
    When the predetermined cell is detected in the hematopoietic stem cell-containing liquid flowing in the first processing channel by the predetermined cell detection unit, the laser irradiation unit is configured to irradiate the detected predetermined cell with the destructive laser beam. Control means for controlling
    A specific cell removing apparatus.
  12. 　請求項１１記載の特定細胞除去装置であって、
    　前記レーザ照射手段は、前記破壊レーザ光の他に前記第１処理用流路に細胞検出用のプローブレーザ光を照射可能な手段であり、
    　前記所定細胞検出手段は、前記プローブレーザ光の照射により蛍光発現する第１マーカーを用いて前記所定細胞を検出する手段であり、
    　前記制御手段は、前記プローブレーザ光を前記第１処理用流路に照射するよう前記レーザ照射手段を制御すると共に前記所定細胞検出手段により前記所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する手段である、
    　特定細胞除去装置。     The specific cell removing device according to claim 11, wherein
    The laser irradiation means is means capable of irradiating the first processing flow path with a probe laser light for cell detection in addition to the destructive laser light,
    The predetermined cell detecting means is means for detecting the predetermined cell using a first marker that is fluorescently expressed by irradiation with the probe laser light,
    The control unit controls the laser irradiation unit to irradiate the probe laser light to the first processing flow path, and the predetermined cell detected when the predetermined cell is detected by the predetermined cell detection unit. Is a means for controlling the laser irradiation means to irradiate the destructive laser light.
    Specific cell removal device.
  13. 　請求項１２記載の特定細胞除去装置であって、
    　前記マーカーはＣＤ３４またはＣＤ３８であり、
    　前記所定細胞検出手段は、
    　　前記マーカーがＣＤ３４のときには、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第１マーカーとによって蛍光発現を生じない細胞を前記所定細胞として検出する手段であり、
    　　前記マーカーがＣＤ３８のときには、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第２マーカーとによって蛍光発現を生じる細胞を前記所定細胞として検出する手段である、
    　特定細胞除去装置。     The specific cell removing device according to claim 12,
    The marker is CD34 or CD38;
    The predetermined cell detection means includes
    When the marker is CD34, 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 first marker A means for detecting a cell that does not cause fluorescence expression as the predetermined cell,
    When the marker is CD38, cells in the hematopoietic stem cell-containing liquid are detected based on scattered light with respect to the probe laser light irradiation, and among the detected cells, irradiation with the probe laser light and the second marker A means for detecting a cell that produces fluorescent expression as the predetermined cell.
    Specific cell removal device.
  14. 　請求項１１記載の特定細胞除去装置であって、
    　前記所定細胞検出手段は、短パルス光を少なくとも直線上に波長が順次異なる波長別光群として前記第１処理用流路に照射し、該照射による反射光に基づいて前記造血幹細胞含有液体中の細胞の画像を形成し、該形成した細胞の画像に基づいて該細胞が前記所定細胞であるか否かを判定することにより前記所定細胞を検出する手段である、
    　特定細胞除去装置。     The specific cell removing device according to claim 11, wherein
    The predetermined cell detecting means irradiates the first processing flow channel with short pulse light as a wavelength-specific light group at least on a straight line, and in the hematopoietic stem cell-containing liquid based on reflected light by the irradiation. A means for detecting the predetermined cell by forming an image of the cell and determining whether the cell is the predetermined cell based on the formed cell image;
    Specific cell removal device.
  15. 　請求項１４記載の特定細胞除去装置であって、
    　前記所定細胞検出手段は、フェムト秒ないしナノ秒オーダーの間隔で繰り返し短パルス光を出力する短パルス光源と、前記短パルス光源からの短パルス光を分光して前記波長別光群とする分光部と、前記波長別光群を前記撮影用流路に照射する照射部と、前記照射により反射した反射波長別光群を集光して反射パルス光とする集光部と、前記反射パルス光を入力して時間の経過に伴って順次波長の異なる光を出力する波長選別用光ファイバと、前記波長選別用光ファイバから出力される波長毎の光の強度に基づいて細胞の画像を形成する細胞画像形成部と、前記形成された細胞の画像に基づいて該細胞が前記所定細胞であるか否かを判定する判定部と、を有する手段である、
