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WO2014097991A1 - Appareil et procédé de détection de cellules rares, système d'observation de cellules rares, et dispositif d'expansion de masse cellulaire - Google Patents

Appareil et procédé de détection de cellules rares, système d'observation de cellules rares, et dispositif d'expansion de masse cellulaire Download PDF

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Publication number
WO2014097991A1
WO2014097991A1 PCT/JP2013/083480 JP2013083480W WO2014097991A1 WO 2014097991 A1 WO2014097991 A1 WO 2014097991A1 JP 2013083480 W JP2013083480 W JP 2013083480W WO 2014097991 A1 WO2014097991 A1 WO 2014097991A1
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Prior art keywords
cell
rare
chamber
rare cell
detection
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PCT/JP2013/083480
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English (en)
Japanese (ja)
Inventor
久美子 星
淳吾 荒木
恒子 千代田
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コニカミノルタ株式会社
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Priority to JP2014553112A priority Critical patent/JPWO2014097991A1/ja
Publication of WO2014097991A1 publication Critical patent/WO2014097991A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/58—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving urea or urease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the present invention relates to a rare cell detection apparatus, a rare cell detection method, a rare cell observation system, and a cell deployment device.
  • Circulating cancer cells, circulating stem cells, and circulating endothelial cells in the blood are cells that are very rarely present in whole blood (hereinafter, also referred to as rare cells or target cells) depending on the disease state.
  • the detection of these rare cells is very difficult to detect, although the clinical usefulness is clear.
  • various cell separation methods have been applied to detect rare cells and commercialized, but in any case, the target cells to be detected are rare. It is still an important issue to reduce the loss of target cells (rare cells) while improving the effectiveness of detection results.
  • specimens whose target cells have been detected by a cell detection device using a scanner or the like are generally moved to another observation device such as a microscope or an inspection device to investigate details such as the shape observation of the target cells. Has been done.
  • Patent Document 1 a cell suspension containing a large amount of cells other than the target cell so as to reduce the loss of the target cell is developed on a flat surface device (slide) to detect the target cell, Then, it is known to move the device to another observation apparatus such as a microscope and investigate the target cell in detail (Patent Document 1).
  • a reticle mark is formed on a slide so that the position of the slide can be corrected even if there is a physical movement to another apparatus.
  • Patent Document 1 since a plurality of cells spread in a plane are optically scanned on a slide formed entirely flat, a cell misalignment occurs even during cell detection, There is a possibility that the position of the cells may change due to drying, aggregation or the like before observation, and in that case, position detection is required again.
  • Patent Document 1 when observing rare cells, it is not possible to accurately check whether the target rare cells are present or not, and whether or not observation is possible at an appropriate position. , There was a possibility that it would be a cause of detection deterioration such as detection leakage.
  • the present invention detects a target rare cell from a plurality of cells developed on a cell deployment device, and the detected rare cell is a predetermined one on the cell deployment device.
  • the detected rare cell is a predetermined one on the cell deployment device.
  • Another object of the present invention is to provide a cell deployment device that can be suitably used for a rare cell detection apparatus, a rare cell detection method, and a rare cell observation system.
  • a rare cell detection device reflecting one aspect of the present invention.
  • a rare cell detection device that detects a target rare cell based on a fluorescent label from a plurality of cells accommodated in each chamber in a device for cell deployment in which a plurality of chambers are formed,
  • a light source for irradiating the device for cell expansion containing a plurality of cells with excitation light;
  • a rare cell detector that optically detects fluorescence from a phosphor labeled with a rare cell, which emits light by excitation light applied to the cell deployment device from the light source;
  • a chamber detection unit for optically detecting detection light based on excitation light emitted from the light source to the device for cell deployment;
  • a rare cell observation system reflecting one aspect of the present invention.
  • a rare cell detection apparatus according to any of the above, and an observation apparatus that is equipped with the cell expansion device and observes rare cells, When observing the rare cells detected by the rare cell detection device with the observation device, the rare cells are accommodated based on information in which chamber the rare cells detected by the rare cell detection device are contained.
  • the specified chamber is specified, and the rare cells in the specified chamber can be observed.
  • a device for cell deployment reflecting one aspect of the present invention includes a plurality of chambers formed in a device body, and a part of the device body. A reticle mark is formed.
  • a specific rare cell is selected from a cell suspension developed in a cell expansion device having a plurality of chambers for containing cells. It is possible to quickly detect and specify in which chamber of the cell expansion device the rare cell is housed.
  • the rare cell at a specific position on the cell deployment device once detected by the rare cell detection device is transferred to the observation device to confirm again or to confirm the shape.
