JP5580117B2 - Cell analyzer - Google Patents

Description

  The present invention relates to a cell analyzer.

  Biological tissues in multicellular organisms maintain a harmonious function as a whole, with various cells sharing roles. Or, if some of the cells become cancerous (we will refer to them collectively as cancer, including tumors), a neoplasm that is different from the surrounding area will appear, but the cancer area and normal far away from it. The tissue part is not necessarily separated by a certain boundary, and the area around the cancer is also affected in some way. Therefore, in order to analyze the function in the organ tissue, it is necessary to separate and analyze a small number of cells present in a narrow region as easily as possible in a short time and with a minimum loss.

  In the field of regenerative medicine, an attempt is made to regenerate a target tissue, and thus an organ, by isolating an organ stem cell from the tissue and reculturing it to induce differentiation.

In order to identify or separate cells, it is necessary to distinguish them according to some sort of index. In general, cell differentiation
1) Visual morphological cell classification: For example, examination of bladder cancer and urethral cancer by examination of atypical cells appearing in urine, classification of atypical cells in blood, cancer examination by cytology in tissues, etc. Can give.
2) Cell classification by cell surface antigen (marker) staining by the fluorescent antibody method: Cell surface antigens generally called CD markers are stained with a specific fluorescently labeled antibody. Cell sorting by cell sorters, flow cytometers and tissues Used for cancer screening by staining. Of course, these are widely used not only for medical purposes but also for cell physiology research and industrial cell utilization.
3) Alternatively, with regard to the separation of stem cells, there is an example in which cells containing stem cells are roughly separated using a fluorescent dye incorporated into the cells as a reporter, and then the target stem cells are separated by actually culturing. This is because an effective marker for stem cells has not yet been established, and the target cells are substantially separated by using only those actually cultured and differentiated.

  The separation and recovery of specific cells in the culture medium in this way is an important technique in biological and medical analysis. When the cells are separated based on the difference in specific gravity of the cells, they can be separated by a velocity sedimentation method. However, if there is almost no difference in specific gravity between cells that distinguishes unsensitized cells from sensitized cells, the cells are separated one by one based on information stained with fluorescent antibodies or visual information. There is a need to.

  An example of this technique is a cell sorter. The cell sorter isolates and drops cells after fluorescence staining in a charged droplet in units of one cell, and based on the presence or absence of fluorescence in the droplet and the amount of light scattering, In the process of dropping, a high electric field is applied in any direction in the normal direction to the direction of drop, and the drop direction of the drop is controlled and fractionated into multiple containers placed underneath. (Non-patent document 1: Kamarck, ME, Methods Enzymol. Vol. 151, p150-165 (1987)).

  However, this technology is expensive, requires a large device, requires a high electric field of several thousand volts, requires a large amount of sample concentrated to a certain concentration, and creates droplets. There are problems such as the possibility of damaging the cells at the stage of performing, and the inability to directly observe the sample. In order to solve these problems, in recent years, a cell sorter has been developed in which a fine flow path is created using a micro-processing technique, and cells flowing in a laminar flow in the flow path are separated while directly observing under a microscope (non- Patent Document 2: Micro Total Analysis, 98, pp. 77-80 (Kluwer Academic Publishers, 1998); Non-Patent Document 3: Analytical Chemistry, 70, pp. 1909-1915 (1998)). However, the cell sorter created using this microfabrication technique has a slow response speed of sample separation to the observation means, and in order to put it to practical use, a separation processing method that does not damage the sample and has a faster response is required. there were. In addition, if the cell concentration in the sample solution to be used is not increased to a certain level in advance, the separation efficiency of the device cannot be sufficiently increased even at a dilute cell concentration. If the sample is concentrated in a separate device, it is difficult not only to recover the concentrated solution without loss, but also in the complicated pretreatment stage, the collected cells are contaminated. The problem is that an undesirable problem occurs in terms of use.

  In order to solve such problems, the present inventors use micromachining technology to fractionate a sample based on the microstructure of the sample and the fluorescence distribution in the sample, and damage the collected sample. And a cell analysis / separation apparatus capable of easily analyzing and separating a cell sample without any problems (Patent Document 1: JP 2003-107099; Patent Document 2: JP 2004-85323; Patent Document 3: WO 2004 / 101731).

