Single particle analysis method, and system for the analysis

Abstract

To purify and concentrate differentiated cells derived from an ES cell or an iPS cell or the like without damaging the cells.SOLUTION: An apparatus for analyzing and separating fine particles includes a flow channel cartridge, light irradiation means, detection means for detecting a target fine particle, and force generating means. In the cartridge, a sample reservoir connected to a first flow channel, a fourth branch flow channel and a fifth branch flow channel connected to the first flow channel, a third A reservoir connected to the fourth branch flow channel, a third B reservoir connected to the fifth branch flow channel, and a fourth reservoir reserving unfractionated fine particles are formed, each of the reservoirs has means for making pneumatic pressure inside each reservoir and pneumatic pressure of a pneumatic pressure control system in each apparatus become the same, and the apparatus controls a flow of flow channels inside the cartridge by controlling pneumatic pressure inside each reservoir by each pneumatic pressure control system in the apparatus.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention aims to damage differentiated cells derived from ES cells, iPS cells, etc. of animals, including humans. Furthermore, it relates to a method for separating and analyzing cells and cell clusters (cell spheroids) individually. [Background technology]

[0002] In the field of regenerative medicine, the key to applying pluripotent stem cells to regenerative medicine and drug discovery is the technology to differentiate them into the desired cells. However, in this case, if undifferentiated cells are mixed in with the differentiated cell group to be transplanted, they can cause tumors. Therefore, technology to completely remove them has become important. Moreover, this removal process must be sterile. Concentration of specific cells is generally performed using a cell sorter, as explained below, but this is not suitable for regenerative medicine technology due to the lack of a sterile process and the significant damage it causes to cells. Therefore, new equipment suitable for practical use is currently required.

[0003] First, we will explain the conventional technology of cell sorters for cell collection. A cell sorter is a device that separates and concentrates target cells, but it also functions as a flow cytometer for identifying target cells. Flow cytometers are commonly used to analyze various types of cells and particles contained in liquids. Conventional flow cytometers are typically made of quartz and include an optically transparent flow cell through which a liquid containing cells to be individually identified flows. The flow of cells through this flow cell is typically concentrated in the center of the flow channel by a sheath liquid that concentrically surrounds the flow of cells. The center of the flow channel is illuminated with a laser beam, and as cells pass through the illuminated area, light scattering occurs depending on the size, shape, and refractive index of the cells. The wavelength of this laser light is determined in combination with the type of fluorescent dye to detect the fluorescence of cells specifically stained with the fluorescent dye. In this way, detecting the scattered light and fluorescence of individual cells using multiple photodetectors at different wavelengths enables multifaceted cell analysis. The above-mentioned flow cytometer technology is described in Patent Document 1.

[0004] Patent Documents 1 and 2 describe cell or particle separation methods used in general cell sorters. This method involves ejecting sample liquid as droplets into the air from a droplet-forming nozzle, electrically charging the droplets containing the target cells, and separating them using an electric field. Patent Document 3 discloses a method in which a sheath flow is passed around the sample liquid flowing through a flow cell, and an electric field is applied to the sample liquid to shift charged particles from the sample flow toward the sheath flow, thereby separating and measuring them. Patent Document 4 describes a method in which a pressure pulse is applied to particles flowing through a flow cell to separate them into a channel other than the steady flow channel within the flow cell. Patent Document 5 describes a technique in which a sheath flow is applied to the surrounding area of ​​a flow cell to constrict particles flowing through the flow cell, shifting the flow of the particles and separating them. Patent Document 6 discloses a method for separating charged cells in a liquid using an electric field using gel electrodes installed on both sides of the channel within the flow cell. Patent Document 7 discloses a method in which a pressure pulse is applied using a bubble that forms a meniscus perpendicular to the particle flow, shifting the flow and separating them. Patent Document 8, like Patent Document 5, applies a pressure pulse, but describes a method of ejecting droplets containing the target particles into a container. Patent Document 9 describes a method of measuring particles in a sample liquid flow constricted by a sheath flow, and if it is determined that the particles are the target particles, introducing them into a separate flow path using a pulsed flow to separate them. This technology is a sorting technology within a fixed microchannel, not a sorting technology within a disposable, replaceable channel. A method is known in which magnetic particles coated with antibodies are used to attract magnetic particles to specific cells and separate them using a gradient magnetic field (Patent Document 10). Patent Documents Patent Document 11 describes a flow cytometer technology that uses a disposable, replaceable microchannel cartridge that achieves contamination-free performance ideal for regenerative medicine. Furthermore, Patent Document 12 describes a method for separation within a disposable, replaceable microchannel cartridge. Patent Document 13 describes a method that has been further improved to reduce the influence of flow within the microchannel. Patent Documents 14 and 15 describe a technology for separating cells within a disposable, replaceable microchannel chip, in which a magnetically driven valve is installed within the channel to separate cells.

[0005] Next, we will explain the technology for dispensing cells one by one. Patent Document 16 describes a technology in which cells are sucked up with a hollow pipette, moved to a different location, and then dispensed by expelling. Patent Document 17 describes a technology in which the presence or absence of cells is confirmed by image recognition, and then the cells are dispensed by dripping them droplet by droplet using pressure pulse drive by a piezoelectric element. Furthermore, Patent Document 23 describes a dispensing method in which the dispensing head can be replaced in order to perform dispensing under sterile conditions.

[0006] Next, we will explain the technology that can analyze the genes of cells at the individual unit. In 1999, Vogelstein and Kinzler proposed a technology called digital PCR. This technology allows the detection of the target gene among impurities. This technique is an excellent method for detecting gene sequences with high sensitivity. This technique is described in Non-Patent Document 1. This technique enables highly sensitive detection by dividing the PCR reaction space into minute compartments, thereby eliminating the influence of contaminants.

[0007] Patent Document 19 describes a technique for forming an emulsion of droplets in oil using fluorinated oil, a technique for performing digital PCR within the emulsion particles of droplets in oil, and a method for adding different PCR reaction reagents within the emulsion particles by fusing different emulsion particles one-to-one.

[0008] Patent Document 20 describes a technique for measuring the flow velocity of particles passing through a channel. This method involves splitting the same light source into two, irradiating the flow in the channel with light at two points spaced a certain distance apart in the direction, and evaluating the flow velocity based on the time difference between the two points.

[0009] Patent Document 21 describes a method in which pulsed air is sent at the same time as fine particles flow down from a nozzle into the atmosphere to blow away unnecessary fine particles, and necessary fine particles fall downward to be collected.

[0010] Patent Document 22 describes a technique for forming droplets in oil with cells entrapped in them, and also describes a method for adding different PCR reaction reagents into emulsion particles by fusing different emulsion particles one-to-one. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 3,710,933 [Patent Document 2] U.S. Patent No. 3,826,364 [Patent Document 3] Japanese Unexamined Patent Publication No. 64-3541 [Patent Document 4] Japanese Patent Application Publication No. 1-170853 [Patent Document 5] International Publication No. 98 / 10267 [Patent Document 6] International Publication No. 2004 / 101731 [Patent Document 7] U.S. Patent No. 6,808,075 [Patent Document 8] International Publication No. 2006 / 076195 [Patent Document 9] U.S. Patent No. 4,756,427 [Patent Document 10] International Publication No. 96 / 28732 [Patent Document 11] U.S. Patent No. 8,248,604 [Patent Document 12] Patent No. 5382852 [Patent Document 13] International Publication No. 2011 / 086990 [Patent Document 14] U.S. Patent No. 8,822,207 [Patent Document 15] U.S. Patent No. 8,993,311 [Patent Document 16] U.S. Patent Application Publication No. 2005 / 0136528 [Patent Document 17] U.S. Patent No. 8,834,793 [Patent Document 18] Patent No. 4927719 [Patent Document 19] U.S. Patent No. 7,968,287 [Patent Document 20] Japanese Patent Application Laid-Open No. 2006-300565 [Patent Document 21] U.S. Patent No. 6,657,713 [Patent Document 22] US published patent US20150057163 [Patent Document 23] Patent Application No. Hei 11-295323 [Non-patent literature]

[0012] [Non-Patent Document 1] BERT VOGELSTEIN AND KENNETH W. KINZLER "Digital PCR" Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9236-9241, August 1999 [Non-patent document 2] White et al. “Digital PCR provides sensitive and absolute calibration for high throughput sequencing” BMC Genomics 2009;10:116 [Non-patent document 3] Jim F. Huggett et al. “The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR” Clinical Chemistry 2013Jun;59(6):892-902. [Non-patent document 4] Nao Hirata et al. “A Chemical Probe that Labels Human Pluripotent Stem Cells” Cell Reports 6, 1165-1174, March 27, 2014 [Non-patent document 5] Richard Williams et al. “Amplification of complex gene libraries by emulsion PCR” NATURE METHODS VOL.3 NO.7 2006 545-550 [Non-patent document 6] SIGMA-ALDRICH Product Information SeqPlex DNA Amplification KitCatalog Number:SEQXE [Non-Patent Document 7] SIGMA-ALDRICH Product Information SeqPlex RNA Amplification KitCatalog Number:SEQR Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention addresses several challenges in cell analysis for regenerative medicine:

[0014] 1) Issues with sorting As a sorting method suitable for regenerative medicine, we will mainly explain the issues involved in sorting technology within disposable replaceable microchannel cartridges, which are suitable for contamination-free, damage-free, and aseptic sorting.

[0015] 1-1) Issues with sorting for aseptic processing For regenerative medicine applications, the sorting process must not damage the collected cells and must be aseptic. To meet these requirements, a sorter that performs sorting within a disposable flow path cartridge is more suitable than a method that performs sorting in the atmosphere. Patent Document 13 describes a sorting method within a disposable flow path cartridge in which cells are sorted by controlling the flow of cells within the flow path cartridge using air pressure. In this method, the air in the reservoir on the flow path cartridge is connected to the air in the air pump outside the flow path cartridge via a filter. This filter prevents airborne bacteria and foreign matter from mixing with the liquid in the flow path cartridge. However, if the pore size of this filter is small, the air conductance decreases, which can lead to a problem of reduced sorting power. Patent Documents 14 and 15 describe a technology for sorting cells by installing a magnetically movable valve in the flow path of a disposable, replaceable flow path cartridge and driving the valve by turning an external electromagnet on and off. In this case, the sorting force is a magnetic force unrelated to aerodynamic forces, making sorting possible in a sealed space and achieving aseptic sorting. However, the problem is that the disposable flow path cartridge itself is expensive due to the complex moving parts built into it that are not suitable for mass production.

[0016] 1-2) Challenges in adjusting sample concentration Patent Document 11 describes a flow cytometer technology that uses a disposable, replaceable flow path cartridge. This technology applies the same air pressure to the sheath fluid reservoir and the sample fluid reservoir, allowing the flow rate to be changed while maintaining a constant concentration of the sample flow. However, if a sample particle liquid is placed in the disposable, replaceable flow path cartridge and the particle liquid concentration is too high or too low, the sample must be recovered and the concentration adjusted again.

[0017] 1-3) Technical challenges in sorting large cells or cell clusters Mass cell culture is common in regenerative medicine. However, cells are often cultured in the form of cell clusters (cell spheroids) rather than individually, often exceeding 100 micrometers in size. In contrast, the cell sorter principles described in Patent Documents 1 and 15 aim to separate droplets containing single cells from a nozzle into the atmosphere, but it is difficult to stably form droplets containing cell clusters larger than 100 μm. Patent Document 21, however, discloses a technology that overcomes this limitation. This method uses a continuous, filamentous stream of liquid, rather than droplets, flowing from a nozzle into the atmosphere. Pulsed air is then blown in from the side to remove unwanted cells, allowing the desired cells to fall and be collected. However, because this method does not involve cell sorting in a closed space, it is susceptible to contamination by airborne bacteria and is not suitable for aseptic processing due to the lack of a replaceable flow path. In the cell collection and sorting technology in a disposable replaceable flow path cartridge described in Patent Document 13, when the cross-sectional size of the flow path is 80 μm in width and 80 μm in depth, Although it has been confirmed that cell spheroids up to 100 μm in size can be sorted, it is impossible to flow cell spheroids larger than 100 μm through the flow channel. Therefore, a technology for aseptically sorting large cell aggregates (cell spheroids) in a disposable cartridge suitable for regenerative medicine applications has not yet been realized.

