WO2015053393A1

WO2015053393A1 - Imaging cell sorter

Info

Publication number: WO2015053393A1
Application number: PCT/JP2014/077198
Authority: WO
Inventors: 安田　賢二; 賢徹金; 服部　明弘; 英之寺薗
Original assignee: 公益財団法人神奈川科学技術アカデミー; 一般社団法人オンチップ・セロミクス・コンソーシアム
Priority date: 2013-10-10
Filing date: 2014-10-10
Publication date: 2015-04-16
Also published as: JPWO2015053393A1

Abstract

The present invention provides a cell concentration and purification device having: a function to continuously concentrate cells; a function to subsequently and continuously arrange the cells on a specific region on a path; a function to simultaneously recognize the shape and the emission of fluorescence of the cells one cell at a time based on an image; and a function to separate and purify the cells by recognizing the cells on the basis of the information of the shape and the emission of fluorescence of the cells.

Description

Imaging cell sorter

The present invention relates to a cell recovery apparatus.

In living tissues in multicellular organisms, various cells share the role and maintain a harmonious function as a whole. 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.

Also, in the field of regenerative medicine, attempts have been made to regenerate target tissues, and thus organs, by isolating organ stem cells from tissues and reculturing them 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.

Such separation and recovery of specific cells in the culture medium 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.

For example, there 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 (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 not only difficult to recover the concentrated solution without loss, but it is also desirable for regenerative medicine where cells are contaminated in a complicated pretreatment stage. The problem was that no problems occurred.

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 that can easily analyze and separate a cell sample (Patent Document 1: JP 2003-107099; Patent Document 2: JP 2004-85323; Patent Document 3: WO 2004 / 101731). These are well-practical cell sorters at the laboratory level, but for general use for regenerative medicine, new technological development is required for pretreatment such as liquid transport, recovery, and sample preparation. is there.

Currently, the detection of cancerous tissue has improved dramatically by improving MRI (nuclear magnetic resonance imaging) and CT (computed tomography), but for the identification of benign and malignant tumors, the method exceeds the 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 the peripheral blood are called peripheral blood circulating cancer cells (Circulating Tumor Cells: CTCs), and it is considered that there are several hundred 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.

For gene diagnosis and expression analysis, the polymerase chain reaction (hereinafter abbreviated as PCR) is a method for amplifying a specific base sequence from a mixture of various types of nucleic acids. In PCR, DNA templates such as complementary DNA reverse transcribed from genomic DNA or messenger RNA in a mixture of various types of nucleic acids, two or more primers, a thermostable enzyme, a salt such as magnesium, and four types After adding deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP), the step of separating the nucleic acid into single strands, the step of binding the primer to the separated nucleic acid, and the primer bound by a thermostable enzyme A specific nucleic acid sequence can be amplified by repeating the hybridization step using a nucleic acid as a template at least once. In PCR, a thermal cycle is used by raising and lowering the temperature of a reaction vessel used for a DNA amplification reaction. There are various temperature change mechanisms used there, for example, a mechanism for changing the temperature of the reaction vessel containing the sample by a heater or Peltier element, heat exchange using hot air, and a heater block with different temperatures for the reaction vessel. Alternatively, there are a mechanism for changing the temperature by alternately contacting the liquid bath, and a mechanism for changing the temperature by flowing a sample in a flow path having regions of different temperatures. For example, a light cycler (Light Cycler) manufactured by Roche Corporation is one of the fastest commercially available devices. The light cycler introduces a sample, a DNA polymerase, a DNA piece serving as a primer, and a fluorescent labeling dye for measurement into each of a plurality of glass capillary tubes, and the temperature of a minute droplet in the capillary tube is set to 55 degrees and 95 degrees, for example. These two temperatures are changed by blowing warm air at the same temperature as the droplet to be changed, and at the same time, this glass capillary tube is irradiated with excitation light of a fluorescent dye, and the obtained fluorescence intensity is measured. It has a mechanism that makes it possible.

It is possible to change the temperature of the sample repeatedly by these methods.

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

In clinical practice, despite the importance of confirming the presence of CTCs in relation to cancer metastasis, diagnostic criteria using this as an index for cancer metastasis have not been established. The reason for this is that due to the diversity and rarity of the sample itself, the conventional method of examining the presence or absence of rare mutant genes in the sample after taking the collected blood as a uniform tissue has a very high detection sensitivity. The point required is mentioned.

In particular, until now, we have analyzed the intracellular genes and expressions without confirming whether the cancer cells in the blood that have been fluorescently labeled are in a cell mass with other cells or are isolated cells. We obtained information as a population average including information on cells other than cancer cells. Therefore, there has been a problem that accurate information on the target cancer cells cannot be obtained.

Moreover, in addition to the means for collecting in units of one cell, it was necessary to perform diagnosis with a higher S / N ratio by means of genetic diagnosis and expression analysis in a small amount of cells after sorting and concentrating rare cells .
Further, the current analysis means using cells has a problem that it does not have a means for analyzing whether the target cell is in a state of apoptosis or the like at the time of cell recovery.

In addition, for cells having a hard shell on the surface of cells such as spores of Bacillus anthracis, in order to analyze the cell contents, the contents of the cell must be used unless the cell shell can be removed by any method. Had a problem of not eluting into the sample solution. For this reason, spore cells are usually germinated by culturing, and by sprouting, the contents of the cells can be eluted into the sample solution even in the same procedure as normal cells. In this case, cell analysis is performed, but in this case, cell culture requires at least several hours, and if it is long, one day and night culture is required. Problems such as complications and contamination have occurred. In addition, as a technique for high-speed analysis, there is a destruction technique such as crushing cells by putting a mixture of a crushing medium such as a glass ball and a sample into a crushing container and randomly colliding them by applying ultrasonic vibration etc. However, there is a problem in that effective crushing efficiency cannot be obtained despite the occurrence of heating of the sample solution due to vibration, and a large amount of sample solution and sample are required. When cells are disrupted, there is a problem of sample recovery efficiency.

When the size of the sample flowing through the microchannel is large, it is necessary to increase the height of the channel. However, when the sample is observed with an image, there is a problem that the image is blurred.

Also, when the sample flows horizontally for a long time in the horizontally arranged flow path, there is a risk that the sample gradually settles and eventually blocks the flow path.

When purifying and collecting fine particles such as cells, when applying external force to the cells by repulsion, it was difficult to collect the cells in one place in the flow path.

When applying an external electric field at a single pinpoint, there was a risk that sufficient external force could not be applied to the sample and cells could not be moved sufficiently.

The specific numerical index for purifying cardiomyocytes by image recognition was unclear in terms of automation.

The narrowing of the sample aqueous solution using the side sheath liquid has a risk of causing dilution of the sample aqueous solution.

When collecting the sample fine particles in the aqueous solution, if the ionic strength of the electrolyte of the sample solution exceeds a certain level, it may be difficult to move by the electric field.

On the other hand, as a method for confirming cancer cells, there is an identification technology (Japanese Patent Laid-Open No. 2011-257241) based on image recognition of cell clusters that has already been filed by the present inventors. The multinucleation phenomenon could not be confirmed by the above technique. In addition, the above technique cannot be evaluated in combination with a staining method using an existing cancer cell marker. As for means for simultaneously acquiring microscopic images of a plurality of wavelengths, the present inventors have devised an absorption imaging spectroscopy technique as an absorption microscope, and have applied for them as JP2012-0555267 and WO2012 / 060163. These techniques did not include a plurality of fluorescence intensity measurement techniques.

In JP2011-257241-A, as a means for detecting the presence or absence of cancer cells in blood, fluorescent dyes are used for antibodies that selectively bind to molecules (cancer markers) present only on the surface of cancer cells. A technique for detecting a cancer cell by causing a cancer cell to emit fluorescence when a cancer cell is present in the blood by reacting with a blood cell is shown. As the means and device configuration, after irradiating the blood containing cancer cells flowing through the microchip with fluorescence excitation light, the fluorescence emitted from the cancer cells is reflected only by light of a specific wavelength and other wavelengths are reflected. The light passes through a mirror (dichroic mirror) that passes through multiple times, extracts fluorescence with a specific wavelength width at each step, and measures the amount of fluorescence with a photodetector for each fluorescence of that wavelength width. However, since the amount of fluorescence from cancer cells that should be detected when passing through the dichroic mirror also attenuates according to the number of passes through the dichroic mirror, the fluorescence intensity measured at each wavelength width is caused by the passage through the dichroic mirror. In particular, it was difficult to detect faint fluorescence at a later stage wavelength, and it was difficult to quantitatively detect multiple stained cancer marker molecules. In addition, in order to irradiate cancer cells with multiple excitation lights with different wavelengths in order to simultaneously use many fluorescent dyes, and to simultaneously detect emitted multiple wavelengths of fluorescence, the fluorescence wavelength and the excitation wavelength overlap. Since the light must be decomposed for each wavelength by passing through a plurality of dichroic mirrors, the device configuration becomes complicated, and each wavelength band needs to be narrowed, not only the signal fluorescence is further attenuated, but also the wavelength In principle, it was difficult to separate light with close bands.

In view of such a situation, the present inventors provide a cell analyzer capable of rapidly identifying the type and state of cancer cells flowing in the blood with metastatic ability and the number (concentration).
That is, the present invention provides the following apparatuses, systems, and methods.

(1) (A) a first device for concentrating, staining, and washing a cell sample solution from a subject;
(B) a second device for concentrating, separating and purifying the stained cell sample solution from the first device;
(C) a third device for performing gene analysis / expression analysis of purified cells in the cell sample solution from the second device;
(D) a fourth device for continuously feeding the cell sample solution over the first to third devices;
(E) A cell analyzer system comprising a control / analyzer that controls the operation of each of the devices and analyzes the cell sample,
(a) the first device is
A chamber with a filter for concentrating, staining, and washing cells in the cell sample solution;
Containers for storing the cell sample solution, staining solution and washing solution, respectively;
A mechanism for sequentially introducing each liquid in each container into the chamber;
With
(b) the second device is
A cell sorter chip having a flow path for flowing a cell sample solution containing cells including target cells, wherein the flow path includes a first flow path in which the cells are concentrated, and detection of the concentrated cells. A cell sorter chip including a second flow path branched from the first flow path, wherein the target cells are selected, and
An external force is applied to the cells flowing through the channels so that the cells flowing through the channels are concentrated in the first channel and converged in a desired direction in the second channel. A mechanism to give
An optical system including light irradiation means for irradiating the cells flowing in the second flow path, and a high-speed camera for acquiring an image of the cells at an image capture rate of at least 200 frames / second;
With
(c) the third device is
A reaction vessel to which a sample solution is added and reacted;
A heat exchange tank for exchanging heat with the reaction tank;
A temperature control mechanism for controlling the temperature of the heat exchange tank;
A cell analyzer system comprising:
(2) Contents of the cells conveyed by the fourth device for feeding the cell sample solution before the third device for performing gene analysis / expression analysis of purified cells in the cell sample solution A cell disruption mechanism that elutes the sample solution by cell disruption,
The control / analyzer transports the cell sample solution from the second device to the cell disruption mechanism by the fourth device for feeding the cell sample solution, and the sample solution is disrupted by the cell disruption mechanism. However, the cell analyzer system according to (1), wherein each unit is controlled so that the fourth apparatus is transported to the third apparatus by the fourth apparatus.
(3) The cell disruption mechanism is
A container for containing the cell sample;
A crushing rotating body for crushing cells in the container,
An abrasive for crushing cells in the container,
The cell sample and the abrasive are added to the inside of the container, and the cell sample is crushed by the movement of the crushing rotating body in which rotation and revolution are strictly controlled. Cell analyzer system.
(4) The cell disruption mechanism further includes a rotating shaft,
When the crushing rotating body is pressed from above by the rotating shaft, the crushing rotating body rotates inside the container, and the frictional force and slippage between the crushing rotating body and the rotating shaft are different from those of the crushing rotating body. The cell analyzer system according to (3), which is controlled by the pressure between the rotating shafts.
(5) The cell crushing mechanism can generate a force for pressing the crushing rotating body in a direction perpendicular to the side surface of the container by shifting the rotating shaft of the crushing rotating body and the rotating shaft of the rotating shaft. The cell analyzer system according to (4) above, comprising a mechanism.
(6) The cell crushing mechanism is a mechanism that allows the crushing rotating body to be lifted from the inside of the container and removed by the magnetic force of the rotating shaft, electrostatic force, or suction force due to gas pressure difference. The cell analyzer system according to (4), comprising:
(7) In the cell disruption mechanism described above, contamination between different cell samples can be eliminated by providing a drive mechanism equipped with a plurality of containers capable of automatic replacement of the containers. The cell analyzer system according to any one of (6).
(8) In the cell crushing mechanism, the crushing rotator is sealed and sealed inside the unused container by a confidential seal, and the container and the crushing rotator are placed at the time of crushing the cell sample. The cell analyzer system according to any one of the above (3) to (7), characterized in that it can be guaranteed that it is not contaminated.
(9) (i) A cell sorter chip including a flow path for flowing a cell sample solution containing cells including target cells, wherein the flow path includes a first flow path in which the cells are concentrated, and the concentration A cell sorter chip including a second flow path branched from the first flow path, wherein the detection of the cells and the selection of the target cells are performed,
(ii) The cells flowing through each of the channels are concentrated in the first channel and converged in a desired direction in the second channel. A mechanism for applying external force to the
(iii) an optical system including a light irradiation means for irradiating the cells flowing in the second flow path, and a high-speed camera for acquiring an image of the cells at an image capture rate of at least 200 frames / second;
(iv) a control / analysis unit that controls the operation of each unit and analyzes an image of each cell captured by the optical system;
An image detection type single cell separation / purification apparatus comprising:
(10) The apparatus according to (9), wherein the external force is ultrasonic radiation pressure, gravity, electrostatic force, or dielectric electrophoresis force.
(11) The device according to (9) or (10), wherein the cell sample containing the target cell is derived from blood.
(12) The device according to any one of (9) to (11), wherein the target cell includes a cancer cell.
(13) The control / analysis unit binarizes the cell image obtained from the optical system, and is selected from the group consisting of the luminance centroid, area, perimeter, major axis, and minor axis of the binarized image. The device according to any one of (9) to (12) above, wherein each of the cells is detected and identified at a single cell level by at least one index.
(14) The cells in the cell sample solution are fluorescently labeled, the optical system further includes fluorescence detection means, and information on the fluorescence image of the cells is used as an additional index by the control / analysis unit. The apparatus according to (13) above.

Furthermore, the present invention provides the following on-chip cell sorter and on-chip cell sorter system.
(15) One sample channel having the same length and cross-sectional area and two buffer liquid channels disposed symmetrically on both sides of the sample channel are arranged to merge, and after the merge, the center of the same length and cross-sectional area is again downstream. The ratio of the cross-sectional area of the sheath liquid reservoir that is distributed to the recovery flow path and the two waste liquid flow paths on both sides and covers the inlets of the three upstream flow paths and the sample liquid reservoir that fills the sample therein However, it is arranged so that the ratio of the number of channels is 2: 1, so that even if the liquid flows, the liquid level of both is the same. Is arranged so that the ratio of the cross-sectional area of the reservoir for the reservoir is 2: 1, and has a mechanism for identifying cells by a high-speed camera and fluorescence detection upstream of the junction, so that the gel electrode is in contact with the junction symmetrically The electric field is only applied to the cells that are placed in the I applied it was characterized by the on-chip cell sorter.
(16) In the reservoir of the cell sorter, a stopper disposed on the top surface of the sheath liquid reservoir, means for applying compressed air through the stopper, means for continuously supplying liquid to the sheath liquid reservoir and the sample reservoir, The cell sorter according to (15) above, further comprising an electric sensor capable of measuring the liquid level height in both the sheath liquid reservoir and the reagent reservoir.
(17) In the reservoir of the cell sorter, the individual containers for storing the reagent liquid and the two sheath liquids are respectively arranged at the upstream inlets of the three flow paths, respectively, (18) An on-chip cell sorter that selectively collects cells undergoing cell division based on the presence or absence of an image of a nucleus in a cell image.
(19) In the image processing type cell sorter, in order to prevent blurring of the image, the time to ignite once in each frame at the shooting frame rate speed of the high-speed camera,

Flash time = Pixel size / flow velocity
An on-chip cell sorter system characterized by deciding on the relationship of
(20) An on-chip cell sorter system characterized by using an optical system that can maintain the depth of field and the depth of field up to the height of the microchannel by combining an objective lens having a numerical aperture of 0.3 or less and a zoom lens. .
(21) An on-chip cell sorter system characterized in that the sample liquid is arranged so as to flow vertically from above to below.
(22) An on-chip cell sorter system characterized in that the inner wall surface of the microchannel facing the surface on which the electrode for generating repulsive force is arranged on the sample fine particles has a convex shape.
(23) A pair of gel electrodes having a structure in which struts are repeatedly arranged at a certain distance that can prevent the leakage of the liquid into the flow path due to the surface tension of the liquid when the gel is in a sol state. An on-chip cell sorter system characterized by being arranged.
(24) From the shape of the cells obtained by image recognition,

, An on-chip cell sorter system characterized by purifying cells with R less than 1.1 as cardiomyocytes.
(25) An on-chip cell sorter system characterized in that oil having a specific gravity lighter than water and not mixed with water is used for the side sheath liquid.
(26) An on-chip cell sorter system characterized by using a solution in which the conductivity of the sample aqueous solution is 10 ² μS / cm or less when collecting the sample fine particles in the aqueous solution.

[1] A cell sorter chip including a first flow path for flowing a sample liquid containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells. A cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image When,
Based on the result of cell identification by the image analysis, a target cell flowing through the first flow path or a cell other than the target cell in a second region substantially coincident with the first region upstream from the branch point An external force is applied to the cell to shift the traveling direction of the cell by applying an external force to the target cell recovery channel, and an external force applying mechanism for guiding cells other than the target cell to the waste liquid recovery channel;
An on-chip cell sorter system comprising: a control unit that controls operations of the optical system and the external force applying mechanism.
[2] In the above [1], the time lag between the timing of obtaining the cell identification result from the digital image by the optical system and the timing of applying the external force by the external force applying mechanism is minimized. The on-chip cell sorter system described.
[3] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells. A cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image When,
A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point. Alternatively, an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel. An external force application mechanism for
A control unit for controlling the operation of the optical system and the external force application mechanism,
An on-chip cell sorter system, wherein the optical system includes a microscope having an objective lens having a numerical aperture of 0.3 or less and a zoom lens optically coupled to the objective lens.
[4] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells. A cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image When,
A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point. Alternatively, an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel. An external force application mechanism;
A control unit for controlling the operation of the optical system and the external force application mechanism,
The cell sorter chip is arranged so that the first flow path is substantially parallel to the direction of gravity so that the sample liquid flows substantially vertically downward from the upstream side to the downstream side of the first flow path. An on-chip cell sorter system.
[5] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells. A cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image When,
A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point. Alternatively, an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel. An external force application mechanism;
A control unit for controlling the operation of the optical system and the external force application mechanism,
The external force applying mechanism includes a gel electrode or a metal electrode for applying an electric force to fine particles including cells flowing through the first flow path, and the conductivity of the sample solution is 10 ² μS / cm or less. Chip cell sorter system.
[6] An external force for aligning cells in the sample solution flowing in the first channel in the third region upstream of the first region upstream of the first channel is applied to the cells. The on-chip cell sorter system according to any one of the above [1] to [5], further comprising a further external force applying mechanism applied to the.
[7] The ON according to [6], wherein the further external force applying mechanism that applies an external force for aligning the cells in the sample solution to the cells applies an external force by an electric force or a sheath flow. Chip cell sorter system.
[8] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells. A cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
An optical system for acquiring a digital image of a cell in the sample solution flowing in the first flow path in a first region upstream from the branch point and identifying the target cell by digital analysis of the image When,
A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point. Alternatively, an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel. An external force application mechanism;
A control unit for controlling the operation of the optical system and the external force application mechanism,
further,
A reservoir fluidly connected to the upstream side of the first flow path and storing a buffer solution for sheath liquid;
A sample solution introduction channel fluidly connected to the upstream side of the channel for introducing a sample solution containing cells into the first channel,
An on-chip-chip, wherein a tip end portion of the sample solution introduction channel on the side fluidly connected to the first channel extends to a downstream side of the introduction portion of the buffer solution to the first channel. Cell sorter system.
[9] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells. A cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
A first external force applying mechanism for applying to the cells an external force for aligning the cells in the sample solution flowing through the first flow path in a preliminary region upstream from the branch point;
A digital image of the cells in the sample solution flowing in the first flow path in a first region upstream of the branch point and downstream of the preliminary region is obtained, and the digital analysis of the image An optical system for identifying a target cell;
A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point. Alternatively, an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel. A second external force application mechanism;
A controller that controls the operation of the optical system and the first and second external force application mechanisms,
The first external force applying mechanism is a comb-shaped electrode for generating repulsive force on microparticles containing cells in a sample solution arranged along one surface of the first flow path, and the first flow path An on-chip cell sorter system in which the cross section perpendicular to the flow path is tapered or convex toward the center of the surface opposite to the surface on which the electrodes are arranged in order to promote alignment of the fine particles .
[10] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells. A cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
A first external force applying mechanism for applying to the cells an external force for aligning the cells in the sample solution flowing through the first flow path in a preliminary region upstream from the branch point;
A digital image of the cells in the sample solution flowing in the first flow path in a first region upstream of the branch point and downstream of the preliminary region is obtained, and the digital analysis of the image An optical system for identifying a target cell;
A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point. Alternatively, an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel. A second external force application mechanism;
A controller that controls the operation of the optical system and the first and second external force application mechanisms,
The second external force applying mechanism is a gel electrode disposed so as to come into contact with the sample liquid on both side surfaces of the first flow path, and when the gel is in a sol state, the sol liquid is In order not to leak into the flow path, the sol liquid is provided on both side surfaces along the first flow path at regular intervals so that the surface tension of the sol liquid can prevent the sol liquid from leaking into the flow path. An on-chip cell sorter system configured to contact the sample solution through an array of slits.
[11] A cell sorter chip including a first flow path for flowing a sample solution containing cells including target cells, wherein the first flow path is a downstream branch point and includes the target cells. A cell sorter chip branching into a target cell recovery flow path for recovering the liquid and a waste liquid recovery flow path for recovering a liquid containing cells other than the target cells;
A first external force applying mechanism for applying to the cells an external force for aligning the cells in the sample solution flowing through the first flow path in a preliminary region upstream from the branch point;
A digital image of the cells in the sample solution flowing in the first flow path in a first region upstream of the branch point and downstream of the preliminary region is obtained, and the digital analysis of the image An optical system for identifying a target cell;
A target cell that flows in the first flow path based on the result of cell identification by the image analysis in a second region that substantially matches or is downstream of the first region upstream from the branch point. Alternatively, an external force is applied to a cell other than the target cell to shift the traveling direction of the cell, and the target cell is guided to the target cell recovery channel and a cell other than the target cell is guided to the waste liquid recovery channel. A second external force application mechanism;
A controller that controls the operation of the optical system and the first and second external force application mechanisms,
The first external force application mechanism includes a pair of flow paths for flowing a side sheath liquid for generating a side sheath flow that is fluidly connected to the upstream side of the first flow path. An on-chip cell sorter system that is light in specific gravity and does not mix with water.
[12] The time lag between the timing of obtaining the cell identification result from the digital image by the optical system and the timing of applying the external force by the second external force applying mechanism is minimized. [9] The on-chip cell sorter system according to any one of [11] to [11].
[13] In any one of the above [1] to [12], the external force applying mechanism that guides the cell to each recovery flow path includes a gel electrode or a metal electrode for applying an electric force to the cell. The on-chip cell sorter system described.
[14] The target cell is a cardiomyocyte,
From the shape of the cells obtained by image recognition with the above optical system,

The on-chip cell sorter system according to any one of [1] to [13] above, wherein a cell having R of less than 1.1 is identified as a cardiomyocyte by
[15] A method for selecting target cells in a sample solution using the on-chip cell sorter system according to any one of [1] to [14].