    　特定細胞除去装置。     The specific cell removing device according to claim 14,
    The predetermined cell detection means includes a short pulse light source that repeatedly outputs short pulse light at intervals of femtoseconds to nanoseconds, and a spectroscopic unit that separates the short pulse light from the short pulse light source into the wavelength-specific light group 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 reflected pulsed light. A wavelength selecting optical fiber that sequentially outputs light having different wavelengths as time passes, and a cell that forms an image of a cell based on the intensity of light for each wavelength output from the wavelength selecting optical fiber An image forming unit; and a determination unit that determines whether or not the cell is the predetermined cell based on the image of the formed cell.
    Specific cell removal device.
  16. 　請求項１１記載の特定細胞除去装置であって、
    　前記第１流路デバイスの後段に配置され、前記第１流路デバイスを用いて破壊された所定細胞を分離した後の前記造血幹細胞含有液体中に残存する所定細胞を破壊処理するための第２処理用流路と、前記造血幹細胞含有液体から破壊された所定細胞を分離するために前記第２処理用流路の後段に形成された第２分離用流路と、を有する第２流路デバイスを備え、
    　前記所定細胞検出手段は、前記第１処理用流路に流れる前記造血幹細胞含有液体中の前記所定細胞を検出する他に前記第２処理用流路に流れる前記造血幹細胞含有液体中の前記所定細胞も検出する手段であり、
    　前記レーザ照射手段は、前記第１処理用流路に前記破壊レーザ光を照射可能な他に前記第２処理用流路に前記破壊レーザ光を照射可能な手段であり、
    　前記制御手段は、前記所定細胞検出手段により前記第１処理用流路に流れる前記造血幹細胞含有液体中に前記所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する他に、前記所定細胞検出手段により前記第２処理用流路に流れる前記造血幹細胞含有液体中に前記所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する手段である、
    　特定細胞除去装置。     The specific cell removing device according to claim 11, wherein
    The second for disposing the predetermined cells remaining in the hematopoietic stem cell-containing liquid after separating the predetermined cells that have been disposed using the first flow path device and are disposed downstream of the first flow path device. A second flow path device having a processing flow path and a second separation flow path formed at a subsequent stage of the second processing flow path for separating predetermined cells destroyed from the hematopoietic stem cell-containing liquid. With
    The predetermined cell detecting means detects the predetermined cell in the hematopoietic stem cell-containing liquid flowing in the first processing flow path, and in addition to detecting the predetermined cell in the hematopoietic stem cell-containing liquid flowing in the second processing flow path. Is also a means to detect
    The laser irradiating means is a means capable of irradiating the destructive laser light to the second processing flow path in addition to irradiating the destructive laser light to the first processing flow path,
    The control means irradiates the detected predetermined cells with the destructive laser light 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. In addition to controlling the laser irradiation means, the predetermined cells detected when the predetermined cells are detected in the hematopoietic stem cell-containing liquid flowing in the second processing channel by the predetermined cell detection means. A means for controlling the laser irradiation means to irradiate a destructive laser beam;
    Specific cell removal device.
  17. 　請求項１６記載の特定細胞除去装置であって、
    　前記レーザ照射手段は、前記破壊レーザ光の他に前記第１処理用流路および前記第２処理用流路に細胞検出用のプローブレーザ光を照射可能な手段であり、