  Currently, detection of cancerous tissue has improved dramatically by improvements in MRI (Nuclear Magnetic Resonance Imaging) and CT (Computerized Tomography), but for the identification of benign and malignant tumors, a method that exceeds evaluation by biopsy Does not exist. As a problem of malignant tumors, it is known that cancer cells metastasize to other organs by their ability to infiltrate blood vessels or lymph vessels from their own tissues. Malignant tumor cells that circulate in this way are called peripheral blood circulating cancer cells (Circulating Tumor Cells: CTCs), and it is thought that there are about several hundreds of cancer cells in 100,000 cells of blood cells (including red blood cells). ing. In recent years, anticancer agents for specific targets have been developed one after another, and if the type of malignant tumor in the blood can be identified, it has become possible to select an anticancer agent that effectively destroys the cells. If technology to monitor CTCs flowing in the blood is realized, the presence of malignant tumor cells that cause metastatic cancer flowing in the blood can be quantitatively measured, thereby quantifying the effect of the administered anticancer drug. This is the realization of the world's first method that can be continuously evaluated and can not only prevent the administration of unnecessary and excessive anticancer drugs, but also detect the presence or absence of recurrence.

JP 2003-107099 A JP 2004-85323 A International Publication No. 2004/101731 Pamphlet

Kamarck, M.E., Methods Enzymol. Vol.151, p150-165 (1987) Micro Total Analysis, 98, pp.77-80 (Kluwer Academic Publishers, 1998) Analytical Chemistry, 70, pp.1909-1915 (1998)

  Despite the importance of confirming the presence of CTCs in relation to cancer metastasis in the clinical setting, diagnostic criteria using this as an index of cancer metastasis have not been established. The reason is that because of the diversity and rarity of cancer itself, a huge shape database is used to identify cancer cells with various shapes from the collected blood, and to identify them as cancer cells only from their shapes. Developing an algorithm that needs to be constructed and thus reliably and selectively recovers cancer cells has been a very difficult task.

  Therefore, in particular, until now, we used to identify and identify cancer cells in the blood using fluorescent labels. However, there is a method to guarantee this whether or not a fluorescent label specific to a specific cancer has been selected properly. not exist. For this reason, there was a “false negative” problem in CTC measurement that cannot be detected even though blood cancer cells are present.

  In view of such a situation, the present inventors provide a method and an apparatus for selectively recovering cancer cells flowing in the blood with metastatic ability.

  The present inventors have found that normal cells in the blood flow in the blood in a single isolated cell state, whereas blood cancer cells, in addition to those in the blood in a single isolated cell, Focusing on the fact that the cells are flowing in the blood in the state of a cell population or cell mass, identify whether the cells in the blood are a cell population or a cell mass, and if it is a cell population or a cell mass, select it selectively Have developed a method for recovering the cell population and a cell analyzer for use in the method.

  That is, the present invention provides the following apparatus system and method.