  • the observation and shape confirmation can be performed quickly and efficiently.
  • FIG. 1 is a schematic cross-sectional view of a cell deployment device according to an embodiment of the present invention.
  • FIG. 2 is a schematic plan view showing the chamber chip from which the flow channel forming frame is removed from the cell deployment device shown in FIG.
  • FIG. 3A is a schematic enlarged plan view showing a state in which a cell suspension is housed in a chamber of a cell deployment device.
  • FIG. 3B is a schematic diagram showing the relationship between the irradiation size of excitation light and the separation distance between adjacent chambers.
  • FIG. 3C is a schematic diagram showing the relationship between the irradiation size of the excitation light and the area of the upper opening of the chamber.
  • FIG. 4 is a schematic diagram showing a configuration of a main part of the rare cell detection device according to one embodiment of the present invention, and is a schematic diagram in the case where transmitted light is adopted as detection light of the chamber.
  • FIG. 5 shows a fluorescence signal for detecting a rare cell and a detection light signal for detecting a chamber position when the rare cell is optically detected by the rare cell detection apparatus shown in FIG. It is the graph represented to one.
  • FIG. 6A is a schematic diagram illustrating a configuration of a main part of a rare cell detection device according to another embodiment of the present invention, and is a schematic diagram in a case where reflected light is employed as detection light of the chamber.
  • FIG. 6B is a schematic diagram illustrating a configuration of a main part of a rare cell detection device according to still another embodiment in which reflected light is used as detection light for the chamber.
  • FIG. 7 is a schematic diagram showing a configuration of a main part of a rare cell detection device according to still another embodiment of the present invention, in which two-wavelength irradiation is employed and chamber autofluorescence is employed as chamber detection light.
  • FIG. 8 is a graph showing the experimental results of the rare cell detection device shown in FIG. 6B.
  • FIG. 9 is a schematic diagram showing the relationship between the position adjustment by the observation device and the cell deployment device when observing the cell deployment device in which the rare cell is detected by the rare cell detection device with another observation device.
  • FIG. 10 is a block diagram showing an outline of the rare cell detection method according to the present invention.
  • the cell suspension when optically detecting rare cells contained in a cell suspension, the cell suspension is not developed on a device having a flat surface, but a plurality of chambers (recessed portions) are preliminarily formed. ) Cell suspension is spread on the device formed, and the cells are dispersed and stored in each chamber. By dispersing and storing cells in the chamber in this way, it is possible to observe and confirm rare cells during detection of rare cells, during device transfer, and with other devices different from the cell detection device. In this case, the rare cells are regulated so as not to move carelessly on the device.
  • the chamber is detected together with the detection of the rare cells, and the position information of the rare cells and the position information of the chamber are obtained. It is possible to specify in which chamber on the device the rare cells are housed.
  • the rare cell position specifying unit in the computer of the rare cell device for example. And the information is stored in a recording medium.
  • the device in which the rare cell is detected once by the rare cell detection device is transferred to another device and observed or confirmed again, it is applicable based on the information stored in the recording medium. By calling the position of the chamber where the rare cells are present, it can be observed quickly.
  • the rare cell detection method includes the steps shown in FIG. 10, for example. That is, a suspension development step 100 for developing a cell suspension containing rare cells in a cell development device having a plurality of chambers, a rare cell detection step 200A for optically detecting the presence of rare cells, It has a chamber detection step 200B for optically detecting the chamber itself in which the turbid liquid is stored, and a rare cell position specifying acquisition step 300 for specifying in which chamber the rare cell is stored.
  • the rare cell detection step 200A and the chamber detection step 200B can be performed by separate scanning, but are preferably performed by simultaneous scanning. With such a rare cell detection method, it is possible to specify in which chamber the rare cells are housed.
  • the rare cell detection step 200A and the chamber detection step 200B are preferably performed by simultaneous scanning. With such a rare cell detection method, workability is good because detection of rare cells and detection of rare cells are performed simultaneously.
  • the irradiation size of the excitation light is not less than the same size as the cell, and the length of the irradiation size in the scanning direction is between adjacent chambers in the scanning direction of the chamber. It is preferable that the length of the irradiation size in the direction perpendicular to the scanning direction is equal to or less than the distance between the centers in the direction perpendicular to the scanning direction of the chamber. Moreover, it is preferable that the irradiation size of the said excitation light is below the area of the upper opening of the said chamber. Thus, if the irradiation size of excitation light is set, it is effective for the detection of the target cell and the chamber, and accurate information can be obtained.