    　前記所定細胞検出手段は、前記プローブレーザ光の照射により蛍光発現する第１マーカーを用いて前記第１処理用流路に流れる前記造血幹細胞含有液体中の所定細胞を検出する第１検出手段と、前記プローブレーザ光の照射により蛍光発現する前記第１マーカーとは異なる第２マーカーを用いて前記第２処理用流路に流れる前記造血幹細胞含有液体中の所定細胞を検出する第２検出手段と、を有する手段であり、
    　前記制御手段は、前記プローブレーザ光を前記第１処理用流路に照射するよう前記レーザ照射手段を制御すると共に前記第１検出手段により所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御し、前記プローブレーザ光を前記第２処理用流路に照射するよう前記レーザ照射手段を制御すると共に前記第２検出手段により所定細胞が検出されたときに該検出された所定細胞に前記破壊レーザ光を照射するよう前記レーザ照射手段を制御する手段である、
    　特定細胞除去装置。     The specific cell removing device according to claim 16, wherein
    The laser irradiation means is means capable of irradiating the first processing flow path and the second processing flow path with a probe laser light for cell detection in addition to the destructive laser light,
    The predetermined cell detecting means detects a predetermined cell in the hematopoietic stem cell-containing liquid flowing in the first processing channel using a first marker that is fluorescently expressed by irradiation with the probe laser light; and Second detection means for detecting predetermined cells in the hematopoietic stem cell-containing liquid flowing in the second processing flow path using a second marker different from the first marker that is fluorescently expressed by irradiation with the probe laser light; Means having
    The control means controls the laser irradiation means so as to irradiate the probe laser light to the first processing flow path, and applies the detected predetermined cells to the detected predetermined cells when the first detection means detects the predetermined cells. The laser irradiation unit is controlled to irradiate the destructive laser beam, the laser irradiation unit is controlled to irradiate the probe laser beam to the second processing channel, and a predetermined cell is detected by the second detection unit. Means for controlling the laser irradiation means to irradiate the destructive laser light to the predetermined cells detected when
    Specific cell removal device.
  18. 　請求項１７記載の特定細胞除去装置であって、
    　前記第１マーカーは、ＣＤ３４であり、
    　前記第２マーカーは、ＣＤ３８であり、
    　前記第１検出手段は、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第１マーカーとによって蛍光発現を生じない細胞を前記所定細胞として検出する手段であり、
    　前記第２検出手段は、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第２マーカーとによって蛍光発現を生じる細胞を前記所定細胞として検出する手段である、
    　特定細胞除去装置。     The specific cell removing device according to claim 17,
    The first marker is CD34;
    The second marker is CD38;
    The first detection means detects a cell in the hematopoietic stem cell-containing liquid based on scattered light with respect to the probe laser light irradiation, and among the detected cells, the probe laser light irradiation and the first marker A means for detecting a cell that does not cause fluorescence expression as the predetermined cell,
    The second detection means detects a cell in the hematopoietic stem cell-containing liquid based on the scattered light with respect to the probe laser light irradiation, and includes the probe laser light irradiation and the second marker among the detected cells. A means for detecting a cell that produces fluorescent expression as the predetermined cell.
    Specific cell removal device.
  19. 　請求項１１記載の特定細胞除去装置であって、
    　前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路である、
    　特定細胞除去装置。     The specific cell removing device according to claim 11, wherein
    The alignment channel is a wavy channel that meanders at a predetermined width within a range of 10 to 100 micrometers and a predetermined depth within a range of 10 to 20 times with a predetermined width within a range of 100 to 500 micrometers. Is,