Furthermore, the present invention provides the following on-chip cell sorter system, a method for identifying candidate cells of blood cancer cells from a cell sample solution derived from a subject using the system, and an optical module.
<1> a cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject;
An optical system including a bright-field light source and a fluorescent light source for irradiating the cells;
A detection system for simultaneously acquiring a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells;
Control / analyzing means for identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image;
An on-chip cell sorter system comprising: means for selectively recovering the identified multinucleated cells and / or cell clusters.
<2> The control / analysis means is
i) one or more data selected from the group consisting of the size (area) of the cell, the perimeter, and the value of R indicating the degree of unevenness of the surface of the cell obtained by the area and the perimeter, and ii) 1 selected from the group consisting of the fluorescence wavelength and intensity spectrum of the labeling substance bound to the cell, and the barycentric coordinates and area of each of one or more regions fluorescently stained in the cell. The on-chip cell sorter system according to the above <1>, wherein two or more data are acquired, and multinucleated cells and / or cell clusters flowing in the flow path are identified based on the data.
<3> The on-chip cell sorter system according to the above <1> or <2>, further comprising means for measuring a nucleic acid sequence of a gene derived from the selectively collected multinucleated cells and / or cell clusters.
<4> Any one of the above <1> to <3>, comprising an image dividing mechanism having a function of dividing and displaying the bright field image and the fluorescent image on the light receiving surface of one high-speed camera at the same time. The on-chip cell sorter system according to item.
<5> The on-chip cell sorter system according to <4>, further including a mechanism that adjusts the magnification of the bright field image and the fluorescent image so that the magnification of the image differs.
<6> The on-chip cell sorter system according to any one of the above <1> to <5>, which is used for identifying candidate cells of blood cancer cells.
<7> A method for identifying candidate cancer cells in blood from a cell sample solution derived from a subject using the on-chip cell sorter system according to any one of <1> to <6> above. ,
The following steps:
(1) A step of identifying and selectively recovering cell clusters (clumps) that do not exist in healthy blood as blood cancer cell candidates,
(2) a step of identifying and selectively recovering multinucleated cells that do not exist in healthy blood as blood cancer cell candidates;
(3) a step of identifying and selectively collecting giant cells that do not exist in healthy blood as blood cancer cell candidates, and / or (4) in (1), (2), or (3) above In addition, the method comprising the step of identifying and selectively recovering from cancer cells by analysis combined with the presence of fluorescence intensity of a fluorescent antibody against one or more biomarkers of cancer cells.
<8> The method according to <7>, wherein the fluorescent antibody is an EpCam antibody, a K-ras antibody, or a cytokeratin antibody.
<9> Further below:
As for (1) above, evaluation by R> 1.3 using a bright field image, or the number and distribution of nuclei by the cell size and the fluorescence image using a bright field image (that is, the distance between the centers of gravity of adjacent nuclear images Are separated from each other by 3 μm or more),
For (2) above, R <1.3 is determined by the bright field image, and the number and distribution of nuclei (that is, the distance between the centroids of the images of adjacent nuclei is within 3 μm from each other) To do,
As for (3) above, it is determined by R <1.3 by the bright field image and the cell size is converted to a diameter exceeding 20 μm, or a combination of the above (1) to (3). The method according to the above <7> or <8>, which comprises determining those having the above matching conditions as cancer cells.
<10> a first dichroic mirror with an angle adjustment function capable of adjusting the direction in which light is reflected in three dimensions;
A filter system in which light including image data reflected by the dichroic mirror is introduced;
An image size adjustment system comprising a movable shielding plate for adjusting the image size, into which light is introduced through the filter system,
Light introduced through the image size adjustment system is introduced, a second dichroic mirror with an angle adjustment function capable of three-dimensionally adjusting the direction in which the light is reflected, and light introduced through the second dichroic mirror An optical module for use in an optical bright field / fluorescence microscope system comprising an optical lens system for correcting differences in imaging position,
An optical module configured such that the image can be enlarged and reduced by the optical lens system, and an image having a different magnification rate can be generated between a bright-field image and a fluorescent image.
<11> A bright field image of the cells in a sample solution containing fluorescently stained cells, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells, which are used to simultaneously acquire 10>. The optical module according to 10>.

Furthermore, the present invention provides the following on-chip cell sorter system.
<1> a cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject;
An optical system including a bright-field light source for irradiating the cells, one or more fluorescent light sources, an optical fiber that conducts light of each wavelength, and a condensing lens that focuses the light on the observation target at the irradiation site; ,
An optical fiber corresponding to each of one or more fluorescence wavelengths that conducts fluorescence for detecting the fluorescence intensity of the cells in the sample solution flowing through the flow path of the cell sorter chip, and a specific disposed at the subsequent stage of the optical fiber A detection system that simultaneously acquires the fluorescence intensity of the fluorescent labeling substance bound to the cells, comprising a bandpass filter and a fluorescence detector that pass the fluorescence wavelength of
A detection system for simultaneously acquiring a bright field image of the cell and a fluorescence image of the cell;
Control / analyzing means for controlling the operation of each system and identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image;
An on-chip cell sorter system comprising means for selectively collecting the identified object.

Furthermore, the present invention provides the following on-chip cell sorter system.
(1) The area of nuclei of 150 μm ² or more of cells (cluster) is measured from the acquired image (2) The area of 250 μm ² or more of cells (cluster) is measured from the acquired image (3) of cells (cluster) Any one of the presence of three or more nuclei measured from the acquired image, or a combination of the above three conditions in AND, that is, (1) and (2), or (1) and (3), Alternatively, an on-chip cell sorter system characterized by using (2) and (3) or (1) and (2) and (3) as criteria for determining the presence of blood cancer cells.

Furthermore, the present invention provides the following on-chip cell sorter system.
When the ratio R of the perimeter derived from the perimeter of the cell (cluster) and the area as a circular approximation (1 / R) is 0.9 or more, the cell (cluster) is an isolated single cell, (1 / R ) Is less than 0.9, the on-chip cell sorter system is characterized in that a cell (cluster) is composed of a cluster of two or more cells as a criterion for determining the number of cells in the cell cluster.

In order to acquire microscopic images in multiple different wavelength bands simultaneously with one high-speed camera light receiving surface,
In the optical branching module of one unit, which is the minimum structural unit, a pair of optical path systems for input / output of image light on the both bottom surfaces of the container and two pairs on the side surface for branching a two-dimensionally developed optical path system Four optical path holes for introducing mirror-reflected light, and six optical path covers that can be freely attached to and detached from each other are arranged, and light is introduced into the holes of the optical path for introducing two-to-four mirror-reflected light on these side surfaces. In this way, two mirror holders with detachable movable adjustment function are arranged, and each of these mirrors can finely adjust the traveling direction of the reflected light, and this makes it possible to connect with the camera. The image position can be freely moved, and the mirror holder can be equipped with a total reflection mirror, high-pass filter, low-pass filter, etc. Two removable adjustable adjustments A removable optical path window is arranged between the mirror holders with a function, the sectional area of the transmitted light can be adjusted, and a removable filter 4304 is arranged. The width can be adjusted more precisely,
In order to connect the above units and observe images of a plurality of wavelengths in combination, incident light introduced so as to have an area equal to or smaller than (total area of light receiving surface of image acquisition camera system to be measured / parallel light introducing module) A parallel light introducing module for introducing an image optical image in which an optical window filter that cuts out a cross-sectional area of
In the first-stage optical branching module connected to the parallel light introducing module, the (wavelength) high-pass filter or the (wavelength) low-pass filter b is introduced into the mirror holder,
The transmitted light of the incident light branched into two wavelengths is reflected by the total reflection mirror, and is the same as the first filter of the optical branching module of the second stage (first stage). On the other hand, the light reflected by the filter of the first-stage optical branching module is introduced into the first filter of the second-stage optical branching module, and similarly up to the n-th optical branching module. Similarly, wavelength band splitting is performed, and for each branch wavelength, positioning to adjust the direction of the optical path so that the images in each wavelength band do not overlap with the light receiving surface of the camera can be performed with each mirror holder with detachable movable adjustment function Can
In particular, in the last-stage optical branching module, a total reflection mirror is used for the first filter, and for the second filter, the first filter in the previous-stage optical branching module is used,
The high-pass filter or low-pass filter is arranged in the order of the monotonically increasing or monotonically decreasing wavelength for the branch wavelength, and the same filter is used for the first filter in the previous stage and the second filter in the next stage. In addition, the image branching display device is characterized in that a total reflection mirror is used for the first-stage second filter and the last-stage first filter.

An imaging cell sorter that observes and separates cells in water droplets that are generated in the air and dropped on the water-repellent substrate.
A mechanism for producing and dropping water droplets of an optimal size by discharging the sample solution at a constant pressure from the thin tube at the tip of the water droplet forming module with a cell reservoir, in the sample solution,
Water droplets are desired by charging the electrostatic field coil that covers from the area where the water just before the water drops are linked to the reservoir to the area where the water drops are just formed, to the opposite charge to the water charge. A mechanism that can be charged with an electric charge,
A mechanism in which the formed water droplets are dropped onto an optically transparent water-repellent insulating substrate having a Teflon (registered trademark) resin processed on the surface of glass or the like, and rolled down in the direction of inclination of the substrate, ,
A mechanism in which a high-speed camera capable of measuring a bright field image and a fluorescent image on the back surface of the substrate on the path where the water drops fall and an optical measurement module capable of measuring scattered light intensity, fluorescent intensity, etc. are arranged,
By analyzing the acquired information with the analysis control module, a mechanism to determine whether the target cell is in a water drop,
After identifying the type of the target cell according to the determination, the charge opposite to the water droplet is transferred to a specific one of the one or more water droplet movement direction control electrostatic field guides for changing the position from the direction in which the water droplet falls. A mechanism to change the drop direction of the water droplets, and to guide to the fractional water droplet reservoir in the subsequent stage,
A mechanism that can apply the electrostatic field guide for each water droplet movement direction control to the electrode in accordance with the analysis result of the analysis control module, and each electrode is a static for the water droplet movement direction control when not controlled by this mechanism. The electric field guide has a mechanism that does not affect the movement of water drops by grounding,
In order to enhance the initial positioning system for the dropping direction of the dropped water droplets, a similar electrostatic field guide for controlling the water droplet movement direction is arranged, or a pair of rail-shaped three-dimensional structures sandwiched between the bottom surfaces of water repellent surfaces such as Teflon coat are added. To do
Having a substrate tilt control mechanism that can freely control the drop speed of water droplets, that is, the measurement time, and using a substrate composed of a combination of different tilts,
The observation area and the water droplet fractionation start area have means that can arrange water drops closely at equal distances in the falling direction by making the inclination horizontal (perpendicular to gravity) so that the water drops move at a constant speed. And
A mechanism for controlling the temperature of water droplets that have made minimal contact in a water-repellent state by adjusting the temperature of the substrate;
In order to selectively collect target cells in the water droplet according to the reaction result, the reaction liquid reservoir water droplet formation mechanism is used to collide the reaction liquid with the sample water droplet and observe and measure the mixed liquid droplet at a certain post-reaction time. A cell sorter that creates reaction droplets with weakly applied reverse charges to the sample water droplets, and can guide the reaction solution to the sample water droplets by the same electrostatic field guide for controlling the direction of water droplet movement.

The present invention also includes a process of collecting blood and a cell cluster (cluster) having an area of about 250 μm ² or more in the blood, which is collected and cultured, or subjected to genetic mutation test or expression analysis test. The procedure for identifying this cell cluster, and if the identified cell is a metastatic cancer cell in the blood, by confirming the gene mutation in each cluster unit, each cluster is the same mutant. The progression of the cancer is presumed to be early in the beginning of metastasis from the primary cancer, and there are many different mutation points while the history of mutations in each cluster is the same. If there is a procedure, it is possible to estimate that the location of metastatic cancer has spread to many areas, and if the cell cluster flowing in the blood can be identified as a tissue piece of the organ, the disease of the organ is estimated. A procedure for recovering cells that are larger in size than normal, especially for phagocytic leukocytes such as macrophages, and examining and identifying genes of heterologous cells such as bacteria in the cells, and B cells From the procedure to diagnose what the immune system is reacting by selectively recovering those whose internal shape has become complicated due to activation of, and those whose size has increased and clarifying the antigen of the B cell A cell cluster diagnostic technique is provided.

The present invention also allows the patient's blood to be refluxed using a cell cluster (cluster) removal mechanism such as a membrane filter that removes a cell cluster (cluster) having a cross-sectional area of approximately 250 μm ² or more that does not exist in healthy blood. A metastatic cancer treatment device that suppresses the onset and progression of metastatic cancer by effectively removing metastatic cancer cells in blood by a physical technique.

More specifically, the present invention relates to the following on-chip cell sorter system, a method for identifying cancer cell candidate cells in a subject-derived cell sample solution, an optical branching module, an imaging cell sorter, and a cell cluster in blood. Disclosed are a medical treatment apparatus for preventing metastatic cancer progression by physical removal, and a method for diagnosing metastatic cancer, organ abnormality, or infection using detection of a cell mass in blood.

[1] An on-chip cell sorter system used for identifying cancer cell candidate cells,
A cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject;
An optical system including a bright-field light source and a fluorescent light source for irradiating the cells;
A detection system for simultaneously acquiring a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells;
Control / analyzing means for identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image;
Means for selectively recovering the identified multinucleated cells and / or cell clusters,
The above control / analysis means is provided by the following (i) to (iii):
(I) The area of the nucleus of the lump or cluster obtained from the fluorescence image of the nucleus of the lump of cells or clusters is 150 μm ² or more.
(Ii) The area of the lump or cluster obtained from a bright field image of a lump of cells or clusters is 250 μm ² or more, or (iii) the number of nuclei in the lump of cells or clusters is 3 or more If one condition is met, or (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii) are met , Judge that cancer cells are likely to be present in the cell sample solution,
On-chip cell sorter system.
[2] The control / analysis means is
About the perimeter ratio R derived as a circular approximation from the perimeter and area of cells (clusters),
(I) When (1 / R) is 0.9 or more, the cell (cluster) is an isolated cell, or (ii) When (1 / R) is less than 0.9, the cell (cluster) is 2 cells. Consisting of a cluster of cells,
As described above, the on-chip cell sorter system according to the above [1], wherein the value of (1 / R) is used as a criterion for determining the presence or absence of clusters in the cell sample solution.
[3] An on-chip cell sorter system used for identifying cancer cell candidate cells,
A cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject;
An optical system including a bright-field light source and a fluorescent light source for irradiating the cells;
A detection system for simultaneously acquiring a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells;
Control / analyzing means for identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image;
Means for selectively recovering the identified multinucleated cells and / or cell clusters,
The control / analysis means is
About the perimeter ratio R derived as a circular approximation from the perimeter and area of cells (clusters),
(I) When (1 / R) is 0.9 or more, the cell (cluster) is an isolated cell, or (ii) When (1 / R) is less than 0.9, the cell (cluster) is 2 cells. Consisting of a cluster of cells,
As described above, an on-chip cell sorter system in which the value of (1 / R) is used as a criterion for determining the presence or absence of clusters in the cell sample solution.
[4] The on-chip cell sorter system according to any one of the above [1] to [3], further comprising means for measuring a nucleic acid sequence of a gene derived from the selectively recovered multinucleated cells and / or cell clusters.
[5] Any one of the above [1] to [4] provided with an image dividing mechanism having a function of dividing and displaying the bright field image and the fluorescent image on the light receiving surface of one high-speed camera at the same time. The on-chip cell sorter system according to item.
[6] The on-chip cell sorter system according to [5], further including a mechanism that adjusts the magnification of the bright field image and the fluorescence image so that the magnification of the image differs.
[7] The on-chip cell sorter system according to any one of [1] to [6] above, wherein the cell derived from the subject is a cell derived from blood of the subject.
[8] A method for identifying candidate cells of cancer cells in a cell sample solution derived from a subject using the on-chip cell sorter system according to any one of [1] to [7],
Flowing a sample solution containing fluorescently-stained cells from a subject through the flow path of the cell sorter chip;
Irradiating the cells with light from a bright-field light source and a fluorescent light source,
Obtaining a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells;
Based on the bright field image, the fluorescence intensity, and the fluorescence image, a step of identifying a multinucleated cell and / or cell cluster flowing through the flow path, and selectively identifying the identified multinucleated cell and / or cell cluster Including the step of collecting,
In identifying the multinucleated cells and / or cell clusters,
(I) The area of the nucleus of the lump or cluster obtained from the fluorescence image of the nucleus of the lump of cells or clusters is 150 μm ² or more.
(Ii) The area of the lump or cluster obtained from a bright field image of a lump of cells or clusters is 250 μm ² or more, or (iii) the number of nuclei in the lump of cells or clusters is 3 or more If one condition is met, or (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii) are met A method for determining that cancer cells are likely to be present in the cell sample solution.
[9] An optical branching module,
A substantially rectangular parallelepiped housing;
An optical path system including a pair of mirrors for input / output of a pair of image light provided symmetrically on the bottom surface in the housing;
Two-to-four openings for introducing mirror reflected light provided on the side surface of the casing, two on the side in the longitudinal direction and one on the side in the short side An aperture formed,
6 optical path covers that can be freely attached to and detached from each of the 2 to 4 openings,
A pair of removable movable mirror holders capable of adjusting the mirror so that light is introduced into each of the two to four openings;
A detachable optical path window arranged between the pair of mirrors and capable of adjusting a cross-sectional area of the transmitted light,
A detachable filter disposed between the pair of mirrors and capable of adjusting a wavelength bandwidth of light;
An optical branching module.
[10] The optical branching module according to [9], wherein a plurality of the optical branching modules are connected and used.
[11] The optical branching module according to the above [10], which is used to simultaneously acquire a plurality of microscope images of different wavelength bands with one camera light receiving surface.
[12] The optical branching module according to any one of [9] to [11], wherein one of the pair of mirrors of some of the optical branching modules can be removed and used.
[13] The bright field image of the cell in the sample solution containing the fluorescence-stained cell, the fluorescence intensity of the fluorescent labeling substance bound to the cell, and the fluorescence image of the cell are used to simultaneously acquire the above [ The optical branching module according to any one of 9] to [12].
[14] A water droplet formation module comprising a sample solution reservoir for holding a sample solution containing cells and a thin tube connected to the reservoir;
An electrostatic field coil for charging water droplets formed by the water droplet forming module;
A substrate having a water-repellent surface for dropping the formed water droplets, the substrate being optically transmissive,
A substrate tilt control mechanism for adjusting the tilt angle of the substrate surface;
An optical measurement module including a camera capable of measuring a bright-field image and a fluorescent image, disposed on the opposite side of the substrate surface on which the water droplets are dropped, and the water droplets formed on the water-repellent surface can be controlled thereon An electrostatic field guide for controlling the direction of movement of water droplets, which is one or a plurality of guides for movement, and is charged with a charge opposite to that of the water droplets;
A fractionated water droplet reservoir disposed in the subsequent stage of each of the plurality of water droplet movement direction control electrostatic field guides;
An electric field switching mechanism for electrostatic field guide for controlling the direction of movement of water droplets connected to the electrostatic field guide for controlling the direction of movement of water droplets, the mechanism for controlling the electric field charged in the electrostatic field guide;
Analysis that is connected to the optical measurement module and the switching mechanism, analyzes image data acquired by the optical measurement module, determines the presence or absence of target cells in the water droplets, and controls the operation of the switching mechanism based on it An imaging cell sorter for observing and separating cells in water droplets, comprising a control module.
[15] A plurality of electrostatic field guides for controlling the direction of movement of the water droplets are provided, at least one of which is for improving the initial water droplet positioning accuracy, and the other guides include water droplets containing target cells and water droplets not containing them. For sorting,
[14] The guide for improving the initial water droplet positioning accuracy is located upstream of the movement of the water droplet, and the guide for selecting the water droplet containing the target cell and the water droplet not containing the target cell is located downstream thereof. ] The imaging cell sorter described in the above.
[16] The above [14] or [15], wherein the optical measurement module is arranged so as to be able to acquire an image of cells in the water droplet moving on a guide for increasing the initial water droplet positioning accuracy. ] The imaging cell sorter described in the above.
[17] Applying a charge opposite to the water droplet to a specific one of the guides for selecting the water droplet containing the target cell and the water droplet not containing the target cell to control the moving direction of the water droplet, The imaging cell sorter according to any one of the above [14] to [16], which is configured to guide the water droplets to a fractional water droplet reservoir disposed in the above.
[18] The imaging cell sorter according to any one of [14] to [17], further covered with a cover for preventing evaporation of the water droplets.
[19] A metastasis cancer treatment apparatus having a cell mass (cluster) removal mechanism including a membrane filter that removes a cell mass (cluster) having a cross-sectional area of about 250 μm ² or more that is not present in healthy blood, the blood of a patient A metastatic cancer treatment device that suppresses the onset and progression of metastatic cancer by effectively removing the metastatic cancer cells in the blood by a physical technique.
[20] Diagnosis of cell cluster of metastatic cancer, organ abnormality, or infectious disease using selection of cell cluster (cluster) having an area of 250 μm ² or more in a blood sample derived from a subject and detection of the cell cluster in blood An on-chip cell sorter system according to any one of the above [1] to [7] used in a diagnostic method for
The above diagnostic method is
This cell cluster is identified from the selected cell mass by culture, gene mutation test, or expression analysis test,
When the identified cell is a metastatic cancer cell in the blood, the gene mutation is confirmed in each cluster unit, and when each cluster is the same mutant, the degree of progression of cancer is If the metastasis from primary cancer is presumed to be early, or if the history of mutations in each cluster is the same but there are many different mutation points, the location of the metastatic cancer Is estimated to be in many areas,
If the cell cluster that flows in the blood can be identified as an organ tissue fragment, organ disease is presumed, or for phagocytic leukocytes including macrophages, cells larger in size than normal are collected, and By examining and identifying genes of heterologous cells such as bacteria, selectively recovering those whose internal shape has been complicated by the activation of B cells and those having increased in size, and clarifying the antigens of the B cells Guess what the immune system is reacting to,
On-chip cell sorter system.
[21] A cell cluster diagnostic method for metastatic cancer, organ abnormality, or infectious disease using detection of a cell mass in blood,
In a blood sample derived from a subject, a cell cluster (cluster) having an area of 250 μm ² or more is selected,
This cell cluster is identified from the selected cell mass by culture, gene mutation test, or expression analysis test,
When the identified cell is a metastatic cancer cell in the blood, the gene mutation is confirmed in each cluster unit, and when each cluster is the same mutant, the degree of progression of cancer is If the metastasis from primary cancer is presumed to be early, or if the history of mutations in each cluster is the same but there are many different mutation points, the location of the metastatic cancer Is estimated to be in many areas,
If the cell cluster that flows in the blood can be identified as an organ tissue fragment, organ disease is presumed, or for phagocytic leukocytes including macrophages, cells larger in size than normal are collected, and By examining and identifying genes of heterologous cells such as bacteria, selectively recovering those whose internal shape has been complicated by the activation of B cells and those having increased in size, and clarifying the antigens of the B cells Guess what the immune system is reacting to,
Cell cluster diagnostic method.
[22] A method for confirming the presence of abnormal cells in blood,
Measuring a cell size in a blood sample from a subject containing blood cells excluding red blood cells,
Based on the result of the measurement step, a step of obtaining a cell size distribution spectrum of the blood sample, and whether the cell size distribution spectrum has one peak or two or more peaks in the cell size distribution spectrum. A method comprising the step of identifying.
[23] A step of selectively recovering cells larger than the threshold from the blood sample with the peak on the large cell side as a threshold when it is identified that there are two or more peaks as a result of the identification step The method according to [22], further comprising:
[24] The above [22] or [23], wherein in the step of measuring the cell size and the step of collecting the cells, an on-chip cell sorter chip having a flow path for flowing the blood sample is used. The method described in 1.
[25] The method according to [24] above, wherein the on-chip cell sorter chip is part of the on-chip cell sorter system according to any one of [1] to [7].
[26] The method according to any of [22] to [25] above, which is used for cytodiagnosis of metastatic cancer, organ abnormality, or infectious disease.
[27] a water droplet formation module comprising a sample solution reservoir for holding a sample solution containing cells and a thin tube connected to the reservoir;
A substrate having a water-repellent surface formed by joining regions having two or more different inclination angles for dropping the formed water droplets;
A substrate tilt control mechanism for adjusting a tilt angle of a surface having two or more different tilt angles of the substrate;
A measurement module for measuring the state of the water droplet and the state inside the water droplet;
Means for separating water droplets in a plurality of traveling directions based on the measurement results;
An imaging cell sorter for observing and separating cells in water droplets.
[28] The means for separating the water droplets in a plurality of traveling directions includes one or more guides for controllably moving the water droplets formed on the water repellent surface,
The imaging cell sorter according to [27] above, wherein the guide is an electrostatic field guide for controlling the direction of movement of a water droplet charged with a charge opposite to that of the water droplet.