[1] A cell analyzer system for selectively separating and purifying cancer cells in blood,
A first flow path for flowing a sample liquid containing cells in blood derived from a subject, and a cell detection region on the first flow path for detecting cells in the sample liquid flowing through the first flow path A cell separation region disposed downstream of the cell detection region on the first flow channel, and a selective branching of cells branched from the first flow channel downstream of the cell separation region. A cell sorter chip comprising at least two branch channels;
Whether the cells are in an isolated cell state, a cell population state or a cell mass state at a light irradiation means for irradiating the cells flowing through the cell detection region and an image capture rate of at least 200 frames / second An optical system including a high-speed camera for acquiring image information to determine
Based on the result of the discrimination, in the cell separation region, an external force capable of controlling which of the at least two branch channels the cell is guided to flow is selectively applied to the cell. Means for applying;
With
A cell analyzer system wherein the presence of the cell population or cell mass suggests the presence of cancer cells in the blood.
[2] The apparatus system according to [1], wherein the external force is ultrasonic radiation pressure, gravity, electrostatic force, or dielectric electrophoresis force.
[3] The image of the cell obtained from the optical system is binarized, and at least one index selected from the group consisting of the luminance centroid, area, perimeter, major axis, and minor axis of the binarized image, The apparatus system according to the above [1] or [2], wherein each of the cells is detected at a single cell level to identify whether the cell is an isolated single cell, a cell population or a cell mass.
[4] The cells in the cell sample solution are fluorescently labeled, the optical system further includes fluorescence detection means, and information on the fluorescence image of the cells is used as an additional index by the control / analysis unit. The apparatus system according to [3] above.
[5] A method for quantitative analysis of cancer cells in blood,
Preparing a sample solution containing cells in blood from a subject;
The sample solution is subjected to cell separation using a cell sorter chip,
The cell separation chip comprises:
A first flow path for flowing the sample solution;
A cell detection region on the first flow path for detecting cells in the sample solution flowing through the first flow path;
A cell separation region disposed downstream of the cell detection region on the first flow channel, and at least a cell branching from the first flow channel downstream of the cell separation region for performing selective recovery of cells. A process comprising two branch channels;
An optical system including a light irradiation means and a high-speed camera irradiates the cells flowing through the cell detection region with light to obtain an image of the cells at an image capture rate of at least 200 frames / second, whereby the image information is Determining whether the cell is in an isolated cell state, a cell population state or a cell mass state;
A step of selectively applying an external force to the cells in the cell separation region based on the result of the discrimination, inducing the cells to flow through one of the at least two branch channels,
With
A cell analysis method wherein the presence of the cell population or cell mass suggests the presence of cancer cells in the blood.
[6] The method according to [5], wherein the external force is ultrasonic radiation pressure, gravity, electrostatic force, or dielectric electrophoretic force.
[7] The image of the cell obtained from the optical system is binarized, and at least one index selected from the group consisting of the luminance centroid, area, circumference length, major axis, and minor axis of the binarized image, The method according to [5] or [6] above, wherein each of the cells is detected at a single cell level to identify whether the cell is an isolated single cell, a cell population or a cell mass.
[8] The cells in the cell sample solution are fluorescently labeled, the optical system further includes fluorescence detection means, and information on the fluorescence image of the cells is used as an additional index by the control / analysis unit. The method according to [7] above.
[9] The device system according to any one of [1] to [4], which is used for cancer diagnosis and / or drug administration planning.
[10] The method according to any one of [5] to [7] above, which is used for cancer diagnosis and / or drug administration planning.

  According to the present invention, it is possible to identify whether or not a small amount of the test subject cells in body fluids (eg, blood, lymph, saliva, urine) are clustered (whether they are isolated single cells), It is possible to selectively collect and purify a target cell as a cell population or a cell mass, and to realize analysis of accurate gene information and expression information of the target cell.

  In addition, when a cell population or a cell mass is present in blood, it is highly likely that the cell is a cancer cell. Therefore, the apparatus system and method of the present invention can be used for diagnosis of cancer, formulation of a drug administration plan for a cancer patient, etc. Can be used for.

It is a schematic diagram which shows notionally an example of the cell separation and refinement | purification (cell sorter) module of the cell analyzer system of this invention. It is a schematic diagram which shows notionally an example of the optical detection system of the cell separation and refinement | purification (cell sorter) module of the cell analyzer system of this invention. It is a schematic diagram which shows typically the example of the shape of the cell population collected or a cell lump.

In one embodiment, the present invention provides a cell analyzer system for selectively separating and purifying cancer cells in a body fluid (particularly blood). This device system is typically
(1) Cell sorter chip for cell separation,
(2) an optical system including light irradiation means (for example, a laser) and a camera (for example, a CCD camera) for detecting cells flowing in the flow path of the cell sorter chip;
(3) a means for applying an external force to the cell to separate the cells flowing through the flow path of the cell sorter chip (for example, a comb-shaped electrode); Control means for controlling the operation of the means for giving is provided.
The control means may be integrated with a means for applying an external force to the cells.

  Thus, according to the cell analyzer of the present invention, image data of about 10,000 images of cells per second is obtained from cells flowing through the microchannel formed on the chip substrate, and the analysis result of the image information is obtained. Based on this, it is possible to purify and collect cell populations or cell masses in real time with a maximum time capacity of 10,000 cells per second.

  The cell sorter chip used in the cell detection system of the present invention typically has a first flow path for flowing a sample liquid containing bodily fluid cells derived from a subject, and the sample liquid flowing through the first flow path. A cell detection region on the first flow channel for detecting cells in the cell, a cell separation region disposed downstream of the cell detection region on the first flow channel, and the downstream of the cell separation region. At least two branch channels branched from one channel are provided.

  In the cell detection area, the camera used in the optical system has an image capture rate of at least 200 frames / second so that it can be discriminated whether the cells are in an isolated cell state, a cell population state or a cell mass state. It is preferable that the camera is a high-speed camera that can capture an image.