  • the rare cell observation system includes a rare cell detection device and an observation device for observing specific rare cells, and when observing rare cells detected by the rare cell detection device with the observation device,
  • a chamber is identified based on information on which chamber contains the rare cell detected by the rare cell detection device, and the rare cell in the identified chamber is observed.
  • a plurality of chambers are formed in the device body, and a reticle mark is formed in a part of the device body. According to the cell deployment device having such a configuration, it can be effectively used for detection and observation of rare cells.
  • FIG. 1 shows a cross section of a cell deployment device according to an embodiment of the present invention.
  • the cell deployment device 10 is detachably attached to a rare cell detection apparatus described later, and the rare cells are detected via the cell deployment device 10.
  • the cell deployment device 10 is transferred to another apparatus such as a microscope as needed to observe or confirm the rare cells.
  • the cell deployment device 10 includes a chamber chip 1 in which a plurality of chambers 6 are formed, and a chamber chip 1 in which a flow path 5 is formed above the chamber 6.
  • the flow channel forming frame 2 provided integrally, the inlet 3 provided in the flow channel forming frame 2, and the cell suspension flowing into the flow channel 5 from the inlet 3 are discharged from the flow channel 5. It has an outlet 4 and the like.
  • the chamber chip 1 is also called a microchamber array (MCA), and a plurality of chambers 6 are arranged on the upper surface thereof at a predetermined interval as shown in FIG.
  • FIG. 2 shows an example in which the chambers 6 are formed on a straight line with predetermined intervals in the vertical and horizontal directions.
  • the arrangement of the chambers 6 is not limited to that shown in FIG. 2, and can be variously arranged.
  • the chambers arranged in a row in parallel with the scanning direction are shifted in the scanning direction for each column.
  • the chambers 6 can also be arranged in a staggered manner.
  • the chamber 6 refers to a concave micropore (microwell) that can “store” and “hold” one or more cells, and is preferably bottomed (that is, not a through-hole).
  • “Storing” means that when the cell suspension is introduced into the flow path 5 on the cell chip 1 for cell deployment, the introduced cells are accommodated in the chamber 6. This means that the cells stored in the chamber 6 do not come out of the chamber 6 due to the sending of staining liquid or washing liquid to the flow path 5 of the cell chip 1 for cell deployment.
  • the inner diameter B of the upper opening of the chamber 6 is preferably 20 ⁇ m to 500 ⁇ m.
  • the inner diameter B of the upper opening of the chamber 6 is in the range of 20 ⁇ m to 500 ⁇ m, it is possible to suitably store and hold rare cells in the chamber 6.
  • the chamber 6 may be mixed in different sizes, but it is preferable that at least cells can be stacked up to a single layer or two layers.
  • the depth of the chamber 6 is preferably varied depending on the inner diameter B of the chamber 6, and can be appropriately determined according to the number of cells to be stored. Specifically, it is preferable to appropriately determine the depth of the chamber so that about 10 to 15 cells can be stored. Typically, the depth of the chamber 6 is 20 ⁇ m to 500 ⁇ m.
  • the shape of the chamber 6 is an inverted frustoconical shape with a flat bottom in FIG. 1, but is not limited to this.
  • An inverted polygon cone or a rectangular parallelepiped may be used.
  • the bottom of the chamber 6 is typically flat, but may be curved.
  • the same material as a conventionally known microplate can be used, for example, polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), cyclic olefin copolymer. And polymers such as (COC).
  • the chamber chip 1 may be a combination of a plurality of materials in which a molded polymer is bonded to a substrate made of metal, glass, quartz glass, or the like.
  • the material of the chamber chip 1 is translucent. Must be sex.
  • the flow path forming frame 2 can be made of the same material as that of the chamber chip 1.
  • the part which covers at least the upper surface of the chamber chip 1 needs to have translucency.
  • a reticle mark R used as a reference point for position detection is provided at a part of the cell deployment device 10, for example, at the corner of the chamber chip 1 disposed below.
  • the reticle mark R may be formed of any object.
  • the reticle mark R is not limited to the upper surface of the chamber chip 1 as described above, and may be provided on the upper surface of the flow path forming frame 2. Further, the number of reticle marks R is not limited to one, and two or more reticle marks R may be provided.
  • the chamber chip 1 and the flow path forming frame 2 can be attached and detached from the viewpoint of ease of observation and maintenance.
  • the height 7 of the flow path 5 is preferably 50 ⁇ m to 500 ⁇ m.
  • the flow path 5 is provided between the chamber chip 1 and the flow path forming frame 2 by covering the periphery of the chamber chip 1 with the flow path forming frame 2.
  • the flow path forming frame 2 and the flow path 5 are not essential. That is, the cell deployment device can be configured with only the chamber chip 1.