According to the present invention, it is possible to purify a minute amount of target cells in blood in units of one cell, and to analyze accurate gene information and expression information of the target cells.

According to the present invention, it is possible to identify whether or not the cells to be examined are clustered (whether or not they are isolated single cells).

According to the present invention, it is possible to determine whether apoptosis has occurred in a cell.

According to the present invention, only the target cells can be separated and purified and collected in real time.

According to the present invention, it is possible to measure the intracellular state of only the collected cells at the single cell level, and perform genome analysis and expression analysis at the single cell level.

According to the present invention, only the collected cells can be re-cultured.

According to the present invention, detailed cell information such as a difference in cell size, a size ratio between the nucleus inside the cell and the cytoplasm, etc. can be acquired, and the cell can be purified by discrimination based on the result.

According to the present invention, it is possible to analyze a substance in a cell at high speed and minimize contamination with respect to cells having spores such as anthrax.

According to the present invention, by collecting cells that are dividing in the blood, it is possible to collect cells having a dividing ability such as blood cancer cells and stem cells.

The present invention makes it possible to effectively recover multinucleated cells and cell clusters that are candidates for cancer cells circulating in the blood.
According to the present invention, it becomes possible to simultaneously excite cells labeled with fluorescent antibodies of multiple wavelengths with excitation light of multiple wavelengths and simultaneously detect the emitted multiple fluorescences, and effectively recover target cells. It becomes possible.
According to the present invention, cancer cells and diseased organ tissue sections can be identified and recovered quantitatively from image data from blood cells.
According to the present invention, metastatic cancer cell clusters can be removed from blood cells to prevent the progression of metastatic cancer.

It is a schematic diagram which shows notionally an example of the means in the apparatus corresponding to the whole process of the cell analysis performed using the cell analyzer apparatus of this invention, and each process. It is a figure which shows typically one example of the whole structure of the cell analyzer system of this invention in FIG. It is a figure which shows typically an example of a structure of the cell concentration / staining / decolorization module in FIG. It is a figure which shows typically an example of a structure of the image detection type 1-cell separation and refinement | purification (cell sorter) module in FIG. It is the figure which showed typically one example of the structure of the 1-cell genome analysis and expression analysis module in FIG. It is a figure which shows typically an example of a structure of the liquid feeding module in FIG. It is a schematic diagram which shows notionally an example of the means in the apparatus corresponding to the whole process in the case of including the cell destruction process in the cell analysis performed using the cell analyzer apparatus of this invention, and each process. It is a figure which shows typically an example of the crushing mechanism comprised in the cell destruction process in the process shown in FIG. 7 comprised from a container, a rotary body, and a rotating shaft. It is a figure which shows typically the various variations of the basic cell crushing mechanism shown in FIG. The mechanism which ensures the adhesiveness of a container and a rotary body is illustrated. It is a schematic diagram which shows the example of the various shapes of the rotary body of a cell crushing mechanism used in this invention, and a rotating shaft. It is the figure which showed typically the whole process of the sample crushing of the cell crushing process in this invention. The conceptual diagram of the automation structure for the continuous crushing of the cell in the cell crushing process in this invention is shown. It is a figure which shows typically an example of the chip | tip structure of the cell sorter module in this invention. It is a figure which shows typically an example of the chip | tip structure of the sample reservoir area | region of the cell sorter module in this invention. It is a figure which shows typically an example of a structure of an image detection type 1-cell separation and refinement | purification (cell sorter) module. FIG. 16 is a diagram schematically showing an example of a cell purification process in the image detection type 1-cell separation / purification (cell sorter) module of FIG. 15. It is a figure which shows typically the image recognition of the disappearance process of the nucleus at the time of the cell division which is one of the identification indexes of the cell purification in the image detection type 1-cell separation / purification (cell sorter) module of FIG. It is a figure which shows typically an example of the light emission timing of the high-speed flash light source for preventing the image blurring in an image detection type 1 cell separation and refinement | purification (cell sorter) module. It is a figure which shows typically an example of a structure of the optical system for preventing the image blurring in an image detection type | mold 1 cell separation and refinement | purification (cell sorter) module (FIG. 19A). FIG. 19 shows a comparison between an image of fine particles observed with a conventional optical system (FIG. 19B) and an example of an image of fine particles observed with an optical system of the present invention (FIG. 19C). It is a figure which shows typically an example of a structure of the optical system for preventing the image blurring in an image detection type | mold 1 cell separation and refinement | purification (cell sorter) module (FIG. 19A). FIG. 19 shows a comparison between an image of fine particles observed with a conventional optical system (FIG. 19B) and an example of an image of fine particles observed with an optical system of the present invention (FIG. 19C). It is a figure which shows typically an example of a structure of the optical system for preventing the image blurring in an image detection type | mold 1 cell separation and refinement | purification (cell sorter) module (FIG. 19A). FIG. 19 shows a comparison between an image of fine particles observed with a conventional optical system (FIG. 19B) and an example of an image of fine particles observed with an optical system of the present invention (FIG. 19C). It is a figure which shows typically an example of the structure which has arrange | positioned the arrangement | positioning of a cell sorter chip so that the flow of a liquid may become a perpendicular direction. It is a figure which shows typically an example of the structure which has arrange | positioned the arrangement | positioning of a cell sorter chip so that the flow of a liquid may become a perpendicular direction. It is a figure which shows typically an example of a structure of the part where the sample solution and buffer solution of a cell sorter chip merge. It is a figure which shows typically an example of a structure of the chip | tip incorporating the mechanism which performs cell sorting among cell sorter systems. It is a figure which shows an example of arrangement | positioning of the electrode for giving external force in the flow path of a cell sorter system. It is a figure which shows the example of arrangement | positioning of the electrode for giving the external force in the flow path of a cell sorter system. It is a schematic diagram which shows an example of the process of the cell purification in the flow path of a cell sorter system. It is a schematic diagram which shows an example of arrangement | positioning of the gel electrode which gives an external electric field in the flow path of a cell sorter system. FIG. 4 is a schematic diagram showing an example of a process for identifying cardiomyocytes and fibroblasts to be separated by an image in an image recognition type cell sorter system. It is a schematic diagram which shows an example of a structure of the cell sorter system which combined water and oil. It is a schematic diagram which shows an example of a structure of the joint area | region of the water and oil of the cell sorter system which combined water and oil. It is the graph which showed the relationship between the electrolytic mass (electric conductivity) in aqueous solution, and the cell separation possible speed. It is the figure which showed typically an example of the structure of the analysis system which performs a fluorescence intensity measurement and high-speed bright-field microscopic image acquisition simultaneously. It is the figure which showed typically an example of the specific structure of the structure of the analysis system which performs the fluorescence intensity measurement shown in FIG. 32, and high-speed bright-field microscopic image acquisition simultaneously. It is the figure which showed typically an example of the structure of the analysis system which performs fluorescence intensity measurement, high-speed bright-field microscopic image acquisition, and high-speed fluorescence microscopic image acquisition simultaneously. It is the figure which showed typically an example of the apparatus structure which acquires high-speed bright-field microscopic image acquisition and high-speed fluorescence microscopic image acquisition simultaneously with one high-speed camera light-receiving surface. It is the figure which showed typically an example about the example and analysis information of the image which acquired high-speed bright-field microscopic image acquisition and high-speed fluorescence microscopic image acquisition simultaneously with one high-speed camera light-receiving surface. It is the photograph which showed the example of the image which acquired simultaneously the high-speed bright-field microscopic image acquisition and the high-speed fluorescence microscopic image acquisition which carried out the nuclear fluorescent dye with one high-speed camera light-receiving surface. Schematic example of a device configuration for simultaneously irradiating cells with multiple wavelengths of fluorescence excitation light using an optical fiber array, simultaneously acquiring the amount of fluorescence emitted from multiple wavelengths, and simultaneously acquiring fluorescent images of multiple wavelengths It is the figure shown in. It is the spectrum figure which showed typically an example which selected the several fluorescence excitation light for irradiating a cell, and the several fluorescence wavelength set to detect. It is the figure which showed typically the structure of the present Example which showed an example in FIG. FIG. 41A is a distribution diagram of the area of cell nuclei when blood cancer cells are measured by the apparatus of the present invention, and a bright field photograph and a fluorescence photograph of cells in each distribution region. FIG. 41B is a graph showing the ratio of the peripheral length of cells (clusters) measured by the apparatus of the present invention and the peripheral length derived by circular approximation from the area of the cells (clusters) when blood cancer cells are measured. . It is the table | surface which showed cell (population) area when the blood cancer cell was measured with the apparatus of this invention, and distribution of the number of nuclei. A diagram (A) schematically showing an example of a unit structure of an apparatus configuration that simultaneously acquires microscopic images in a plurality of different wavelength bands with one light-receiving surface of a high-speed camera, and an example of a combination of these units. FIGS. 43B and 43C and FIG. 43C are examples showing an example of image acquisition of a plurality of images captured by the camera shown in FIG. 43B. A diagram (A) schematically showing an example of a unit structure of an apparatus configuration that simultaneously acquires microscopic images in a plurality of different wavelength bands with one light-receiving surface of a high-speed camera, and an example of a combination of these units. FIGS. 43B and 43C and FIG. 43C are examples showing an example of image acquisition of a plurality of images captured by the camera shown in FIG. 43B. FIG. 44A is a diagram schematically illustrating an example of a configuration of an imaging cell sorter that observes and separates cells in water droplets. 44B is a diagram of the configuration of the chip illustrated in FIG. 44A as viewed from above. Figure (A) schematically showing an example of a process for detecting and analyzing a cell cluster in human blood, and an example of an integrated measurement analysis system for realizing the process shown in Figure 45A It is the figure (B and C) shown typically. Figure (A) schematically showing an example of a process for detecting and analyzing a cell cluster in human blood, and an example of an integrated measurement analysis system for realizing the process shown in Figure 45A It is the figure (B and C) shown typically. It is the figure which showed typically an example of the method of removing the cell lump (cluster) in the blood beyond a fixed size as a metastatic cancer treatment technique. As an explanation of a method for diagnosing metastatic cancer in blood, the distribution spectrum of blood cell size is determined for each of normal blood (FIG. 47A) and blood in which metastatic cancer cells are flowing (FIGS. 47B and C). It is the figure shown typically. 44 schematically shows a method using a substrate having an acceleration region and a constant velocity moving region by having two inclination angles in the means for moving the water droplet dropped on the water repellent substrate described above with reference to FIG. FIG.

The cell analyzer of the present invention generally comprises:
(1) Cell concentration / staining / decolorization part that continuously performs processes including cell concentration, fluorescent antibody labeling (or staining and washing with reversible fluorescent labeling markers such as aptamers if necessary) ,
(2) Obtain image data of about 10,000 images of cells per second from cells flowing through the microchannel formed on the chip substrate, and purify 10,000 cells per second in real time based on the analysis result of the image information An image detection type 1 cell separation / purification (cell sorter) unit,
(3) a 1-cell genome analysis / expression analysis unit that measures the intracellular state at the 1-cell level;
(4) a liquid feeding part for carrying the sample liquid between the above parts,
(5) A control analysis unit for controlling the operation of each unit and performing the analysis.

A typical embodiment of the cell analyzer of the present invention is characterized in that the three modules (1) to (3) are continuously combined in the order described above, and the cells are continuously connected by the flow path. Therefore, a small amount of cells can be eliminated by contamination or manipulation.

By using the cell analyzer of the present invention, the presence or absence of fluorescent labeling of the cells is detected and confirmed at the single cell level, and the fluorescence-labeled cells are confirmed to be isolated single cells that are not clustered. In addition, it can be determined whether apoptosis is occurring in the cells. Therefore, according to the cell analyzer of the present invention, cells can be separated and purified based on an index that cannot be identified by the conventional scattered light detection type cell sorter technique.

According to the cell analyzer of the present invention, cells that are stained accurately in units of 1 cell are selectively collected, and cell states such as apoptosis of the cells to be collected are confirmed, and fluorescence information and cell state information of each cell are confirmed. In addition, cell genetic information and expression information can be analyzed.

In the cell concentration / staining / decoloring part (1) above, a small amount of cells contained in the reaction solution continuously sent from the previous module by the non-contact force is continuously captured and concentrated, and a certain number of cells is obtained. When the cell label staining solution is introduced and the cells are stained, the unbound reagent is washed away, and then the cells are delivered to the next module at a constant concentration. In this cell concentration / staining / decolorization part, for example, cell capture / concentration using the feature that cells gather by “dielectrophoretic force” created by an alternating electric field by a metal electrode created in a microchannel as a non-contact force These elemental technologies are used.

Further, in the means for performing separation / purification in units of one cell based on the result of the image detection of (2) above, details such as the difference in cell size, the ratio of the nucleus inside the cell to the cytoplasm, etc. Cell information is acquired as image information, and the cell is purified based on the result. 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 that can be controlled at high speed, such as an LED light source or a pulsed laser light source, for a period of 1 / 10th of the shutter speed. Thus, the fine shape of the cell can be obtained.

When constructing a cell sorter on a chip as the separation / purification means described above, in the conventional cell sorter, the cells introduced into the cell sorter were separately concentrated through a concentration step such as a centrifuge, and so on. There was a problem with Nation. Therefore, in the present invention, functions other than the optical system are performed in a closed manner using only the chip. That is, it was set as the structure which concentrates a cell directly on a chip | tip, and the liquid feeding part and the culture tank of the isolate | separated cell were also formed on the chip | tip. As a result, not only contamination and cell loss do not occur, but also the procedure is simplified and the usability of shortening the processing time can be improved. Moreover, by making it closed, it is not necessary to consider contamination even in cases where it is essential to prevent contamination of other specimen tissue-derived cells, such as stem cell separation or clinical examination. In this way, the present invention makes it possible to completely prevent cross-contamination of the device by making the main part of the cell sorter into a chip, and to separate cells without cross-contamination essential in the medical field, particularly in the field of regenerative medicine. Provide a system.

The cells assumed as detection targets in the present invention are bacteria for small ones and animal cells (for example, cancer cells) for large ones. Therefore, the cell size typically ranges from about 0.5 μm to about 30 μm. When a channel incorporating both a cell concentration function and a cell separation function is formed on one surface of a substrate and cell concentration and separation are continuously performed, the first problem is the channel width (cross-sectional shape). In addition, the channel 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, the most typical size is about 5 to about 10 μm in the thickness direction for bacteria, and about 10 to about 50 μm in the thickness direction for animal cells.

The cell analyzer of the present invention typically comprises a cell concentrating unit having a function of concentrating cells, a cell arrangement unit having a function of separating and purifying cells, and a cell separation / purification in the same chip. And an optical analysis unit for identifying and judging cells to be separated and purified. Typically, a sample solution that has not been subjected to concentration treatment is introduced into the cell concentration section from one inlet, and the sample solution is discharged from a discharge section disposed downstream of the cell concentration section. In addition to such a basic configuration, there may be provided means for concentrating the cells by applying an external force to the cells toward the concentrated cell recovery port installed in the concentration unit side wall. As the external force at this time, ultrasonic radiation pressure, gravity, electrostatic force, dielectric electrophoretic force and the like can be used, but are not limited thereto. In this case, an arrangement is used in which these external forces can be applied in a direction perpendicular to the flow of the sample solution in the concentration section and in the direction of the concentrated cell recovery port.

In the cell separation / purification unit, all cells should flow in one of the two channels that are branched into two downstreams by applying external force to the cells so that the cells are arranged in the center of the channel where the cells are flowing. Subsequently, by applying an external force to only the cells to be collected out of the arranged cells and moving the flow position of the cells, the external force is applied to the flow path branched into the above two. Only when the cells are introduced into another channel. Here, specifically, as the external force, means for arranging cells into nodes of standing waves by ultrasonic radiation pressure can be used. Alternatively, a means for arranging cells at the position of the apex of the wedge can be used by combining wedge-shaped electrode arrays. Alternatively, means for arranging cells between two counter electrodes using a pair of bearded electrodes can be used. Moreover, since the cells can be arranged in a straight line without adding a side sheath solution by using this means, the above-described invention that the cell solution concentrated in the previous stage is diluted is described. It will be possible to solve the problems that have been solved.

The cell detection function of the cell analyzer of the present invention resides in the image detection type 1-cell separation / purification part (2) above. When a cell is captured as an image and evaluated, a part to be observed by a CCD camera is provided upstream of the flow path branching part, and a cell separation region is provided downstream of the part if necessary. Irrespective of the image, the cells passing through the flow path are irradiated with a laser or the like, and when the cells cross and the scattered light or the cells are modified with fluorescence, the fluorescence can be detected with a photodetector. In this case as well, a separation channel point that becomes a cell separation region is installed downstream of the detection unit.