  A typical embodiment of the cell analyzer of the present invention is that the cell is an isolated single cell by fine discrimination evaluation of the shape based on the image of the image detection type 1 cell separation / purification (cell sorter) unit, or Distinguish a small amount of cell population / cell mass from a cell sample that is continuously conveyed by a flow path and minimize loss due to contamination and manipulation by determining whether it is a cell population / cell mass. Even it can be recovered.

  Further, the cell analyzer of the present invention comprises means for irradiating a target cell population with excitation light having a specific narrow-band wavelength range that matches the excitation light wavelength of a fluorescent dye having a wavelength of 350 nm to 560 nm. A means for quantitatively measuring the fluorescence intensity of the fluorescent dye emitted by the excitation light in a wavelength band where the excitation light does not overlap, and by using this fluorescence measurement means, in the target cell population or cell mass, The presence or absence of fluorescent labeling of the cells can be detected and confirmed at the single cell level, and the fluorescently labeled cell population or cell mass can be determined and selectively recovered. Therefore, according to the cell analyzer of the present invention, it is possible to accurately separate and purify cells according to the difference in the presence or absence of their clustering based on an index that could not be identified by the conventional scattered light detection type cell sorter technology. it can. Here, the excitation light and the fluorescence wavelength used in the group to measure fluorescence simultaneously measure two or more wavelengths simultaneously using a wavelength band that does not overlap with each other by incorporating an optimum bandpass filter optical element, By adding a different fluorescent dye to each of the labels that specify a plurality of cell states and types, the type of each cell can be identified from the combination of the presence or absence of labeling of the plurality of labels.

  For example, normal cells in the blood flow in the blood in a single isolated cell state, whereas blood cancer cells, in addition to those that flow in the blood in a single isolated cell, It flows in the blood in the form of cell clumps. Thus, the presence of a cell population or cell mass in the sample fluid indicates the presence of cancer cells in the subject's blood.

  Also, in the means for performing separation / purification by determining the presence or absence of cell clustering based on the result of image detection, information such as the difference in cell size and the number of cell populations is acquired as image information. Based on the results, the cells are purified. For image acquisition, a high-speed camera is used, the light emission of the light source is adjusted in accordance with the shutter cycle of the high-speed camera, and light from the light source is emitted for a certain period of time during which each shutter is released. For example, if the shutter speed is 1 / 10,000th of a second, illuminate the target cells with a light source capable of high-speed light emission control, such as an LED light source or a pulsed laser light source, for a period of 1 / 10th of the shutter speed. Thus, it is possible to acquire a fine shape such as the presence or absence of grouping of cell samples.

  In the present specification, “body fluid” is used in the meaning normally used in the art, and includes blood, lymph, saliva, urine, semen and the like.

  The cells assumed as detection targets in the present invention are bacteria for small cells, animal cells (for example, cancer cells) for large cells, and cell populations / cell masses for cancer cells in particular. Accordingly, the cell sample size typically ranges from about 0.5 μm to about 100 μm. For this reason, when a channel incorporating a cell separation function is formed on one surface of a biochip substrate to continuously perform cell concentration and separation, the first problem is the channel width (cross-sectional shape). Further, the flow path is formed in a substantially two-dimensional plane using a space of about 10 to about 100 μm in the thickness direction of the substrate on one of the substrate surfaces. In terms of cell size, about 5 to about 10 μm in the thickness direction for bacteria and about 100 to about 500 μm in the thickness direction for animal cells and cell populations / cell masses are the most typical sizes.

  The cell analyzer system of the present invention typically includes a cell separation / purification unit (corresponding to the above (3) and (4)) having a function of separating and purifying cells in a biochip, and a stage preceding the separation unit. An optical analysis unit (corresponding to the above (2)) for discriminating and judging cells to be separated and purified is included.

  In the cell separation / purification unit, for example, in the flow path in which the cells flow, the external force is applied to different cells in different directions, and the cells to be collected in one of the two flow paths branched downstream are Other cells can be induced in the other channel.

In another embodiment, the present invention provides a method for quantitative analysis of cancer cells in blood. This method is typically
(1) preparing a sample solution containing cells in blood derived from a subject;
(2) A step of subjecting the sample solution to cell separation using a cell sorter chip,
(3) An optical system including a light irradiation means and a high-speed camera irradiates the cells flowing through the cell detection region with light to obtain an image of the cells at a predetermined image capture rate, whereby the image information is Determining whether the cell is in an isolated cell state, a cell population state or a cell mass state; and (4) selectively applying external force to the cell in the cell separation region based on the determination result. And applying and flowing the cells through one of the at least two branch channels.