  • the cell suspension is blood such as human, which may contain rare cells, lymph fluid, tissue fluid, body cavity fluid, etc., and may be appropriately diluted with a diluent or the like.
  • the cell suspension is not limited to those derived from living organisms, and may be a cell dispersion prepared by suspending cells artificially for testing and research.
  • rare cells include cancer cells.
  • the rare cells are any of CTC (circulating tumor cells or circulating cancer cells), CEC (circulating vascular endothelial cells) and CEP (circulating vascular endothelial progenitor cells).
  • CTC circulating tumor cells or circulating cancer cells
  • CEC circulating vascular endothelial cells
  • CEP circulating vascular endothelial progenitor cells
  • One or more types of cells may be used.
  • the diameter of various cells contained in such a cell suspension is preferably 10 ⁇ m to 100 ⁇ m, but is preferably the same as the diameter of the chamber 6 or smaller than the diameter of the chamber 6.
  • the target rare cell (reference numeral 24 in FIG. 3A) in the cell suspension for example, a specific cancer cell is stored in the chamber 6 through the flow path 5 and held.
  • a staining solution containing a phosphor capable of staining only specific cancer cells is stored in the chamber 6 through the flow path 5 and held.
  • the stained rare cells 24 can be detected by optical means.
  • FIG. 3A shows a state in which the target rare cell 24 is mixed with other cells and accommodated in each chamber 6, and the rare cell 24 is a reticle mark in the cell expansion device 10.
  • FIG. This indicates that the current is present in the chamber 6 'located second from the R position (reference point) in the X direction and first in the Y direction.
  • the excitation light is scanned relative to the cell deployment device 10.
  • the excitation light 25 is applied from the upper left end in the figure where the reticle mark R is formed.
  • the scanning is sequentially performed to scan the lower stage while reversing the scanning direction, thereby efficiently scanning all the chambers 6, 6... 6 on the cell deployment device 10. can do. In this way, necessary information can be detected.
  • the spot shape (irradiation shape) of the excitation light 25 can be selected as appropriate, and for example, any of a circle, an ellipse, a rectangle, and a polygon can be adopted.
  • Irradiation size of the excitation light 25 (Here, the irradiation size of the excitation light is a dimension in two directions perpendicular to each other indicating the spread of the excitation light, or an irradiation area when the cell expansion device is irradiated with the excitation light.)
  • the length in the scanning direction of the excitation light 25 (the dimensions in two directions perpendicular to each other indicating the spread of the excitation light 25), as shown in FIG. 3B, the length in the scanning direction of the excitation light 25 (the arrow X direction in FIG. 3B).
  • the depth G is preferably equal to or greater than the size of the rare cell 24, and is preferably equal to or less than the distance P between the centers between the chambers 6 adjacent to each other in the scanning direction (arrow X direction) (G ⁇ P). .
  • the length I in the direction (arrow Y direction) perpendicular to the scanning direction (arrow X direction) is the scanning direction.
  • the distance J is equal to or less than the distance J between the chambers 6 and 6 adjacent to each other in the direction perpendicular to (arrow X direction) (arrow Y direction) (I ⁇ J).
  • the excitation light 25 does not straddle a plurality of chambers, and each chamber can be easily made individually. Can be detected.
  • the irradiation size of the excitation light 25 (dimensions in two directions perpendicular to each other indicating the spread) is, for example, about 10 ⁇ m ⁇ 100 ⁇ m.
  • the irradiation size (area) of the excitation light 25 is preferably equal to or smaller than the area of the upper opening of the chamber 6. Specific surface area of the excitation light 25 is 78 ⁇ m 2 ⁇ 2.0 ⁇ 10 5 ⁇ m 2 approximately. If the excitation light 25 made of a circle, an ellipse, a rectangle, or the like has such an area, it can be easily reduced that the excitation light 25 is simultaneously irradiated across a plurality of chambers.
  • the inner diameter of the upper opening of the chamber 6 is preferably 20 ⁇ m to 500 ⁇ m. If the inner diameter of the upper opening of the chamber 6 is set in such a range, the cells can be suitably accommodated and held in the chamber.
  • the spot diameter K is preferably the same as or larger than that of the rare cell, and the same diameter or larger than the upper opening of the chamber. It is preferable that the diameter is slightly larger than that.
  • spot diameter K is set in such a range, it is preferable to obtain accurate information in one chamber. Further, it is possible to prevent the excitation light 25 from straddling between the two chambers 6 and 6 adjacent to each other in the Y direction.