When the cells are separated by the sorting unit which is a cell separation region, as a means for moving the cells by applying external force to the cells from the outside, for example, when using a dielectrophoretic force, a pair of comb-shaped electrodes is used. Install and provide a flow path that can separate and drain cells. In the case of electrostatic force, a voltage is applied to the electrode to change the position of the cell in the flow path. At this time, since the cell is generally charged negatively, it moves toward the positive electrode.

In the present invention, since the introduction pressure of the sample liquid into the chip is a driving force for the movement of the liquid, the waste liquid outlet (outlet 213) of the cell concentrating unit 215 and the purified cell of the cell sorting unit 217 It is desirable that the pressure at the outlet (cell recovery unit 224) and the outlet of the waste liquid of the cell sorting unit (waste liquid recovery unit 223) be substantially the same (see FIG. 4B). For this purpose, a flow path resistance adjusting portion for pressure adjustment is arranged immediately before each outlet such as a thin flow path or a long S-shaped flow path.

The cell recognition and separation algorithm has the following characteristics.

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, it is an image processing method, but a high capture rate 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, when used 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.

In order to achieve the above object, the means for high-speed single-cell genome analysis / expression analysis of (3) used in the present invention, for example, the reaction control device used, For a plurality of temperatures to be changed, means for changing a plurality of liquids having different heat capacities at a high speed at high speed using a liquid having a large heat capacity maintained at each temperature as a heat exchange medium, and a liquid having a large heat capacity And a microreaction tank in which heat exchange with the sample liquid is performed quickly. Specifically, the reaction control device used in the present invention includes a reaction vessel that has a structure and material suitable for heat exchange, and a reaction that circulates a liquid having a temperature suitable for each reaction outside the reaction vessel. Liquid is transferred from any liquid reservoir tank to the outside of the reaction tank to rapidly change the temperature of the tank heat exchange tank, heat sources that maintain the temperature of the liquid with high accuracy, and the temperature of the micro reaction tank And a mechanism for preventing the mixing of liquids at different temperatures when the valve system is switched.

Advantages of controlling the temperature of the reaction vessel with the refluxing liquid include the following points. First, the temperature overshoot problem can be solved. Since the temperature of the liquid that is constantly refluxing is constant, the temperature of the reaction vessel surface and the temperature of the liquid are instantaneously equilibrated. Since the heat capacity of the reaction vessel and the sample is insignificant compared to the liquid being refluxed, the liquid flows continuously even if heat is locally deprived from the liquid. Basically does not occur. Of course, the temperature of the reaction vessel does not exceed the temperature of the liquid. It is possible to change the temperature by 30 degrees or more within 0.5 seconds by sequentially pouring liquids of different temperatures into the reaction tank heat exchange tank.

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

(System configuration of cell analyzer)
FIG. 1 illustrates an example of a procedure from collection of a blood sample to analysis performed using the cell analyzer of the present invention.

The blood sample collected from the patient is directly introduced into the cell concentration / staining part. Here, only the cellular components are extracted from the blood, and after adding a fluorescent labeling agent such as a fluorescent cancer marker to react with the sample cells, the excess fluorescent labeling agent that did not react is washed away. The cells are introduced into the image detection type 1-cell separation and purification unit in a form adjusted to the optimum cell concentration and solution for the next image detection type 1-cell separation and purification unit.

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. Thereby, it can be confirmed by a conventional labeling technique whether the cell is a target cell. 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 emitting fluorescence are isolated cells or become cell clusters with other cells 2) Determine whether the cells emitting fluorescence are in a healthy state or in a state such as apoptosis in which the cell nucleus and cell shape are deformed, and depending on the purpose, Recover healthy cells or recover cells that are undergoing apoptosis, and perform gene analysis and expression analysis on the next stage so that gene analysis and expression analysis can be performed separately for each cell morphology. Can be introduced into the department. In particular, in the case of a cell mass, since cells other than the target cell are mixed, collection is not performed even when there are cells that emit fluorescence.

In addition, the cells identified and purified at this stage can be re-cultured in a contamination-free manner in purified cells, in addition to being introduced into the gene analysis / expression analysis section.

The gene analysis / expression analysis unit converts the introduced cells into a small amount of cells, as one cell that is identified as the same cell based on information from the image detection type 1 cell separation and purification unit, or a group of the same cells Gene identification or expression identification is performed in units.

FIG. 2 shows an example of an overall image of the cell analyzer system 1 that realizes the procedure shown in FIG. The apparatus system 1 includes a concentration / staining / decoloring module 10 that introduces a blood sample and pretreats cells, an image detection type 1-cell separation / purification module 20 that identifies and purifies cells in units of cells, and is purified. 1-cell genome analysis / expression analysis module 30 for performing gene analysis and expression analysis of the collected cells, liquid feeding module 40 for transporting samples between modules, and controlling the operation of the entire system and analyzing the analysis results A control / analysis module (computer) 50 is provided.

3 to 6 show an example of the configuration of each module in the example shown in FIG.

First, FIG. 3 shows an example of the configuration of the cell concentration / staining / decoloring module 10 that introduces a blood sample derived from a subject (eg, cancer patient) and pretreats the cells. In the example of FIG. 3, the cell concentration / staining / decoloring module 10 is integrally disposed on the chassis 114, and each solution of the sample cell sample, the staining agent, and the cleaning agent is held in the module. There is a container or reservoir (101, 102, 103), and a chamber 107 in which a concentration / decolorization filter 106 is disposed on the bottom surface on which the solution is disposed on the turntable 105 by a dispensing head 104 attached to the rotary arm 115. (Together with the concentration chamber 108). First, a sample cell sample such as blood is introduced into the concentration chamber 108, and the liquid component is discharged through the filter to the waste liquid collection tube 110 by the pressure pump 109, thereby concentrating the cells. Next, the dyeing solution is introduced using the dispensing head 104 and reacted for a predetermined time, and then the dyeing solution is discharged again by the pressure pump 109. Next, by introducing a decolorizing agent into the concentration chamber 108, excess dyeing agent is washed and discharged. After that, generally, a diluent also serving as a cleaning agent is introduced to dilute the cells to a desired concentration, and the cells are introduced into the collection tube 112 through the collection head 111 having the collection tip 113 at the tip. Yes.

FIG. 4 shows an example of the configuration of the image detection type 1-cell separation / purification module 20 for identifying and purifying cells in units of one cell. As shown in FIG. 4A, the image detection type 1-cell separation / purification module 20 includes a light source 201, a mirror 202, a condensing lens 203, a dichroic mirror 204, a filter 205, a light detection element 206 for fluorescence detection, a high-speed camera 207, And a cell sorter chip 209 for introducing a cell sample. In the module of FIG. 4A, a light source 201 such as a pulse laser or a high-intensity LED light source, a light detection element 208 such as a photodiode that detects passage of cells with scattered light, and fluorescence are detected for cells passing through the cell sorter chip 209. A plurality of pieces of information can be detected simultaneously by a high-sensitivity light detection element 206 such as a photomultiplier, a high-speed camera 207, or the like. 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 the processing using an image and the 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.

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

The cell separation and purification module is configured on the chip. FIG. 4B schematically shows an example of a cell sorter chip 209 configured on such a chip. A microchannel 211 is provided inside the chip substrate 210, and an opening communicating with the channel is provided on the upper surface to serve as a supply port for a sample and a necessary buffer solution (medium). The flow path can be created by so-called injection molding in which a plastic such as PMMA is poured into a mold, or can be created by bonding a plurality of glass substrates. The size of the chip is, for example, 50 × 70 × 1 mm (t), but is not limited thereto. 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 210. In addition, the cells envisaged in the present invention are bacteria at a small size, and animal cells at a large size, such as cancer cells. Therefore, the cell size is typically in the range of about 0.5 μm to 30 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 done. 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 211 is formed on one of the substrate surfaces in a substantially two-dimensional plane in a space typically 10 to 100 μm in the thickness direction of the substrate. The appropriate size is 5 to 10 μm in the thickness direction for bacteria and 10 to 50 μm in the thickness direction for animal cells based on the cell size.

First, on the chip 209, the sample solution is introduced from the inlet 212 into the microchannel 211 by a syringe pump or cell introduction means that does not generate a pulsating flow such as air pressure. The sample liquid containing the cells introduced into the microchannel 211 flows along the streamline of the particle flow 218 before application toward the outlet 213 downstream, and is discharged. At this time, means for continuously applying an external force to the cells is introduced so that the cells are concentrated toward the cell concentrate inlet 214 arranged in a part of the side wall of the microchannel 211. The cells are concentrated along the cell flow 219 after application, and a cell concentrate having a concentration 100 times or more the cell concentration introduced at the inlet 212 is introduced into the cell concentrate inlet 214.

As the external force applied to the cells at this time, ultrasonic radiation pressure, gravity, electrostatic force, dielectric electrophoretic force can be used. For example, when ultrasonic radiation pressure is used, a traveling wave of ultrasonic waves is generated in the direction of the cell concentrate inlet 214 and orthogonal to the flow of the sample liquid, and is applied by the ultrasonic radiation pressure after application. A cell stream 219 can be generated. As for the ultrasonic wave introduction means, a PZT-based piezoelectric element may be adhered to the surface of the chip 209, or a comb-shaped electrode array is disposed on the surface of the piezoelectric element so that surface acoustic waves are generated in the cell concentration unit 215 This may be applied to the surface of the cell concentrating unit 215, and the fact that the soaked ultrasonic waves are introduced into the cell concentrating unit 215 may be used. When gravity is used, the spatial arrangement of the chip 209 may be adjusted so that the direction of the concentrated liquid inlet 214 is perpendicular to the flow of the sample liquid and the direction of the concentrated liquid inlet 214 is the direction of gravity, or The chip 209 may be arranged on a disc that can rotate, perpendicular to the flow of the sample solution, and the cell concentrate in the same direction as the radial direction of the disc. When electrostatic force is used, an electrode is arranged on the side wall of the microchannel 211 so that the cell receives an external force toward the side wall. In that case, the cell of the target cell It may be determined which charge is applied depending on whether the surface potential is positive or negative. However, when an electrostatic force is generated, bubbles are generated from the electrode when the potential of the electrode surface to which current is applied exceeds a certain potential such as a peroxygenation potential or a perhydrogenation potential. Therefore, the flow path distance of the micro flow path 211 must be flexibly adjusted according to the type and strength of the external force applied to the cell, for example, sufficiently long in the case of electrostatic force It must be a thing. When the dielectrophoretic force is used as an external force, the dielectrophoretic force is applied in the cell concentration unit 215 so as to be orthogonal to the flow of the sample liquid and in the direction of the concentrate inlet 214. An electrode may be disposed.

Next, as shown in FIG. 4C, the concentrated cell liquid into which the cell concentrated liquid has been introduced into the concentrated liquid inlet 214 is arranged in a line along the flow in the solution at the converging unit 216. Specifically, it has means for generating an external force so that cells are attracted to the central part of the flow path of the converging unit 216 by using a dielectric electrophoretic force or a standing wave mode of ultrasonic radiation pressure. is there. The cells arranged in a straight line in the center in this way are measured in the cell detection region 218 arranged in the preceding stage of the cell sorting unit 217, and after determining the type of each cell, from upstream to downstream. Guided to one of the two downstream areas, ie, the first outlet 221 and the second outlet 222, which are branched in the cell sorting part branching part 220, depending on whether or not an external force is applied in a direction perpendicular to the flow of It becomes.

As an example of the actual configuration of the converging unit 216, as an example of the configuration of the electrode arrangement in the case where the external force is realized by the dielectric electrophoretic force, the wedge-shaped electrodes (converging V-shaped comb electrodes) 225 are alternately arranged. And applying an alternating voltage to the converging V-shaped comb electrode contact allows the cell to be directed toward the position of the wedge-shaped apex, thereby applying an external force to the cell. As a result, the position of the wedge-shaped apex Cells can be continuously concentrated. What is important in the electrode of this embodiment is the shape of the electrode arranged in the flow path, that the electrode has an angle toward the downstream side, and that this electrode has a sharp tip instead of a straight line, and Because of the comb-shaped electrode array that has the shape of the axis, the cell that receives the dielectric electrophoretic force is affected by the flow regardless of whether it receives a repulsive force or an attractive force. The cells are guided and arranged at the acute electrode tip portion by the resultant force of the force swept downstream and the force applied to the cell toward the sharp tip portion. In other words, by placing the position of the apex of the acute angle of the electrode array at the position where the cells in the flow channel are to be concentrated, the cell is forced to flow downstream by the flow and the dielectric electrophoresis directed toward this acute angle tip direction. The resultant force and force gather at the acute angle tip.

FIG. 5 illustrates an example of the configuration of a one-cell genome analysis / expression analysis module 30 that performs gene analysis and expression analysis of purified cells. The reaction tank 301 is composed of a thin plate of aluminum, nickel, or gold having a plurality of depressions. The thickness of the thin plate in the depression region is about 10 to 30 microns, and the region between adjacent depressions has a thickness of 100 to 500 microns in order to ensure overall strength. The reaction vessel 301 is fixed to the bottom surface of a square or circular reaction vessel frame, and has a structure that can be easily detached from the reaction vessel heat exchange vessel 302. The temperature of the liquid introduced into the reaction tank heat exchange tank 302 is overheated by a heat source disposed inside the liquid reservoir tank 303. An agitation mechanism is provided to quickly remove heat from the surface of the heat source and to make the temperature inside the liquid reservoir tank 303 uniform. The liquid in the liquid reservoir tank is guided inside the flow path by the pump 304. The liquid is led to the reaction tank heat exchange tank 302 by the switching valve 305 or directly returned to the liquid reservoir tank 303 by being led to the bypass flow path. If necessary, the temperature of the liquid is slightly controlled by the auxiliary temperature control mechanism 306 so as to suppress temperature fluctuation in the liquid reservoir tank 303. The basic configuration of the reaction tank heat exchange tank 302 includes an inlet A (307) and an inlet B (308) for introducing liquids having different temperatures. As for the number, the number corresponding to the plurality of temperatures for which the temperature of the sample solution is to be changed will be prepared as a plurality of two temperatures or more. For example, when it is desired to achieve a three temperature system, the number is three. It is not limited to two. Further, in order to return the liquid in the reaction tank heat exchange tank 302 to the liquid reservoir tank 303, a plurality of outlets, outlet A (309) and outlet B (310) are provided. The number is not limited to two. Various types of reaction vessels can be used, and reaction vessel A, reaction vessel B, reaction vessel C, and reaction vessel D are shown as an example. Here, as the liquid in the reaction tank heat exchange tank 302, water may be used, but a liquid having a large heat capacity and low viscosity may be used. For example, liquid ammonia. Moreover, about the liquid of the reaction tank heat exchange tank 302, by using the liquid whose boiling point is higher than water, the sample liquid is surely set to 100 degrees, or the liquid having a lower freezing point than water is used. It is also possible to reliably change the temperature up to the freezing point of water while preventing freezing of the circulating liquid.

In addition, the reaction vessel frame can measure the change in fluorescence intensity of the fluorescent dye in the sample solution, which changes due to the reaction of the sample solution 311 in the reaction vessel 301, for each of the one or more reaction vessels 301. An optical window that transmits the excitation light of the fluorescent dye and the fluorescence is arranged, and the fluorescence detector 312 is arranged to measure the temporal change of the measured fluorescence intensity of each reaction vessel 301. it can. In the embodiment of FIG. 5, each of the plurality of fluorescence detectors 312 includes an excitation light irradiation mechanism and a fluorescence detection mechanism, and each of a plurality of reaction vessels 301 to which different primers or different sample solutions are dropped. It is the structure which can measure the PCR amplification information of which is different independently. The fluorescence intensity data acquired by the fluorescence detector 312 is recorded by the control analysis unit 313 and has a function of estimating the amount of DNA or mRNA in the sample solution obtained by the PCR reaction. Further, the control analysis unit 313 obtains the switching information of the switching valve 305, thereby estimating whether the temperature change of the sample liquid 311 after the valve switching has reached the target temperature from the change in fluorescence intensity over time, And a mechanism for controlling valve switching based on the result. This is due to the fact that the fluorescence quenching based on the movement of water molecules universally possessed by fluorescent dyes depends on the liquid temperature, so that the amount of change in fluorescence intensity per unit time becomes small or zero. This is an estimate, and is particularly effective in confirming the achievement of a high temperature state in which DNA is denatured.

Further, in this embodiment, one detector is arranged in each reaction vessel 301. However, the fluorescence intensity change in a plurality of reaction vessels can be changed by combining a fluorescent example light source and a camera capable of quantitative fluorescence detection such as a cooled CCD camera. You may measure. Alternatively, when fewer detectors than the number of reaction vessels 301 are used, the fluorescence intensity of all reaction vessels can be measured by combining a mechanical drive mechanism that can move at high speed on the XY plane with the detectors. You may do it.

It is convenient to freeze and dry the reagents necessary for the reaction. It is possible to prepare a freeze-dried reagent at the bottom of the reaction vessel. Further, if a plug-like freeze-dried reagent is formed inside a dispensing tip used when dispensing a sample, the reagent can be dissolved in the sample by moving the sample up and down. Alternatively, it is also possible to dissolve the freeze-dried reagent by forming a freeze-dried reagent on the surface of the fiber ball on which nylon fibers or the like are bundled, and inserting the sample into a sample inside the reaction vessel and stirring.

Since it is inconvenient to directly handle the reaction tank 301 composed of a thin film, it is convenient to fix the reaction tank 301 to the reaction tank frame. The reaction vessel frame is preferably formed of a heat-insulating material such as polystyrene, polycarbonate, PEEK, acrylic, and the like, and the reaction area of the reaction vessel 301 can be controlled quickly and with high accuracy. Is desirable. When mounting the reaction tank 301 to the reaction tank heat exchange tank 302, there is a method in which a thread is formed on the surface of the reaction tank frame and the reaction tank frame is screwed. In order to maintain watertightness, it is desirable to attach a seal to the opening. Alternatively, it is possible to employ a tapered reaction vessel frame and attach it only by pressure.

Next, a specific example of the valve switching mechanism is shown. There are an inlet valve A and an inlet valve B for introducing a liquid into the reaction tank 301, and an outlet valve A and an outlet valve B for introducing the liquid to the outside. The liquid guided from the inlet valve A returns from the outlet valve A to the liquid reservoir tank, and the liquid guided from the inlet valve A returns from the outlet bawl B to another liquid reservoir tank. By alternately repeating these two states, the sample in the reaction vessel can be reacted. As a more preferable valve switching method, in addition to the above two states, the inlet valve B and the outlet valve A, or the inlet valve A and the outlet valve B are simultaneously opened for a moment so that liquids of different temperatures are mixed. And the temperature control of the liquid reservoir tank of each system becomes easy. The conditions for PCR are, for example, reaction buffer 1.0 μL, 2 mM dNTP (dATP, dCTP, dGTP, dTTP) 1 μL, 25 mM magnesium sulfate 1.2 μL, 10% fetal bovine serum 0.125 μL, SYBR Green I 0.5 μL, primer Two types can be used: 0.6 μL each, sterilized water 3.725 μL, KOD plus polymerase 0.25 μL, and genomic DNA 1.0 μL. As the temperature condition, first, 95 ° C. for 10 seconds, and then temperature change of 95 ° C. for 1 second and 60 ° C. for 3 seconds can be measured in 40 cycles.

FIG. 6 illustrates an example of a configuration of a liquid feeding module 40 that transports a sample between the modules. It has a dispensing head 401 and a dispensing tip 402 for exchanging liquid between each module arranged on the chassis 406, and controls the height direction of the dispensing head in the Z-axis direction. The Z-axis movement guide 403 and the Z-axis movement motor 404 as functions and the arm rotation motor 405 as an arm rotation mechanism have a function of controlling the position of the dispensing head 401 on the XY plane.

FIG. 7 shows cell expression of a sample in which a nucleic acid component in a cell is not easily eluted into a sample solution by a shell covering a cell such as an anthrax spore in a cell analysis performed using the cell analyzer of the present invention. An example of a procedure from collection of a sample into which a procedure for crushing a shell covering a cell is introduced before an analysis procedure is shown. By adding this procedure for cell disruption, the present cell analyzer can analyze cells such as spores of Bacillus anthracis by means similar to the means for analyzing blood cells described above.

FIG. 8 schematically shows an example of a basic structure for automatically crushing a shell of a spore or the like covering a small amount of sample cells in order to analyze intracellular genes and expression information for cells having spores such as anthrax. Indicate. A minute sample 802 is dispensed into the container 801, and a rotating body 803 for crushing is placed inside the container 801. In order to rotate the rotating body 803 inside the container 801, the rotating body 803 is pressed against the container 801 by the rotating shaft 804. As the rotating body 803 rotates and revolves inside the container, the sample 805 in the minute sample is ground by the abrasive 806. After the process is completed, the processed sample 805 can be easily recovered by removing the rotating body 803. Since the rotating body 803 and the container 1 have a simple structure, there is no problem even if they are handled as consumables.

FIG. 9 schematically shows various variations of the basic cell disruption mechanism shown in FIG. In order to crush the sample with high efficiency, it is necessary that the rotating body touches the container closely. In the example shown in FIG. 9A, the container 811 including the rotating body 810 is held by a flexible structure 812 such as rubber. Since the tip portion 814 of the shaft 813 is cut obliquely, when the shaft 813 is pressed against the rotating body 810, the rotating body 810 presses the container 811 downward and laterally, and the flexible structure 812 By deforming, pressure is absorbed. As a result, the sample can be crushed while holding the rotating body 810 and the container 811 closely without giving excessive stress to the rotating shaft 813. As shown in FIG. 9B, as a method of releasing stress, it is possible to incorporate a spring mechanism 815 that deforms vertically and laterally inside the rotating shaft.