    Specific cell removal device.
  20. 　請求項１１記載の特定細胞除去装置であって、
    　前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで流路を形成する少なくとも１つの面には２０～５０回の範囲内の所定繰り返し回数の凹凸が形成された直線状の流路である、
    　特定細胞除去装置。     The specific cell removing device according to claim 11, wherein
    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 at least one surface forming the flow channel has a range of 20 to 50 times. Is a linear flow path in which irregularities of a predetermined number of repetitions are formed,
    Specific cell removal device.
  21. 　請求項１１記載の特定細胞除去装置であって、
    　前記第１分離用流路は、最小間隔が造血幹細胞の直径の３倍より小さくなるように複数の円柱形状のポストを整列してなるポストアレイが形成された流路である、
    　特定細胞除去装置。     The specific cell removing device according to claim 11, wherein
    The first separation channel is a channel in which a post array formed by aligning a plurality of columnar posts so that the minimum interval is smaller than three times the diameter of the hematopoietic stem cells.
    Specific cell removal device.
  22. 　患者から採取した造血幹細胞を含有する造血幹細胞含有液体から少なくとも特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去用流路であって、
    　前記造血幹細胞含有液体中に存在する細胞が一列に流れるようにする整列用流路と、前記造血幹細胞含有液体中の特定細胞を破壊処理するために前記整列用流路の後段に形成された第１処理用流路と、前記造血幹細胞含有液体から破壊された特定細胞を分離するために前記第１処理用流路の後段に形成された第１分離用流路と、が一体形成された第１流路デバイスを備える、
    　特定細胞除去用流路。     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,
    Flow path for specific cell removal.
  23. 　請求項２２記載の特定細胞除去用流路であって、
    　前記第１分離用流路で破壊された特定細胞が分離された後の前記造血幹細胞含有液体中に残存する特定細胞を破壊処理するための第２処理用流路と、前記造血幹細胞含有液体から破壊された特定細胞を分離するために前記第２処理用流路の後段に形成された第２分離用流路と、が一体形成された第２流路デバイスを備える、
    　特定細胞除去用流路。     A flow path for removing specific cells according to claim 22,
    A second treatment channel 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, and the hematopoietic stem cell-containing liquid. A second flow path device integrally formed with a second separation flow path formed at a subsequent stage of the second processing flow path to separate the destroyed specific cells;
    Flow path for specific cell removal.
  24. 　請求項２３記載の特定細胞除去用流路であって、
    　前記第１流路デバイスと前記第２流路デバイスとが一体形成されている、
    　特定細胞除去用流路。     The specific cell removal flow path according to claim 23,
    The first flow path device and the second flow path device are integrally formed,
    Flow path for specific cell removal.
  25. 　請求項２２記載の特定細胞除去用流路であって、
    　前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで１０～２０回の範囲内の所定繰り返し回数で蛇行する波状の流路である、
    　特定細胞除去用流路。     A flow path for removing specific cells according to claim 22,
    The alignment channel is a wavy channel that meanders at a predetermined width within a range of 10 to 100 micrometers and a predetermined depth within a range of 10 to 20 times with a predetermined width within a range of 100 to 500 micrometers. Is,
    Flow path for specific cell removal.
  26. 　請求項２２記載の特定細胞除去用流路であって、
    　前記整列用流路は、１００～５００マイクロメートルの範囲内の所定幅で１０～１００マイクロメートルの範囲内の所定深さで流路を形成する少なくとも１つの面には２０～５０回の範囲内の所定繰り返し回数の凹凸が形成された直線状の流路である、
    　特定細胞除去用流路。     A flow path for removing specific cells according to claim 22,
    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 at least one surface forming the flow channel has a range of 20 to 50 times. Is a linear flow path in which irregularities of a predetermined number of repetitions are formed,
    Flow path for specific cell removal.
  27. 　請求項２２記載の特定細胞除去用流路であって、
    　前記第１分離用流路は、最小間隔が造血幹細胞の直径の３倍より小さくなるように複数の円柱形状のポストを整列してなるポストアレイが形成された流路である、
    　特定細胞除去用流路。     A flow path for removing specific cells according to claim 22,