  • FIG. 4 shows a configuration of a main part of the rare cell detection device 32 according to an embodiment of the present invention.
  • one-wavelength irradiation including one light source 26 for scanning the excitation light 25 is performed. It has been adopted.
  • a laser light source is employed as the light source 26.
  • the rare cell 24 is detected by receiving the fluorescence 54 from the phosphor labeled with the rare cell 24 by the rare cell detection unit 27.
  • the detection of the chamber 6 at each position shown in FIG. 2 is also performed simultaneously with the detection of the rare cell 24.
  • the detection light can be used.
  • the detection light for detecting the chamber 6 is not particularly limited, 1) Transmitted light 30 of the excitation light 25 irradiated from the light source 26 to the cell deployment device 10 and transmitted through the cell deployment device 10 (FIG. 4), 2) Reflected light 61 (FIGS. 6A and 6B) of the excitation light 25 irradiated from the light source 26 to the cell deployment device 10 and reflected by the cell deployment device 10; 3) The material autofluorescence 63 (FIG. 7), in which the cell deployment device 10 is irradiated from the light source 74 and the material of the cell deployment device 10 emits light based on the irradiated excitation light 77. Any of the above can be used for detection. The position of the chamber 6 can be easily detected by using any one of these detection lights.
  • the rare cell detection device 32 in FIG. 4 has a light source 26 that irradiates the cell deployment device 10 with the excitation light 25. Further, in the rare cell detection device 32, the phosphor labeled with the rare cell 24 is emitted in the cell deployment device 10 by irradiation of the excitation light 25, and the fluorescence 54 having the specific wavelength is allowed to pass through the dichroic mirror 29. Thus, the rare cell detector 27 is set to detect the fluorescence signal of the fluorescence 54.
  • the excitation light 25 irradiated from the light source 26 to the cell deployment device 10 and transmitted light 30 transmitted through the cell deployment device 10 is used to detect the chamber 6.
  • the detection light is used.
  • the detection light signal of the transmitted light 30 is set to be detected by the chamber detection unit 28.
  • the rare cell detection unit 27 shown in FIG. 4 is not particularly limited, but in this embodiment, a PMT (photomultiplier tube) is employed.
  • the chamber detector 28 is not particularly limited, but a photodiode is employed in this embodiment.
  • reference numerals 34, 36, and 38 are condenser lenses
  • reference numeral 46 is a pinhole member
  • reference numerals 48 and 52 are emission filters.
  • the positional information of the rare cell 24 is obtained based on the fluorescence signal. Further, by obtaining the detection light signal of the chamber 6 detected by the transmitted light 30, the position information of the chamber is obtained.
  • the wavelength of the transmitted light 30 transmitted through the cell deployment device 10 is the same as that of the excitation light 25. Then, based on the position information of the rare cell 24 and the position information of the chamber 6, the chamber in which the rare cell 24 is accommodated can be specified by the rare cell position specification acquisition unit 56 a of the computer 56. These pieces of information are stored in a recording medium 56b in the computer 56.
  • FIG. 5 is a graph showing the fluorescence signal of the fluorescence 54 obtained by the rare cell detection unit 27 of the rare cell detection device 32 in FIG. 4 and the detection light signal obtained by the chamber detection unit 28. It is.
  • the horizontal axis in the graph represents the scanning distance (position) from the scanning start position (reference point).
  • the solid line indicates the fluorescence signal due to the fluorescence 54 from the rare cell 24, and the dotted line indicates the detection light signal due to the transmitted light 30 from the cell deployment device 10.
  • the position where the peak of the fluorescence signal indicated by the solid line indicates the detection of the rare cell 24, and the low detection light signal indicated by the dotted line indicates the detection of the chamber 6. ing.
  • the rare cell 24 is accommodated in the chamber 6 at each position of about 300 ⁇ m, 600 ⁇ m, 900 ⁇ m, 1200 ⁇ m, and 1500 ⁇ m from the reference point, and the position of the rare cell 24 is It can be identified by reading the position dimension on the horizontal axis.
  • chamber of the cell deployment device 10 the rare cells 24 are housed in this manner is specified by the rare cell position specifying acquisition unit 56a of the computer 56, and the information is stored in the recording medium 56b.
  • the cell deployment device 10 in which the detection of the rare cell 24 is completed is transferred from the rare cell detection device 32 to another device for observation and confirmation. Even so, it becomes possible to immediately observe the rare cell 24 based on the recorded information.
  • the cell deployment device 10 when the cell deployment device 10 is mounted in another apparatus, it may be mounted in a posture that is biased with respect to the reference position. Since the reticle mark R is formed on the cell deployment device 10, correct information when the reticle mark R is arranged at a correct position can be obtained by detecting the reticle mark R.