FIG. 10 shows the possibility of rotating bodies and rotating shafts of various shapes in the cell disruption mechanism used in the present invention. In addition to the shaft whose tip is cut obliquely (FIG. 10a), it may be recessed in a gently curved surface (FIG. 10b), bowl-shaped (FIG. 10c), or the like. Further, the rotating body does not have to be a true sphere, and may have a structure in which the shaft and the rotating body are gently engaged with each other (FIG. 10d). The hemisphere may be rotated with a rotating shaft cut diagonally (FIG. 10e). Further, it may have an egg-shaped rotating body (FIG. 10f) or a protruding structure that meshes with the rotating shaft (FIG. 10g). It is also possible to rotate a dish-like rotating body with a shaft (FIG. 10h).

FIG. 11 shows an example of the cell disruption step in the present invention. A rotating body 831 and an abrasive 832 are enclosed in the container 830 (FIG. 11a). Immediately before crushing, the seal 833 is broken (FIG. 11b), and a sample 834 containing cells is dispensed into the container 830 (FIG. 11c). By rotating the rotating body 831 while pressing it with the rotating shaft 835 (FIG. 11d), the cells in the sample are crushed by the abrasive 832 and the component 836 is eluted (FIG. 11e). After the crushing operation is completed, the sample can be easily collected by removing the rotating body 831 from the container 830 (FIG. 11f). As a method for removing the rotating body, negative pressure, magnetic force, and electrostatic force can be used, and such a mechanism can be incorporated in the rotating shaft. Of course, a special mechanism may be prepared separately.

FIG. 12 shows a conceptual diagram of a mechanism that can be used when the cell disruption process in the present invention is automated. A plurality of containers 840 are integrally formed, and a rotating body is sealed in advance in each container. In order to break the seal, it is possible to directly press and tear the rotating shaft (FIG. 12A), or to break with the opening cutter 841 attached to the rotating shaft (FIG. 12B). The relative position of the shaft and the container can be automatically changed, and a plurality of samples can be crushed one after another.

FIG. 13 shows an example of the on-chip cell sorter chip of the present invention that can also be used in the cell analyzer system of the present invention. In the cell sorter chip 1301, three axial flow paths are arranged symmetrically on the upstream side (1302, 1304, 1306) and downstream side (1303, 1305, 1307) on the chip substrate. At the merging point of the road, the three flow paths merge while maintaining a laminar flow, and maintain the state as it is and branch to the three downstream flow paths. Therefore, the upstream side 1302 of the central flow path through which the sample flows is changed to the downstream central flow path 1303, and the upstream side flow path 1304 to the downstream side 1305, and the upstream flow path 1306, respectively, for the two side sheath flows. To the downstream flow path 1307. In addition, the inlets of the three upstream channels are connected to

inlet openings

1308, 1309, and 1310, respectively. In particular, the inlet opening 1308 upstream of the flow path through which the sample flows is connected to the sample reservoir 1322, typically (but not limited to) by adding a small annular cap (or stopper). The

inlet openings

1309 and 1310 of the flow path for flowing the sheath liquid stored in the sheath liquid reservoir 1311 are separated from each other and are arranged so that the sample solution does not diffuse. The downstream reservoir is also arranged in the same manner as the upstream side, and the waste liquid reservoir 1312 is connected to the

outlet openings

1313 and 1314 of the flow path through which the two side sheath liquids flow. Is connected to the outlet opening 1315 of the recovered purified sample, and typically (but not limited to) a small annular cap is added to the outlet opening 1315 to recover the recovered purified sample. Does not diffuse into the waste reservoir.

In addition, the flow rate is generated using a gravity type that utilizes the difference between the liquid level of the sample reservoir and the sheath liquid reservoir and the liquid level of the waste / recovered liquid reservoir, or pressurized air with a cap attached to the upper surface of the reservoir. When pressure is applied to the liquid surface to generate a flow velocity, an ideal laminar flow is generated at the confluence, so that the three flow velocities are the same, so that the upstream and downstream from the confluence For each of the three flow paths on the side, it is preferable that the cross-sectional shape of the flow path and the distance from the merging point to the solution inlet coincide with each other. Further, the ratio of the cross-sectional area of the side sheath flow (or waste liquid) reservoir to the cross-sectional area of the inner sample / recovery sample reservoir is 1 (sample / recovery reservoir): 2 (side sheath liquid reservoir / waste liquid reservoir). It is desirable. This is because if the change in the liquid level of each reservoir is different, the rate of decrease in the liquid level will be different, which will eventually destroy the generation of laminar flow at the confluence. It is. Therefore, since the flow rate of the liquid flow per unit time is 1: 2 in the sheath liquid having two inlets with respect to the sample inlet 1, the cross-sectional area of each reservoir is adjusted so that the liquid level is the same. The ratio was set to 1: 2. To make this universal, it is desirable that the ratio of the total cross-sectional areas of the flow paths coupled to the respective reservoirs matches the ratio of the cross-sectional areas of the respective reservoirs.

Next, an electrode is arranged at a point where three laminar flows without walls join, where all six channels join. The electrode is typically composed of a gel electrode. As the gel, for example, an agarose gel in which NaCl is dissolved so that the electrolyte becomes a current carrier is used. The gel is placed in a Y-shaped channel 1316 for gel filling so that the gel tip can be in contact with the agarose gel in a sol state from the inlet 1317 so that the gel can be directed to the outlet 1318. The gel does not penetrate into the cell sorter flow path and stops at the boundary line due to surface tension. As an advantage of using a gel electrode, by inserting a wire 1319 such as a platinum wire connected to a power source 1320 to apply an electric field to this gel introduction point, at the gel electrode boundary in contact with the flow path, a normal metal electrode Even if the bubbles are raised to a voltage higher than the voltage generated in the flow path, no bubbles are generated and a current can be applied. The on-off of the electric field application can be adjusted using the switch 1321, for example.

FIG. 14 schematically shows an example of the cross section of the upstream reservoir, particularly in the AA cross section of FIG. A flow path 1409 is embedded in the cell sorter chip 1401. In a typical embodiment, the upper surface of the outer sheath fluid reservoir 1403 is blocked by the cap 1402, so that air pressure at an appropriate flow rate is supplied from the pressurized air introduction pipe 1405. As can be seen from the figure, the flow path 1409 through which the sample flows is connected to the sample reservoir 1404 so that the sample liquid and the sheath liquid are not mixed. In a preferred and typical embodiment, the ratio of the cross-sectional area between the sample reservoir and the sheath fluid reservoir is 1: 2 because the ratio of the number of channels is 1: 2, and the ratio of each reservoir connected to each channel is The liquid level is adjusted to be the same.

A mechanism for supplying liquid can be added so that a larger amount of sample can be processed. This includes a sample solution introduction tube 1406 or a sheath liquid introduction tube 1407, and a water level measurement sensor 1408 using conductivity measurement on the wall surface of each reservoir. As a result, when the water level falls below a certain level, the sample solution can be supplied through the tube until it reaches a certain height. The water level measurement sensor 1408 can be configured by electrodes or electrode pairs disposed at the lower limit of the water level and the upper limit of the water level, which are desired to be set.

FIG. 15 shows an example of another configuration for handling a large amount of sample in the cell sorter of the present invention. Three large reservoirs 1502 are arranged on the chip 1501 upstream of each of the three flow paths, and these are distributed more flexibly using the distribution valve 1505 from the air pressure application device 1503 through the pressure sensor 1504. can do. Regarding sample collection, both the sorted (purified) sample and the waste liquid are collected in the sorted sample collection reservoir 1506 and the waste liquid collection reservoir 1507, respectively, disposed at positions below the chip.

FIG. 16 schematically shows a procedure for collecting an actual sample in the chip. The sample solution stream 1601 flowing from the upstream is sandwiched between the two side

sheath solution streams

1602 and 1603 and proceeds to the cell monitoring region 1604 while maintaining the arrangement. Therefore, the shape discrimination of each cell, the presence or absence of a fluorescent label, etc. are confirmed, and cell separation is performed downstream based on the results. When the cells to be collected flow, they flow as they are to the downstream sorted sample collection flow channel 1606, and when the cells or fine particles to be discarded flow, they are arranged to face each other regardless of whether the charge is positive or negative. In addition, by applying a voltage to the two gel electrodes 1605, the gel can move to one of the two side sheath flows 1607 and be eliminated.

FIG. 17 is a schematic diagram for explaining one of the indices of cell recovery in the image processing cell sorter. Usually, the cell is in the G0 cycle and has a nucleus (FIG. 17A (a)), which is clearly image-recognized as a black sphere inside the cell (FIG. 17B (a)). On the other hand, since the nucleus of the cell in the division phase has disappeared (FIG. 17A (b)), the nucleus cannot be confirmed even if the cell is image-recognized (FIG. 17B (b)). With conventional labeling techniques such as antibody labeling, it is difficult to confirm the state of the cell. In the present invention, in addition to confirming the shape of the cell by such image recognition, the presence or absence of a nucleus in the cell Dividing cells can be recovered. In general, most normal cells flowing in the blood are already terminally differentiated, but by collecting cells that have undergone cell division in the blood according to the present invention, blood cancer cells, stem cells, etc. It becomes possible to collect cells having the ability to divide.

FIG. 18 is an example of an operation timing chart when using a flash light source when actually operating the image recognition type cell sorter of the present invention. When observing cells moving through the chip using a high-speed camera, in order to prevent blurring, the spatial resolution of one pixel of the camera is calculated based on the magnification of the objective lens, and the moving speed of the sample flow is determined. If this is the case, it is only necessary to calculate the amount of movement for one pixel size at that flow rate and turn on the flashlight only for the time required for the movement. That is,

Flash time = Pixel size / flow velocity
Thus, the flash may be emitted once in each shutter interval. For example, the pixel size of a 1/2000 second camera is 12 μm × 12 μm, and the pixel resolution when viewed with a 20 × objective lens is 0.6 μm / pixel. If an LED light source that can perform flash firing at a high speed is used, it is possible to actually obtain an image without blurring.

The present invention also provides an on-chip cell sorter system that describes exemplary aspects below. In these systems, control of each part (for example, image acquisition and analysis by an optical system, external force addition by an external force application device, etc.) is performed using a control device including a personal computer or the like as in the above embodiment. be able to.

FIG. 19 is a diagram schematically showing an example of the configuration of an optical system for preventing image blur in the image detection type 1-cell separation / purification (cell sorter) module. Usually, when observing target particles using an optical system of a microscope, the magnification ratio of the object image is determined only by the magnification of the objective lens. It depends on the magnification and numerical aperture of the objective lens. The higher the magnification of the objective lens, the shallower the focal depth and depth of field of the optical system.

When observing an object in a microchannel, if the object is a cell, samples of various sizes from small to several tens of microns in size can flow. The width and depth of the path must be large enough for the largest sample size to flow. However, in order to recognize the image and discriminate the sample type, it is preferable that the resolution of the image is high. In general, in order to increase the magnification with an optical microscope, it is usual to use an objective lens with a higher numerical aperture. However, using such a means reduces the depth of focus, and as a result, the image is captured in the flow path. There was a problem that the depth of field would also be shallow. In order to identify the sample from a higher-definition image with an image recognition type cell sorter, in order to increase the magnification of the target sample and to make the depth of field about the height of the flow path, first the focus of the objective lens It is only necessary to select an objective lens having a numerical aperture at which the depth and depth of field are about the height of the flow path, and to insert a zoom lens after the objective lens. Specifically, as shown in FIG. 19A, an objective lens and a zoom lens may be combined and this magnified image may be captured by an image acquisition device such as a CCD camera.

Specifically, for example, as shown in FIG. 19B, when a 10 × objective lens with a numerical aperture (NA) of 0.3 is used to measure the imageable height at this time, the image can be obtained up to about 15 μm without any problem. You can get that. On the other hand, with an objective lens with a numerical aperture of 20 times 20 times and an objective lens with a numerical aperture of 0.6 and magnification of 40 times, an image with a blur of only about 5 μm can be acquired.

From the above results, it was confirmed that a depth of focus up to a depth of about 15 μm could be secured by using an objective lens with a numerical aperture of 10 × and a zoom lens with a numerical aperture of 0.28 was placed behind this objective lens. The measured result is shown in FIG. 19C. As can be seen, the image is a combination of an objective lens 10 × and a zoom lens 1 × combined image, an objective lens 10 × combined with a zoom lens 2 × image, and an objective lens 10 × combined with a zoom lens 4 × image. It can be seen that when the depth and the depth of field are combined and the image is not blurred, the image can be sufficiently observed even when the magnification of the zoom lens is different up to 25 μm.

This result is obtained by combining a 10 × objective lens with a 4 × zoom lens when an image processing cell sorter system obtains an image with the same magnification as that conventionally observed with a 40 × objective lens. An example of a configuration that can obtain an image that is optimal for cell sorting and that has no blur in the height direction of the flow path is shown.

In FIG. 20, since the conventional cell sorter chip is horizontally arranged, the cell sorter chip is arranged vertically to prevent the sample from sinking to the bottom of the horizontally flowing channel and causing clogging. The configuration is such that the settling direction and the flow direction are the same. The cell sorter chip 2001 in the cell sorter system is vertically arranged as shown in FIG. 20, and the upstream of the flow is installed upward, and the downstream is installed downward. The buffer introduction device 2003 and the sample solution introduction device 2006 are arranged. The sample solution and the buffer solution whose pressures are controlled by the pressure sensor 2004 are introduced into each solution reservoir 2002 connected to the upper surface of the chip. Here, when introducing into a plurality of reservoirs from one introduction device, the flow of each flow path can be finely adjusted by finely adjusting the open / closed state of the distribution valve 2005 according to the pressure and state of each reservoir. . Next, the liquids introduced from the reservoir are joined via the sample liquid channel 2007 or the buffer channel 2008, and the quantitative measurement of the amount of fluorescence and the amount of light scattering such as image acquisition of the cell type or labeling measurement such as fluorescence. In accordance with the determination based on the above result, the cell is moved vertically in the flow direction in the flow path by the sorting external force applying mechanism 2009, and as a result, the cells can be collected by the plurality of

downstream reservoirs

20101, 20102, and 20103.

FIG. 21 shows an example of another configuration of the cell sorter arranged in the vertical type shown in FIG. As shown in FIG. 21, the cell sorter chip 2101 in the cell sorter system is arranged vertically as in FIG. 20, and the upstream of the flow is installed upward and the downstream thereof is installed downward. The sample liquid and the buffer solution, the pressure of which is controlled by the pressure sensor 2104 from the sample liquid introduction device 2106, are introduced into the solution reservoir 2102 connected to the upper surface of the chip. The sample solution introduced after passing through is introduced into the flow path 2107 while being sandwiched between the buffer solutions. Next, the sort of external force application mechanism 2109 sorts the cells in the flow path in accordance with the determination based on the result of quantitative measurement of the amount of fluorescence and light scattering, such as image acquisition of fluorescence or other labeling measurement. As a result, the plurality of

downstream reservoirs

21101 and 21102 can be collected.

FIG. 22 is a diagram schematically showing an example of the configuration of the portion where the sample solution and the buffer solution of the cell sorter chip shown in FIG. 21 merge. The sample liquid is introduced into the flow path 2107 by the capillary tube 21061, the buffer liquid is introduced into the reservoir 2102 connected to the upper surface of the end point of the flow path 2107, and the buffer liquid is arranged to be introduced into the flow path 2107. Has been. In this configuration, the outlet of the capillary tube 21061 is disposed downstream of the reservoir portion, so that the sample solution can be introduced into a desired position in the buffer flow path. When sorting cells, it is desirable to arrange the capillary tube 21061 so as to approach one side of the flow path 2107. In this embodiment, the flow of the sample solution is arranged in the buffer channel using the capillary tube. However, if the sample solution can be introduced into the place where the buffer flow is generated, the capillary tube is not particularly used. However, the same effect can be obtained by combining the flow paths configured by microfabrication.

FIG. 23 is a diagram schematically showing an example of a chip configuration incorporating a cell sorting mechanism in the cell sorter system. A sample introduction port 23002 is arranged upstream in the chip 23001. In the sample solution introduced from here, target particles 23003 to be collected and unnecessary particles 23004 to be discarded are mixed. Here, the sample solution flows through the microchannel 23005 and is first introduced into the particle alignment mechanism 23006. In this particle alignment mechanism, a particle alignment external force input (electric force or sheath flow) 23007 is arranged. When the particle alignment mechanism is arranged by an electric force, a gel electrode is introduced on both sides of the flow path 23005 to introduce an electric field. In the case of being aligned in the sheath flow, a buffer solution is introduced. Next, for the samples arranged in a line, the type of particles is identified by the particle detection mechanism 23008, and the particles are separated by the particle purification mechanism 23009 which is the next step. In this particle detection mechanism 23008, a particle purification external force input ((gel or metal) electrode + electric force) 23010 is arranged, and an electric force can be applied to the position of the particle purification mechanism 23009 using a gel electrode or a metal electrode. it can. For example, when the target particle to be collected has arrived at the particle purification mechanism 23009, the particle is allowed to flow downstream without applying an external force and collected at the collection port 23011. When particles other than the particle to be collected have arrived, By applying an external force, the particles can be guided to

unnecessary particle reservoirs

23012 and 23013.

FIG. 24 is a diagram showing an example of the arrangement of electrodes for applying an external force in the flow path of the cell sorter system. As shown in the schematic view from the upper surface of FIG. 24A and the schematic view from the cross section of FIG. 24B, the metal thin film electrode first layer 24002 and the metal thin film electrode The two comb-shaped electrode portions of the two-layer 24003 are arranged so as to be slightly shifted from each other with the insulating film layer 24004 interposed therebetween, and the second-layer electrode 24003 is in direct contact with the sample channel 24005. FIG. 24 (c) shows a photograph of an example in which the comb-shaped electrode array is actually arranged on the bottom surface of the channel with such a configuration.

FIG. 25 is a diagram showing an example of the arrangement of electrode arrays for applying an external force in the flow path of the cell sorter system. FIG. 25 (a) shows a case where the sample particles 25004 introduced into the sample introduction port 25001 flow through the micro flow channel 25002, and the metal thin film stacked parallel type having the configuration as shown in FIG. When an alternating electric field is generated in the metal thin film stacked parallel comb electrode 25003 when the comb electrode 25003 is disposed, the sample particles 25004 are directed to the upper surface by the dielectric electrophoretic force. At this time, as shown in FIG. 25 (d), when the cross-sectional shape of the flow path receives repulsive force from the bottom surface and the particles progress to the top surface, the semicircular cross-sectional configuration is such that the particles gather at a specific top surface position. It has become. Although a semicircular shape is used here, a triangular cross-sectional configuration may be used. FIG. 25 (b) uses a metal thin film laminated type V-shaped comb electrode 25005. Similarly, by generating repulsive force from the bottom electrode 25007, particles are induced to the top wall, Depending on the shape, the fine particles can be arranged in a line in the center of the flow path. FIG. 25 (c) shows an arrangement of fine particles by adding a buffer fluid sheath channel 25006 from both sides of the channel 25002. FIG.

FIG. 26 is a schematic diagram showing an example of a cell purification process in the flow path of the cell sorter system. The following cell purification process after cell identification is described. When the target particles 26001, unnecessary particles (negatively charged) 26002, unnecessary particles (positively charged) 26003 are arranged in a line and flow through the microchannel 26004, where the target particles are in the region of the particle purification electrode 26005, Since the electrode is OFF, the target particles are guided to the flow 26006 to the target particle recovery port. On the other hand, when the external electric field is turned on when the unnecessary particles arrive, unnecessary cells are attracted to the anode or the cathode, respectively, are removed from the center of the flow path, and are guided to the

flows

26007 and 26008 to the unnecessary particle reservoir.

FIG. 27 is a schematic diagram showing an example of the arrangement of gel electrodes that give an external electric field in the flow path of the cell sorter system, and a photograph of an example of an actual chip. As shown in FIGS. 27A and 27C, the electrode gel liquid junction part 27004 is arranged adjacent to the microchannel 27001. Here, the electrode gel liquid junction part 27004, which is a boundary separating the flow path 27001 and the gel, introduces the gel from the electrode gel inlet 27002 to the electrode gel passage 27003 and fills the electrode gel outlet 27005. In such a case, the gel is prevented from leaking out by surface tension so that the gel does not flow out to the channel 27001. Specifically, the side surface of the flow path 27001 has a structure in which a large number of columns are arranged instead of walls, and the gel and the liquid flowing through the channel are in contact with each other through the space between the columns. The width is preferably 500 μm or less. Further, the end point of the gel filled with the electrolyte is connected to the metal wire 27006, and bubbles and the like generated when an electric field is applied to the electrode are generated not at the flow path but at the position of the electrode wire. This electrode is connected to a DC voltage source 27007 and controls ON / OFF based on the observation result of particles flowing by the voltage application switching mechanism 27008. For example, when an electric field is applied when the sample particle 27709 to be collected flows, the position of the particle in the flow path can be changed, whereby the fine particles can be purified. Although the gel electrode is used here, the metal thin film electrode 27010 may be used when the applied voltage does not reach the potential at which bubbles are generated (FIGS. 27B and 27D). By constructing such an array of gel end points of the gel electrode in contact with the sample liquid in the flow path through which the sample particles flow, sufficient external force can be applied compared to the application of an external electric field at a single pin point. It can be given to the sample and the cells can be moved sufficiently.

FIG. 28 is a schematic diagram showing an example of a process for identifying cardiomyocytes and fibroblasts to be separated according to an image in the image recognition type cell sorter system. In cardiomyocytes, the surface is very smooth (smooth surface) as can be seen from the image (upper: original image) taken by the image acquisition mechanism of the cell sorter in FIG. It can be seen that the surface is very uneven (rough surface). This image is binarized like the image in the middle of FIG. 28 (binarized image) to clarify the image of the cell boundary surface. Here, the length l of the boundary line of the boundary is measured from the number of pixels of the boundary, and at the same time, the area S of the filled inner surface is calculated from the number of filled pixels. here,

The surface roughness (R) can be quantitatively quantified by comparing the actual perimeter length with the perimeter length when converted from the area into a circle. Actually, the numerical value of R is shown in the lower part of FIG. 28, but by performing more detailed measurement, when the value of R is less than about 1.1, this cell is a cardiomyocyte, and when the value is larger than this, It is known to be another cell. In this way, by using a specific numerical value R for purifying cardiomyocytes by image recognition as an index, it becomes possible to identify cells based on the difference in unevenness of the cell surface.