    The first separation channel is a channel in which a post array formed by aligning a plurality of columnar posts so that the minimum interval is smaller than three times the diameter of the hematopoietic stem cells.
    Flow path for specific cell removal.
  28. 　患者から採取した造血幹細胞を含有する造血幹細胞含有液体から少なくとも特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去方法であって、
    （ａ）前記造血幹細胞含有液体中に存在する細胞が一列となるように前記造血幹細胞含有液体を流し、
    （ｂ）細胞が一列になって流れる前記造血幹細胞含有液体に細胞検出用のプローブレーザ光の照射と第１マーカーとによる蛍光発現を用いて、前記造血幹細胞は含まないが前記特定細胞は含み且つ前記特定細胞の可能性が認められる細胞は含む範囲の細胞としての第１所定細胞を検出し、
    （ｃ）該検出した第１所定細胞に細胞破壊用の破壊レーザ光を照射して前記第１所定細胞を破壊し、
    （ｄ）前記造血幹細胞含有液体から破壊された第１所定細胞を分離し、
    （ｅ）破壊された第１所定細胞を分離した後の前記造血幹細胞含有液体に前記プローブレーザ光の照射と前記第１マーカーとは異なる第２マーカーによる蛍光発現を用いて、前記造血幹細胞は含まないが前記特定細胞は含み且つ前記特定細胞の可能性が認められる細胞は含む範囲の細胞としての第２所定細胞を検出し、
    （ｆ）該検出した第２所定細胞に細胞破壊用の破壊レーザ光を照射して前記第２所定細胞を破壊し、
    （ｇ）前記造血幹細胞含有液体から破壊された第２所定細胞を分離する、
    　特定細胞除去方法。     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 destruction;
    (D) separating the first predetermined cells destroyed from the hematopoietic stem cell-containing liquid,
    (E) The hematopoietic stem cell is contained in the hematopoietic stem cell-containing liquid after separating the destroyed first predetermined cells using irradiation of the probe laser light and fluorescence expression by a second marker different from the first marker. 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,
    Specific cell removal method.
  29. 　請求項２８記載の特定細胞除去方法であって、
    　前記ステップ（ｂ）は、前記第１マーカーとしてＣＤ３４を用い、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第１マーカーとによって蛍光発現を生じない細胞を前記第１所定細胞として検出するステップであり、
    　前記ステップ（ｅ）は、前記第２マーカーとしてＣＤ３８を用い、前記プローブレーザ光の照射に対する散乱光に基づいて前記造血幹細胞含有液体中の細胞を検出すると共に該検出した細胞のうち前記プローブレーザ光の照射と前記第２マーカーとによって蛍光発現を生じる細胞を前記第２所定細胞として検出するステップである、
    　特定細胞除去方法。     The method for removing specific cells according to claim 28, wherein
    The step (b) uses CD34 as the first marker, detects cells in the hematopoietic stem cell-containing liquid based on scattered light in response to irradiation with the probe laser light, and detects the probe laser light among the detected cells. Detecting a cell that does not produce fluorescence expression as a result of irradiation with the first marker as the first predetermined cell,
    The step (e) uses CD38 as the second marker, detects cells in the hematopoietic stem cell-containing liquid based on scattered light in response to irradiation with the probe laser light, and detects the probe laser light among the detected cells. Detecting a cell that causes fluorescence expression by irradiation of the second marker and the second marker as the second predetermined cell,
    Specific cell removal method.
  30. 　患者から採取した造血幹細胞を含有する造血幹細胞含有液体から少なくとも特定細胞を除去して該患者に移植する自家造血幹細胞移植に用いられる特定細胞除去方法であって、
    （ａ）前記造血幹細胞含有液体中に存在する細胞を一列に流し、
    （ｂ）細胞が一列になって流れる前記造血幹細胞含有液体に短パルス光を少なくとも直線上に波長が順次異なる波長別光群として照射すると共に該照射による反射光に基づいて前記造血幹細胞含有液体中の細胞の画像を形成し、前記形成した細胞の画像に基づいて該細胞が前記特定細胞であるか否かを判定することによって特定細胞を検出し、
    （ｃ）該検出した特定細胞に細胞破壊用の破壊レーザ光を照射して前記特定細胞を破壊し、
    （ｄ）前記造血幹細胞含有液体から破壊された特定細胞を分離する、
    　特定細胞除去方法。     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;
    (D) separating the broken specific cells from the hematopoietic stem cell-containing liquid,
    Specific cell removal method.
PCT/JP2014/080473 2013-11-21 2014-11-18 Apparatus for selecting and removing cell, and method for selecting and removing cell WO2015076251A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2013-241092 2013-11-21
JP2013241077 2013-11-21
JP2013-241077 2013-11-21
JP2013241092 2013-11-21