  • FIG. 6A shows a rare cell detection device 58 according to another embodiment of the present invention.
  • the cell deployment device 10 is irradiated from the light source 26 as detection light for detecting the chamber 6.
  • the reflected light 61 of the excitation light 25 reflected by the cell deployment device 10 is used.
  • the wavelength of the excitation light 25 and the wavelength of the reflected light 61 are the same wavelength.
  • the reflected light 61 irradiated from the light source 26 toward the cell expansion device 10 and reflected through the cell expansion device 10 is received.
  • a chamber detection unit 28 is arranged.
  • a condensing lens 34, an emission filter 48, a pinhole member 46, and a condensing lens 36 are disposed between the cell deployment device 10 and the rare cell detection unit 27.
  • the excitation light 25 from the light source 26 is made incident on the cell deployment device 10 via the condensing lens 38, and the rare cells 24 are caused to enter by the incident excitation light 25.
  • the fluorescently labeled fluorescent material is caused to emit light, and the fluorescent light 54 having the specific wavelength is guided to the rare cell detection unit 27 through the condensing lens 34, the emission filter 48, the pinhole member 46, and the condensing lens 36.
  • the rare cell detector 27 can detect the fluorescence signal of the cell 24.
  • the rare cell detection device 58 guides the reflected light 61 of the cell deployment device 10 of the excitation light 25 irradiated from the same light source 26 to the chamber detection unit 28 simultaneously with the detection of the fluorescence signal of the rare cell 24.
  • the detection light signal of the chamber 6 can be detected by detecting by the chamber detection unit 28.
  • the position information of the chamber 6 can be obtained by detecting the reflected light 61. Furthermore, in this rare cell detection device 58, as in the case of the rare cell detection device 32 shown in FIG. 4, the rare cell 24 is located in the chamber 6 based on the positional information of the rare cell 24 and the positional information of the chamber 6. It is specified by the rare cell position specifying unit 56a of the computer 56 whether it is stored in the storage medium 56b, and stored in the recording medium 56b. Therefore, even if the cell deployment device 10 once the rare cell 24 is detected is transferred to another apparatus, the information can be called from the recording medium 56b.
  • the optical system in the case of obtaining the position information of the chamber 6 using the reflected light 61 is not limited to the mode disclosed in FIG. 6A.
  • the rare cell detection unit 27 and the chamber detection unit 28 are configured separately, but can be combined into one as shown in FIG. 6B.
  • FIG. 6B shows a rare cell detection device 59 according to still another embodiment in which the rare cell detection unit 27 and the chamber detection unit 28 shown in FIG. 6A are used as one detection unit 270.
  • a single detection unit 270 in place of the rare cell detection unit 27 can detect the fluorescent light 54 of the phosphor labeled with the rare cell 24 and can detect the reflected light 61 from the chamber 6. .
  • a single detection unit 270 is provided, a dichroic mirror 62 is provided between the light source 26 and the cell deployment device 10, and the dichroic mirror 62 and the detection unit are further provided.
  • a filter switching unit 480 is provided between the H.S. The filter switching unit 480 can selectively use two different emission filters.
  • the fluorescence signal of the rare cell 24 and the detection light signal of the chamber 6 can be obtained by performing scanning twice.
  • the fluorescence signal of the rare cell 24 can be detected by the detection unit 270 by installing a filter for extracting the fluorescence 54 labeled with the rare cell 24 in the filter switching unit 480 and performing the first scan. If a filter for extracting a detection light signal from the reflected light 61 from the cell deployment device 10 is installed in the filter switching unit 480 and the second scan is performed, detection by the reflected light 61 from the cell deployment device 10 is performed.
  • the light signal can be detected by the detection unit 270.
  • FIG. 7 shows a rare cell detection device 70 according to still another embodiment of the present invention.
  • excitation light from the second light source 74 is used as detection light for detecting the chamber 6.
  • the material autofluorescence 63 is employed, in which the material of the cell deployment device 10 emits light based on 77.
  • a first light source 26 used for detecting the rare cell 24 and a second light source 74 used for detecting the autofluorescence 63 included in the material of the chamber 6 are provided. It is equipped.
  • the excitation light 25 is emitted from the first light source 26, and the excitation light 77 having a wavelength different from the wavelength of the excitation light 25 is emitted from the second light source 74. Then, the wavelength of the autofluorescence 63 emitted from the material of the cell deployment device 10 is set to be different from the fluorescence wavelength of the fluorescence 54 emitted from the fluorescent substance that fluorescently labels the rare cells 24.