FIG. 29 is a schematic diagram showing an example of the configuration of a cell sorter system in which water and oil are combined. In this case, the sheath liquid reservoir 1311 is filled with oil having a specific gravity lower than that of water such as silicon oil, and when a sample aqueous solution is dropped into the sample inlet opening 1308, water containing the sample is obtained only in the channel 1302. Oil flows in the

flow paths

1304 and 1306 on both sides. In addition, since water and oil are not mixed, it is not necessary to isolate the entrance of the sheath liquid and the sample liquid with a cap or the like.

FIG. 30 is a schematic diagram illustrating an example of a configuration of a water and oil merging region of a cell sorter system in which water and oil are combined. As shown in the description of FIG. 29, oil is filled and the sample aqueous solution is introduced into the sample aqueous solution introduction port 3001, and the oil in the reservoir is introduced as it is into the remaining

oil introduction ports

3002 and 3003. The introduced sample aqueous solution and oil merge at the merge region 3004. However, when the sample aqueous solution 3005 is arranged as shown in FIG. 30B, the sample aqueous solution 3005 can be narrowed down by the oil 3006 as shown in FIG. . As an advantage, since water and oil are not mixed until the end, the sample aqueous solution can be easily recovered without diluting.

FIG. 31 is a graph showing the relationship between the electrolytic mass (conductivity) in an aqueous solution under various solution conditions and the cell separation rate. As can be seen from the figure, it is desirable to have a solution composition with an ionic strength such that the conductivity of the aqueous sample solution is 10 ² μS / cm or less. By doing so, it becomes easy to move the fine particles in the sample liquid by the electric field. Specifically, a solution composition that maintains osmotic pressure while lowering ionic strength is particularly important when sorting cells alive. For example, molecules that do not directly contribute to the increase in ionic strength, such as sugars and polymers, are desirably used as sample solutions during cell purification.

FIG. 32 is a diagram schematically showing an example of the configuration of an analysis system that simultaneously performs fluorescence intensity measurement and high-speed bright-field microscopic image acquisition. Monochromatic light for observation emitted from a bright field light source 3200 such as an LED flash light source synchronized with a frame interval of a high-speed camera is collected by a condenser lens 3201, and the flow of target cells in blood as described above flows. The cells in the cell sorting unit 3202 including the cell and the cell sorting chip incorporating the cell sorting mechanism are irradiated. The cells in the channel can be focused with the objective lens 3203. Here, a depth-of-field improving technique incorporating the zoom lens system may be incorporated. Stained with fluorescent excitation light irradiated to the objective lens from a plurality of fluorescent

light sources

3204, 3206, 3208 such as monochromatic lasers, and fluorescent antibodies bound to the cells in the flow channel, and nuclear staining fluorescent dyes (DAPI, Hoechst33258, etc.) Fluorescence can be generated from the formed nucleus. The intensity of the obtained fluorescence can be quantitatively measured by a

fluorescence detection system

3205, 3207, 3209 including a fluorescence intensity measurement system such as a photomultiplier tube or a photodiode. Here, although three systems have been described as examples of the excitation light and fluorescence detection systems, a plurality of fluorescences can be excited by a single excitation light, so that a plurality of free combinations are possible. In this way, a bright field image of a cell can be simultaneously acquired by the high-speed camera 3210 while performing fluorescence detection of the cell.

FIG. 33 is a diagram schematically showing an example of a specific configuration of the analysis system that simultaneously performs the fluorescence intensity measurement and the high-speed bright-field microscopic image acquisition shown in FIG. In this example, a high-intensity LED flash light source that emits monochromatic light in the infrared region is used as a light source for a bright field (high-speed camera), and lasers of 375 nm, 488 nm, and 515 nm are used as excitation light sources for fluorescent dyes. The introduction of fluorescence into a fluorescence detector using a dichroic mirror is arranged so that the wavelength increases monotonically from short wavelength light to long wavelength light as shown in FIG. The wavelength region is located in the high speed camera. As a result, the fluorescence intensity at various wavelengths and the bright field image of the cells in the cell sorter chip disposed in the microchip holder can be simultaneously measured.

FIG. 34 is a diagram schematically showing an example of the configuration of an analysis system that simultaneously performs fluorescence intensity measurement, high-speed bright-field microscopic image acquisition, and high-speed fluorescent microscopic image acquisition in the apparatus system shown in FIG. Monochromatic light for observation emitted from a bright field light source 3400 such as an LED flash light source synchronized with a frame interval of a high-speed camera is collected by a condenser lens 3401, and the flow of target cells in blood as described above flows. The cells in the cell sorting unit 3402 including the path and the cell sorting chip incorporating the cell sorting mechanism are irradiated. The cells in the channel can be focused with the objective lens 3403. Here, a depth-of-field improving technique incorporating the zoom lens system may be incorporated. With fluorescent excitation light irradiated to the objective lens from a plurality of fluorescent

light sources

3404, 3406, 3408 such as monochromatic lasers, the fluorescent antibody bound to the cells in the flow path, or a nuclear staining fluorescent dye (DAPI, Hoechst33258, etc.) Fluorescence can be generated from stained nuclei and the like. The intensity of the obtained fluorescence can be quantitatively measured by a

fluorescence detection system

3405, 3407, 3409 including a fluorescence intensity measurement system such as a photomultiplier tube or a photodiode. Here, although three systems have been described as examples of the excitation light and fluorescence detection systems, a plurality of fluorescences can be excited by a single excitation light, so that a plurality of free combinations are possible. Further, an image dividing system 3410 for dividing an optical microscopic image into a bright field image and a fluorescent image and simultaneously acquiring a plurality of images with a single high-speed camera light receiving element will be described later with reference to FIG. Then, while detecting the fluorescence intensity of the cells in this way, a bright field image of the cells can be simultaneously acquired by the high speed camera 3411.

FIG. 35 is a diagram schematically showing an example of an apparatus configuration that simultaneously acquires high-speed bright-field microscopic image acquisition and high-speed fluorescence microscopic image acquisition with a single high-speed camera light-receiving surface. The image data that has entered from the input optical path 3501 is first introduced into the first image dividing unit 3510. Here, a dichroic mirror 3511 with an angle adjustment function that can finely adjust the direction of reflection three-dimensionally, a specific wavelength is set as a cutoff wavelength, and a long wavelength region or a short wavelength region is reflected from that wavelength to filter system 3512. Can be introduced. Here, the filter system 3512 includes an ND filter for intensity adjustment for aligning the bright field or fluorescence intensity to some extent on a high-speed camera, or a bandpass filter for obtaining a fluorescent image in a sharper wavelength band. Next, a plurality of divided images are finely adjusted three-dimensionally in the direction of reflection through an image size adjustment system 3513 including a movable shielding plate for reducing the image size so that the light receiving surface of the high-speed camera does not overlap. Through a dichroic mirror 3514 with an adjustable angle function and an optical lens system 3515 for correcting a difference in image formation position from an image of another path caused by a path difference of an optical system including a wavelength difference of light to be further handled. The second image dividing unit 3520 having the same configuration is introduced. Furthermore, the image 3502 that is introduced into the third image dividing unit 3530 having the same configuration and finally output is such that microscopic images composed of monochromatic light of different wavelengths do not overlap on the light receiving surface of the high-speed camera. They are arranged in a cut-out size. Here, in this figure, three image dividing units having the same configuration are combined, but two or four or more may be used in combination. In addition, as shown in FIG. 35, the division system is “a dichroic mirror 3511 with an angle adjustment function capable of finely adjusting the reflection direction in three dimensions, a filter system 3512, and a movable shield for cutting and reducing the image size. A combination of an image size adjustment system 3513 composed of a plate, a dichroic mirror 3514 with an angle adjustment function capable of finely adjusting the reflection direction in three dimensions, and an optical lens system 3515 for correcting a difference in image formation position as a set. For this reason, as described above, the necessary number of divided image images can be combined by adjusting the number of connections in the divided system freely and flexibly as described above.

FIG. 36 is a diagram schematically illustrating an example of an image obtained by simultaneously acquiring a high-speed bright-field microscopic image and one high-speed fluorescence microscopic image with a single high-speed camera light-receiving surface, and an example of analysis information. The image 3601 at the time of input to the divided system is data of the cell 3600 in which information of light of a plurality of wavelengths is superimposed. By passing through the divided system shown in FIG. 35, the bright field image 3610 and The fluorescence image 3620 of the nucleus can be acquired simultaneously on one light receiving surface 3602. As can be seen here, the extra area on both sides of the input image is cut out by the image size adjustment system in the division system, so that a plurality of images can be acquired with a size that does not overlap on the light receiving surface. . Here, the position of each image on the light-receiving surface of the high-speed camera can be freely adjusted by adjusting the surface position of a plurality of dichroic mirrors with an angle adjustment function that can be finely adjusted in three dimensions. Further, by using the optical lens system 3515 for enlarging or reducing the image of a bright field image or fluorescent image, it is possible to form images with different magnifications on one high-speed camera light receiving surface 3602. This is because the magnification factor is reduced for bright-field images, especially for measurements including the surrounding conditions of cells, and the magnification factor is increased for fluorescent images for confirming the minute state in the cell. is there. An optical system combining images with different magnifications is a divided optical system shown in FIG. 35, “a dichroic mirror 3511 with an angle adjustment function that can finely adjust the reflecting direction in three dimensions, a filter system 3512, and a small image size. An image size adjustment system 3513 composed of a movable shielding plate, a dichroic mirror 3514 with an angle adjustment function capable of finely adjusting the reflection direction in three dimensions, and an optical lens system 3515 for correcting a difference in imaging position. When the combined configuration is modularized and used as a set, it is not limited to the application to an imaging cell sorter, but is a general optical bright field / It can also be incorporated into a fluorescence microscope system.

For a plurality of obtained images, only the cell image is extracted from the bright-field image, for example, by subtracting the image data when the previously recorded cells do not flow from the flowed cell image. be able to. Therefore, the cell size (area) and the cell perimeter can be obtained from the total number of pixels in the area where the data remains after subtraction or the total number of pixels at the boundary of the area where the data remains. . Furthermore, using these two data, it is possible to obtain the degree R of the irregularities of the cell shown in the above-described equation 1. Here, if R is about 1.3 or more, the cell cluster can be determined only from the cell cluster and the bright field image.

Similarly, a fluorescence image 3621 of the nucleus is obtained from the fluorescence image (nuclear staining) 3620, and the total number of nuclei, the area of the nucleus, and the fluorescence intensity, that is, the integrated value of luminance (comparable to the photomal data) is obtained. Can do. Further, since the bright field image and the fluorescent image are only taken at different wavelengths at the same place, the coordinate axes of both are the same. Therefore, although the shape of a cell cannot be measured with a fluorescent image, the relative position of a stained nucleus in a cell can be estimated using coordinates in a bright field image. Here, FIG. 36 shows an example of a normal isolated single cell having one nucleus, but data can be similarly obtained from a cell cluster or a multinucleated cell. Also, in the above-described system in which a plurality of images with different magnification rates are formed, the relative coordinate system is similarly combined with a relative coordinate system that takes into account the difference in magnification rate with the same origin (image center) as the center. If used, the same processing can be performed.

FIG. 37 is a photograph showing an example of an image obtained by simultaneously obtaining a high-speed bright-field microscopic image and a high-speed fluorescence microscopic image stained with nuclear fluorescence with a single high-speed camera light-receiving surface. As mentioned above, here, by matching the relative coordinates of the two images in advance, the position of the nucleus that can be identified by the fluorescence image can be determined at any part of the cell image or cell cluster image of the bright field image. Whether nuclei are distributed can be collated using relative coordinates of each other. By comparing the relative coordinates, it can be seen that in normal cells, one nucleus shines with fluorescence in a smooth cell surface and a normal size cell. On the other hand, in cancer cells, multinucleation is an indicator of one cancer cell, but as shown in the photograph, multiple nuclei glow in cells that have become larger than normal cells by comparing relative coordinates. You can see that In addition, in the blood in animals with cancer metastasis, the fluorescence of multiple nuclei in the cluster is observed in the blood of animals with cancer metastasis due to the fluorescence of cell clusters and the nucleus of each cell in the cluster. It can be seen from the fluorescence of such a plurality of nuclei that it is a cluster. Also, from the bright field image of the cluster, R> 1.3 at this time, and it can be seen that the identification of the cluster can be determined using only the bright field image without using the fluorescent image. Thus, using the apparatus of the present invention, (1) a method for identifying and selectively recovering cell clusters (lumps) that do not exist in healthy blood as blood cancer cell candidates, (2) healthy To identify and selectively collect polynuclear cells that do not exist in healthy blood as cancer cell candidates in blood, and (3) identify and select giant cells that do not exist in healthy blood as cancer cell candidates (4) In addition to (1), (2) or (3) above, cancer cell biomarkers (for example, EpCam antibody, K-ras antibody, site) measured by fluorescence intensity measurement shown in FIG. Blood cancer cells instead of conventional molecular biomarkers by a method that identifies and selectively recovers cancer cells by analysis combined with the presence of fluorescence intensity of fluorescent antibodies against one or more of keratin antibodies) ,"cell It is possible to identify and select and collect a new biomarker such as an image of the shape, grouping state, or internal structure such as multinucleation. In addition, blood cancer cell candidates collected by the above method can be combined with the above-described gene analysis means such as the PCR analysis technology for microcells, and finally measured for gene mutations. If it is a cancer cell, it is possible to finally identify the characteristics of the cancer cell. For (1), evaluation by R> 1.3 using a bright-field image, or the number and distribution of nuclei by size and fluorescence image by a bright-field image (that is, the distance between the centers of gravity of adjacent nuclei images is 3 μm It can be determined by the fact that they are far apart. For (2), R <1.3 is determined by the bright field image, and the number and distribution of nuclei (that is, the distance between the centroids of the images of adjacent nuclei is within 3 μm from each other). can do. As for (3), it can be determined from the bright field image that R <1.3 and that the cell size is more than 20 μm in terms of diameter. Alternatively, a combination of (1) to (3) above can be determined as a cancer cell if there is one or more matching conditions.

FIG. 38 shows an example of simultaneously irradiating a plurality of wavelengths of fluorescence excitation light to cells such as blood flowing in a microchip simultaneously using a fiber optic array, It is the figure which showed typically an example of the apparatus structure for acquiring a fluorescence image simultaneously.

The apparatus of the embodiment of FIG. 38 includes an excitation light source unit (3801-3807) including a fluorescent excitation light source for generating six different monochromatic excitation lights and a bright-field microscopic image light source. Each excitation light source is connected to a controller 3808-3814 that can individually control the light emission timing, light emission time, and light emission intensity. As the excitation light source, in the case of continuous irradiation, a light source combining a filter with an ordinary broadband light source such as a xenon lamp or a mercury lamp, or a laser light source such as a semiconductor excitation solid state laser or a He-Ne laser may be used. One of the most suitable is an LED light source that has a narrow wavelength range of output light, can control the intensity stably, is small, and can easily control pulse emission in less than a millisecond. A controller 3808-3814 connected to the excitation light source can control the intensity and output time of the excitation light source, and can irradiate light from continuous light to pulsed light. For one of the embodiments of the pulse exposure method using a pulse light source, the technique described in FIG. 18 can be used. Of course, the excitation light source can be used for the purpose of exciting fluorescence, but one or more of them can be used as a bright field light source for acquiring a bright field microscopic image. In particular, when a light source having a wide output light wavelength width such as a xenon lamp is used as an excitation light source, the sample can be irradiated with only light having a specific wavelength by the excitation light filter 3821.

At the excitation light exit of each excitation light source, each filter 3821 that optimizes the optical bandwidth of the excitation light of each light source 3801 to 3807 is arranged, and each lens 3822 is arranged at the subsequent stage. Each excitation light generated by each excitation light source is focused and irradiated to the end face of each optical fiber 3824 for each excitation light, and the excitation light is introduced into each independent optical fiber 3824. These optical fibers are bundled, irradiated from the opposite end face, and irradiated through the condensing microlens 3826 to the sample flowing through the microchannel in the microchip 3827. Here, for example, the shape of the flow path of a typical microchip may be the one described in FIG. The diameter of the optical fiber is typically 100 microns, and the width of the microchannel through which the sample cells in the microchip pass is typically 10 to 100 microns. For this purpose, a microlens 3826 for condensing excitation light is disposed immediately above the microchip. By adjusting the focal position of the microlens 3826, it is possible to irradiate only the very narrow region of about 1 micron diameter of the microchannel in the microchip with the excitation light focused, and the irradiation region diameter is 100 microns. It is also possible to irradiate the entire microchannel width as a measure.

When the above-mentioned focused excitation light is irradiated to a sample such as a fluorescently stained cancer cell that has flowed through the microchannel, fluorescence of a specific wavelength is emitted from the sample such as a cancer cell in a spherical wave shape. . The fluorescence emitted in one hemispherical direction (in the embodiment of FIG. 38 in the direction of the upper surface of the chip) is guided to the end face where the optical fibers 3825 prepared for the number of fluorescence wavelength bands to be measured are bundled by the microlens 3826. Further, the light is guided to a fluorescence intensity detection unit that performs fluorescence intensity detection via an optical fiber.

In the present embodiment, for example, a fluorescence intensity detection unit comprising fluorescence detectors 3815-3820 for detecting fluorescence intensities in six different fluorescence wavelength regions is mounted in this embodiment, and is emitted from the end face of each optical fiber 3825. Fluorescence can be measured by first guiding the fluorescence to the lens 3822 disposed at the end point of each optical fiber, and then guiding the fluorescence to the filter 3823 of each fluorescence wavelength to be measured and the fluorescence detector 3815-3820. . As a fluorescence detector, it is suitable to use a photomultiplier tube that can detect weak light and easily quantify the amount of received light. However, both the excitation light source and the fluorescence detector are suitable for the measurement target. It is desirable to select an appropriate device such as a semiconductor element having a photoelectric conversion function such as an aparancia photodiode. In addition, a fluorescent filter 3823 that can be exchanged according to measurement conditions can be mounted on the detection port of the fluorescence detector.

The detected fluorescence amount is analyzed and processed by the fluorescence detection control unit 3832, and a specific cell shape or cell cluster obtained from the specific fluorescence or the high-speed camera 3830 when a specific fluorescence or a combination of fluorescence is detected. When the above state is observed, a feedback signal (pulse voltage) for cell sorting is sent to the microchip 3827. For example, when a cancer cell that has been fluorescently stained passes through the microchannel and the amount of fluorescence detected by the fluorescence detector exceeds a threshold amount set in advance by the fluorescence detection control unit 3832, a feedback signal is transmitted to the microchip. As a result, a voltage is applied to the electrodes mounted on the microchip to recover the target cancer cells.

Fluorescence emitted in the remaining hemispherical direction (in the embodiment of FIG. 38 in the direction of the lower surface of the chip) passes through the objective lens 3828, and then simultaneously acquires a bright-field microscopic image and a fluorescent microscopic image with a single high-speed camera light-receiving surface. A bright-field microscopic image and a plurality of fluorescent microscopic images are obtained by passing through a device 3829 (multi-view system; device details are, for example, the configuration of the embodiment shown in FIG. 35 as an example) and being captured by the high-speed camera 3830. All can be acquired simultaneously. Since the high-speed camera 3830 is connected to the light source control unit 3831 and the light source control unit 3831 is further connected to an individual excitation light source controller 3808-3814, the timing of the imaging of the high-speed camera and the excitation light source pulse irradiation can be synchronized. Thus, a clear cell image without shape distortion can be acquired (for example, the configuration of the embodiment shown in FIG. 18 is an example of the details of synchronization).

FIG. 39 shows an example of six types of fluorescence excitation light sources and one type of bright-field microscopic image acquisition light source mounted on the apparatus of FIG. Here, an example of 6 wavelengths of fluorescence excitation, 1 wavelength of bright-field microscopic image light source, and 6 wavelengths of fluorescence detection is shown, but the number can be easily increased by increasing the number of light sources, detectors, and optical fibers. . In FIG. 39, the excitation light source central wavelengths are 370, 440, 465, 498, 533, 618 nm, the bright field microscopic image light source is 750 nm, and the fluorescence center wavelengths are 488, 510, 580, 610, 640, and 660 nm. This makes it possible to flexibly select and detect standard fluorescent reagents such as DAPI, Hoechst33258, EGFP, FAM, HEX, TRITC, Texas Red, Cy3, Cy5, etc. A bright-field microscopic image can also be obtained.

The feature of the apparatus system configuration of the present embodiment is that a group of dichroic mirrors are arranged in the optical path system between the objective lens 3828 and the multi-view system 3829, the wavelength bands are divided, and the excitation light sources 3801-3807 and Instead of arranging the fluorescence detectors 3815-3820, as shown in the embodiment of FIG. 38, an optical fiber array is arranged on the opposite side of the objective lens, and each optical fiber of the optical fiber array has one wavelength band. Excitation light source 3801-3807 and fluorescence detector 3815-3820 are arranged. This avoids the problem that fluorescent light emitted from a sample such as cancer cells has been attenuated when passing through a dichroic mirror multiple times, and also eliminates the need for light separation for each wavelength. The simultaneous use of excitation light and fluorescence was facilitated. Furthermore, the amount of fluorescence was detected using the fluorescence emitted in the hemispherical direction opposite to the objective lens, which could not be detected by the conventional apparatus configuration, and the fluorescence detected in the past was emitted in the remaining hemispherical direction. Thus, all of them can be used for acquiring a fluorescence microscopic image without attenuating, so that it is possible to simultaneously measure a fluorescence amount and a clear fluorescent microscopic image.

FIG. 40 is a diagram schematically showing the configuration of the present embodiment whose example is shown in FIG. Monochromatic light for observation emitted from a bright-field light source 4000 such as an LED flash light source synchronized with the frame interval of the high-speed camera is collected by the lens 4001 and the flow path through which target cells in the blood flow as described above. Then, the cells in the cell sorting unit 4002 including the cell sorting chip incorporating the cell sorting mechanism are irradiated. The cells in the channel can be focused with the objective lens 4003. Here, a depth-of-field improving technique incorporating the zoom lens system may be incorporated.

Fluorescent light sources

4004, 4006, 4008 such as monochromatic lasers and the like, and fluorescence excitation light irradiated to the cell sorting unit 4002 from above, fluorescent antibodies bound to cells in the flow path, and nuclear staining fluorescent dyes (DAPI, Hoechst33258, etc.) ) Can generate fluorescence from nuclei stained with). The intensity of the obtained fluorescence can be quantitatively measured by a fluorescence detection system 4005, 4007, 4009 including a fluorescence intensity measurement system such as a photomultiplier tube or a photodiode. Here, although three systems have been described as examples of the excitation light and fluorescence detection systems, a plurality of fluorescences can be excited by a single excitation light, so that a plurality of free combinations are possible. Further, an image division system for dividing an optical microscopic image into a bright-field image and a fluorescent image and acquiring a plurality of images at the same time with a single high-speed camera light-receiving element is described in detail later in FIG. Through 4010, a bright field image of a cell can be simultaneously acquired by the high-speed camera 4011 while detecting the fluorescence intensity of the cell in this way.

In this embodiment, the description is based on the combination with the image detection unit. However, naturally, it does not include the image detection unit using the high-speed camera, and the simultaneous detection of the multiple excitation light and the multiple fluorescence using only the optical fiber array. It can also be used as a system.

As mentioned above, although each embodiment was described with reference to drawings, this invention is not limited to these embodiment, and it cannot be overemphasized that various deformation | transformation are possible within the range of the mind of this invention.

FIG. 41A (a) shows an example of a distribution diagram of cell nucleus areas when blood cancer cells are measured by the apparatus of the present invention. Here, the area of each nucleus is compared between an image of cells in blood when cancer tissue is actually transplanted and an image of cells in healthy blood for comparison. From this result, it can be seen that the presence of cancer cells can be clearly identified as cancer cells when the area of the fluorescence image of the nucleus of about 150 μm ² or more is observed. Further, (b) to (g) of FIG. 41A show the bright field image and the fluorescence image of typical cells at the respective nucleus sizes in the graph of FIG. (A). It can be confirmed that the cells are clustered in the region where the area of the fluorescence image of the nucleus of 150 μm ² or more is observed. However, this is when measured by integrating the total area in the image for the nuclei in the cell clusters, in about 150 [mu] m ² or less of conditions, and also shows that it is impossible to distinguish cancer cells and normal cells, The above about 150 μm ² or more is one of the sufficient conditions for the existence of cancer cell clusters. This index is also effective for identifying multinucleated cells that are characteristic of cancer cells.

FIG. 41B is a graph showing the ratio of the peripheral length of the blood cancer cells measured by the apparatus of the present invention to the peripheral length derived by circular approximation from the area of the cells (cluster). An example is shown. Here, with respect to an isolated single cell flowing through blood to a cell cluster of 5 cells or more, the ratio of the perimeter of the cell (1 / R) with the number of cells in the cluster as the X axis using R shown in Formula 1. ) Is plotted on the Y axis, the isolated one cell has (1 / R)> about 0.9, and the cluster of two or more cells has (1 / R) <about 0.9. From the result (1 / R), when this is about 0.9 or more, it can be determined that the cell is an isolated cell, and when it is less than about 0.9, two or more cells are clumped. It can be seen that it is a cell cluster.

FIG. 42 shows a table showing the distribution of cell (population) area and number of nuclei when blood cancer cells are measured with the apparatus of the present invention. Here, when comparing the area of each cell in a blood cell (Positive) when a cancer tissue is actually transplanted and a healthy blood cell (Control) for comparison, the bright field is compared. Based on the observation results of a cluster of cells (microscopic image) on a microscopic image, the presence of cancer cells is clearly identified as a cancer cell cluster if the area of the bright field image of cells of approximately 250 μm ² or more. You can see that you can. However, this means that about 250 [mu] m ² following conditions when the area measured by the image for bright-field image of the cell clusters, and also shows that it is impossible to distinguish cancer cells and normal cells, above about 250 [mu] m ² This is just one of the sufficient conditions for the existence of cancer cell clusters.

In addition, when the number of nuclei in a mass of cells (clusters) measured by a fluorescence microscopic image was measured, the number of nuclei shining with fluorescence was 3 or more. However, only an image of healthy blood cells (Control) for comparison could not be found. From this, when the number of nuclei in a lump of cells (cluster) is measured at 3 or more, it can be determined that this cell (cluster) is a cancer cell. However, in this case as well, it is shown that cancer cells and normal cells having a nucleus number of 2 or less cannot be distinguished, and the number of nuclei in the cell (cluster) is Three or more is one of the sufficient conditions for the existence of cancer cell clusters.

From these results, the following three judgment conditions,
(1) The area of the nucleus of about 150 μm ² or more of the cell (cluster) is measured from the acquired image (2) The area of about 250 μm ² or more of the cell (cluster) is measured from the acquired image (3) The cell (cluster) The presence of three or more nuclei in (1) is measured from the acquired image, or a combination of the above three conditions in AND, that is, (1) and (2), or (1) and (3 ), (2) and (3), or (1), (2) and (3) may be used as criteria for determining the presence of blood cancer cells.

FIG. 43 schematically shows a more specific configuration for practical application of the device configuration for simultaneously acquiring microscopic images in a plurality of different wavelength bands with one high-speed camera light-receiving surface for the device shown in FIG. FIG. 43A in FIG. 43A is a schematic view of the entire internal configuration of one unit of the optical branching module, which is the minimum configuration unit, as viewed from above. This apparatus is a two-dimensionally developed optical path system viewed from the top, and as shown in the figure, a pair of image light input / output optical path systems symmetrically on both bottom surfaces of a rectangular parallelepiped container, and On the side surface, 2 to 4 holes for introducing optical paths of mirror reflected light are arranged, and six optical path covers 4301 that can be freely attached and detached are arranged on each side. Two detachable movable adjustment function mirror holders 4302 are arranged on these side surfaces so that light is introduced into the holes of the two to four mirror reflected light introduction optical paths. Adjustment enables the traveling direction of the reflected light to be moved minutely, so that the imaging position of the camera can be freely moved. The mirror holder can be provided with a total reflection mirror, a high pass filter, a low pass filter, and the like. A detachable optical path window 4303 is arranged between the two mirror holders 4302 with detachable movable adjustment function, and the cross-sectional area of the transmitted light can be adjusted. Further, a detachable filter 4304 is provided, and the wavelength bandwidth of light can be finely adjusted by a band pass filter or the like.

FIG. 43B schematically shows one of the embodiments for observing a combination of images of a plurality of wavelengths by actually connecting the units shown in FIG. 43A. First, an image optical image to be observed is introduced from the parallel light introduction module 4305 to the first light branching module 4300. The optical path cover 4301 in the optical path introduced here is removed, and is fixed to the parallel light introducing module 4305 in such a manner as to maintain hermeticity by the connecting portion. The parallel light introducing module 4305 incorporates a lens system so that incident light is introduced into the light branching module 4300 as parallel light, and the cross-sectional area of the incident light to be introduced is set at the front stage of the lens system. A small optical window filter may be arranged. In this case, the cross-sectional area of the incident light cut out by the optical window is an image acquisition camera that is finally measured in order to project an independent image corresponding to the number of modules to be connected so as not to overlap the light receiving surface on the observation camera. In the system 4306, the area is adjusted to be equal to or smaller than (total area of the light receiving surface / parallel light introducing module).

Next, in the first-stage light branching module 4300, it is introduced into the mirror holder 4302 in which the (wavelength) high-pass filter or the (wavelength) low-pass filter b is incorporated, and the incident light is branched into two wavelengths. The transmitted light is reflected by the total reflection mirror a, and is introduced into the second filter b of the second-stage light branching module 4300 (the same filter as the first filter of the preceding-stage (first-stage) light-branching module). The light reflected by the filter b of the first-stage light branch module 4300 is introduced into the first filter c of the second-stage light branch module. Similarly, in this embodiment, the same wavelength band branching is performed up to the fifth stage optical branching module, and the direction of the optical path is adjusted for each branch wavelength so that the images of the respective wavelength bands do not overlap on the light receiving surface of the camera. Positioning is performed by each detachable movable adjustment function mirror holder 4302. In particular, in the final-stage light branching module 4300, the total reflection mirror a is used for the first filter, and the first filter e in the preceding-stage (fourth-stage) light branching module 4300 is used for the second filter. Here, b, c, d, and e, which are high-pass filters or low-pass filters, are arranged in order of monotonically increasing or monotonically decreasing wavelengths (b <c <d <e or b> c> d> e), the same filter is used for the first filter in the previous stage and the second filter in the next stage, and a total reflection mirror is used in the second filter in the first stage and the first filter in the final stage. Shall be used.

FIG. 43C schematically shows how an actually input image is projected on the camera system 4306. First, the acquired image (left figure) is 1 / of the size of the light receiving surface of the camera system by an optical window filter in the parallel light introducing module 4305 that cuts off the cross-sectional area of the incident light introduced in the previous stage of the lens system. The image is cut into a size that allows an image having an area of 5 or less to pass through and is introduced into the optical branching module 4300 (center view). Furthermore, by adjusting the position of the mirror holder of each light branch module, the image forming positions on the light receiving surface of each light branch module are arranged so that there is no overlapping portion (right diagram). As a result, images of each optical wavelength bandwidth created by the five optical branching modules are simultaneously formed on the light receiving surface of one camera, and particularly when a high-speed camera is used, a high-speed image of one image. Only by processing, comparative analysis of images of a plurality of wavelengths can be performed at one time.

FIG. 43D shows another application example in which an optical branching module is combined. Here, the second mirror holder of the optical branching module can be removed and another camera system can be connected here, or the light intensity measurement 4307 can be connected to measure the light intensity of a specific wavelength bandwidth. it can.

FIG. 44 is a diagram schematically showing an example of the configuration of an imaging cell sorter that observes and separates cells in water droplets. The apparatus shown in FIG. 44A produces and drops water droplets of an optimal size by discharging cells in the sample solution from the thin tube at the tip of the water droplet forming module 4401 with a cell reservoir at a constant pressure. Can do. In addition, by charging the electrostatic field coil 4402 covering the region from the region where the water just before the water droplet is formed to the reservoir to the region where the water droplet is just formed to the opposite charge to the water droplet to be charged, Water droplets can be charged with a desired charge. For example, when it is desired to charge a water droplet with a negative charge, the coil may be charged with a positive charge. The formed charged water droplet 4403 is dropped on an optically transparent water-repellent insulating substrate having a Teflon resin processed surface such as glass, and rolls down in the tilt direction of the substrate. A high-speed camera capable of measuring bright field images and fluorescent images and an optical measurement module 4405 capable of measuring scattered light intensity, fluorescent intensity, etc. are arranged on the back of the substrate on the path where the water drops fall, and the acquired information is analyzed. By analyzing with the control module 4410, it can be determined whether the target cell is in a water droplet. After identifying the type of the target cell based on the above determination, a charge opposite to the water droplet is given to one specific path among the plurality of water droplet movement direction control electrostatic field guides 4406 for changing the position from the water droplet falling direction. The electric charge is applied so as to hold, and the falling direction of the water droplet is changed and guided to the fraction water droplet reservoir 4407 at the subsequent stage. In order to fractionate a plurality of targets, a plurality of water droplet movement direction control electrostatic field guides 4406 and a fractional water droplet reservoir 4407 may be arranged in accordance with the types of the targets. The electrodes of the electrostatic field guides for controlling each water droplet movement direction are arranged in the lower stage of the substrate 4404, and each of these electrodes is connected to the electric field switching mechanism 4409 for electrostatic field guides for controlling the water droplet movement direction. A control charge from 4410 can be applied. The analysis control module analyzes the acquired image and calculates the cell (cluster) area and perimeter, the nucleus area and the number in the cell cluster by calculation, and the calculation result obtained (for example, a computer). It can be configured by a mechanism (for example, a computer and a power source) that identifies the types of cells to be collected in combination and applies an electric field to an electrode of a specific guide to give an electric charge. The switching mechanism can be composed of a removable inset groove for fixing the substrate and an array of electrical contacts for applying electric charges from the analysis control module, which are aligned with the position of the electrode line of the substrate guide. . Further, when not controlled by this mechanism, each electrode has a mechanism that does not affect the movement of the water droplet by grounding the electrostatic field guide for controlling the direction of water droplet movement. In addition, in order to improve the initial positioning accuracy in the dropping direction of the dropped water droplet, a similar water droplet movement direction control electrostatic field guide 4406 is disposed, or a pair of rails sandwiched between the lower surfaces of a water repellent surface such as a Teflon coat These three-dimensional structures may be added. Since the guide is placed under a water-repellent coat such as Teflon, any material can be used as long as it can build a physical structure. In order to prevent the structure from changing by sterilization, it is desirable to use a material whose deformation and rigidity do not change significantly at a temperature of 150 ° C. or higher. Further, it is desirable that it is optically transparent or constant absorption in the wavelength region used for observation. Specifically, glass, SU8, PDMS, etc. are mentioned.

One of the features of this technology is that it has a substrate tilt control mechanism 4408 that can freely control the drop speed of water droplets, that is, the measurement time. In the present embodiment, a mechanism in which a tilt mechanism is added to a flat plate is shown, but a substrate having a combination of different tilts may be used. For example, by using a steeper slope for the transport and separation of water drops and a gentle slope in the measurement area, it is possible to observe the water drops in a state where the distance between adjacent water drops is short and the moving speed is slow. Can do. In particular, in the observation region and the water droplet fractionation start region, the water droplets can be arranged closely at equal distances in the falling direction by making them horizontal (perpendicular to gravity) so that the water droplets move at a constant speed. Further, if the substrate is lengthened under such a particularly horizontal condition, the distance for sequentially observing various measurements under the condition that the water droplet moving speed is the same can be made sufficiently long. Further, by adjusting the temperature of the substrate, it is possible to control the temperature of water droplets that have made minimal contact in a water-repellent state. Further, a temperature / humidity control container 4411 and a temperature / humidity control mechanism 4412 are added to prevent evaporation of water droplets. For the temperature / humidity control container 4411 and the temperature / humidity control mechanism 4412, for example, an acrylic optically transparent container or a light-shielding metal container may be used, and temperature control by a Peltier element is possible. It can comprise with the air blower etc. to the said container.

In addition, as a feature of the present technology, it is possible to selectively collect target cells in the water droplets according to the reaction result by colliding the reaction liquid with the sample water droplets and observing and measuring the mixed droplets at a certain post-reaction time. . Here, the reaction liquid reservoir water droplet formation mechanism 4413 creates a reaction liquid to which a charge opposite to that of the sample water droplet is weakly applied, and the liquid droplet movement direction control electrostatic field guide 4414 can guide the reaction liquid to the sample water droplet. .

FIG. 44B is a diagram schematically showing the apparatus from the top in order to explain a mechanism for actually separating and collecting water droplets. As can be seen from the figure, the water droplets falling on the straight line are arranged so as to overlap at the end points of the plurality of branching water droplet movement direction control electrostatic field guides 4406. In addition, each guide 4406 is arranged so that a water droplet falls obliquely with a resultant force of gravity with an angle in the tilt direction, and when a charge opposite to the water droplet is applied to the guide, Is configured to move. Also, a fractional water droplet reservoir 4407 serving as a U-shaped water droplet receiver for capturing water droplets is disposed below. Further, in order to make it easy to apply a charge to each guide, each guide extends to the end of the substrate, and by arranging there, it is possible to easily apply a charge to each guide. It has a configuration.

FIG. 45A is a diagram schematically showing an example of a process for detecting a cell cluster in human blood and analyzing it for diagnosis. 45B and C are diagrams schematically showing an example of an integrated measurement analysis system for realizing the process shown in FIG. 45A. This said, the experimental results from that cell mass with approximately 250 [mu] m ² or more areas in healthy blood (clusters) is not detected, the cell mass with approximately 250 [mu] m ² or more areas in unstained (cluster) Metastatic cancer in blood by selecting from the image with the (multi-) imaging cell sorter of the invention, collecting this, and identifying this cell cluster by re-culture, gene mutation test, or expression analysis test Used as a diagnostic device to clarify whether it is a cell, a cell mass generated by damage to an organ such as the liver or kidney, or a white blood cell enlarged by an infection It is possible to do. In particular, for the detection of metastatic cancer in the blood, by confirming the gene mutation in each cluster unit, if each cluster is the same mutant, the degree of cancer progression is determined from the primary cancer. If there are many different mutation points while the history of mutations in each cluster is the same, the location of the metastatic cancer is spread over many regions. Can be estimated. In addition to metastatic cancer diagnosis, if the cell cluster that flows in the blood can be identified as a liver tissue fragment, the liver disease can be estimated, and similarly, it can be identified from other organ sections It can be estimated that these are diseases of each organ. In addition, for phagocytic leukocytes such as macrophages, it is possible to diagnose infections in a short time by collecting cells that are larger in size than normal, and examining and identifying genes of heterologous cells such as bacteria in the cells. is there. Or, by selectively recovering those whose internal shape has become complicated or increased in size due to activation of the B cell, and clarifying the antigen of the B cell, it is diagnosed what the immune system is reacting to It is also possible to perform. FIG. 45B schematically shows the components of the system for enabling the measurement. In the first stage, the process of removing red blood cells from the blood and concentrating the white blood cells, in the next stage, the process of identifying and collecting the cell mass based on the image, and as the final stage, the cell cluster at each single cluster unit level It is possible to identify the type of cell ferment by gene mutation assay or expression analysis. FIG. 45C shows an example of a configuration created to actually realize the concept of FIG. 45B. The cell pretreatment mechanism is arranged in the upper stage, the imaging cell sorter is arranged in the middle stage, and the droplet type PCR apparatus is arranged in the lower stage.

FIG. 46 is a diagram schematically showing an example of a technique for removing cell clusters (clusters) in blood of a certain size or more as a metastatic cancer treatment technique. This is because the cell blood (cluster) removing mechanism 4601 such as a membrane filter that removes a cell mass (cluster) having a cross-sectional area of about 250 μm ² or more that does not exist in healthy blood due to the above discovery is used to circulate the patient's blood. Therefore, a technique that suppresses the onset and progression of metastatic cancer by effectively removing metastatic cancer cells in the blood by physical methods without using drugs such as anticancer drugs is schematically shown. It is what. In this embodiment, although the membrane filter to remove about 250 [mu] m ² or more of the cross-sectional area of the cell mass (cluster), if a technique for removing about 250 [mu] m ² or more of the cross-sectional area of the cell mass (cluster), physical A non-contact capture technique such as a ladder array for selective capture or capture by ultrasonic radiation pressure may be used. In addition, as a basis for the metastatic cancer inhibitory effect of this technology, the establishment of implantation / metastasis of cancer cells that have been clustered (clustered) can be performed in the blood with a weakly isolated single cell that easily causes apoptosis. This stems from an exponential increase in potential for flowing cancer cells.

FIG. 47 shows the distribution spectrum of blood cell size in the case of measuring the particle size distribution of leukocytes in blood for diagnosis of metastatic cancer in blood, normal blood (FIG. 47A), metastatic cancer cells. FIG. 47B schematically shows each of the blood (FIGS. 47B and 47C) flowing. Conventionally, the presence of abnormal cells such as cancer cells was measured based on the size of each cell relative to the absolute indicator of cell size. To more effectively discriminate, the cell size distribution in the blood is plotted on the X axis. The distribution curve (spectrum) is obtained with the quantity (or the ratio of the quantity to the whole) as the Y axis, and when the distribution has a monotonous single peak as shown in FIG. 47A, normal blood It is determined. On the other hand, there are two obvious size distribution peaks as shown in FIG. 47B, or a size distribution curve that cannot be explained by one size peak as shown in FIG. 47C, and the second peak is a cell. When the size is on the larger side, it can be determined that abnormal cells such as metastatic cancer cells are flowing in the blood. In particular, in order to confirm the normality of the size distribution, distribution curves such as a normal distribution and a Gaussian distribution can be used for comparison reference. Moreover, not only for discrimination, but also for cell recovery, based on the index obtained by this measurement, the value of the abnormal cell size peak larger than the peak of normal cells is used as an index (threshold), and blood is again In this case, the cell size spectrum is measured in the first step, and the obtained abnormal cell size peak is obtained in the second step. Accurate cell recovery can be realized in two steps for recovering cells that are equal to or greater than the threshold value (peak value of distribution: see arrows in FIG. 47B, C abnormal cell distribution portion). The peak of abnormal cells corresponds to a giant cell or a cell cluster depending on the case.