[0018] 1-4) Issues regarding the purification of differentiated cells in regenerative medicine When transplanting cell sheets composed of differentiated cells into patients in regenerative medicine, tumor-causing undifferentiated cells must be removed. Conventional differentiation induction does not achieve a 100% differentiation rate, but rather involves the presence of at least 1% undifferentiated cells. Methods for removing these cells include the so-called conventional cell sorting method (Patent Documents 1 and 2), which uses a cell sorter to separate only differentiated cells, and the method described in Patent Document 10, which uses antibody magnetic beads to remove undifferentiated cells. Conventional cell sorting using droplet separation in the atmosphere involves the problem of significant cell damage due to high-speed flow and surface collisions during collection. The problem with antibody magnetic beads is that it is impossible to remove undifferentiated cells that are not present on the surface of the cell mass.

[0019] 1-5) Necessity and challenges of emulsion sorting It is becoming increasingly recognized that pathological tissues are a mixture of diverse cells, and that analysis cannot be performed using population average data. Therefore, digital PCR, which is less susceptible to the influence of impurities by dividing the PCR reaction space into minute parts, is attracting attention. This method includes emulsion method and multi-chamber method. The emulsion method is advantageous for analyzing more than 100,000 particles, while the multi-chamber method is advantageous for analyzing less than 10,000 particles. On the other hand, over the past 10 years, Next-generation DNA sequencers, which have achieved the miracle of improved performance and cost performance, are creating new markets in the future. High-purity sample purification is required for these next-generation sequencers (Non-patent Documents 2 and 3). As a tool for this purification, sorting of emulsion droplets after digital PCR is required. Therefore, we will introduce the following emulsion droplet sorting method. We will explain the challenges of sorting droplets.

[0020] In the present invention, cells are incorporated into the microreaction space of the emulsion, and a gene containing a partial sequence of interest is isolated by digital PCR targeting a partial sequence of the gene in the cells. The emulsion droplets are specifically fluorescently marked. Then, emulsion particles containing the target gene are separated using this fluorescence as an indicator. Pretreatment equipment and technology are then provided to amplify all the genes of a single cell contained within the particle and perform detailed analysis using a sequencer. Since DNA contamination must be avoided in the first place, the technology must be able to process the data within a disposable, replaceable chip. The challenges with this method are as follows: Regarding digital PCR technology using an emulsion of droplets in oil, a method using fluorinated oil is described in Patent Document 19. Known examples of fluorinated oils include Fluorinert, which has a high specific gravity and causes droplets in the fluorinated oil to float to the top of the oil. When sorting these emulsion particles in a disposable, replaceable flow path cartridge, the emulsion floats on top of the reservoir on the disposable flow path cartridge. When oil flows out of the bottom of the reservoir into the flow path and the top surface of the oil drops, emulsion droplets adhere to the inner wall of the reservoir, resulting in a significant loss of approximately 80% of the total emulsion.

[0021] 1-6) Issues related to flow velocity Cell sorting devices, such as cell sorters, are based on the premise that the flow rate can be controlled to a constant value. This is because they detect particles in the liquid, identify them, and if they are the target particles, they are sorted downstream from the detection position. Since the time it takes for the particles to travel from the detection position to the sorting position varies depending on the flow rate, the flow rate must be constant in order to sort the particles a certain amount of time after detection. The flow rate within the channel can be adjusted by applying pressure, but the relationship between pressure and flow rate varies depending on the viscosity of the buffer that suspends the sample and the viscosity of the sheath fluid that is flowed together with the sample liquid to narrow it down. For this reason, the types of buffer and sheath fluid available for commercially available cell sorters are generally limited by the manufacturer. In the current situation described above, certain types of cells often require specific culture media for measurement in a live state. Therefore, flow rate adjustment technology is required to accommodate not only specific buffers but also various buffers with different viscosities, including culture media.

[0022] 2) Challenges with single-cell analysis methods In regenerative medicine, when transplanting differentiated cell populations, they must be free of undifferentiated cells, which can cause tumors. Therefore, it is necessary to detect and remove undifferentiated cells, even if they are present at a low density of approximately one per 1,000,000 differentiated cells. Therefore, quality control of differentiated cells is crucial. This requires analysis of individual cells or cell clusters. For this analysis, digital PCR, which utilizes a detection reaction within a microspace to eliminate the influence of contaminating cells, as described in Non-Patent Document 1, is suitable. Patent Document 22 describes a method for incorporating individual cells into emulsion droplets and analyzing them using a detection reaction within that microspace. In this method, the PCR reaction is performed after lysing the cells in the emulsion. The detection sensitivity of conventional PCR is at a level that allows the detection of target genes at a concentration approximately 1 / 1000 of that of non-target genes. In contrast, with digital PCR, there is no influence of contaminants, so the specificity limit is 1 million subdivisions in the reaction space. This method makes it possible to detect targets at a precision of about 1 / 1 million. The proportion of individuals with a gene sequence can be quantified. Just because a gene contains a child sequence does not mean that other sequences are common. Therefore, it is necessary to separate genes containing the target gene sequence individually, amplify the entire gene, and determine the entire sequence using a next-generation sequencer. To implement this method, reagents for whole gene amplification must be added to emulsion droplets selected by fluorescent signal after the PCR reaction. Patent documents 19 and 22 both describe a method in which different droplets are fused one-to-one within a channel. According to these methods, if the other emulsion droplet to be fused contains reagents for the next reaction, another whole gene amplification reaction can be performed after fusion. However, these methods are less reliable because the position of each droplet is not controlled. On the other hand, a method for detailed analysis of single cells without using emulsions is to separate the cells in a disposable replaceable flow channel cartridge, then directly dispense the single cells into a multi-well plate and analyze them by PCR reaction or the like. In this case, as described in Patent Document 17, there is a method in which a piezo is used to eject the dispensing liquid from a dispensing nozzle. The problem with this method is that the pressure pulses from the piezo damage the cells. Since cells that have been damaged and died cannot be analyzed for RNA, for example, dispensing the cells in the same way as cell sorting is required. Low damage is also required. [Means for solving the problem]

[0023] In view of the above circumstances, the present inventors have conducted extensive research into a method for analyzing single cells or cell spheroids that is optimal for regenerative medicine, a method for removing undifferentiated cells from differentiated cells, and an apparatus for performing the same. As a result, they have surprisingly found that the above-mentioned problems can be solved by the following apparatus or method. The present invention is based on this finding.

[0024] Therefore, the present invention provides [1] A flow path cartridge having a flow path formed on a transparent substrate; a light irradiation means for irradiating light onto particles in the sample liquid flowing through the flow channel; a detection means for detecting scattered light or fluorescence emitted from the particles when irradiated with the light, identifying the particles based on the signal strength of the scattered light or fluorescence, and detecting the target particles; a force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detecting means, wherein the cartridge is formed with a reservoir for sample liquid (sample reservoir) connected to a first flow path, a fourth branch flow path and a fifth branch flow path connected to opposite sides of the first flow path, a 3A reservoir for sending out a pulsed flow connected to the fourth branch flow path, a 3B reservoir connected to the fifth branch flow path for sorting and recovering the particles by changing the flow of the particles toward the fifth branch flow path using a pulsed flow generated by the force generating means flowing from the fourth branch flow path toward the fifth branch flow path, and a fourth reservoir connected to the downstream side of the first flow path for storing particles not sorted, and wherein the inside of each reservoir is sealed from the outside by being covered with a sealing cover. [2] An apparatus for analyzing and separating particles, comprising: a flow path cartridge having a flow path formed on a transparent substrate; a light irradiating means for irradiating light onto particles in a sample solution flowing through the flow path; a detecting means for detecting scattered light or fluorescence emitted from the particles when the light is irradiated, identifying the particles based on the signal strength, and detecting target particles; and a force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detecting means, wherein the cartridge comprises a sample solution reservoir (sample reservoir) connected to a first flow path, a fourth branch flow path and a fifth branch flow path connected to both sides of the first flow path so as to face each other, a third A reservoir for sending out a pulse flow connected to the fourth branch flow path, and a pulse flow generated by the force generating means flowing from the fourth branch flow path in the direction of the fifth branch flow path to change the flow of particles in the fifth branch flow path. a third reservoir connected to a fifth branch flow path for separating and recovering the particles by changing the polarity of the first flow path, and a fourth reservoir connected to the downstream side of the first flow path for storing the particles not separated, each reservoir being sealed from the outside by being covered with a sealing cover, and a means for equalizing the air pressure in each reservoir with the air pressure of each air pressure control system within the device, wherein the device for analyzing and separating particles according to [1] is characterized in that the flow in the flow path within the cartridge is controlled by controlling the air pressure in each reservoir with the air pressure control system within the device. [3] An apparatus for analyzing and separating particles, comprising: a flow path cartridge formed on a transparent substrate with a flow path; a light irradiating means for irradiating light onto particles in a sample liquid flowing through the flow path; a detecting means for detecting scattered light or fluorescence emitted from the particles when the light is irradiated, identifying the particles based on the signal strength, and detecting the target particles; and a force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detecting means, wherein the cartridge includes a first flow path for the sample liquid connected to the first flow path. a fourth branch channel and a fifth branch channel connected to both sides of the first channel so as to face each other; a third A reservoir for sending out a pulsed flow connected to the fourth branch channel; a third B reservoir connected to the fifth branch channel for sorting and recovering the particles by changing the flow of the particles to the fifth branch channel by a pulsed flow generated by the force generating means and flowing from the fourth branch channel to the fifth branch channel; and a fourth reservoir connected to the downstream side of the first channel for storing particles that have not been sorted. The apparatus for analyzing and separating microparticles according to [1], characterized in that the reservoirs are covered with a cover for sealing, thereby sealing the inside of each reservoir from the outside, and the sealing covers of the 3A reservoir for sending out the pulse flow and the 3B reservoir for collecting the microparticles are stretchable and deformable membranes, and the apparatus has an actuator for displacing the sealing cover membrane by applying a mechanical force from the outside to the sealing cover membrane, and when the microparticles pass through the sorting area, the actuator operates to push down the sealing cover membrane of the 3A reservoir at high speed and to pull up the sealing cover of the 3B reservoir, thereby generating a pulse flow in the branch flow path and sorting the microparticles, [4] A flow path cartridge including a first flow path formed on a substrate, into which a sample liquid containing particles is introduced, second and third flow paths arranged on both sides of the first flow path and into which a sheath liquid is introduced, a first confluent flow path where the first to third flow paths are joined, a first reservoir as a reservoir for the sample liquid, a second reservoir as a reservoir for the sheath liquid, and a reservoir (fourth reservoir) for storing waste liquid, a light irradiation means for irradiating light onto the particles flowing in the first confluent flow path, and a means for detecting and analyzing scattered light or fluorescence generated from the particles. the flow path cartridge has first to third flow paths on the upstream side of a first confluent flow path, the first flow path is connected to a first reservoir, the second and third flow paths are connected to a second reservoir, and the first confluent flow path includes a flow path pattern that is connected to a fourth reservoir on the downstream side, and the flow path cartridge has a function of adjusting the narrowing width of the sample flow and the sample flow rate by adjusting the pressure of each of the first reservoir, the second reservoir, and the fourth reservoir; [5] A particle analyzing and separating device comprising: a flow path cartridge having a flow path formed on a transparent substrate; light irradiating means for irradiating light onto particles in a sample solution flowing through the flow path; detection means for detecting scattered light or fluorescence emitted from the particles when the light is irradiated and identifying the particles based on the signal intensity to detect target particles; and force generating means for changing the flow direction of the particles flowing through the flow path of the cartridge based on a signal from the detection means, wherein the cartridge comprises a sample solution reservoir (sample reservoir) connected to a first flow path; a fourth branch flow path and a fifth branch flow path connected to both sides of the first flow path so as to face each other; a third A reservoir for sending out a pulse flow connected to the fourth branch flow path; and a pulse flow generated by the force generating means flowing from the fourth branch flow path to the fifth branch flow path, thereby changing the flow of the particles to the fifth branch flow path, thereby sorting the particles. a third B reservoir connected to a fifth branch flow path for collecting the particles, and a fourth reservoir connected to the downstream side of the first flow path for storing the unsorted fine particles, each reservoir being covered with a sealing cover to seal the interior of each reservoir from the outside, each flow path having a width and depth of 150 micrometers or more, and an irradiated laser beam size in the flow path width direction being 100 micrometers or more; [6] A differentiated cell purification method using the device according to any one of [1] to [5], which removes undifferentiated cells that are mixed in at a lower density than the differentiated cells from a group of differentiated cells differentiated from undifferentiated cells, characterized in that, based on a signal from the detection means, a flow path change process is performed by applying a force that changes the flow direction to the undifferentiated cells, and a flow path change process is not performed by applying a force that changes the flow direction to the differentiated cells, thereby removing the undifferentiated cells from the differentiated cells contained in the fourth reservoir that has been passed through, thereby recovering the cell liquid and improving the recovery rate of the differentiated cells. [7] The differentiated cell purification method according to [6], characterized in that the same undifferentiated cell removal treatment is repeated for the recovered cell solution. [8] A device for dispensing microparticles contained in a sample liquid one by one, the dispensing nozzle is an automatically replaceable transparent hollow pipette, the amount of liquid dispensed is 0.3 μL or less, and the presence and number of particles 10 μm or larger are detected by image recognition of the entire amount of liquid dispensed by the hollow pipette at each dispense; a dispensing device for dispensing a specified number of particles into a multi-well plate; [9] An apparatus for analyzing and separating particles, comprising: a flow path cartridge having a flow path formed on a transparent substrate; light irradiating means for irradiating light onto particles in a sample liquid flowing through the flow path; detection means for detecting scattered light or fluorescence emitted from the particles when the light is irradiated, identifying the particles based on the signal intensity, and detecting target particles; and force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detection means, wherein the cartridge comprises a reservoir (sample reservoir) for the sample liquid connected to the flow path, and a reservoir connected to the flow path for separating and recovering particles from the flow path by changing the direction of flow with the force generating means, wherein the inner wall of the sample reservoir or the reservoir for recovering the separated particles is covered with a water-repellent material, or is covered with an adapter made of a water-repellent material.