  • the excitation light 25 irradiated from the first light source 26 for detecting the rare cell 24 passes through the dichroic mirror 86 and is further reflected by the dichroic mirror 81.
  • the light is incident on the device 10 for cell deployment, and thereby, the phosphor that fluorescently labels the rare cell 24 is caused to emit light.
  • the fluorescent light 54 having a specific wavelength emitted by the phosphor is taken into the rare cell detection unit 27 via the dichroic mirror 81, the dichroic mirror 82, the emission filter 48, the pinhole member 46, the condensing lens 36, and the like. It is done.
  • the rare cell 24 is detected based on the fluorescence signal of the fluorescence 54 taken into the rare cell detection unit 27.
  • the excitation light 77 emitted from the second light source 74 for detecting the chamber 6 passes through the condenser lens 83 and is reflected by the dichroic mirror 86 and the dichroic mirror 81. Then, it is incident on the device 10 for cell deployment. Then, the autofluorescence 63 emitted from the material of the cell deployment device 10 passes through the dichroic mirror 81, is reflected by the dichroic mirror 82, and enters the chamber detection unit 28 via the condenser lens 79. Thereby, the detection light signal of the chamber 6 can be detected by the chamber detection unit 28. In the chamber position detection using the autofluorescence of the cell deployment device 10, for example, the chamber can be detected by detecting different amounts of fluorescence depending on the material thickness difference between the chamber and the other portions. .
  • the chamber in which the rare cell 24 is accommodated is specified by the rare cell position specifying unit 56 a of the computer 56.
  • the data is stored in the recording medium 56b in the computer.
  • the position information of the chamber 6 is also obtained by detecting the autofluorescence 63 of the cell deployment device 10, and based on the position information of the rare cell 24 and the position information of the chamber 6. Thus, it can be specified in which chamber the rare cell 24 is housed.
  • the detection light for detecting the chamber 6 is either a case where the reflected light 61 is used as shown in FIG. 6A or 6B, or a case where the autofluorescence 63 is used as shown in FIG. If the graph as shown in FIG. 5 is created, a tendency similar to that when the transmitted light 30 in FIG. 4 is used can be obtained.
  • FIG. 8 is a graph showing experimental results by the rare cell detection device 59 of FIG. 6B in which the reflected light 61 is adopted as the detection light of the chamber 6.
  • the solid line indicates the detection light signal due to the reflected light 61
  • the dotted line indicates the fluorescence signal due to the fluorescence 54.
  • the position of the chamber 6 can be detected by searching for a peak point where the amount of reflected light decreases. Further, in the fluorescence signal by the fluorescence 54, the position of the rare cell can be detected by searching for a position where the light amount of the fluorescence 54 increases.
  • the wavelength of the detection light signal (reflected light 61) indicated by the solid line is 488 nm
  • the wavelength of the fluorescent signal (fluorescence 54) indicated by the dotted line is 647 nm.
  • the rare cell detection device according to each of the above embodiments is used to transfer the cell deployment device 10 from which the rare cell 24 has been detected to another device for observation or confirmation again, the computer. Since the information is stored in the 56 recording media 56b, the position of the chamber 6 where the corresponding rare cell 24 is present is called based on the stored information, and the called information is read by other observations. If the observation apparatus is moved to an appropriate position based on the position adjustment apparatus of the apparatus, the corresponding rare cell 24 can be observed quickly.
  • the observation device 76 is provided with a position adjusting device 90. And the observation part which consists of a lens etc. of the observation apparatus 76 is enabled to move to an XYZ direction by the information taken in from the recording medium 56b to the position adjustment apparatus 90.
  • FIG. 9 the observation device 76 is provided with a position adjusting device 90.
  • the observation part which consists of a lens etc. of the observation apparatus 76 is enabled to move to an XYZ direction by the information taken in from the recording medium 56b to the position adjustment apparatus 90.
  • the observation unit of the observation device 76 is moved to observe the desired rare cell 24.
  • the rare cell 24 once detected by the rare cell detection device can be quickly observed. .
  • the optical element relating to the rare cell detection device and the observation device can take various forms.
  • single-color fluorescence may be used to detect the target rare cells, but in order to detect non-specific cells other than the target rare cells, two or more colors of fluorescence are used so that each cell emits light in a different color. Can also be used. Moreover, it is preferable to employ a longer wavelength side than the wavelength of the excitation light 25 for the fluorescence wavelength of the phosphor labeled with the rare cells 24 in order to avoid the influence of autofluorescence.