  In the cell analyzer system or cell analysis method of the present invention, when a cell is captured and evaluated as an image, for example, a site (cell detection region) to be observed with a CCD camera is provided upstream of the flow path branching portion. Accordingly, a cell separation region is provided downstream thereof. Regardless of the image, if the cells passing through the flow path are irradiated with laser or the like and the cells are modified with fluorescence, the fluorescence can be detected with a photodetector. Also in this case, it can be set as the structure which installs a cell separation area | region downstream of a detection part.

  When separating cells in the cell separation region, as a means for moving the cells by applying an external force to the cells flowing in the cell separation region, for example, when using a dielectrophoretic force, a pair of comb-shaped electrodes is installed, A flow path that can be separated and discharged can be provided. In the case of electrostatic force, it is possible to change the position of the cell in the flow path by applying a voltage to the electrode. At this time, since the cell is generally charged negatively, it moves toward the positive electrode. Alternatively, when ultrasonic radiation pressure is used as an external force, cells can be guided to the sound pressure node.

  In the present invention, for example, the pressure for introducing the sample liquid into the chip can be used as the driving force for moving the liquid.

  The cell recognition and separation algorithm has the following characteristics.

  When evaluating cells by capturing them as images, for example, a part of the cell detection area of the flow path can be observed with a CCD camera, and the measurement range can be extended to the surface to identify and track the cells by image recognition. Perform more reliable cell separation. What is important at this time is the image capturing speed. With a typical camera with a video rate of 30 frames / second, cell dropout occurs in the image. With a minimum uptake rate of about 200 frames / second, cells flowing at a significant rate in the flow path can be recognized.

  Next, as an image processing method, the fact that the capture rate is high cannot perform very complicated image processing. First, cell recognition, as described above, varies depending on the moving speed of the cell and the cell, and in some cases, the cell is overtaken. For this reason, when each cell first appears in the image frame, the cell is numbered, and thereafter, management is performed with the same number until it disappears from the image frame. That is, the number is used to manage the situation in which the cell image moves in a plurality of consecutive frames. The cells in each frame move from the upstream side to the downstream side in order, and the cells between the frames are linked under the condition that the moving speed of the numbered specific cells recognized in the image falls within a certain range. Let In this way, each cell can be traced reliably even if the cell is overtaken.

  This makes it possible to recognize cells. For cell numbering, the cell image is first binarized and its center of gravity is obtained. The luminance center of gravity, area, perimeter length, major axis, minor axis of the binarized cells are obtained, and each cell is numbered using these parameters. It is possible to automatically save each cell image as an image at this point because it is beneficial to the user.

  Next, although it is a case where it uses for a cell isolation | separation, it is necessary to isolate | separate only a specific cell among the numbered cells. The separation index may be information such as the luminance center of gravity, area, circumference length, major axis, minor axis, or the like, or information using fluorescence may be obtained by using fluorescence detection separately from the image. In any case, the cells obtained by the detection unit are separated according to the numbering. Specifically, the movement speed (V) of the numbered cells is calculated from the image captured every predetermined time, and the distance from the detection unit to the selection unit with respect to the cell movement speed (V) (L), By setting the application timing from (L / V) to (L / V + T) depending on the application time (T), the cells are electrically distributed and separated when the cells of the target number come between the electrodes.

  Hereinafter, although embodiments of the present invention will be described in more detail with reference to the drawings, these are merely examples, and the scope of the present invention is not limited to these embodiments.

(System configuration of cell analyzer)
FIG. 1 shows an example of a cell sorter chip 100 for separating a blood sample using the cell analyzer of the present invention.

  A blood sample collected from a patient, for example, can be used to selectively lyse red blood cells with drugs, or remove the red blood cell layer collected at the bottom layer by centrifugation and collect only the white blood cell layer remaining on the upper layer. Prepare by method. The sample solution thus prepared is introduced into the cell sorter chip in the direction of arrow 101 as a cell sample.

  Next, in the image detection type 1-cell separation and purification unit, as the primary detection, the presence or absence of fluorescence emission based on the fluorescent label at the 1-cell level is confirmed. This makes it possible to confirm whether the cell is a target cell by a conventional labeling technique using fluorescence. In addition, by analyzing in real time the image collected by the high-speed camera for the target cells that emit fluorescence, (1) the cells that emit fluorescence are isolated cells, or cell clusters with other cells and And (2) discriminating whether the cells emitting fluorescence are in a healthy state or in a state such as apoptosis in which the cell nucleus and cell shape are deformed, and depending on the purpose Thus, based on the analysis of information that can be obtained from images, it is possible to collect healthy isolated cells, a cell population or a cell mass in blood, or cells undergoing apoptosis.