[10] The particle analyzing and separating device according to [9], wherein the water-repellent material is a fluororesin.

[11] A method for forming droplets in oil by merging oil into a flow path in which a sample liquid is flowing through flow paths that merge from the left and right, characterized in that a component with a high specific gravity is mixed into the sample liquid to make the specific gravity of the droplets higher than that of the oil.

[12] The method for forming droplets in oil according to

[11] , wherein the component with a high specific gravity is polytungsten, bromoform, or iodomesilene.

[13] A particle measurement or particle sorting device including a flow path cartridge formed on a transparent substrate, a light irradiation means for irradiating light onto a part of the flow path through which particles in a sample liquid flow, a detection means for detecting scattered light or fluorescence generated when each of the particles passes through the light irradiation area, and a means for identifying a target particle based on each light signal, the particle measurement or particle sorting device including a means for measuring the flow velocity of each particle based on the light signal, and a means for adjusting the flow velocity of the particles so that it is constant.

[14] The particle measurement or particle sorting device according to

[13] , wherein the means for measuring the flow velocity is an irradiation optical system that irradiates two irradiation lights of different wavelengths onto positions at a fixed distance apart in the flow direction in the flow path, and the means for adjusting the flow velocity of the particles to be constant is a means for measuring the time change in pulse waveform of scattered light intensity of each wavelength generated when the particles pass through, calculating the individual flow velocity of the particles based on the time difference between the peak values ​​of each pulse waveform, and adjusting the flow velocity to be constant so that the average flow velocity of the particles is constant.

[15] A method for measuring or sorting particles using the particle measuring or particle sorting device according to

[13] or

[14] , characterized in that the particles are suspended in an arbitrary buffer.

[16] The method for measuring or separating fine particles according to

[15] , wherein the buffer is an aqueous liquid or an oily liquid.

[17] A system for genetic analysis of cells, comprising: a means for forming droplets in oil containing cells, a cell lysis reagent, and a PCR reaction reagent; a means for carrying out a PCR reaction after the cell lysis reaction; a means for separating the droplets in the oil by fluorescent labeling after the PCR reaction; and a means for dispensing the separated droplets in the oil one by one.

[18] The gene analysis system according to

[17] , wherein the cell is a single cell or a single cell spheroid.

[19] The means for forming droplets in the oil is a disposable flow path cartridge, the width of the flow path for cells in the disposable flow path cartridge is at least 100 μm or more, and the size of the droplets formed in the oil can be adjusted to 40 μm to 100 μm. The genetic analysis system according to

[17] or

[18] ,

[20] The genetic analysis system according to any one of

[17] to

[19] , characterized in that the dispensing means is a dispensing means for dispensing into a multi-well plate, and the liquid discharged from the dispensing nozzle is pushed out while in contact with the inner wall of the multi-well plate or the liquid present in the multi-well plate.

[21] The dispensing nozzle of the dispensing means is an automatically replaceable transparent hollow pipette, the amount of the dispensed liquid is 0.3 μL or less, and image recognition of the entire amount of the dispensed liquid of the hollow pipette is performed for each dispensing, and the liquid is discharged when there is one particle of 10 μm or more.

[17] to

[20] The genetic analysis system described herein,

[22] A genetic analysis method comprising the steps of: forming droplets in oil containing cells, a cell lysis reagent, and a PCR reaction reagent; performing a PCR reaction after the cell lysis reaction; fractionating the droplets in the oil by fluorescent labeling after the PCR reaction; and dispensing the fractionated droplets in the oil one by one.

[23] The genetic analysis method according to

[22] , wherein the cell is a single cell or a single cell spheroid.

[24] The genetic analysis method according to

[22] or

[23] , wherein the step of forming droplets in oil uses a disposable flow path cartridge, the width of the flow path through which the cells flow is at least 110 μm, and the size of the droplets in oil formed is 40 μm to 100 μm.

[25] The genetic analysis method according to any one of

[22] to

[24] , characterized in that the step of dispensing droplets in oil is dispensing into a multi-well plate, and the liquid discharged from the dispensing nozzle is pushed out while in contact with the inner wall of the multi-well plate or the liquid present in the multi-well plate.

[26] The dispensing nozzle in the dispensing process of droplets in oil is an automatically replaceable transparent hollow pipette, the amount of dispensing liquid is 0.3 μL or less, and image recognition of the entire amount of dispensing liquid in the hollow pipette is performed for each dispensing, and dispensing is performed only when there is one particle of 10 μm or more

[22]

[0025] The genetic analysis method according to any one of the preceding claims, and

[27] An emulsion droplet forming device for forming droplets in oil containing cells, characterized in that the flow path for forming the droplets in oil is located in a disposable flow path cartridge, the cartridge is formed with a plurality of reservoirs connected to the flow path, the size of the flow path for the cells is at least 110 μm or more, and the size of the formed droplets in oil can be adjusted in the range of 40 μm to 100 μm by controlling the air pressure above the liquid in each reservoir. Regarding. [Effects of the Invention]

[0025] According to the present invention, it is possible to perform aseptic sorting in a closed space, which is necessary in regenerative medicine. Furthermore, it is possible to sort undifferentiated cells mixed in with differentiated cells induced to differentiate from iPS cells or ES cells. Efficient removal of differentiated cells 6 We have achieved zero undifferentiated cells per individual. In addition, it is possible to sort cell clusters (cell spheroids) with a size of 100 μm or more, and it is now possible to remove undifferentiated cells contained in the cell clusters on a cluster-by-cluster basis. It has become possible to perform genetic analysis on a cell spheroid basis. [Brief explanation of the drawings]

[0026] [Figure 1] (A) Shows the closed system state with the reservoir in the disposable replaceable flow channel cartridge covered. (B) Shows a method for controlling the liquid flow in the flow channel inside the cartridge from the outside with the reservoir in the disposable replaceable flow channel cartridge covered. (C) Shows the measurement system for analyzing particles in the flow channel in (B) above. [Figure 2](A) A top view of a disposable, replaceable flow path cartridge for sorting is shown. (B) An AA' cross-sectional view of a disposable, replaceable flow path cartridge for sorting is shown. (C) A BB' cross-sectional view of a disposable, replaceable flow path cartridge for sorting is shown. (D) A CC' cross-sectional view of a disposable, replaceable flow path cartridge for sorting is shown. (E) A DD' cross-sectional view of a disposable, replaceable flow path cartridge for sorting is shown. [Figure 3] 1 shows a flow path pattern in a disposable replaceable flow path cartridge for sorting. [Figure 4] (A) A top view of the structure when the disposable replaceable flow path cartridge for sorting shown in Figure 2 is used for sorting emulsion droplets. (B) A cross-sectional view of (A) taken along the line AA'. [Figure 5] (A) Two types of laser irradiation positions for evaluating flow velocity in a disposable replaceable flow path cartridge for sorting. (B) Signal measurement method for evaluating flow velocity. [Figure 6] 1 shows a disposable, replaceable fluidic cartridge for forming cell-containing droplets in oil. [Figure 7] FIG. 7 is an explanatory diagram of the flow path pattern of FIG. 6. [Figure 8] FIG. 1 is a top view of a disposable, replaceable flow channel cartridge that simultaneously forms four types of cell-containing droplets in oil. [Figure 9] (A) Schematic diagram of a method for dispensing droplets in oil. (B) Shows how droplets in oil are discharged from a dispensing pipette. (C) External view of the device. [Figure 10] This shows the case where pulsed air pressure is used as the sorting force by connecting a syringe pump and an electromagnetic valve in series. [Figure 11] This shows a case where the force pressing the elastic cover membrane as the sorting force is changed by an electromagnetic actuator. [Figure 12] (A) shows an example of measured flow rate when adjusting the flow rate by changing the pressure of the sample liquid reservoir in the disposable replaceable flow cartridge. (B) shows an example of measured flow rate when adjusting the width of the sample flow by changing the pressure of the sample liquid reservoir in the disposable replaceable flow cartridge. [Figure 13] (A) An example of the disposable replaceable flow channel cartridge structure when measuring flow velocity using side-scattering signals from two lasers with different wavelengths. (B) An example of the device optical system when measuring flow velocity using side-scattering signals from two lasers with different wavelengths. [Figure 14] An example of particle flow velocity distribution is shown below. [Figure 15] (A) An example of evaluation of the efficiency of undifferentiated cell removal treatment in regenerative medicine (when the undifferentiated cell ratio is 20%). (B) An example of evaluation of the efficiency of undifferentiated cell removal treatment in regenerative medicine (when the undifferentiated cell ratio is <0.3%). [Figure 16] Shown is a camera image of a 10 μm particle in a dispensing pipette. [Figure 17] This is a flowchart summarizing the procedures for analyzing single cells (Case 1), single cell clumps (Case 2), emulsion droplets containing single cells (Case 3), and emulsion droplets containing single cell clumps (Case 4). [Figure 18] (A) A diagram showing pressure control using constant pressure pump control to maintain constant steady flow rates in the sample fluid reservoir, sheath fluid reservoir, and waste fluid reservoir in Figure 2. (B) A diagram showing a system for pressure control for the sorting fluid reservoir 16 and collection reservoir 17 in Figure 2, connecting a compressor or vacuum pump, an electropneumatic regulator, a buffer tank, and further an electromagnetic valve. [Figure 19] FIG. 10 is a diagram showing one embodiment of a means for equalizing the air pressure in the air pressure control system with the air pressure in the reservoir. DETAILED DESCRIPTION OF THE INVENTION

[0027] 1) Embodiments of the means for solving problems in sorting When applying a cell sorting device to regenerative medicine, a method of sorting cells in a closed system within a disposable, replaceable flow path cartridge is suitable. Furthermore, for genetic analysis of single cells using digital PCR, a technique for sorting emulsion droplets within a disposable, replaceable flow path cartridge, which does not cause DNA contamination between samples, is suitable. However, there are several issues that need to be resolved. An embodiment that resolves these issues will be described.