Publications (1)

Publication Number Publication Date
WO2015076251A1 true WO2015076251A1 (en) 2015-05-28

Family

ID=53179513

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2014/080473 WO2015076251A1 (en) 2013-11-21 2014-11-18 Apparatus for selecting and removing cell, and method for selecting and removing cell

Country Status (1)

Country Link
WO (1) WO2015076251A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017042137A (en) * 2015-08-28 2017-03-02 国立大学法人 奈良先端科学技術大学院大学 Cell sorting apparatus
US10357771B2 (en) 2017-08-22 2019-07-23 10X Genomics, Inc. Method of producing emulsions
US10544413B2 (en) 2017-05-18 2020-01-28 10X Genomics, Inc. Methods and systems for sorting droplets and beads
US11660601B2 (en) 2017-05-18 2023-05-30 10X Genomics, Inc. Methods for sorting particles
US11833515B2 (en) 2017-10-26 2023-12-05 10X Genomics, Inc. Microfluidic channel networks for partitioning

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01311270A (en) * 1988-06-09 1989-12-15 Omron Tateisi Electron Co Cell screening device
JPH01313758A (en) * 1988-06-13 1989-12-19 Omron Tateisi Electron Co Cell sorting device
JPH027866A (en) * 1988-06-21 1990-01-11 Matsushita Electric Ind Co Ltd Source voltage converter circuit
JP2003530544A (en) * 2000-01-04 2003-10-14 アイメトリクス インコーポレイティッド Intravascular image processing detector
JP2010008082A (en) * 2008-06-24 2010-01-14 Olympus Corp Multiphoton excitation measuring instrument
JP2010252785A (en) * 2009-03-31 2010-11-11 Kanagawa Acad Of Sci & Technol Device for concentrating and separating cell
JP2013217672A (en) * 2012-04-04 2013-10-24 Sharp Corp Particle pretreatment device and particle pretreatment method

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01311270A (en) * 1988-06-09 1989-12-15 Omron Tateisi Electron Co Cell screening device
JPH01313758A (en) * 1988-06-13 1989-12-19 Omron Tateisi Electron Co Cell sorting device
JPH027866A (en) * 1988-06-21 1990-01-11 Matsushita Electric Ind Co Ltd Source voltage converter circuit
JP2003530544A (en) * 2000-01-04 2003-10-14 アイメトリクス インコーポレイティッド Intravascular image processing detector
JP2010008082A (en) * 2008-06-24 2010-01-14 Olympus Corp Multiphoton excitation measuring instrument
JP2010252785A (en) * 2009-03-31 2010-11-11 Kanagawa Acad Of Sci & Technol Device for concentrating and separating cell
JP2013217672A (en) * 2012-04-04 2013-10-24 Sharp Corp Particle pretreatment device and particle pretreatment method