  FIG. 2 shows an example of the optical system of the cell analyzer system 1 that realizes the procedure shown in FIG.

  The image detection type 1-cell separation / purification module includes a light source 201, a mirror 202, a condenser lens 203, a dichroic mirror 204, a filter 205, a light detection element 206 for fluorescence detection, a high-speed camera 207, and a photodiode 208 for scattered light detection. And a cell sorter chip 100 for introducing a cell sample. In the module of FIG. 2, a light source 201 such as a pulse laser or a high-intensity LED light source, a light detection element 208 such as a photodiode that detects the passage of cells with scattered light, and fluorescence are detected with respect to cells passing through the cell sorter chip 209. A plurality of pieces of information can be detected simultaneously by a high-sensitivity light detection element 206 such as a photomultiplier, a high-speed camera 207, or the like. As for the light emitted from the light source, continuous light may be emitted, but in order to increase the spatial resolution of the image without blurring, pulse light is generated in synchronization with the shutter cycle of the high-speed camera 207. Thus, it is possible to acquire an image having a higher term time resolution with shorter light irradiation.

  Here, it goes without saying that processing using an image and processing using fluorescence or scattered light may be used in combination. Further, the image data obtained by the high-speed camera 207 can be displayed on the monitor of the computer 50 for use by the user. In addition, when there are a plurality of fluorescences to be observed, the filter 205 is appropriately adjusted to transmit a plurality of excitation lights, and a wavelength that does not overlap with the fluorescence wavelength for fluorescence detection in the lower stage is selected to light the cells. And a plurality of devices to which devices from the dichroic mirror 204 to the filter 205 and the fluorescence detector 206 are added in accordance with the type of fluorescence to be observed. In addition, by using this configuration, it is possible to use fluorescence observation results for cell images as data.

  The cell recognition and separation algorithm has the following characteristics.

  When evaluating cells as images, more reliable cell separation can be achieved by providing a part where the flow path part after merging is observed with a CCD camera, expanding the measurement range to identify the cells by image recognition, and tracking them I do. What is important at this time is the image capturing speed. With a typical camera with a video rate of 30 frames / second, cell dropout occurs in the image. With an uptake rate of at least 200 frames / second, cells flowing at a significant rate in the flow path can be recognized.

  Next, as an image processing method, the fact that the capture rate is high cannot perform very complicated image processing. First, cell recognition, as described above, varies depending on the moving speed of the cell and the cell, and in some cases, the cell is overtaken. For this reason, when each cell first appears in the image frame, the cell is numbered, and thereafter, management is performed with the same number until it disappears from the image frame. That is, the number is used to manage the situation in which the cell image moves in a plurality of consecutive frames. The cells in each frame move from the upstream side to the downstream side in order, and the cells between the frames are linked under the condition that the moving speed of the numbered specific cells recognized in the image falls within a certain range. Let In this way, each cell can be traced reliably even if the cell is overtaken.

  This makes it possible to recognize cells. For cell numbering, the cell image is first binarized and its center of gravity is obtained. The luminance center of gravity, area, perimeter length, major axis, minor axis of the binarized cells are obtained, and each cell is numbered using these parameters. It is possible to automatically save each cell image as an image at this point because it is beneficial to the user.

  Next, in the case of use for cell separation, among the numbered cells, only specific cells must be separated. The separation index may be information such as the luminance center of gravity, area, circumference length, major axis, minor axis, or the like, or information using fluorescence may be obtained by using fluorescence detection separately from the image. In any case, the cells obtained by the detection unit are separated according to the numbering. Specifically, the movement speed (V) of the numbered cells is calculated from the image captured every predetermined time, and the distance from the detection unit to the selection unit with respect to the cell movement speed (V) (L), By setting the application timing from (L / V) to (L / V + T) depending on the application time (T), the cells are electrically distributed and separated when the cells of the target number come between the electrodes.

  An example of the configuration for separation and purification of cells is as follows. A series of microfabricated channels arranged in a two-dimensional manner on a planar chip, from concentration to arrangement and purification of cells in the sample solution, and force applied to the cells incorporated in the chip Consists of means.