  • the mode in which information is recorded on a recording medium in a computer in the rare cell detection device and information is read from the recording medium by connecting another device such as an observation device has been described. May be separate from the rare cell detection device, and the cell deployment device itself is provided with an information recording unit to store the information obtained by the rare cell detection device in the information recording unit, Of course, other devices may be configured to read out necessary information from the information recording unit when the device for cell deployment is mounted.
  • CTC blood circulating cancer cells
  • the cell suspension may be peripheral blood itself, or peripheral blood diluted with an appropriate buffer such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • peripheral blood is diluted with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the cell suspension was previously fluorescently labeled with blood circulating cancer cells (CTC) and leukocytes.
  • the preparation method of the cell suspension previously fluorescently labeled is as follows. First, paraformaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 1 ml of the cell suspension so as to be 4%, gently mixed, and allowed to react in the dark at room temperature for 15 minutes. Here, a sufficient amount of phosphate buffered saline (PBS) is added and mixed, washed by centrifugation, and then phosphate buffered saline (PBS) containing 0.1% Tween is used for cell membrane permeabilization.
  • PBS phosphate buffered saline
  • a cell deployment device 10 (chamber depth 50 ⁇ m, chamber diameter 100 ⁇ ) is provided with a flow path 5 (width 5 mm) and a flow path forming member (flow path height 500 ⁇ m) 2 and a blocking solution (PBS containing 3% BSA). The solution was fed at a high flow rate (16 mLmin). Thereafter, the blocking agent is removed from the flow path 5 with PBS, the cell suspension is introduced into the flow path 5 at the same flow rate, and then intermittent liquid feeding (10 ⁇ L of liquid stopped for 10 seconds) is repeated. Then, the cells were collected in the chamber 6.
  • ⁇ Cell Detection Method 1 (Fluorescence 54 and reflected light 61 are detected by the same irradiation from the light source 26)
  • the cell deployment device 10 containing the cells is irradiated with a He-Ne laser (wavelength 633 nm) that excites Alexa647 labeled for blood circulation cancer cell (CTC) labeling, and the fluorescence signal of Alexa647 is transmitted to the rare cell detection unit 27.
  • PMT and the reflected light 61 of the chamber irradiated by the light source 26 is measured by the chamber detector 28 (PD).
  • the position information is obtained from the pulse signal of the automatic stage, and the blood circulation
  • the detection light signal (reflected light) of the chamber was detected together with the cancer cell (CTC) signal.
  • ⁇ Cell detection method 2 (FIG. 7)> A cell-expanding device 10 containing cells is irradiated with a He-Ne laser (wavelength 633 nm) for exciting Alexa647 labeled for blood circulation cancer cell (CTC) labeling from a light source 26, and the fluorescence signal of Alexa647 is rare cells. Measurement was performed using a PMT (photomultiplier tube) as the detection unit 27. Furthermore, when the irradiation light was measured at 405 nm and the autofluorescence of the device for cell deployment was measured with a PD (photodiode) as the chamber detection unit 28, the signal of the blood circulating cancer cell (CTC) and the signal of the chamber 6 were measured. A detection light signal (autofluorescence) was detected.

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Abstract

Cette invention concerne un appareil de détection de cellule rare qui détecte une cellule rare (24), sur la base d'un marqueur fluorescent, parmi les multiples cellules qui se trouvent dans chacune des multiples chambres (6) formées dans un dispositif d'expansion de masse cellulaire (10), ledit appareil de détection de cellule rare comprenant : une source de lumière (26) pour exposer le dispositif à une lumière d'excitation (25) ; une section détection de cellule rare (27) pour détecter optiquement une lumière fluorescente (54), qui est émise par le phosphore qui marque la cellule rare (24) à l'aide de la lumière d'excitation (25) ; une section détection de chambre (28) pour détecter optiquement la lumière de détection provenant de la lumière d'excitation (25) à laquelle le dispositif d'expansion de masse cellulaire (10) a été exposé ; et une section détermination/acquisition de position de cellule rare (56a) pour acquérir des informations sur la position de la cellule rare, et les positions des chambres (6) respectivement fournies par la section détection (27) et de la section détection (28)pour déterminer ainsi la chambre (6) du dispositif d'expansion de masse cellulaire (10) dans laquelle se trouve la cellule rare (24). Ainsi, l'acquisition des informations sur la position de la cellule rare permet l'observation de la terre rare et la confirmation sur la forme de la cellule rare et autre de manière immédiate même si le dispositif est transporté dans un autre appareil.
PCT/JP2013/083480 2012-12-18 2013-12-13 Appareil et procédé de détection de cellules rares, système d'observation de cellules rares, et dispositif d'expansion de masse cellulaire WO2014097991A1 (fr)

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