[0028] 1-1) Embodiments for solving the problem of sorting for aseptic processing The apparatus for analyzing and separating particles of the present invention comprises a flow path cartridge formed on a transparent substrate, a light irradiating means for irradiating light onto particles in a sample liquid flowing through the flow path, a detecting means for detecting scattered light or fluorescence emitted from the particles when the light is irradiated, and identifying the particles based on the signal strength of the scattered light or fluorescence to detect the target particles, and a force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detecting means, wherein the cartridge comprises a reservoir for sample liquid (sample reservoir) connected to a first flow path, and a first a fourth branch flow path and a fifth branch flow path connected to opposite sides of the flow path; a third A reservoir for sending out a pulsed flow connected to the fourth branch flow path; a third B reservoir connected to the fifth branch flow path for sorting and recovering the particles by changing the flow of the particles toward the fifth branch flow path using a pulsed flow generated by the force generating means that flows from the fourth branch flow path toward the fifth branch flow path; and a fourth reservoir connected to the downstream side of the first flow path for storing particles that have not been sorted, and each of the reservoirs is covered with a sealing cover, thereby sealing the inside of each reservoir from the outside. The device for analyzing and separating particles of the present invention will be described with reference to FIGS. 1(A) and 1(B). Figure 1(A) shows a simple flow channel cartridge with two reservoirs, with the cartridge airtightly sealed. The figure shows a method for controlling the flow of a channel. Reservoirs 2A and 2B are formed on substrate 1 and are connected by a channel 4 formed within substrate 1. Each reservoir is airtightly capped with covers 3A and 3B. These covers are made of deformable rubber. The cartridge is made of a transparent resin, such as COP, COC, or PMMA. The cover is preferably made of a Teflon (registered trademark)-based elastomer, which is a water-repellent, stretchable resin, but ordinary silicone rubber may also be used. In other words, in the device for analyzing and separating microparticles of the present invention, each reservoir is covered with a cover, thereby sealing the interior of each reservoir from the outside, allowing, for example, cells to be analyzed and / or separated in a sterile manner. Furthermore, the feature of the reservoirs being covered with a cover can be applied to all of the devices described herein. The flow path cartridge used in the present invention has a reservoir covered with a cover as described above, but when in use, a hollow needle-shaped tube can be airtightly inserted through the cover to communicate the air pressure control system in the device with the inside of the reservoir. This hollow needle-shaped tube is an example of a means for equalizing the air pressure in the reservoir and the air pressure in each of the air pressure control systems in the device. For example, FIG. 1(B) shows a state in which hollow needles 8A and 8B are airtightly inserted through covers 3A and 3B, respectively. The hollow needles are preferably made of stainless steel. The hollow needles 8A and 8B are connected to syringe pumps 9A and 9B, respectively. In this state, the syringe pump 9A is pressed. When the syringe pump 9B is pulled to create a positive pressure and the syringe pump 9C is pulled to create a negative pressure, the sample solution 5A in the cartridge flows through the flow path 4 and moves to the waste liquid 4. By using a hollow needle-shaped tube to airtightly penetrate the cover of each reservoir and then installing the cartridge in the device, the air pressure in the air pressure control system within the device and the air pressure in the reservoirs can be made equal. That is, as one means for equalizing the air pressure in the air pressure control system and the air pressure in the reservoirs, multiple hollow needle-shaped tubes connected to multiple air pressure control systems within the device can be airtightly penetrated through the cover of each reservoir and then installed in the device, thereby adjusting the air pressure. This means for equalizing air pressures can be applied to all of the devices described herein. As a means for equalizing the air pressure in the air pressure control system and the air pressure in the reservoir other than the above method, the structure shown in Figure 19 can be used. As shown in the figure, the cover has a two-layer structure consisting of a cover 401 with a hole and a cover 402 that covers the hole, and the contact between the two layers ensures airtightness. As shown in FIG. 19(B), for example, when pressure is applied from above by a rod 403, portions of the two-layer cover separate, breaking the airtight seal, and the air pressure in the reservoir and the space above the cover becomes the same. When equalizing the air pressure in the air pressure control system and the air pressure in the reservoir in this manner, it is important to isolate the air pressure control system and the reservoir space from the external environment, for example, to keep the reservoir space 404 sterile. For this purpose, as shown in FIG. 19B, the rod can be surrounded by a side wall 405 made of a deformable material such as rubber, and the side wall can be brought into contact with the perforated cover 401, thereby isolating the air pressure control system and the reservoir space from the external environment. This means for equalizing air pressure can be applied to all of the devices described herein. In this specification, the air pressure control system within the device can be an atmospheric pressure control system or a pump and valve control system. The air pressure controlled by the air pressure control system can be negative, positive, or normal pressure, and can be adjusted as needed. The features of airtightly penetrating the reservoir covers using hollow needle-like tubes and the features of the air pressure control system within the device can be applied to all aspects of the device described herein. The pressure on the positive pressure side and the negative pressure side is monitored, and the flow rate is controlled by the difference between these values. FIG. 1(C) illustrates the above-mentioned flow path cartridge and the optical system and control system for irradiating light onto particles such as cells flowing through the flow path in the cartridge and detecting optical signals from the particles. Laser light emitted from the laser light source 35-1 irradiates the flow path 4. The moment a particle passes through the irradiated area, scattered light and fluorescent light are generated in pulses. These laser lights are focused by the objective lens 151. The scattered light and the fluorescent light are detected by a plurality of wavelengths in the optical system downstream of the objective lens. The scattered light is detected by a detector 161 by selecting the same wavelength as the irradiated laser light using a dichroic mirror 154 and a bandpass filter 157. A light shielding plate 160 is installed in front of the detector 161. The laser transmitted light is removed. The fluorescence is split into multiple wavelength regions with wavelengths longer than the irradiated laser light by dichroic mirrors 155, 156 and band pass filters 158, 159, and detected by different detectors 162, 163, respectively. The optical signal output from the detector is an analog signal, so it is converted into a digital quantity by an AD converter 164 and sent to a control computer 169, which records and displays the results. The control computer controls an actuator 170 that drives an air pump 9A connected to the upstream reservoir and an air pump 9B connected to the downstream reservoir. The speed of the particles flowing in the flow path 4 is controlled by controlling an actuator 171 that drives the pump 9B. The light intensity of the irradiated laser 35 is controlled by controlling a driver circuit 35-2 of the laser light source 35-1 from the control computer 169.

[0029] 2(A), (B), (C), (D), and (E), we will explain a method for externally controlling the liquid flow inside a disposable, replaceable flow path cartridge for sorting. The air pressure applied to the sample liquid reservoir (sample reservoir: first reservoir) 11, the sheath liquid reservoir (second reservoir) 12, and the waste liquid reservoir (reservoir for storing unsorted particles: fourth reservoir) 21 controls the flow rate of particles such as cells from upstream to downstream. Since the purpose is to sort particles, a steady air pressure should be applied. The "air pressure" applied to these sample liquid reservoir, sheath liquid reservoir, and waste liquid reservoir is one of the "air pressure control systems" in the present invention. In contrast, the sorting force must be a short-term pulse pressure applied only to specific particles flowing through the main channel 22. In other words, in the present invention, a force generating means can be used that applies a force that changes the flow direction based on a signal from the detection means. Figure 3 is an enlarged view of the channel pattern in Figure 2, and shows that particles determined to be sorted by signal light from particles generated in the laser light irradiation region 35 (detection region) are sorted in the sorting region, which is the intersection of the main channel 22 and the sorting channels (24L, 24R). By adjusting the timing of the particles passing through the reservoir 16, the target particles are isolated by adjusting the timing so that a sorting force is generated. An example of a force-generating means for generating pulsed air pressure for sorting is shown in Figure 10. This figure corresponds to the BB' cross section of Figure 2(A). In this method for generating pulsed air pressure, as shown in Figure 10, a constant-pressure air pump and a high-speed electromagnetic valve are connected in series. The constant-pressure air pump 90 applies positive pressure (in this specification, all pressures are expressed as differential pressure values ​​from atmospheric pressure, with atmospheric pressure as the reference pressure), and the constant-pressure air pump 91 applies negative pressure. When valves 92 and 93 are opened for a short period of time, pulsed air pressure acts on the reservoirs 16 and 17, generating a sorting pulse flow. The "constant-pressure air pump and high-speed electromagnetic valve" are also considered to be part of the "air pressure control system" of the present invention. The "constant-pressure air pump and high-speed electromagnetic valve" are also considered to be part of the "force-generating means" for applying pulsed pressure for sorting. Next, Figure 11 shows an example of a "force generating means" that generates a pulsed air pressure different from that shown in Figure 10. In this method, the tops of reservoirs 16 and 17 are covered and sealed with elastic, expandable membranes. These membranes 110 and 111 are fixed to electromagnetic actuators (100 and 101) installed on the device, respectively. When particles determined to be sorted pass through the sorting area, electromagnetic actuators 100 and 101 are displaced for a short period of time. At this time, the force of electromagnetic actuator 100 moves the membrane downward, while the force of electromagnetic actuator 101 moves it upward. The pulsed operation of the electromagnetic actuators generates a pulsed sorting flow. That is, the sorting flow can be generated by the actuators rapidly pushing down the sealing cover membrane of reservoir 3A and lifting the sealing cover of reservoir 3B. When the electromagnetic actuators complete their movement, the sorting pulse flow collects the particles sorted by the sorting pulse flow and takes them into collection reservoir 17. The important thing here is to prevent the occurrence of a flow that would cause the microparticles to flow backward when the pulse flow ceases. Backflow is prevented by increasing the actuator's movement amount in proportion to the number of sorting operations. It is desirable to keep air out of reservoirs 16 and 17. This is because, when no air is present, there is no process of air compression or expansion, and the time response speed of the pressure exerted on the liquid in the reservoir is fast. Note that even when an "electromagnetic actuator" is used for reservoirs 3A and 3B as a force generation means for sorting, a steady air pressure, etc. is applied to the sample liquid reservoir, sheath liquid reservoir, and waste liquid reservoir by an "air pressure control system." For example, the sample fluid reservoir, sheath fluid reservoir, and waste fluid reservoir in Figure 2 Pressure control is a control to keep the flow rate of a steady flow constant, so it is a constant pressure pump control. An example of this constant pressure pump control is a syringe pump, as shown in Figure 1. Other examples include As shown in FIG. 18(A), a method using a compressor 204 and an electropneumatic regulator 202 is also available. In the case of a syringe pump, pressure fluctuations can be ignored, but in the case of an electropneumatic regulator, pressure fluctuations always exist. In order to mitigate these fluctuations, a buffer tank 203 is connected. By combining a compressor, electropneumatic regulator and buffer tank, it is possible to control a constant pressure of 0.1 kPa or less with an accuracy of ±0.025 kPa. The cross-sectional size of the flow path is 80 μm wide and deep. In a channel with a depth of 80 μm, a constant pressure in the range of 0.1 kPa to 30 kPa is usually used for steady flow control to allow the liquid to flow, so a pressure adjustment accuracy of ±0.025 kPa is sufficient performance. FIG. 18(B) shows a pressure control for the sorting fluid reservoir 16 and the collection reservoir 17 in FIG. The figure shows a system connecting a compressor or vacuum pump, an electropneumatic regulator, a buffer tank, and an electromagnetic valve. For the pressure control system for the sorting liquid reservoir 16, the compressor pressure is set to a range of 100 kPa, and the electropneumatic regulator control pressure is set to 50 kPa. For the pressure control system for the collection reservoir 17, the vacuum pump is set to a range of -100 kPa, and the electropneumatic regulator control pressure is set to -50 kPa. This generates a pulse flow sufficient for sorting, allowing the microparticles to be separated.