  The cell separation and purification module is configured using a cell sorter chip 100. A microchannel 102 is provided inside the chip substrate, and an opening communicating with the channel is provided on the upper surface to serve as a supply port for a sample and a necessary buffer solution (medium). The flow path can be created by so-called injection molding in which a plastic such as PMMA is poured into a mold, or can be created by bonding a plurality of glass substrates. The size of the chip is, for example, 50 × 70 × 1 mm (t), but is not limited thereto. When PMMA plastic is used so that the cells flowing through the channels and wells in the grooves and through holes carved in the inner surface of the chip can be observed with a high-power optical microscope, for example, a 0.1 mm thick laminate film Is used by thermocompression bonding, and in the case of glass, 0.1 mm glass is similarly optically bonded. For example, using an objective lens having a numerical aperture of 1.4 and a magnification of 100, cells flowing in the flow path through a 0.1 mm laminated film can be observed. In the case of plastic, if the plastic is highly translucent, it can be observed from the upper surface side of the chip substrate 100. In addition, the cells envisaged in the present invention are bacteria such as small ones and animal cells such as large ones, such as cancer cells, and these cell populations or cell masses. Therefore, the cell sample size is typically a wide range of about 0.5 μm to 500 μm, but is not strictly limited to this range, and cells of any size can be used as long as the present invention is effectively used. Can be used. When trying to continuously perform cell concentration and cell separation using a flow channel incorporating one surface of the substrate, the first problem is the flow channel width (cross-sectional shape). In addition, the channel 102 is formed on one of the surfaces of the chip 100 in a substantially two-dimensional plane in a space typically 10 to 500 μm in the thickness direction of the substrate. The appropriate size for bacteria is 5 to 10 μm in the thickness direction, and for animal cells and cell populations / cell clumps is 10 to 500 μm in the thickness direction.

  On the chip 100, first, the sample solution is introduced into the microchannel 102 from the inflow port by a cell introduction means that does not generate a pulsating flow such as a syringe pump or air pressure. The sample solution containing cells introduced into the microchannel flows from upstream to downstream after measuring the cells and determining the type of each cell in the cell detection region arranged in the preceding stage of the cell sorting unit. Depending on whether or not an external force is applied in a direction perpendicular to the cell, it is guided to one of the first outflow port 105 and the second outflow port 106 that are two flow regions branched at the cell sorting unit branching portion.

  FIG. 3 shows an example of an index for discriminating between healthy cell samples 301 and 302 that flow as isolated cells in the blood and cell samples 303 and 304 that flow in the blood as a cell population or cell mass such as cancer cells. In blood, healthy cells exist in the form of isolated one cells, and the two-dimensional shape obtained by acquiring an image of the shape is circular or elliptical. On the other hand, it is natural that a cell sample that is a cell population or a cell mass such as a cancer cell has a size larger than that of a single isolated cell. Since the cells are connected, the shape is not circular or elliptical. Therefore, by quantitatively measuring the size of these cell samples from the acquired image of the cell samples and how far their shape deviates from a circle or ellipse. Whether a cell sample is a cell population or a cell mass can be determined.

  The present invention is useful for separating and purifying a very small amount of target cells in blood with an indicator of whether the cells are clustered, and for performing accurate gene information, expression information analysis, re-culture, etc. of the target cells. is there.

  In addition, when a cell population or a cell mass is present in blood, it is highly likely that the cell is a cancer cell. Therefore, the apparatus system and method of the present invention can be used for diagnosis of cancer, formulation of a drug administration plan for a cancer patient, etc. Useful for.

100 Cell sorter chip 101 Cell sample introduction path 102 Channel 103 External force generator 104 External force direction 105 Flow of recovered cells (1)
106 Flow of recovered cells (2)
201 Laser 202 Mirror 203 Condensing lens 204 Dichroic mirror 205 Filter 206 Photomultiplier 207 for fluorescence detection High-speed camera 208 Photodiode 301, 302 for detecting forward scattered light Normal isolated cell 303, 304 Abnormal blood cell population, cell mass

Claims (12)