FIG. 48 shows a method using a substrate having an acceleration region and a constant velocity moving region by having two inclination angles in the means for moving the water droplet dropped on the water repellent substrate described in FIG. 44 on the surface of the substrate. FIG. In the embodiment shown in FIG. 44, the moving speed of the water droplet is adjusted by adjusting the inclination angle of one flat plate, but only one surface is “acceleration only” or “constant velocity motion only”. In particular, when the movement distance of the water droplet becomes long, the movement speed of the water droplet becomes too high with “acceleration alone”, and higher-speed processing is required for the subsequent measurement and separation. In order to solve this problem, it is desirable to combine a slope that is an “acceleration zone” and a horizontal plane that is a “constant velocity zone”, instead of using a single slope plane. The dropped water droplet 4801 is placed on a water repellent treated substrate whose inclination angle can be adjusted along the water droplet dropping direction 4802. For example, the moving speed is increased by using the inclined surface 4803 having the inclination θ as an “acceleration region”. While rolling down. Then, on the horizontal plane 4804 having the function of adjusting the inclination angle that follows, the water droplet moves at a constant speed as the “constant velocity region”. In addition, after performing processing such as analysis and separation in the constant velocity region, the inclined surface may be newly set as an “acceleration region”. In addition, the “constant velocity region” is basically leveled, but in actuality, when decelerating due to minute resistance on the surface of the water-repellent surface, a slight inclination angle is maintained to maintain the constant velocity of water droplets. You may give it.

The present invention is useful for purifying a minute amount of target cells in blood in units of one cell, and analyzing accurate gene information and expression information of the target cells. The present invention is useful for purifying a very small amount of target cells having spores such as Bacillus anthracis per cell and analyzing accurate gene information and expression information of the target cells at high speed.

The present invention is also useful as a technique for identifying and / or collecting cancer cells circulating in the blood.

1 Cell Analyzer System 10 Cell Concentration / Staining / Decoloring Module 101 Cell Sample Container 102 Staining Agent Container 103 Cleaning Agent Container 104 Dispensing Head 105 Turntable 106 Concentration / Decoloring Filter 107 Concentration Chamber 108 Chamber 109 Pressure Pump 110 Waste Liquid Collection Tube 111 Recovery head 112 Recovery tube 113 Recovery chip 114 Chassis 115 Rotating arm 20 Image detection type 1-cell separation / purification module 201 Laser 202 Mirror 203 Condensing lens 204 Dichroic mirror 205 Filter 206 Photodetector for fluorescence detection 207 High-speed camera 208 Forward scattered light Photodiode for detection 209 Cell sorter chip 210 Chip substrate 211 Microchannel 212 Inlet 213 Outlet 214 With cell concentrate 215 Cell concentration unit 216 Converging unit 217 Sorting unit 218 Cell detection region 219 Flow of cells after application 220 Flow of particles before application 221 Outlet 222 Outlet 223 Waste liquid recovery unit 224 Cell recovery unit 225 V-shaped comb electrode 30 1 cell Genome analysis / expression analysis module 31 First temperature control unit 32 Second temperature control unit 301 Reaction tank 302 Heat exchange tank 303 Liquid reservoir tank 304 Pump 305 Switching valve 306 Auxiliary temperature control mechanism 307 Inlet A
308 Inlet B
309 Outlet A
310 Outlet B
311 Sample liquid 312 Fluorescence detector 313 Control analysis unit 314 Reverse support valve 315 Control signal 40 Liquid feeding module 401 Dispensing head 402 Dispensing tip 403a, b Z-axis moving guide 404 Z-axis moving motor 405a, b Arm rotation motor 406 Chassis 50 Control / analysis module (computer)
801 Container 802 Small amount sample 803 Rotating body 804 Rotating shaft 805 Sample 806 Abrasive 810 Rotating body 811 Container 812 Flexible structure 813 Rotating shaft 814 Tip portion 815 Spring mechanism 820 Curved cut 821 Mortar-shaped cut 822 Engagement structure 823 Semispherical rotator 824 Egg-shaped rotating body 825 Protruding rotating body 826 Bowl-shaped rotating body 830 Container 831 Rotating body 832 Abrasive 833 Seal 883 Sample 835 Rotating shaft 836 Component 840 Integrated container 841 Cutter for opening 1301 Cell sorter chips 1302, 1304, 1306 Upstream Side channel 1303, 1305, 1307 Downstream channel 1308 Sample liquid inlet opening 1309, 1310 Sheath liquid inlet opening 1311 Sheath liquid reservoir 1312 Waste liquid reservoir 1313, 13 4 Sheath liquid outlet opening 1315 Purified sample liquid outlet opening 1316 Flow path for gel filling 1317 Entrance opening 1318 for gel filling Outlet opening 1319 for gel filling Electric wire 1320 Power supply 1321 Switch 1322 Sample liquid Reservoir 1323 Purified sample collection reservoir 1401 Cell sorter chip 1402 Cap 1403 Sheath liquid reservoir 1404 Sample liquid reservoir 1405 Pressurized air introduction pipe 1406 Sample solution introduction tube 1407 Sheath liquid introduction tube 1408 Water level measurement sensor 1409 Flow path 1501 Cell sorter chip 1502 Large reservoir 1503 Air pressure application device 1504 Pressure sensor 1505 Distributing valve 1506 Sorted sample collection reservoir 1507 Waste liquid collection reservoir 1601 Sample solution flow 160 2, 1603 Side sheath flow 1604 Cell monitoring region 1605 Gel electrode 1606 Sorted sample collection flow channel 1607 Side sheath flow 2001 Cell sorter chip 2002 Solution reservoir 2003 Buffer introduction device 2004 Pressure sensor 2005 Distribution valve 2006 Sample solution introduction device 2007 Sample solution flow channel 2008 Buffer channel 2009 Sorting external force application mechanism 20101, 20102, 20103 Sorted sample, waste liquid recovery reservoir 2101 Cell sorter chip 2102 Buffer reservoir 2103 Buffer introduction device 2104 Pressure sensor 2105 Valve 2106 Sample solution introduction device 21061 Sample solution introduction channel 2107 Buffer liquid flow path 2109 Sorting external force application mechanism 21101, 21102 Sorted sample, waste liquid collection reservoir 23001 Chip 23002 Sample inlet 23003 Target particle 23004 Unnecessary particle 23005 Micro channel 23006 Particle alignment mechanism 23007 Particle alignment external force input (electric force or sheath flow)
23008 Particle detection mechanism 23009 Particle purification mechanism 23010 Particle purification external force input ((gel or metal) electrode + electric force)
23011 Target

particle collection ports

23012, 23013 Unnecessary particle reservoir 24001 Support substrate 24002 Metal thin film electrode first layer 24003 Metal thin film electrode second layer 24004 Insulating film layer 24005 Sample flow channel 25001 Sample introduction port 25002 Micro flow channel 25003 Metal thin film stacked type parallel Comb electrode 25004 Sample particle 25005 Metal thin film laminated V-shaped electrode 25006 Sheath channel 25007 Bottom electrode 26001 Target particle 26002 Unnecessary particle (negatively charged)
26003 Unnecessary particles (positively charged)
26004 Micro flow channel 26005 Particle purification electrode 26006 Flow to target

particle recovery port

26007, 26008 Flow to unwanted particle reservoir 27001 Micro flow channel 27002 Electrode gel inlet 27003 Electrode gel passage 27004 Electrode gel liquid junction 27005 Electrode Gel outlet 27006 Metal wire 27007 DC voltage source 27008 Voltage switching mechanism 27008 Sample particle 27010 Metal thin film electrode 3001 Sample

aqueous solution inlet

3002, 3003 Oil inlet 3004 Merged region 3005 Sample aqueous solution 3006 Oil 3200 Bright field light source 3201 Condenser lens 3202 Cell sorting 3203

objective lens

3204, 3206, 3208

fluorescence light source

3205, 3207, 3209 fluorescence detection system 3210 high-speed camera (image detection system)
3400 Bright field light source 3401 Condenser lens 3402 Cell sorting unit 3403

Objective lenses

3404, 3406, 3408 Fluorescence light source 3405.3407, 3409 Fluorescence detection system 3410 Image division system 3411 High-speed camera (image detection system)
3501 Input optical path (image)
3510, 3520, 3530

Image division units

3511, 3521, 3531 Dichroic mirrors with

angle adjustment function

3512, 3522, 3532

Filter systems

3513, 3523, 3533 Image

size adjustment systems

3514, 3524, 3534 Dichroic mirrors 3515, 3525 with angle adjustment function 3535 Optical lens system 3600 Cell 3601 Input image 3602 High-speed camera light receiving surface 3610 Output image 1 (bright field)
3620 Output image 2 (fluorescence: nuclear staining)
3621 Nuclear fluorescence image 3701 High-speed camera light-receiving surface image 3801-3807 Fluorescence excitation light source 3808-3814 Excitation light source controller 3815-3820 Fluorescence detector 3821 Excitation light filter 3822 Lens 3823

Fluorescence filter

3824, 3825 Optical fiber 3826 Condensing microlens 3827 Microchip 3828 Objective lens 3829 Multi-view unit 3830 High-speed camera 3831 Light source control unit 3832 Fluorescence detection control unit 4000 Bright field light source 4001 Condenser lens 4002 Cell sorting unit 4003

Objective lens

4004, 4006, 4008 Fluorescence light source 4005.4007, 4009 Fluorescence detection system 4010 Image Division system 4011 High-speed camera (image detection system)
4300 Optical branching module 4301 Optical path cover 4302 Removable mirror holder with movable adjustment function 4303 Removable optical path window 4304 Removable filter 4305 Parallel light introduction module 4306 Image acquisition camera system 4307 Light intensity measurement system 4401 Water droplet forming module with cell reservoir 4402 Electrostatic field Coil 4403 Charged water droplet 4404 Optically transparent water-repellent insulating substrate 4405

Optical measurement modules

4406 and 4414 Water droplet movement direction control electrostatic field guide 4407 Fractional water droplet reservoir 4408 Substrate tilt control mechanism 4409 Water droplet movement direction control electrostatic field Electric field switching mechanism for guide 4410 Analysis control module 4411 Temperature / humidity control container 4412 Temperature / humidity control mechanism 4413 Reaction liquid reservoir Water drop formation mechanism 4601 Cell mass (cluster) ) Removal mechanism 4801 Water drop 4802 Water drop dropping direction 4803 Slope 4804 with slope θ Horizontal plane

Claims

An on-chip cell sorter system used to identify cancer cell candidate cells,
A cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject;
An optical system including a bright field light source and a fluorescent light source for irradiating the cells;
A detection system for simultaneously acquiring a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of the fluorescent labeling substance bound to the cells, and a fluorescence image of the cells;
Control / analysis means for identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image;
Means for selectively recovering the identified multinucleated cells and / or cell clusters,
The control / analysis means includes the following (i) to (iii):
(I) The area of the nucleus of the lump or cluster obtained from the fluorescence image of the nucleus of the lump of cells or clusters is 150 μm 2 or more.
(Ii) The area of the lump or cluster obtained from a bright field image of a lump of cells or clusters is 250 μm 2 or more, or (iii) the number of nuclei in the lump of cells or clusters is 3 or more If one condition is met, or (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii) are met , Determining that cancer cells are likely to be present in the cell sample solution,
On-chip cell sorter system.
The control / analysis means is
About the perimeter ratio R derived as a circular approximation from the perimeter and area of cells (clusters),
(I) When (1 / R) is 0.9 or more, the cell (cluster) is an isolated cell, or (ii) When (1 / R) is less than 0.9, the cell (cluster) is 2 cells. Consisting of a cluster of cells,
The on-chip cell sorter system according to claim 1, wherein the value of (1 / R) is used as a criterion for determining the presence or absence of clusters in the cell sample solution.
An on-chip cell sorter system used to identify cancer cell candidate cells,
A cell sorter chip provided with a flow path for flowing a sample solution containing fluorescently stained cells derived from a subject;
An optical system including a bright field light source and a fluorescent light source for irradiating the cells;
A detection system for simultaneously acquiring a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of the fluorescent labeling substance bound to the cells, and a fluorescence image of the cells;
Control / analysis means for identifying multinucleated cells and / or cell clusters flowing in the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image;
Means for selectively recovering the identified multinucleated cells and / or cell clusters,
The control / analysis means is
About the perimeter ratio R derived as a circular approximation from the perimeter and area of cells (clusters),
(I) When (1 / R) is 0.9 or more, the cell (cluster) is an isolated cell, or (ii) When (1 / R) is less than 0.9, the cell (cluster) is 2 cells. Consisting of a cluster of cells,
As described above, an on-chip cell sorter system in which the value of (1 / R) is used as a criterion for the presence or absence of clusters in the cell sample solution.
The on-chip cell sorter system according to any one of claims 1 to 3, further comprising means for measuring a nucleic acid sequence of a gene derived from the selectively collected multinucleated cells and / or cell clusters.
The on-chip according to any one of claims 1 to 4, further comprising an image dividing mechanism having a function of dividing and simultaneously displaying the bright field image and the fluorescent image on a light receiving surface of one high-speed camera.・ Cell sorter system.
6. The on-chip cell sorter system according to claim 5, further comprising a mechanism for adjusting the magnification of the bright field image and the fluorescent image so that the magnification of the image differs.
The on-chip cell sorter system according to any one of claims 1 to 6, wherein the subject-derived cell is a blood-derived cell of the subject.
A method for identifying candidate cells of cancer cells in a cell sample solution derived from a subject using the on-chip cell sorter system according to any one of claims 1 to 7,
Flowing a sample solution containing fluorescently-stained cells from a subject through the flow path of the cell sorter chip;
Irradiating the cells with light from a bright-field light source and a fluorescent light source,
Obtaining a bright field image of the cells in the sample solution flowing through the flow path of the cell sorter chip, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells;
Identifying a multinucleated cell and / or cell cluster flowing through the flow path based on the bright field image, the fluorescence intensity, and the fluorescence image, and selectively identifying the identified multinucleated cell and / or cell cluster Including the step of collecting,
In identifying the multinucleated cells and / or cell clusters,
(I) The area of the nucleus of the lump or cluster obtained from the fluorescence image of the nucleus of the lump of cells or clusters is 150 μm 2 or more.
(Ii) The area of the lump or cluster obtained from a bright field image of a lump of cells or clusters is 250 μm 2 or more, or (iii) the number of nuclei in the lump cells or clusters is 3 or more If one condition is met, or (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii) are met A method for determining that cancer cells are likely to be present in the cell sample solution.
An optical branching module,
A substantially rectangular parallelepiped housing;
An optical path system including a pair of mirrors for input / output of a pair of image light provided symmetrically on the bottom surface in the housing;
2 to 4 openings for introducing light of mirror reflected light provided on the side surface of the housing, each having two on the side in the longitudinal direction and one on each side in the short side An aperture formed,
6 optical path covers that can be freely attached to and detached from each of the 2 to 4 openings,
A pair of removable movable mirror holders capable of adjusting the mirror so that light is introduced into each of the two to four openings;
A detachable optical path window disposed between the pair of mirrors and capable of adjusting a cross-sectional area of transmitted light; and
A detachable filter disposed between the pair of mirrors and capable of adjusting a wavelength bandwidth of light;
An optical branching module.
The optical branching module according to claim 9, wherein a plurality of the optical branching modules are connected and used.
The optical branching module according to claim 10, wherein the optical branching module is used to simultaneously acquire a plurality of microscope images of different wavelength bands with one camera light receiving surface.
12. The optical branching module according to claim 9, wherein one of the pair of mirrors of some of the optical branching modules can be removed and used.
A bright field image of the cells in a sample solution containing fluorescently stained cells, a fluorescence intensity of a fluorescent labeling substance bound to the cells, and a fluorescence image of the cells are used to simultaneously acquire the cells. The optical branching module according to any one of the above.
A water droplet formation module comprising a sample solution reservoir for holding a sample solution containing cells and a capillary tube connected to the reservoir;
An electrostatic field coil for charging water droplets formed by the water droplet forming module;
A substrate having a water repellent surface for dropping the formed water droplets, the substrate being optically transmissive,
A substrate tilt control mechanism for adjusting the tilt angle of the substrate surface;
An optical measurement module including a camera capable of measuring a bright field image and a fluorescent image, disposed on the opposite side of the substrate surface on which the water droplets are dropped, and the water droplets formed on the water repellent surface can be controlled thereon One or a plurality of guides for movement, an electrostatic field guide for controlling the direction of movement of a water droplet charged with a charge opposite to that of the water droplet;
A fractionated water droplet reservoir disposed at a subsequent stage of each of the plurality of water droplet movement direction control electrostatic field guides;
An electrostatic field guide electric field switching mechanism for controlling the water droplet movement direction connected to the electrostatic field guide for controlling the water droplet movement direction, the mechanism for controlling the electric field to be charged to the electrostatic field guide;
Analysis that is connected to the optical measurement module and the switching mechanism, analyzes image data acquired by the optical measurement module, determines the presence or absence of target cells in the water droplets, and controls the operation of the switching mechanism based on it An imaging cell sorter for observing and separating cells in water droplets, comprising a control module.
A plurality of electrostatic field guides for controlling the direction of movement of the water droplets are provided, at least one of which is for improving the initial water droplet positioning accuracy, and for the other guides to select water droplets containing target cells and water droplets not containing them. And
The guide for improving the positioning accuracy of the initial water droplet is located upstream of the movement of the water droplet, and the guide for sorting the water droplet containing the target cell and the water droplet not containing the target cell is located downstream thereof. The imaging cell sorter described in 1.
The imaging cell sorter according to claim 14 or 15, wherein the optical measurement module is arranged so as to be able to acquire an image of a cell in the water droplet moving on a guide for improving the positioning accuracy of the initial water droplet. .
A specific one of the guides for selecting the water droplets including and not including the target cells is charged with a charge opposite to that of the water droplets to control the moving direction of the water droplets, and is arranged at the subsequent stage of the guides. The imaging cell sorter according to any one of claims 14 to 16, wherein the imaging cell sorter is configured to guide the water droplet to a fractionated water droplet reservoir.
The imaging cell sorter according to any one of claims 14 to 17, further covered with a cover for preventing evaporation of the water droplets.
A metastatic cancer treatment apparatus having a cell mass (cluster) removal mechanism including a membrane filter that removes a cell mass (cluster) having a cross-sectional area of about 250 μm 2 or more that does not exist in healthy blood, and circulates the patient's blood Therefore, a metastatic cancer treatment device for suppressing the onset and progression of metastatic cancer by effectively removing metastatic cancer cells in the blood by a physical technique.
For selecting a cell cluster (cluster) having an area of 250 μm 2 or more in a blood sample derived from a subject and diagnosing a cell cluster of metastatic cancer, organ abnormality, or infectious disease using detection of the cell cluster in blood The on-chip cell sorter system according to any one of claims 1 to 7, which is used in a diagnostic method,
The diagnostic method comprises:
This cell cluster is identified from the selected cell mass by culture, gene mutation test, or expression analysis test,
When the identified cell is a metastatic cancer cell in the blood, the gene mutation is confirmed in each cluster unit, and when each cluster is the same mutant, the degree of progression of cancer is If the metastasis from primary cancer is presumed to be early, or if the history of mutations in each cluster is the same but there are many different mutation points, the location of the metastatic cancer Is estimated to be in many areas,
If the cell cluster that flows in the blood can be identified as an organ tissue fragment, organ disease is presumed, or for phagocytic leukocytes including macrophages, cells larger in size than normal are collected, and By examining and identifying genes of heterologous cells such as bacteria, selectively recovering those whose internal shape has been complicated by the activation of B cells and those having increased in size, and clarifying the antigens of the B cells Guess what the immune system is reacting to,
On-chip cell sorter system.
A cell cluster diagnosis method for metastatic cancer, organ abnormality, or infectious disease using detection of cell clusters in blood,
In a blood sample derived from a subject, a cell cluster (cluster) having an area of 250 μm 2 or more is selected,
This cell cluster is identified from the selected cell mass by culture, gene mutation test, or expression analysis test,
When the identified cell is a metastatic cancer cell in the blood, the gene mutation is confirmed in each cluster unit, and when each cluster is the same mutant, the degree of progression of cancer is If the metastasis from primary cancer is presumed to be early, or if the history of mutations in each cluster is the same but there are many different mutation points, the location of the metastatic cancer Is estimated to be in many areas,
If the cell cluster that flows in the blood can be identified as an organ tissue fragment, organ disease is presumed, or for phagocytic leukocytes including macrophages, cells larger in size than normal are collected, and By examining and identifying genes of heterologous cells such as bacteria, selectively recovering those whose internal shape has been complicated by the activation of B cells and those having increased in size, and clarifying the antigens of the B cells Guess what the immune system is reacting to,
Cell cluster diagnostic method.
A method for confirming the presence of abnormal cells in blood,
Measuring a cell size in a blood sample from a subject containing blood cells excluding red blood cells,
Based on the result of the measurement step, a step of obtaining a cell size distribution spectrum of the blood sample, and whether the cell size distribution spectrum has one peak or two or more peaks in the cell size distribution spectrum. A method comprising the step of identifying.
In the case where two or more peaks are identified as a result of the identification step, the method further includes a step of selectively collecting cells larger than the threshold value from the blood sample with a peak on the large cell side as a threshold value. The method of claim 22.
24. The method according to claim 22 or 23, wherein in the step of measuring the cell size and the step of collecting the cells, an on-chip cell sorter chip having a flow path for flowing the blood sample is used.
The method according to claim 24, wherein the on-chip cell sorter chip is part of the on-chip cell sorter system according to any one of claims 1 to 7.
The method according to any one of claims 22 to 25, which is used for cytodiagnosis of metastatic cancer, organ abnormality, or infectious disease.
A water droplet formation module comprising a sample solution reservoir for holding a sample solution containing cells and a capillary tube connected to the reservoir;
A substrate having a water-repellent surface formed by joining regions having two or more different inclination angles for dropping the formed water droplets;
A substrate tilt control mechanism for adjusting a tilt angle of a surface having two or more different tilt angles of the substrate;
A measurement module for measuring the state of the water droplet and the state inside the water droplet;
Means for separating water droplets in a plurality of traveling directions based on the measurement results;
An imaging cell sorter for observing and separating cells in water droplets.
The means for separating the water droplets in a plurality of travel directions includes one or more guides for controllably moving the water droplets formed on the water repellent surface;
28. The imaging cell sorter according to claim 27, wherein the guide is an electrostatic field guide for controlling the direction of movement of a water droplet charged with a charge opposite to that of the water droplet.