[0030] 1-2) Embodiments of the means for solving the problem of adjusting the sample concentration Using Figure 2, the sample was sorted using a disposable, replaceable flow path cartridge. The concentration adjustment function will be described below. Figure 2(A) shows a top view of a disposable, replaceable flow path cartridge for sorting. This flow path cartridge has a reservoir (sample reservoir: first reservoir) 11 for sample fluid containing particles such as cells, a reservoir (second reservoir) 12 for sheath fluid, a reservoir (reservoir for storing unsorted particles: fourth reservoir) 21 for waste fluid, a reservoir (third reservoir) 16 for storing fluid for the sorting pulse flow, and a reservoir (third reservoir) 17 for recovering particles. Each reservoir is connected to a flow path via a port on the bottom of the reservoir. The sample fluid is connected to flow path 22 via port 13 on the bottom of reservoir 11. The sheath fluid is connected to flow path 23L via port 15L on the left side of the bottom of reservoir 12 and to flow path 23R via port 15R on the right side. When air pressure is applied to the upper part of the liquid surface of reservoir 11 and reservoir 12, as shown in Figure 3, The sample liquid converges and flows toward the center of the channel by merging with the sheath liquid. When the pressure inside the reservoir 11 for the sample liquid is made higher than the pressure inside the reservoir 12 for the sheath liquid, the flow width W of the sample liquid after merging increases. However, when the pressure inside the reservoir 11 is made lower than the pressure inside the reservoir 12, the flow width W of the sample liquid increases. Then, W becomes smaller. In other words, the ratio of the pressures applied to the reservoir 11 and the reservoir 12 The width of the sample liquid can be adjusted by changing the pressure applied to reservoir 11 and reservoir 12 while keeping the ratio of the pressures applied to reservoir 11 and reservoir 12 constant. The flow rate of the sample liquid can be changed while keeping the flow width constant by changing the pressure applied to each reservoir. When the concentration of particles or cells to be measured in the sample liquid is high, the width of W is adjusted to be small while keeping the flow rate constant. When the concentration is low, the width of W is adjusted to be small. In this case, the width W can be increased while keeping the flow rate constant. This can be achieved by adjusting the pressure applied to the reservoir 12. Figures 12(A) and (B) show the chip shown in Figure 2(A) with the air pressure in the sheath fluid reservoir set to 1.8 kPa (atmospheric pressure reference), the air pressure in the waste fluid reservoir set to -0.7 kPa (atmospheric pressure reference), and the sample fluid reservoir set to The figure shows the relative changes in flow rate and sample flow width when the air pressure inside the sample is changed. The sample buffer is a viscous liquid with a viscosity of 3.6 CP, and the sheath liquid is a viscous liquid with a viscosity of 1.07 CP. This figure shows an example of measurements for a liquid containing a sample. When the air pressure in the sample reservoir is increased to approximately twice the air pressure of the sheath reservoir (1.8 kPa), the flow rate remains nearly constant, while the channel width expands by approximately 2.6 times. In contrast, when the air pressure in the sample reservoir is reduced below the air pressure of the sheath reservoir (1.8 kPa), the flow rate increases while the channel width narrows. Therefore, the following procedure can be used to adjust the flow rate and channel width. First, the ratio of the sample reservoir pressure to the sheath reservoir pressure is adjusted to the desired sample flow rate. Next, the waste reservoir pressure is adjusted to adjust only the flow rate while keeping the sample flow rate constant. In this way, pressure control of the waste reservoir is important to adjust only the flow rate without changing the sample flow rate. This adjustment requires flow rate evaluation, which will be described later.

[0031] In order to apply air pressure to each reservoir in a sterile manner, the force generating means described in 1-1) can be applied. Figures 2(B), (C), (D), and (E) show cross-sectional views of Figure 2(A). The reservoir is airtightly covered with a cover 3. In order to apply pressure to the air above the sample liquid in the reservoir 11, a hollow needle 30 is pierced through the cover of the reservoir 11 as shown in Figures 2(B) and (E). The air inside this needle is passed through an internal pressure chamber 30 of the device, which is adjusted to the pressure of the reservoir 11 on the device side. It is connected to an air pressure control system. It applies pressure to the air above the sheath liquid in the reservoir 12. To achieve this, a hollow needle 31 is pierced through the cover of reservoir 12, as shown in Figure 2(E). The air inside this needle is connected to an air pressure control system regulated to the pressure of reservoir 12 on the device side. A hollow needle 34 is pierced through the cover of waste reservoir 21 on the downstream side. The air inside this needle is connected to an air pressure control system regulated to the pressure of reservoir 21 on the device side. The pressure applied to reservoir 21 must be negative, below atmospheric pressure. Next, the air pressure control in reservoirs 16 and 17 is a pulse pressure control for separating particles from the sample liquid, which differs from the flow rate control using constant air pressure described above. The method of separating particles using pulse pressure, as shown in Figure 10, uses a system in which a constant pressure pump and a normally closed solenoid valve are connected in series, and utilizes the pulse pressure generated when the solenoid valve is in the open state for a short period of time. The particle is identified as being the target particle by signal light such as scattered light or fluorescence generated when it passes through detection region 35, which is the laser irradiation region, and if it is determined to be the target particle, a positive pressure air pulse is applied to reservoir 16 and a negative pressure air pulse is applied to reservoir 17 so that a push flow is generated from channel 24L and a pull flow is generated from channel 24R when it passes through the region where downstream channel 24L and channel 24R intersect. As shown in Figure 2(C), hollow needles 32 and 33 are penetrated into reservoirs 16 and 17, respectively, which are sealed with covers. If the inner diameter of this hollow needle is small, the response to air pressure will be poor, so the inner diameter must be at least a certain size. If the inner diameter is 1 mm or more, the valve will open even in a short time, on the order of milliseconds. The pressure required to separate particles can be transmitted to the liquid in the channel via the air in the reservoir.

[0032] 1-3) Embodiments of a means for solving technical problems for sorting giant cells or cell clusters (cell spheroids) To sort cell aggregates larger than 100 μm, the following issues must be resolved. It is known that the gravitational sedimentation rate increases with cell or cell aggregate size. Therefore, large cell aggregates accumulate at the bottom port of the reservoir within a short time, and if the cell accumulation height exceeds the depth of the channel, they clog the channel. Therefore, preventing gravitational sedimentation is necessary for sorting cell aggregates. Gravitational sedimentation can be prevented by adding a high-density component to the cell suspension buffer to increase its specific gravity. Polyvinylpyrrolidone or gellan gum are effective components for increasing the specific gravity. Cell culture is possible by adding these components to the medium, with negligible cell damage. While the specific gravity of the buffer does not need to be higher than that of the cells, slowing the gravitational sedimentation rate can also prevent cell clogging. Previous experiments have shown that a buffer specific gravity of 1.01 or higher can prevent spontaneous sedimentation of typical cells. Next, we will explain the size of the cross section of the flow channel of the disposable replaceable flow channel cartridge. In order to stably flow cell aggregates of 100 μm in size without clogging the flow channel, the flow channel width must be 150 μm or more and the flow channel depth must be 150 μm or more. Furthermore, it was found that the size of cell aggregates that can flow without clogging in a flow channel cross section size of 80 μm in width and 80 μm in depth is 40 μm. Therefore, The channel cross section must be at least 40 μm larger than the maximum size of the cell mass. Another technical issue with sorting large cells or cell clusters is a lack of sorting force. To compensate for this sorting force, it is effective to apply the force generated by actuators 100 and 101, which generate electromagnetic force directly without using air, to the flowing liquid as a sorting pulse flow, as shown in Figure 11. This method provides faster response time and increased force, making it suitable for sorting large cells and cell clusters.

[0033] 1-4) Embodiments of the means for solving the technical problems related to the purification of differentiated cells in regenerative medicine In regenerative medicine, cell sheets made from differentiated cells derived from iPS cells or ES cells Transplantation into patients is being considered. In this case, the problem is that even a small amount of undifferentiated cells can cause tumors. The differentiation induction rate is not 100%, but at least 1%. In this study, we will explain how to remove these undifferentiated cells aseptically without damaging the cells, and how to efficiently purify differentiated cells. Consider the case where this method is applied to a process for removing undifferentiated cells that are mixed in a differentiated cell population at a density of, for example, 1%. Methods for specifically fluorescently labeling undifferentiated cells include fluorescent antibody labels of surface markers of undifferentiated cells and the method described in Non-Patent Document 4, which utilizes fluorescent compounds that are specifically taken up by undifferentiated cells. Fluorescent antibody labels can stain undifferentiated cells in their disaggregated state. In contrast, fluorescent compounds that are specifically taken up by undifferentiated cells are known to be able to specifically stain undifferentiated cells within tissues, and therefore can specifically stain undifferentiated cells within cell clusters. Consider the case where the suspension of the above-mentioned specific fluorescently stained cell group is used as a sample and the following cell sorting method is applied. When a method for separating differentiated cells is applied to a flow path cartridge formed with a flow path on a transparent substrate, a light irradiation means for irradiating light onto cells in a sample solution flowing through the flow path, a means for detecting fluorescence emitted from undifferentiated cells when irradiated with the light and identifying the undifferentiated cells based on the signal intensity, a force generation means for applying a force to undifferentiated cells flowing through the flow path of the cartridge to change the flow direction based on a signal from the detection means, a reservoir connected to a flow path into which undifferentiated cells flow due to the change in flow direction caused by the force, and a reservoir connected to a flow path into which differentiated cells flow when the flow direction is not changed based on a signal from the detection means, the number of differentiated cells passing through the detection area per unit time is 99 times that of undifferentiated cells, so if undifferentiated cells are removed at a processing speed of 300 cells / second, the recovery speed of differentiated cells will be 99 times that, or approximately 30,000 cells / second. Generally, if the mixed ratio of undifferentiated cells is A%, the recovery efficiency of differentiated cells will be The sorting rate corresponds to (100-A) times the sorting speed of the device. The smaller the value of A, the greater the benefit of improved processing capacity. This sorting method is called negative sorting. Figure 15(A) shows the results for a total cell count of 2 x 10 6 Treatment when the undifferentiated ratio is 20% The results of actual measurements and simulations of efficiency are shown. The sorting processing speed is 100 items / Because the undifferentiated ratio is high, the removal process was performed four times, and the undifferentiated cells were completely removed. The actual measurements and simulations both agree that the cells can be removed. The total time required was 150 minutes, and the undifferentiated ratio was high at 20%, so the efficiency was poor. The number is 10 7 The simulation results for the case where the undifferentiated cell ratio is 0.3% or less are shown in Figure 15(B). Undifferentiated cells are completely removed by two treatments. The higher the proportion of undifferentiated cells, the lower the efficiency.

[0034] 1-5) Embodiments of the means for solving the problem of emulsion sorting The emulsion will be explained assuming droplets in oil. If droplets in the emulsion adhere to the inner wall of the reservoir on the disposable flow path cartridge, it will cause the flow path to become clogged. To prevent droplets in the emulsion from adhering to the inner wall of the reservoir, two methods will be explained below: one to make the inner wall of the reservoir water-repellent, and one to allow droplets to settle naturally under their own weight even if they do adhere.

[0035] Explanation of the method to prevent adhesion by making the inner wall of the reservoir water-repellent Figure 4(A) shows the same disposable, replaceable flow path cartridge as Figure 2(A). The problem with this cartridge is the adhesion of droplets to the inner walls of the sample liquid reservoir (sample reservoir: first reservoir) 11 containing the particles to be sorted, and the inner walls of the reservoir (third reservoir) 17 where emulsion droplets are collected after sorting. To prevent this, Figures 4(A) and 4(B) show structures in which reservoir 11 and reservoir 17 are each covered with a water-repellent material. Here, a hollow Teflon (registered trademark) adapter is inserted into the reservoir to prevent the emulsion from coming into contact with the inner walls of the reservoir. The flow path cartridge is injection molded from transparent resin, and it is not possible to make the entire flow path out of Teflon (registered trademark), so it is advisable to take the above measures. It is also possible to apply a water-repellent coating to the inner walls of the reservoir 17. The water-repellent coating may be a structure in which the walls are covered with a fluororesin.

[0036] Embodiments of a Means for Preventing Adhesion by Increasing the Specific Gravity of Droplets in an Emulsion Fluorinert and other fluorine-based oils have a higher specific gravity than the droplets, so the droplets float in the oil. The reason for this is that the density of Fluorinert is approximately 1.8 g / cm 3 It is about 1.8 times the amount of water. Therefore, the specific gravity can be increased by mixing in a liquid with a specific gravity of 1 or more. Such liquids include sodium polytungstate solution, bromoform solution, and iodomesilene solution. 3 If the density is higher than this, the droplets can be made to settle in fluorine-based oil. The density of a saturated aqueous solution of sodium polytungstate is 3.1 g / cm 3 and the saturated aqueous solution of bromoform is 2.89 g / cm 3 and the density of a saturated aqueous solution of iodomesilene is 3.31 g / cm 3 In order to prevent these components from floating in Fluorinert, the density must be 1.8 g / cm 3 To prevent the emulsion droplets from adhering to the inner wall of the reservoir, the density must be 1.8 g / cm 3 It is not necessary for the density to be higher than 1.2 g / cm3, but it is sufficient that the density is high enough so that the droplets adhering to the inner wall fall downward due to gravity when the buoyancy from the oil is lost as the oil surface moves downward due to the decrease in oil. 3 to 1.8 g / cm 3 It is sufficient if it is in the range.

[0037] 1-6) Embodiments of means for solving problems related to flow velocity Cell sorting devices, such as cell sorters, are based on the premise that the flow rate can be controlled to a constant value. This is because they detect particles in the liquid, identify them, and if they are the target particles, they are sorted downstream from the detection position. Since the time it takes for the particles to travel from the detection position to the sorting position varies depending on the flow rate, the flow rate must be constant in order to sort the particles a certain amount of time after detection. The flow rate within the channel can be adjusted by applying pressure, but the relationship between pressure and flow rate varies depending on the viscosity of the buffer that suspends the sample and the viscosity of the sheath fluid that is flowed together with the sample liquid to narrow it down. For this reason, the types of buffer and source liquid that can be used are generally limited by the manufacturer of commercially available cell sorters. In the current situation described above, certain types of cells often require specific culture media for measurement in a live state. Therefore, flow rate adjustment technology is required to accommodate not only specific buffers but also various buffers, including culture media. Therefore, for cell sorting without cell damage, which is desirable in regenerative medicine, the ability to evaluate flow rate is important because flow rate varies depending on the type of buffer. To this end, the following measures are taken to evaluate the flow velocity. In a method for performing cell sorting in a disposable replaceable flow path cartridge, as shown in 35 and 36 of FIG. 5(A), laser light of two different wavelengths is applied to the flow path 22 through which particles flow. The laser beam is irradiated at different positions along the axis. When a particle passes through the two irradiation areas, two optical signals (35-A and 36-A) resulting from the two laser irradiations are generated for each particle, as shown in Figure 5(B). Detection of these optical signals begins the moment signal 35-A is detected to be above the threshold (TH) set in the detection conditions. In this case, AD conversion of the 35-A signal and the 36-A signal start simultaneously, and the time difference between the times when these optical signals reach their maximum is recorded. This time difference is the distance (ΔL) between the two irradiation positions divided by the velocity V of the particle flow. In other words, by measuring the time difference ΔT for each particle, the velocity V of each particle can be measured. The AD conversion performance of optical signals is such that, when the sampling frequency is 5 MHz, the conversion is performed every 0.2 microseconds. Since the analog time waveform signals are digitized, the time difference between the two signals can be evaluated with a resolution of 0.2 microseconds. If the distance ΔL between the irradiation positions of the two lasers is 100 μm and the flow velocity V is 1 m / s, the time difference will be 100 μseconds, and a resolution of 0.2 microseconds is sufficient. This velocity estimation can be done for individual particles. For example, for fluorescent particles, One signal is scattered light and the other is fluorescent light. In the case of particles that do not emit fluorescence, two signals are used. In this case, two types of scattered light are detected. An optical system is required to detect the scattered light from each of the two types of lasers. An optical system that detects side scattering by total reflection at the end surface of the channel substrate, as described in Patent Document 12, is suitable for use in disposable replaceable channel cartridges. In this case, the reflecting surfaces on both sides of the substrate can be used as optical systems for detecting side scattering resulting from the two laser wavelengths. As described above, once the flow velocity of each particle is known, it becomes possible to derive the time difference from the detection time to the cell sorting area, and apply a sorting force to the cells at the time they pass through the cell sorting area to sort them. Furthermore, when the type of buffer used to suspend particles is changed, or when the viscosity of the buffer changes with temperature, causing the flow rate to change, the air pressure is adjusted to maintain a constant average particle flow rate. In this case, the pressure conditions for each reservoir are initially set to the channel width and flow rate for the buffer used, and particle analysis and sorting are initiated under those conditions. Even under the initial settings, the flow rate may vary depending on the ambient temperature, so the flow rate of each particle is monitored, and the pressure of the waste reservoir is controlled via a control PC so that the average flow rate per unit number is the desired value. Figure 13(A) shows the same disposable replaceable channel cartridge as Figure 2(A), but with a transparent channel substrate. The measurement system shown here is designed to evaluate the cell flow velocity when multiple lasers with different wavelengths are installed. The reflecting surfaces (180 and 181) are inclined at a 45-degree angle to the vertical and have a mirror-finished surface. Figure 13(B) illustrates both the D1D1' cross section at the position of laser irradiation area 35 in Figure 13(A) and the D2D2' cross section at laser irradiation area 36. Two laser irradiation areas (35 and 36) with different wavelengths irradiate the area from the confluence to the sorting channel. Side-scattered light generated when cells pass through the irradiation area is internally reflected downward by the reflecting surfaces (180 and 181). The scattered light that exits the substrate by internal reflection is collected by transparent resin focusing blocks 182 and 183 and guided to photodetectors (188 and 189) by light guides (184 and 185) for detection. In this case, a bandpass filter with a wavelength range that transmits only the wavelength of the first laser light source 35-1 is placed in front of the photodetector (188), and a bandpass filter with a wavelength range that transmits only the wavelength of the second laser light source 36-1 is placed in front of the photodetector (189), thereby eliminating light other than scattered light of each wavelength. As shown in Figure 5(B), the time difference between the maximum values ​​of the time waveforms of the two types of side scattering signals is evaluated for particles flowing through the flow channel, and the flow velocity of each particle is calculated by dividing the distance between the irradiation areas 35 and 36 by the time difference. Figure 14 is a graph showing the histogram distribution of the measured flow velocities of individual cells.

[0038] 2) Embodiments of the means for solving the problem of the method for analyzing the genes of a single cell As a means to solve the problems of the method for analyzing the genes of single cells, there are a method for forming emulsion droplets containing cells, a method for lysing cells inside the emulsion droplets, a method for performing a PCR reaction inside the emulsion droplets, a method for separating and concentrating fluorescent emulsion droplets, and a method for dispensing the separated emulsion droplets individually into a multi-well plate. This paper explains how to mix the emulsion droplets in the multi-well plate with another reaction reagent solution, how to break the emulsion droplets in the multi-well plate, and how to perform a whole gene amplification reaction and analyze the results using a next-generation sequencer. Figure 6 shows the disposable and replaceable flow channel car for forming emulsion droplets containing cells. It has almost the same structure as the cartridge in Figure 2, but the role of each reservoir is different. This cartridge is formed with a reservoir (first reservoir) 11-E for sample liquid containing cells to be incorporated into droplets, a reservoir (second reservoir) 12-E for reaction reagent liquid containing PCR reaction reagents and cell lysis reagents, left and right emulsion oil reservoirs (third A reservoir) 16-E and (third B reservoir) 17-E, and a reservoir (fourth reservoir) 21-E for the formed emulsion droplets. A controlled air pressure is applied to the top of each reservoir, and the size of the droplets in the oil can be adjusted by the air pressure of each reservoir. The air pressure inside the reservoir can be adjusted by a combination of a compressor and an electro-pneumatic regulator, as shown in Figure 18. The pressure adjustment range is from 0.1 kPa to 30 kPa. The pressure applied to the first and second reservoirs is adjusted by the pressure for oil. By changing the pressure applied to the reservoirs (3A and B), the diameter size of the droplets in the oil is The size can be adjusted from 20 μm to 100 μm. However, the viscosity may vary depending on the type of oil. Since the oil pressure varies, it is necessary to set the desired air pressure for each type of oil. Also, to capture cells of approximately 10 μm in size within emulsion droplets, the droplet size should be 40 μm or larger. The upper limit of the maximum droplet size is determined by the size of the channel. Figure 7 shows the flow channels in the microchannel cartridge for forming emulsion droplets. The sample liquid containing the cells 50 is merged with the flow channel 23L-E through which the reaction reagent liquid (PCR reaction reagent and cell lysis reagent) flows in the flow channel 23L-E, so that the cells 50 are aligned in a line, and then the cells are transferred to the flow channel 24L-E. The oil from paths 24R-E is merged. After merging, droplets are formed in the oil, but the droplets containing cells are Two types of droplets are formed: droplets 51 and cell-free droplets 52.

[0039] FIG. 8 shows four sample liquid reservoirs 63, four reagent reservoirs 62, and four oil reservoirs. This is an emulsion forming cartridge in which a reservoir is formed. Emulsion particles are formed by applying air pressure to each reservoir, and this flow path cartridge can form emulsions for four types of samples at once. The type of oil can be either fluorine-based oil or mineral oil, and emulsions of droplets in oil can be formed. The diameter size of the droplets is determined by the ratio of the liquid flow rate and the oil flow rate at the junction with the oil. When forming emulsion droplets containing individual cells of about 10 μm in size, the droplets It is desirable that the size of the flow channel 22-E is at least 40 μm or more. The cross-sectional size of flow channel 22-E may be 100 μm wide and 100 μm deep or less, but when forming emulsion droplets that incorporate spheroids, which are cell clusters with a size of approximately 100 μm, the cross-sectional size of flow channel 22-E must be 200 μm wide and 200 μm deep or more. The cell lysis reagent contains an enzyme that breaks down cell membranes, such as Proteinase K. PCR reaction reagents contain primers corresponding to the gene sequence to be detected, as well as polymerase and fluorescent reagents such as TaqMan probes or Cyber ​​Green. When using Proteinase K for cell lysis, the reaction is carried out by leaving the cells at 36°C for 12 hours.

[0040] Next, the PCR reaction is carried out using, for example, a thermal cycler, repeating thermal cycles between temperatures of 60°C and 95°C about 40 times. After the PCR reaction, the droplets containing the target gene sequence become fluorescent.

[0041] In the next step, only the droplets that have fluorescence are sorted in the aforementioned flow path cartridge. This method is as explained in 1-5). As a result of this sorting, the target gene sequence is The emulsion droplets having rows are obtained as 95% or more of emulsion liquid.

[0042] Next, a method for dispensing the emulsion droplets one by one onto a multi-well plate will be described. Figure 9(A) shows a schematic diagram of the method for dispensing the emulsion droplets. A reagent solution to be reacted with the dispensed droplets is dispensed into the destination multi-well plate 70. This reagent solution is, for example, an aqueous solution containing a reagent for whole gene amplification. The emulsion droplets before dispensing are contained in a tube 71. The tip of a dispensing pipette 73, which is a transparent hollow pipe attached to the end of a syringe pump 74 that controls air pressure, is moved downward along the Z axis and inserted into the emulsion liquid in this tube 71. Next, the piston of the syringe is raised by the amount of dispensing, and the emulsion is then dispensed. The infusion liquid is aspirated into the pipette 73. After aspirating, raise the pipette 73 upward on the Z axis and The camera captures an image of the entire volume dispensed in the pipette, determines whether a droplet is present, and if so, moves the droplet to a well at a specified address on the multi-well plate. 9(B), the dispensing pipette 73 is moved downward to the position of the reagent solution that has already been dispensed. The syringe pump is lowered below the upper surface 81, and the piston 75 of the syringe pump is pushed out to discharge the droplets in the pipette. After the droplets are discharged, the piston 75 is pressed down while the dispensing pipette is pulled up. If the result of image recognition inside the dispensing pipette is that there are zero droplets or two or more droplets, the dispensing pipette is returned to the tube 71. Discharge the emulsion liquid from the dispensing pipette 73 and aspirate it again. Dispense only if necessary. If the dispensing volume is 0.3 μL and the inner diameter of the pipette is 400 μm, there will be 2.4 mm inside the pipette. Next, take a single image of the entire 0.3 μL volume of liquid, with the camera in focus. When considering whether it is possible to take a photograph in this state, if a 1x objective lens is used, The depth of field is 440 μm, and the entire 400 μm diameter inside the pipette can be photographed in focus. Considering the width of the field of view, a 1 / 2-inch camera covers 6.4 mm, so it is possible to photograph the entire 2.4 mm length inside the pipette. Therefore, it can be determined that the entire dispensed volume of 0.3 μL can be photographed with one image shot. However, since the image resolution is 11 μm, it is possible to identify the presence and number of a single cell. The emulsion droplet size is 11 μm, so it is possible to photograph the entire 0.3 μL dispensed volume with one image shot. The optimum size for capturing is 40 μm, so the presence and number of emulsion droplets can be easily identified. Figure 16 shows the standard particle size of 10 μm in the hollow pipette made of transparent resin with an inner diameter of 400 μm. The image was taken with a 1x objective lens and a 1 / 2-inch camera. Individual particles can be identified. It is possible, and the length of the aspirated liquid is 2.4 mm or more. When dispensing emulsion droplets as described above, it is desirable to coat the inside of the dispensing pipette with a water-repellent coating solution in advance to prevent droplets from adhering to the inner wall of the dispensing pipette. The above-described dispenser can also be used to dispense cells in an aqueous solution. In this case, it is desirable to coat the inside of the dispensing pipette with a hydrophilic coating solution to prevent cells from adhering to the inner wall of the dispensing pipette. To perform a detailed analysis of the DNA of one cell contained in one droplet on a multiwell plate, it is advisable to determine the entire sequence using a next-generation sequencer. This sequence analysis requires whole genome amplification. Therefore, the following describes a method for performing a whole genome amplification reaction by disrupting each dispensed emulsion droplet and mixing the extracted DNA with a whole genome amplification reaction reagent solution. First, to disrupt each emulsion and extract the internal DNA, the method using centrifugation and diethyl ether described in Non-Patent Document 5 is applied. However, in order to apply this method to each emulsion droplet, the emulsion solution dispensed individually into the multiwell plate is transferred to a centrifuge tube and applied. Next, to perform a whole genome amplification reaction on the extracted DNA, a whole genome amplification kit for next-generation sequencers (SeqPlex DNA Amplification Kit) from Sigma-Aldrich, for example, is used. ) is used. This protocol is described in Non-Patent Document 6. For RNA analysis, for example, a total RNA amplification kit for next-generation sequencers (SeqPlex DNA Amplification Kit) from Sigma-Aldrich is used. This protocol is described in Non-Patent Document 7. It is listed. Figure 17 summarizes the analytical procedures related to the present invention. These are single cell analysis (case 1), single cell clump analysis (case 2), emulsion droplet analysis containing single cells (case 3), and emulsion droplet analysis containing single cell clumps (case 4). The means of the present invention are essential for the processing procedures in each case. After the above amplification, sequence analysis is performed using a next-generation sequencer. [Industrial Applicability]

[0043] The device for analyzing and separating microparticles of the present invention can aseptically separate cells suitable for regenerative medicine. In addition, since the sample concentration can be easily adjusted, it is possible to adjust the cell concentration in the sample. Furthermore, the device for analyzing and separating particles can easily sort cell spheroids or emulsions. Furthermore, by using the device of the present invention, differentiated cells can be easily purified. According to the present invention, the flow rate of cells or cell clusters can be measured, so cells or cell clusters can be sorted without using a specific buffer. Furthermore, according to the genetic analysis system or genetic analysis method of the present invention, the genetic information of a single cell or one cell cluster (cell spheroid) can be analyzed. It is possible to analyze the children without contamination. Although the present invention has been described above with reference to specific embodiments, modifications and improvements that are obvious to those skilled in the art are within the scope of the present invention. [Explanation of symbols]

[0044] 1...Flow path substrate 2A…Reservoir 2B...Reservoir 3A…Reservoir cover 3B…Reservoir cover 4...Flow path 5A...Sample solution 5B...Sample solution 6A...Air 6B...Air 8A...hollow needle 8B...Hollow needle 9A...Syringe pump 9B...Syringe pump 10...Outer frame of disposable replaceable flow path cartridge 11...Reservoir for storing sample liquid (sample reservoir: first reservoir) 11-E... Sample liquid containing particles to be incorporated into emulsion droplets 12...Sheath fluid reservoir (second reservoir) 12-E: Reagent solution containing cell lysis reagent and PCR reaction reagent 13...Main flow path for sample liquid and connection port 14...Partition between sample liquid and sheath liquid 15L: Connection port for the sheath fluid flow path on the left side 15R: Connection port for the sheath fluid flow path on the right side 16...Sorting fluid reservoir (3A reservoir) 16-E...Emulsion oil reservoir (3A reservoir) 17...Recovery reservoir (3B reservoir) 17-E...Emulsion oil reservoir (3B reservoir) 18...Connection port for sorting fluid reservoir and sorting channel 19...Connection port for the sorting channel of the collection reservoir 20...Connection port to the main waste fluid flow path 21...Reservoir for storing waste liquid (4th reservoir) 21-E…Emulsion droplet reservoir (fourth reservoir) 22...Main flow path (first flow path) 22-E...Main flow path (first flow path) 23L: Left sheath channel (second channel) 23L-E: Left reagent flow path (second flow path) 23R: Right sheath channel (third channel) 23R-E: Right-side reagent flow path (third flow path) 24L...Sorting channel on the pulsed flow push side (fourth channel) 24L-E: Oil flow path for droplet formation (fourth flow path) 24R: Pulse flow pull side sorting channel (5th channel) 24R-E: Oil flow path for droplet formation (5th flow path) 30...Hollow needle that penetrates the cover of the sample liquid reservoir 31...Hollow needle that penetrates the cover of the sheath fluid reservoir 32...Hollow needle that penetrates the cover of the sorting fluid reservoir 33...Hollow needle that penetrates the cover of the collection reservoir 34...Hollow needle that penetrates the waste reservoir cover 35...First laser beam or its irradiation area 35-A: Scattered light signal generated when passing through the laser irradiation area of ​​35 36...Second laser with a different wavelength from the first or its irradiation area 36-A: Scattered light signal generated when passing through the laser irradiation area of ​​36 35-1...First laser light source 35-2...Laser driver circuit 36-1...Second laser light source 40...Teflon (registered trademark) adapter for sample liquid reservoir 41...Teflon adapter for collection reservoir 50...cell 51...Droplets containing cells 52...cell-free droplets 60...Emulsion droplet forming flow path cartridge 61...Oil reservoir for emulsion droplet formation 62...Reagent reservoir 63...Sample liquid reservoir 64...Flow path for emulsion droplet forming oil 65...Reagent flow path 65...Emulsion droplet reservoir 70...Multi-well plate into which emulsion droplets are dispensed 71... Emulsion liquid containing droplets before dispensing 72... Cleaning oil 73...Dispensing pipette (transparent hollow pipe) 74...Pneumatic syringe pump 75...Piston part of pneumatic syringe pump 76...Camera 77...Dispenser control PC 78...Single particle dispenser 80...wells in a multi-well plate 90...Positive pressure syringe pump 91...Negative pressure syringe pump 92...High-speed electromagnetic valve 93...High-speed electromagnetic valve 100...Electromagnetic actuator 101...Electromagnetic actuator 110...Elastic cover 111...Elastic cover 151...Objective lens 152...Laser light 153: Area sandwiched between sorting channels 4-1 and 4-2 154, 155, 156...Dichroic mirrors 157, 158, 159...Bandpass filters 160...Spatial filter for blocking transmitted laser light 161...Photodiode 162, 163...Photomultiplier tube 164...AD converter 169...Control computer 170...Driver circuit for 9A air pump 171...Driver circuit for air pump 9B 172...Driver circuit for irradiation laser light source 35-1 180,181...reflective surface 182, 183...Transparent resin light-collecting block 184, 185...Transparent resin light guide 186...A bandpass filter of a wavelength range that transmits only the wavelength of the first laser 35 187...A bandpass filter of a wavelength range that transmits only the wavelength of the second laser 36 188,189...Photodetector 200...Solenoid valve 201...Solenoid valve driver circuit 202...Electro-pneumatic regulator 203...Air buffer tank 204...Compressor 205...Gas foreign matter removal filter 206...Compressor atmospheric suction pipe 301...Driver circuit for electromagnetic actuator 401...Cover with holes 402...Cover to close the hole 403…rod 403 404...Reservoir space 405...Side wall

Claims

1. a flow path cartridge formed by forming a flow path on a transparent substrate; a light irradiation means for irradiating light onto particles in the sample liquid flowing through the flow channel; a detection means for detecting scattered light or fluorescence emitted from the particles when irradiated with the light, identifying the particles based on the signal strength of the scattered light or fluorescence, and detecting the target particles; a force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detecting means, The cartridge is formed with a reservoir for sample liquid (sample reservoir) connected to the first flow path, a fourth branch flow path and a fifth branch flow path connected to opposite sides of the first flow path, a third A reservoir for sending out a pulsed flow connected to the fourth branch flow path, a third B reservoir connected to the fifth branch flow path for sorting and recovering the particles by changing the flow of particles toward the fifth branch flow path using a pulsed flow generated by the force generating means that flows from the fourth branch flow path in the direction of the fifth branch flow path, and a fourth reservoir connected to the downstream side of the first flow path for storing particles that have not been sorted, and each of the reservoirs is covered with a sealing cover, thereby sealing the inside of each reservoir from the outside.

2. a flow path cartridge formed by forming a flow path on a transparent substrate; a light irradiation means for irradiating light onto particles in the sample liquid flowing through the flow channel; a detection means for detecting scattered light or fluorescence emitted from the particles when irradiated with the light, identifying the particles based on the signal strength of the scattered light or fluorescence, and detecting the target particles; a force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detecting means, a third reservoir connected to the fifth branch channel for separating and recovering the particles by changing the flow of the particles toward the fifth branch channel using a pulsed flow generated by the force generating means that flows from the fourth branch channel toward the fifth branch channel, thereby separating and recovering the particles; and a fourth reservoir connected to the downstream side of the first channel for storing the particles that have not been separated. Each of the reservoirs is covered with a sealing cover, thereby sealing the interior of each reservoir from the outside. The cartridge has a means for equalizing the air pressure in each reservoir with the air pressure of each internal air pressure control system, and the flow of the channels in the cartridge is controlled by controlling the air pressure in each reservoir with the internal air pressure control system.

3. a flow path cartridge formed by forming a flow path on a transparent substrate; a light irradiation means for irradiating light onto particles in the sample liquid flowing through the flow channel; a detection means for detecting scattered light or fluorescence emitted from the particles when irradiated with the light, identifying the particles based on the signal strength of the scattered light or fluorescence, and detecting the target particles; a force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detecting means, The cartridge includes a reservoir for sample liquid (sample reservoir) connected to the first flow path, a fourth branch flow path and a fifth branch flow path connected to both sides of the first flow path so as to face each other, a third A reservoir for sending out a pulse flow connected to the fourth branch flow path, and a force generating means for generating a pulse flow from the fourth branch flow path to the fifth branch flow path. a third-B reservoir connected to the fifth branch flow channel for sorting and recovering the particles by changing the flow of the particles in the direction of the fifth branch flow channel, and a fourth reservoir connected to the downstream side of the first flow channel for storing the particles that have not been sorted, wherein each reservoir is covered with a sealing cover so that the interior of each reservoir is sealed from the outside, the sealing covers of the third-A reservoir for sending out the pulsed flow and the third-B reservoir for recovering the particles are stretchable and deformable membranes, and an actuator is provided for displacing the sealing cover membrane by applying a mechanical force from the outside, and when the particles pass through the sorting area, the actuator is used to rapidly press down the sealing cover membrane of the third-A reservoir and to pull up the sealing cover of the third-B reservoir, thereby generating a pulsed flow in the branch flow channel and sorting the particles.

4. a flow path cartridge formed on a substrate, the flow path cartridge including: a first flow path into which a sample liquid containing fine particles is introduced; second and third flow paths disposed on both sides of the first flow path into which a sheath liquid is introduced; a first confluence flow path where the first to third flow paths are joined; a first reservoir serving as a reservoir for the sample liquid; a second reservoir serving as a reservoir for the sheath liquid; and a reservoir (fourth reservoir) for storing waste liquid; a light irradiation means for irradiating light onto the particles flowing through the first confluent flow path; and means for detecting and analyzing scattered light or fluorescence emitted from the particles, The flow path cartridge has first to third flow paths upstream of a first confluent flow path, the first flow path being connected to a first reservoir, the second and third flow paths being connected to a second reservoir, and the first confluent flow path including a flow path pattern that connects to a fourth reservoir downstream, and the device for analyzing and / or separating microparticles is characterized in that it has the function of adjusting the narrowing width of the sample flow and the sample flow rate by adjusting the pressure of each of the first reservoir, the second reservoir, and the fourth reservoir.

5. a flow path cartridge formed by forming a flow path on a transparent substrate; a light irradiation means for irradiating light onto particles in the sample liquid flowing through the flow channel; a detection means for detecting scattered light or fluorescence emitted from the particles when irradiated with the light, identifying the particles based on the signal strength of the scattered light or fluorescence, and detecting the target particles; a force generating means for changing a flow direction of the particles flowing through the flow path of the cartridge based on a signal from the detecting means, The cartridge is formed with a reservoir for sample liquid (sample reservoir) connected to the first flow path, a fourth branch flow path and a fifth branch flow path connected to both sides of the first flow path so as to face each other, a third A reservoir for sending out a pulsed flow connected to the fourth branch flow path, a third B reservoir connected to the fifth branch flow path for sorting and recovering the particles by changing the flow of the particles toward the fifth branch flow path using a pulsed flow generated by the force generating means flowing from the fourth branch flow path toward the fifth branch flow path, and a fourth reservoir connected to the downstream side of the first flow path for storing particles that have not been sorted, and the interior of each reservoir is sealed from the outside by being covered with a sealing cover, A particle analyzing and separating device, characterized in that the flow path has a width and depth of 150 micrometers or more, and the beam size of the irradiated laser in the flow path width direction is 100 micrometers or more.

6. A differentiated cell purification method using the device according to any one of claims 1 to 5, which removes undifferentiated cells that are mixed in a differentiated cell population differentiated from undifferentiated cells at a lower density than the differentiated cells, A differentiated cell purification method characterized by performing a flow path change process by applying a force to undifferentiated cells to change the flow direction based on a signal from the detection means, and not performing a flow path change process by not applying a force to differentiated cells to change the flow direction, thereby removing undifferentiated cells from the differentiated cells contained in the fourth reservoir that has been passed through, thereby recovering cell liquid and improving the recovery rate of differentiated cells.

7. 7. The differentiated cell purification method according to claim 6, wherein the same undifferentiated cell removal treatment is repeated for the collected cell solution.

8. In this device, which dispenses microparticles contained in a sample liquid one by one, the dispensing nozzle is an automatically replaceable transparent hollow pipette, and the amount of liquid dispensed is 0.3 μL or less. For each dispense, the presence and number of particles 10 μm or larger are detected by image recognition of the entire amount of liquid dispensed by the hollow pipette, and the particle A dispensing device characterized by dispensing a specified number of samples into a multi-well plate.

9. a flow path cartridge formed by forming a flow path on a transparent substrate; a light irradiation means for irradiating light onto particles in the sample liquid flowing through the flow channel; a detection means for detecting scattered light or fluorescence emitted from the particles when irradiated with the light, identifying the particles based on the signal strength of the scattered light or fluorescence, and detecting the target particles; a force generating means for applying a force to the particles flowing through the flow path of the cartridge to change the direction of flow based on a signal from the detecting means, The cartridge is an apparatus for analyzing and separating particles, comprising a reservoir (sample reservoir) for a sample liquid connected to a flow path, and a reservoir connected to a flow path for separating and recovering particles from the flow path by changing the direction of flow using the force generating means, characterized in that the inner walls of the sample reservoir or the reservoir for recovering the separated particles are covered with a water-repellent material or are covered with an adapter made of a water-repellent material.

10. The particle analyzing and separating device according to claim 9 , wherein the water-repellent material is a fluororesin.

11. A method for forming droplets in oil by merging oil into a flow path in which a sample liquid is flowing through flow paths that merge from the left and right, characterized in that a component with a high specific gravity is mixed into the sample liquid to make the specific gravity of the droplets greater than that of the oil.

12. 12. The method for forming droplets in oil according to claim 11, wherein the component with a high specific gravity is polytungsten, bromoform, or iodomesilene.

13. a flow path cartridge formed by forming a flow path on a transparent substrate; a light irradiation means for irradiating a part of the flow path through which the particles in the sample liquid flow; a detection means for detecting scattered light or fluorescent light generated when each of the fine particles passes through the light irradiation region; A particle measurement or particle sorting device including a means for identifying target particles based on each optical signal, A particle measuring or particle sorting device comprising: means for measuring the flow velocity of each particle based on the optical signal; and means for adjusting the flow velocity of the particles so that it becomes constant.

14. The flow velocity measuring means is an irradiation optical system that irradiates two irradiation lights of different wavelengths at positions at a fixed distance apart in the flow direction in the flow channel, and the flow velocity of the particles is adjusted to be constant by measuring the time variation of the pulse waveform of the scattered light intensity of each wavelength that occurs when the particles pass through. The particle measuring or particle sorting device according to claim 13, wherein the flow rate of the particles is measured, the flow rate of the particles is calculated based on the time difference between the peak values ​​of each pulse waveform, and the flow rate is adjusted so that the average flow rate of the particles becomes constant.

15. A method for measuring or sorting particles using the particle measuring or sorting device according to claim 13 or 14, characterized in that the particles are suspended in an arbitrary buffer.

16. The method for measuring or sorting fine particles according to claim 15, wherein the buffer is an aqueous liquid or an oily liquid.

17. a means for forming droplets in oil containing cells, a cell lysis reagent, and PCR reaction reagents; means for carrying out a PCR reaction after the cell lysis reaction; A means for separating droplets in oil by fluorescent labeling after a PCR reaction; and a means for dispensing the collected oil droplets one by one. Cellular genetic analysis system.

18. The genetic analysis system according to claim 17 , wherein the cell is a single cell or a single cell spheroid.

19. The means for forming droplets in the oil is a disposable flow path cartridge, the width of the flow path for cells in the disposable flow path cartridge is at least 100 μm or more, and the size of the droplets formed in the oil can be adjusted to 40 μm to 100 μm. The gene analysis system according to claim 17 or 18,

20. A genetic analysis system according to any one of claims 17 to 19, characterized in that the dispensing means is a dispensing means for dispensing into a multi-well plate, and the liquid ejected from the dispensing nozzle is pushed out while in contact with the inner wall of the multi-well plate or the liquid present in the multi-well plate.

21. The dispensing nozzle of the dispensing means is an automatically replaceable transparent hollow pipette, the amount of dispensed liquid is 0.3 μL or less, and image recognition of the entire amount of dispensed liquid of the hollow pipette is performed for each dispensing, and dispensing is performed when there is one particle of 10 μm or more, as described in any one of claims 17 to 20. Genetic analysis system.

22. forming droplets in oil containing cells, cell lysis reagents, and PCR reaction reagents; performing a PCR reaction after the cell lysis reaction; A step of separating droplets in oil by fluorescent labeling after the PCR reaction; and dispensing the collected oil droplets one by one.

23. The genetic analysis method according to claim 22 , wherein the cell is a single cell or a single cell spheroid.

24. 24. The genetic analysis method of claim 22 or 23, wherein the step of forming droplets in oil uses a disposable flow path cartridge, the width of the flow path through which the cells flow is at least 110 μm, and the size of the droplets in oil formed is 40 μm to 100 μm.

25. 25. Any one of claims 22 to 24, wherein the step of dispensing droplets in oil is dispensing into a multi-well plate, and the liquid discharged from a dispensing nozzle is pushed out while in contact with the inner wall of the multi-well plate or the liquid present in the multi-well plate. Item 1. A genetic analysis method according to item 1.

26. The dispensing nozzle in the dispensing step of the droplets in the oil is an automatically replaceable transparent hollow pipette, the amount of the dispensed liquid is 0.3 μL or less, and image recognition of the entire amount of the dispensed liquid of the hollow pipette is performed for each dispensing, and the nozzle discharges only when there is one particle of 10 μm or more. The genetic analysis method according to any one of claims 1 to 4.

27. In the device for forming droplets in oil containing cells, a flow path for forming droplets in oil is provided in a disposable flow path cartridge, and the cartridge is formed with a plurality of reservoirs connected to the flow path, and the width of the flow path for passing cells is at least 110 μm or more, and the droplets to be formed are This emulsion droplet forming device is characterized by being able to adjust the size of the droplets in the oil in the range of 40 μm to 100 μm by controlling the air pressure above the liquid in each reservoir.

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