A cell analyzer system for selectively separating and purifying cancer cells in blood,
A first flow path for flowing a sample liquid containing cells in blood derived from a subject, and a cell detection region on the first flow path for detecting cells in the sample liquid flowing through the first flow path A cell separation region disposed downstream of the cell detection region on the first flow channel, and a selective recovery of cells branched from the first flow channel downstream of the cell separation region. A cell sorter chip comprising at least two branch channels;
A light irradiation means for irradiating the cells flowing through the cell detection region with light;
An optical system including a high-speed camera for acquiring image information to determine whether the cells are in an isolated cell state, a cell population state or a cell mass state;
A control / analysis unit that detects each cell at a single cell level based on the image information and identifies whether the cell is an isolated single cell, or a cell population or a cell mass,
Based on the result of the identification, in the cell separation region, an external force capable of controlling which of the at least two branch channels to guide and flow the cell is selectively applied to the cell. Means for applying;
With
A cell analyzer system in which the presence of the cell population or cell mass identified by the control / analysis unit suggests the presence of cancer cells in the blood.   The apparatus system according to claim 1, wherein the external force is ultrasonic radiation pressure, gravity, electrostatic force, or dielectric electrophoresis force.   The control / analysis unit binarizes the cell image obtained from the optical system, and at least one selected from the group consisting of the luminance center of gravity, area, perimeter length, major axis, and minor axis of the binarized image. The apparatus system according to claim 1, wherein each cell is detected at a single cell level by an index, and is identified as an isolated single cell, a cell population, or a cell mass.   The cells in the cell sample solution are fluorescently labeled, the optical system further includes fluorescence detection means, and information on the fluorescence image of the cells is used as an additional index by the control / analysis unit, The apparatus system according to claim 3. The light irradiation means irradiates excitation light having a specific narrow-band wavelength width that matches the excitation light wavelength of a fluorescent dye having a wavelength of 350 nm to 560 nm,
The fluorescence detection means quantitatively measures the fluorescence intensity of the fluorescent dye emitted by the excitation light in a wavelength band where the excitation light does not overlap,
The control / analysis unit detects and confirms the presence or absence of fluorescent labeling of the cells at a single cell level in the target cell population or cell mass, and determines the fluorescently labeled cell population or the cell mass. The apparatus system according to claim 4, wherein:   The apparatus system according to any one of claims 1 to 5, wherein the high-speed camera is capable of acquiring image information of the cells at an image capturing rate of at least 200 frames / second. A method for quantitative analysis of cancer cells in blood,
Preparing a sample solution containing cells in blood from a subject;
The sample solution, and subjecting the cell separation using cell sorter chip,
(However, the cell sorter chip is
A first flow path for flowing the sample solution;
A cell detection region on the first flow path for detecting cells in the sample liquid flowing through the first flow path;
A cell separation region disposed downstream of the cell detection region on the first channel, and at least a cell branching from the first channel downstream of the cell separation region for performing selective recovery of cells. With two branch channels )
An optical system including a light irradiating means and a high-speed camera irradiates the cells flowing through the cell detection region with light, and obtains an image of the cells. Determining whether it is a population state or a cell mass state;
Selectively applying an external force to the cells in the cell separation region based on the result of the discrimination, inducing the cells to flow through one of the at least two branch channels,
With
A cell analysis method, wherein the presence of the cell population or cell mass determined in the determining step indicates the presence of cancer cells in the blood.   The method according to claim 7, wherein the external force is ultrasonic radiation pressure, gravity, electrostatic force, or dielectric electrophoretic force.   The cell image obtained from the optical system is binarized, and each of the cells is represented by at least one index selected from the group consisting of the luminance centroid, area, perimeter, major axis, and minor axis of the binarized image. The method according to claim 7 or 8, wherein the cell is detected at a single cell level and is discriminated as an isolated single cell, or a cell population or a cell mass.   10. The cell according to claim 9, wherein the cell in the cell sample solution is fluorescently labeled, the optical system further includes a fluorescence detection unit, and information on the fluorescence image of the cell is used as an additional indicator. Method. The light irradiation means irradiates excitation light having a specific narrow-band wavelength width that matches the excitation light wavelength of a fluorescent dye having a wavelength of 350 nm to 560 nm,
The fluorescence detection means quantitatively measures the fluorescence intensity of the fluorescent dye emitted by the excitation light in a wavelength band where the excitation light does not overlap,
The method according to claim 10, wherein in the target cell population or cell mass, the presence or absence of fluorescent labeling of the cells is detected and confirmed at a single cell level, and the fluorescently labeled cell population or cell mass is determined. .   The method according to claim 7, wherein the high-speed camera is capable of acquiring image information of the cells at an image capture rate of at least 200 frames / second.