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KEN'ICHI SAWADA ET AL., JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol. JIKA MAS, no. 14, 30 September 2000 (2000-09-30), pages 1109 - 1113 *
TRICOT G. ET AL.: "Collection, Tumor Contamination, and Engraftment Kinetics of Highly Purified Hematopoietic Progenitor Cells to Suport High Dose Therapy in Multiple Myeloma", BLOOD, vol. 91, no. 12, 15 June 1998 (1998-06-15), pages 4489 - 4495 *
YASUO MORISHIMA: "Purified Hematopoietic Stem Cell Transplantation", JAPANESE JOURNAL OF CANCER CLINICS, vol. 44, no. 7, July 1998 (1998-07-01), pages 703 - 707 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017042137A (en) * 2015-08-28 2017-03-02 国立大学法人 奈良先端科学技術大学院大学 Cell sorting apparatus
US10544413B2 (en) 2017-05-18 2020-01-28 10X Genomics, Inc. Methods and systems for sorting droplets and beads
US11660601B2 (en) 2017-05-18 2023-05-30 10X Genomics, Inc. Methods for sorting particles
US10357771B2 (en) 2017-08-22 2019-07-23 10X Genomics, Inc. Method of producing emulsions
US10549279B2 (en) 2017-08-22 2020-02-04 10X Genomics, Inc. Devices having a plurality of droplet formation regions
US10583440B2 (en) 2017-08-22 2020-03-10 10X Genomics, Inc. Method of producing emulsions
US10610865B2 (en) 2017-08-22 2020-04-07 10X Genomics, Inc. Droplet forming devices and system with differential surface properties
US10766032B2 (en) 2017-08-22 2020-09-08 10X Genomics, Inc. Devices having a plurality of droplet formation regions
US10821442B2 (en) 2017-08-22 2020-11-03 10X Genomics, Inc. Devices, systems, and kits for forming droplets
US10898900B2 (en) 2017-08-22 2021-01-26 10X Genomics, Inc. Method of producing emulsions
US11565263B2 (en) 2017-08-22 2023-01-31 10X Genomics, Inc. Droplet forming devices and system with differential surface properties
US11833515B2 (en) 2017-10-26 2023-12-05 10X Genomics, Inc. Microfluidic channel networks for partitioning

Similar Documents

Publication Publication Date Title
WO2015076251A1 (en) Apparatus for selecting and removing cell, and method for selecting and removing cell
JP4047336B2 (en) Cell sorter chip with gel electrode
JP6911254B2 (en) Live cell identification and sorting system
US8241238B2 (en) Cell selection apparatus
US10465163B2 (en) Methods, systems, and apparatus for performing flow cytometry
US9638620B2 (en) System for deforming and analyzing particles
JP6317770B2 (en) Apparatus and method for high throughput sperm sorting
EP1846748B1 (en) Method and apparatus for sorting cells
US20220276250A1 (en) Cell analyzer system and cell analysis method
US11193879B2 (en) Use of vibrational spectroscopy for microfluidic liquid measurement
US8693762B2 (en) Inertial particle focusing flow cytometer
US11607688B2 (en) Apparatus and method for sorting microfluidic particles
US11530975B2 (en) Control device, microparticle sorting device and microparticle sorting system using control device, and control method
JP5580117B2 (en) Cell analyzer
JP7476483B2 (en) PARTICLE MANIPULATION METHOD, PARTICLE CAPTURE CHIP, PARTICLE MANIPULATION SYSTEM, AND PARTICLE CAPTURE CHAMBER
EP4185837A1 (en) A system and method thereof for real-time automatic label-free holography-activated sorting of cells
Kim et al. High-throughput residual white blood cell counter enabled by microfluidic cell enrichment and reagent-containing patch integration
JP6621493B2 (en) Apparatus and method for high throughput sperm sorting
Sugino et al. Integration in a multilayer microfluidic chip of 8 parallel cell sorters with flow control by sol–gel transition of thermoreversible gelation polymer
JP5098650B2 (en) Microparticle flow method and analysis method, and microparticle analysis substrate
US11998913B2 (en) Apparatus and method for sorting microfluidic particles
US20240139741A1 (en) Massively parallel cell analysis and sorting apparatus and methods
WO2018025088A2 (en) Use of vibrationnal spectroscopy for dna content inspection
WO2024020264A2 (en) Systems, methods, and apparatus for a microfluidic chip having a microchannel design which asymmetrically focuses particles
JP2014183854A (en) Cell analyzer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14864416

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14864416

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP