Particle purification method, single particle dispensing method, cell cluster analysis method, and device used therefor

Abstract

To provide a method or device for quickly purifying target particles from high concentration particles.SOLUTION: The above challenge is cleared by a method provided herein for purifying target particles, the method comprising a step of sorting target particles from among high concentration non-target particles, and repeating the sorting step three or more times.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a particle purification method, a single particle dispensing method, a cell cluster analysis method, and an apparatus used therefor. [Background technology]

[0002] In order to detect specific cells among many contaminating cells and perform genetic analysis, etc., it is necessary to dispense each particle individually. 8 This paper relates to a method for purifying or dispensing approximately 100 to 1000 cells. Applications also include the separation of circulating tumor cells circulating in the blood of cancer patients. Therefore, this paper relates to a particle separation and dispensing technology that utilizes a disposable, replaceable tip to eliminate cell contamination between different patients. In this specification, "particles" includes cells, particles containing droplets in oil, and the like.

[0003] This paper describes the prior art and problems associated with cell sorting and subsequent aliquoting techniques.

[0004] (1) Conventional Jet-in-Air cell sorter technology As described in Non-Patent Document 1, this is a Jet in Air method in which droplets are formed from a nozzle and cells contained in the droplets are separated on a droplet-by-droplet basis.

[0005] (2) Cell sorting technology using microchannels The sorting speed in microchannel sorting is slow, at around 1000 cells / second or less. However, Patent Document 1 describes a technique for improving throughput by using multiple channels for parallel processing, and a technique for returning processed cells to the channel and processing them again to improve sorting purity. Patent Document 2 describes a technique in which a channel chip has multiple sorting sections and each process is performed consecutively to improve sorting purity. Patent Document 3 describes a method in which a microchannel chip with a reservoir is used to remove unnecessary cells from a cell population by sorting, and the remaining cells are returned to an upstream sample reservoir for sorting. Patent Document 3 describes a sorting technique using a pulsed flow in a microchannel chip with a reservoir. Furthermore, Patent Document 4 describes a method (repeated negative sorting method) in which unnecessary cells are removed by sorting in a microchannel chip, the remaining cell fluid is collected from a waste fluid reservoir, and returned to an upstream sample reservoir, and negative sorting is repeated.

[0006] (3) Single cell dispensing technology If necessary, sorted cells may be dispensed individually into a multi-well plate. This technology and its challenges are described below. Non-Patent Document 2 describes a method for selecting target cells from among contaminating cells and dispensing the cells into a multi-well plate, in which droplets collected using a jet-in-air cell sorter are directly dispensed into the multi-well plate. Patent Document 5 describes a method for identifying target cells using image recognition and dispensing them as droplets into a multi-well plate using piezoelectric pressure. The challenge with this method is that when piezoelectric pressure is used, there is a limit to the size of droplets that can be dispensed. Patent Document 6 describes a technology in which the cells to be dispensed are drawn up from a suspension using a pipette, the inside of the pipette is photographed, and the cells are dispensed only when it is determined that a single cell has been placed inside the pipette.

[0007] (4) Emulsion droplet dispensing technology As mentioned above, there are several techniques for dispensing single cells. However, when dispensing emulsion droplets in oil, emulsion droplets that settle in the oil can be dispensed in the same way as cells. However, the fluorinated oil used in droplet digital PCR or single-cell expression analysis has a high specific gravity. Therefore, emulsion droplets float in the oil, making it difficult to dispense them by dropping them from above. A method for dispensing emulsion droplets in fluorinated oil is described in Patent Document 7.

[0008] (5) Cell analysis technology using flow cytometry Non-Patent Document 3 describes conventional flow cytometry techniques and analytical methods. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 6,976,590 [Patent Document 2] U.S. Patent No. 8,993,311 [Patent Document 3] International Publication No. 2012 / 067259 [Patent Document 4] International Publication No. WO2016 / 182034 [Patent Document 5] International Publication No. 2011 / 154042 [Patent Document 7] International Publication No. 2018 / 052137 [Patent Document 8] British Patent No. 2566002 Specification [Patent Document 6] U.S. Patent No. 07268167 [Patent Document 7] U.S. Patent No. 09,500,664 [Non-patent literature]

[0010] [Non-Patent Document 1] FACS Aria III Brochure [Non-patent document 2] SONY SH800S Brochure [Non-patent document 3] https: / / ls.beckmancoulter.co.jp / files / appli_note / Couter_Practical_Flow_Cytometry.pdf [Non-patent document 4] Szczerba, BM et al., Nature volume 566, pages 553-557(2019) Summary of the Invention [Problem to be solved by the invention]

[0011] (1) Issues with conventional Jet-in-Air cell sorting technology The sorting speed of the Jet in Air cell sorter is several tens of times faster than that of the conventional cell sorter. 4 / sec. However, the number of cells contained in the droplet must be one or less. In the air method, droplets formed at high speed are released into the atmosphere. Just before release, the droplets containing the target cells are charged, and the direction of their flight is changed by the electric field, allowing them to be collected. In this method, the droplets collide with the wall of the collection tube from the atmosphere at high speed, causing significant damage to the cells. Therefore, it is necessary to collect cells with sufficient purity in a single sorting process. In other words, to achieve sufficient purity in a single process, the cells must be diluted. Therefore, the processing time increases as the number of cells increases. Figure 1 shows the results of Poisson distribution analysis to determine the processing time required to achieve a purity of 98% or more after sorting, assuming a sorting speed of 30,000 times / second. From Figure 1, it can be seen that when the total number of cells is 10 8 In this case, the processing time is 30 hours, which is unrealistic. When the total number of cells is so large, dilution is necessary, which poses the problem of prolonged processing time.

[0012] (2) Issues with conventional cell sorting technology using microfluidics Sorting using microchannels tends to leave cells in the channel chip after sorting, so when sorting is repeated, the remaining contaminating cells in the channel chip can have a significant effect on the final purity.

[0013] (3) Issues with single-cell dispensing technology In the method of dispensing droplets collected by a Jet-in-Air cell sorter directly into a multi-well plate, the multi-well plate is moved droplet by droplet in a step-and-repeat manner in the atmosphere to dispense the droplets into different wells. However, if the time between sorting events of the target cells is shorter than the movement time of the destination multi-well plate, the target cells may not be dispensed. Furthermore, the size of the cells that can be dispensed is limited by the size of the droplets formed from the nozzle. Therefore, the size of the cells that can be sorted is limited to the size of the droplets to be sorted. Furthermore, in the method of aspirating the cells from a suspension containing the target cells with a pipette and then dispensing them after confirming the presence of only one cell using image recognition, the dispensing time is long, resulting in low throughput.

[0014] (4) Issues with emulsion particle dispensing technology Emulsion droplets in fluorine-based oil float because the specific gravity of the fluorine-based oil is greater than that of water. Furthermore, in a resin container, the droplets tend to move toward the wall of the container above the fluorine-based oil. Therefore, it is impossible to dispense emulsion droplets one by one by sucking them up with a pipette and then expelling them.

[0015] (5) Issues with emulsion formation methods In a method for forming emulsion droplets in fluorinated oil, the method for recovering emulsion droplets using a reservoir formed on a microchannel chip has the following problem: When the amount of emulsion in the recovery reservoir increases and the liquid level of the fluorinated oil rises, a force resulting from the mass of the oil is generated in a direction that causes the oil to flow backward. As a result, the flow rate slows as the liquid level rises.

[0016] (6) Challenges of cluster cell analysis using flow cytometry Current flow cytometry analysis relies on a distribution derived from data on a large number of cells. Therefore, it is impossible to determine from data on a single cell whether the cell is a large cell or a cluster of cells, nor is it possible to make a quantitative determination. According to Non-Patent Document 3, cell data is an analysis of the relative positions of cell populations on a two-dimensional scatter plot, and there is a problem in that there is no quantitative numerical threshold-based judgment standard. Specifically, a technology is needed to rapidly identify and count the several circulating tumor cells (CTCs) in the blood obtained by the aforementioned repeated sorting, based on numerical values, to determine whether they are a single large cell or CTCs in a cluster of cells. This is because CTCs in neutrophil clusters have been reported to shorten the prognosis of cancer patients (Non-Patent Document 4). Therefore, one object of the present invention is to provide a method or apparatus for purifying target particles from highly concentrated particles in a short time. Another object of the present invention is to provide a method or apparatus for reliably dispensing single particles. Yet another object of the present invention is to provide a method for reliably dispensing emulsion droplets in fluorinated oil. Yet another object of the present invention is to provide a method or apparatus for determining whether cells analyzed in a flow cytometer are single cells or cell clusters. [Means for solving the problem]

[0017] The present inventors have conducted extensive research into a method or apparatus for purifying target particles from highly concentrated particles in a short period of time, and have surprisingly found that target particles can be purified in a short period of time by repeatedly sorting highly concentrated particles. Furthermore, the present inventors have conducted extensive research into a method or apparatus for reliably dispensing single particles, and have surprisingly found that single particles can be reliably dispensed by sorting each particle into a collection reservoir connected to a flow channel and then dispensing the particles from the collection reservoir into another container. Furthermore, the present inventors have conducted extensive research into a method for reliably dispensing emulsion droplets in fluorinated oil, and have surprisingly found that emulsion droplets in fluorinated oil can be reliably dispensed by using fluorinated oil and mineral oil. Furthermore, the present inventors have conducted extensive research into a method or apparatus for determining whether cells analyzed by a flow cytometer are single cells or cell clusters, and have surprisingly found that single cells and cell clusters can be easily distinguished by calculating the ratio of the forward scattered light signal intensity to the non-forward scattered light signal intensity. The present invention is based on this finding. Therefore, the present invention provides [1] A method for purifying target particles, comprising a step of sorting the target particles from among non-target particles at a high concentration, the method being characterized in that the sorting step is repeated three or more times; [2] A method for purifying target particles, wherein in a step of sorting target particles from non-target particles with a high concentration, the initial concentration of non-target particles is 10 8 a method for purifying particles, characterized by repeatedly sorting particles under conditions of particles / mL or more; [3] The total number of particles, including the first target particle, is 10 8 The method for purifying particles according to [1] or [2], [4] The method for purifying particles according to any one of [1] to [3], wherein the particles are cells. [5] The particle purification method according to any one of [1] to [4], wherein the target particles are fluorescently dyed, and after one or more sorting steps, non-target particles are fluorescently dyed and a subsequent sorting step is performed. [6] The method for purifying particles according to any one of [1] to [5], further comprising a sorting step for separating a single particle. [7] A method for dispensing a single particle, comprising the steps of collecting the particles into a collection reservoir connected to a flow path for each sorting step and dispensing the particles from the collection reservoir into another container; [8] A device for purifying target particles, capable of repeatedly sorting target particles, comprising: the device includes a channel chip for separating particles contained in a sample liquid; The channel chip has channels formed in a transparent substrate, and a sample fluid reservoir, a sheath fluid reservoir, a sorting reservoir, a recovery reservoir, and a waste fluid reservoir fluidically connected to the channels. The flow of liquid in the channels is controlled by the air pressure above each reservoir. The channel chip has a confluence channel where an introduction channel from the sample fluid reservoir and a pair of sheath fluid introduction channels arranged on both sides of the introduction channel join together. A light irradiation area for detecting particles is located downstream of the confluence channel, and a light irradiation area for detecting particles is located further downstream of the confluence channel. a pair of opposing branch channels connected to one of the pair of branch channels, a sorting reservoir connected to the other of the pair of branch channels, an upper part of the recovery reservoir being capable of being released to atmospheric pressure, the sample liquid reservoir being capable of being released to atmospheric pressure, the channel chip being capable of moving laterally during repeated sorting, and configured so that liquid transfer between each reservoir, liquid transfer from each reservoir to the outside, and liquid addition to each reservoir from the outside are performed from the upper part of the reservoir; [9] A device for dispensing a single target particle, capable of sorting the target particle, comprising a channel chip for separating particles contained in a sample liquid, wherein the channel chip has a channel formed in a transparent substrate, and a sample liquid reservoir, a sheath liquid reservoir, a sorting reservoir, a recovery reservoir, and a waste liquid reservoir fluidically connected to the channel, and the flow of liquid in the channel is controlled by the air pressure above each reservoir, and the channel chip has an introduction channel from the sample liquid reservoir and a pair of sheath liquid introduction channels arranged on both sides of the channel. a target particle dispensing device including a confluence flow path where flow paths converge, a light irradiation area for detecting particles downstream of the confluence flow path, and a pair of opposing branch flow paths connected from the sides of the confluence flow path further downstream, one of the pair of branch flow paths being connected to a sorting reservoir and the other being connected to a collection reservoir, the upper part of the collection reservoir being capable of being released to atmospheric pressure, and a configuration in which sorting is stopped after sorting one target particle into the collection reservoir and the one target particle is dispensed from the collection reservoir into another container;

[10] The method for dispensing a single particle according to [7], wherein the particles are emulsion droplets in a fluorine-based oil, the fluorine-based oil and the mineral oil are contained in a collection reservoir in advance, and the target emulsion droplets are separated based on a fluorescent signal, the emulsion droplets are taken into the collection reservoir one by one, and are floated up from the bottom of the collection reservoir, the emulsion droplets are trapped at the dome-shaped interface between the fluorine-based oil and the mineral oil in the collection reservoir, and the emulsion droplets are sucked up from the top and dispensed into an external container.

[11] A cell cluster analysis method, which is a data analysis method for a flow cytometer, comprising determining the ratio of the forward scattered light signal intensity to the non-forward scattered light signal intensity detected for each cell, and identifying each cell as a single cell or a cell cluster based on the ratio;

[12] The cluster analysis method according to

[11] , wherein the target cells are circulating tumor cells in the blood, the circulating tumor cells in the blood are fluorescently stained, and clusters or single circulating tumor cells in the blood are analyzed by combining with the fluorescent signal; and

[13] A flow cytometer device, which is a cell cluster analysis device that calculates the ratio of scattered light signals in multiple directions and uses the ratio to identify whether a cell is a single cell or a cell cluster. Regarding. [Effects of the Invention]

[0018] The particle purification method and apparatus of the present invention can solve the problems of the conventional Jet-in-Air cell sorting technology. That is, it is possible to purify the target particles from highly concentrated particles in a short time, for example, when the total number of cells is 10 8 Target cells can be separated from non-target cells within a few hours. In another aspect, the problems of conventional cell sorting techniques using microchannels can be solved. That is, in the automated operation for repeated sorting, the removal and washing of cells remaining in the channel chip can be incorporated any number of times. The single particle dispensing method and device of the present invention can solve the problems of single cell dispensing technology. That is, the single particle dispensing method and device of the present invention can reliably dispense single particles. Specifically, it can solve the problem of target cells not being dispensed when the time between target cell sorting events is shorter than the movement time of the multi-well plate to which the target cells are dispensed. The method and apparatus for dispensing single particles of the present invention can solve the problems of dispensing emulsion particles in fluorine-based oil. That is, it can reliably dispense emulsion droplets in fluorine-based oil. Specifically, it can solve the problem that the droplets float because fluorine-based oil has a higher specific gravity than water, and after floating, they are difficult to pick up with a pipette due to adsorption to the resin wall. Furthermore, it can solve the problem that when the amount of emulsion in the collection reservoir increases and the liquid level of the fluorine-based oil rises, a force due to the mass of the oil is generated in a direction that causes the oil to flow backward. The data analysis method of the present invention can solve the problem of cluster cell analysis in flow cytometry by determining whether each detected cell or cell cluster is a large single cell or a cluster of multiple cells based on quantitative values. [Brief explanation of the drawings]

[0019] [Figure 1] This table shows the results of Poisson distribution analysis of the processing time required to achieve a purity of 98% or more of target cells when using a Jet in Air cell sorter with a sorting speed of 30,000 times per second. [Figure 2] This table compares the processing time required to achieve a target cell purity of 98% or higher using Jet in Air sorting (JS) and repetitive sorting (RS) according to the particle purification method of the present invention. The speed of Jet in Air sorting is 30,000 cells / second, while that of the repetitive sorting of the present invention is 1,000 cells / second. [Figure 3] 1 shows (A) the structure of a chip used in the particle purification method of the present invention, (B) a conceptual diagram of repeated sorting, and (C) a graph showing the results of repeated sorting performed by mixing PC-9 cells with leukocytes. [Figure 4] Graph (A) shows the experimental results of adding 10 PC-9 cells to white blood cells, and then replacing the flow channel chip with a new one after sorting, versus continuing to use the same chip. Graph (B) shows a simulation of cell purification depending on the number of repeated sorting attempts at different white blood cell residual rates (0%, 0.3%, 0.5%, 0.7%, and 1.0%) in the chip. The 0% white blood cell residual rate in graph (B) matches the experimental results when a new chip is used. [Figure 5] This figure shows the structure (A) for airtight connection between the reservoirs and the air pressure control system in the device, and the operation (B) for automatically releasing atmospheric pressure in the air pressure control of the sample liquid reservoir, sheath liquid reservoir, and waste liquid reservoir of the channel chip. [Figure 6] 10 is a diagram showing the lateral movement of the channel chip for the dispensing pipette to access each reservoir after atmospheric pressure is released. In addition to the lateral movement of the channel chip, the dispensing pipette must also move up and down and in a plane. [Figure 7] 10A and 10B are diagrams showing the operation for repeated sorting of the present invention, illustrating the transfer of liquid from a collection reservoir to a sample liquid reservoir of a channel chip. [Figure 8] 1A is a diagram showing dispensing from a collection reservoir to a container (each well in a multi-well plate) in a single particle dispensing method of the present invention, and FIG. 1B is a flowchart of the single particle dispensing method. [Figure 9] 1 is a flowchart showing the single particle dispensing method of the present invention performed after repeated sorting. [Figure 10] This figure shows the operation of the method for dispensing single emulsion droplets of the present invention. First, emulsion droplets are taken into a branch channel in a channel chip using fluorinated oil as a sheath liquid, then the separated emulsion droplets are trapped at a dome-shaped interface between two types of oil, fluorinated oil and mineral oil, in a collection reservoir (see the photograph in Figure 11), and finally the emulsion droplets are dispensed from the collection reservoir into a container outside the device. [Figure 11] Photograph (A) shows that emulsion droplets are adsorbed onto the wall surface of the plastic resin when no mineral oil is present in the single particle dispensing method of the present invention, and photograph (B) shows that when mineral oil is added, the interface between the mineral oil and the fluorinated oil forms a dome shape, and the emulsion droplets are trapped at the top of the dome. [Figure 12] In the single particle dispensing method of the present invention, (A) is a micrograph showing emulsion droplets (40 μm in diameter) trapped on the inside of the upper part of the dome-shaped interface between fluorinated oil and mineral oil from the bottom of the collection reservoir before dispensing, and (B) is a micrograph of emulsion droplets sucked up by the pipette of the dispensing head and dispensed into wells in a 384-well plate together with mineral oil and fluorinated oil. [Figure 13] FIG. 1 is a diagram showing one embodiment of the structure of a channel chip (without a collection reservoir) that forms emulsion droplets in fluorine-based oil, and shows the structure of a system that collects emulsion droplets using a vertically downward tube. [Figure 14] FIG. 1 shows one embodiment of the structure of a channel chip that forms emulsion droplets in fluorine-based oil (a structure without a collection reservoir), and shows a structure in which emulsion droplets are collected using a horizontal tube. [Figure 15] This is a photograph of the droplet formation channel region (A) and the wide channel region downstream of the droplet formation channel region (B) when emulsion droplets are formed using an emulsion formation channel chip with a vertically downward tube recovery method. [Figure 16] Flow cytometry data of a mixture of leukocytes, leukocyte clusters, and cell line PC-9 cells stained with cytokeratin antibodies and nuclear stained with Hoechst. [Figure 17] Histogram distribution of SSC / FSC values of flow cytometry data of a mixture of leukocytes, leukocyte clusters, and cell line PC-9 cells stained with cytokeratin antibodies and nuclear stained with Hoechst (A), histogram distribution of SSC / FSC values of granulocytes, a leukocyte component (B), and histogram distribution of SSC / FSC values of leukocyte clusters (C). [Figure 18] This is a two-dimensional scatter plot of the cytokeratin fluorescent signal intensity and SSC / FSC value of CTCs isolated from the blood of cancer patients. DETAILED DESCRIPTION OF THE INVENTION

[0020] [1] Particle purification method The particle purification method of the present invention is a method for purifying target particles, which includes a step of sorting target particles from a high concentration of non-target particles, and is characterized in that the sorting step is repeated three or more times. As used herein, "particles" include cells. Also, "particles" may be emulsion droplets. Emulsion droplets may contain cells. Furthermore, target particles are particles to be purified, and non-target particles are particles that are different from target particles and are removed by sorting.

[0021] <Sorting process> The number of times the sorting step is performed is not limited as long as it is performed three or more times. Preferably, it is performed three or more times, and more preferably, it is performed four or more times. The upper limit is not particularly limited, but is six or less times, and more preferably five or less times. The number of repeated sorting steps required depends on 1) the number of non-target cells in the initial sample, 2) the number of target cells, 3) the survival rate of non-target cells after sorting, 4) the recovery rate of target cells after sorting, and 5) the final purity of target cells. For example, if the number of non-target cells is 10 8 If the number of target cells is 100, the residual rate of non-target cells after sorting is 1%, the recovery rate of target cells after sorting is 95%, and the final target purity is 98%, the required number of repeated sorting cycles is calculated to be 4. If there are 10 target cells, the required number of cycles is 5.

[0022] In the particle purification method of the present invention, the initial concentration of undesired particles (or the concentration of total particles) is not particularly limited, but may be, for example, 10 6 cells / mL or more, preferably 10 7 cells / mL or more, more preferably 2x10 7 cells / mL or more, more preferably 5x10 7 cells / mL or more, more preferably 7x10 7 cells / mL or more, more preferably 1x10 8 cells / mL or more, more preferably 2 x 10 8 cells / mL or more. In the sorting step, the concentration of non-target particles (or the concentration of total particles) decreases as non-target particles are removed each time the sorting step is repeated.

[0023] The total number of particles in the particle purification method of the present invention is not particularly limited, but for example, 10 6 10 or more, preferably 10 7 More preferably, 2x10 7 10 or more, more preferably 5x10 7 10 or more, more preferably 7x10 7 more preferably 1x10 8 more preferably 2x10 8 There are more than one.

[0024] In the particle purification method of the present invention, the target particles are fluorescently dyed, and after one or more sorting steps, the non-target particles can be fluorescently dyed and the subsequent sorting step can be performed, although this is not limited thereto. Before the sorting step, the target particles and non-target particles can be specifically fluorescently dyed and then sorted. However, it is also possible to fluorescently dye only the target particles and then perform the sorting step. In the purification method of the present invention, a relatively high concentration of non-target particles is used. Therefore, if non-target particles are fluorescently stained before the first sorting, a large amount of antibody must be used. However, if non-target particles are fluorescently stained after one or more sorting steps, the number of non-target particles is reduced, and therefore the amount of antibody used to fluorescently stain the non-target particles can be reduced.

[0025] The particle purification method of the present invention may further include, but is not limited to, a sorting step for collecting a single particle. The "collection of a single particle" can be performed according to the "single particle dispensing method" described below.

[0026] The particle purification method of the present invention can solve the problems associated with Jet-in-Air cell sorters, as will be explained using the results of numerical simulations. Numerical simulations were used to compare the processing time of the method of the present invention, in which a sample solution containing a high concentration of cells is repeatedly sorted until the required purity is reached, with the processing time required to achieve the required purity in a single sorting process by diluting the sample solution containing a high concentration of cells to a low concentration where only a single cell is contained in the droplets to be sorted. Figure 2 shows the results of a comparison of processing times between Jet-in-Air sorting (JS) and the repetitive sorting of the present invention (RS). As in Figure 1, the processing time required to achieve a purity of 98% or more was calculated using Poisson distribution analysis. The speed of Jet-in-Air sorting is 30,000 cells / second, while that of the repetitive sorting of the present invention is 1,000 cells / second. ... 8 In this case, the processing time for Jet-in-Air sorting is 30 hours due to the increase in sample volume caused by dilution. On the other hand, the processing time for repeated sorting is shorter because the sample volume is small and there is no need to dilute the sample. This method shortens the processing time for sorting target cells when the number of non-target cells is very high. It is not necessary to separate a single target cell in a single sorting run. Even under cell concentration conditions where multiple non-target cells are accidentally separated, the target cells can be purified to the required level by repeating sorting.

[0027] The target particle purification device of the present invention is an apparatus for purifying target particles, capable of repeated sorting of target particles. The target particle purification device includes a channel chip for separating particles contained in a sample solution. The channel chip has channels formed in a transparent substrate, and a sample solution reservoir, a sheath solution reservoir, a sorting reservoir, a recovery reservoir, and a waste solution reservoir fluidically connected to the channels. The flow of liquid within the channels is controlled by the air pressure above each reservoir. The channel chip has a confluence channel where an introduction channel from the sample solution reservoir and a pair of sheath solution introduction channels located on either side of the confluence channel converge. A light irradiation area for particle detection is located downstream of the confluence channel. Further downstream, a pair of opposing branch channels are connected to the sides of the confluence channel. One of the pair of branch channels is connected to a sorting reservoir, and the other is connected to a recovery reservoir. The top of the recovery reservoir is open to atmospheric pressure, and the sample solution reservoir is open to atmospheric pressure. The channel chip is configured to be movable laterally during repeated sorting, and to allow liquid transfer between reservoirs, liquid transfer from each reservoir to the outside, and liquid addition to each reservoir from the outside from above the reservoirs.

[0028] The repeat sorting method is described in detail below. The particle separation device includes an exchangeable channel chip with channels formed within a flat substrate; a light irradiation unit that irradiates light onto particles in a sample solution flowing through the channel; a detection unit that detects scattered light or fluorescence emitted from the particles when irradiated with the light and identifies the particles based on their signal strength to detect target particles; a constant-pressure air pump (such as an electropneumatic regulator or a cylinder pump) and a solenoid valve connected to it as a means for applying a pressure pulse to the particles in the sample solution flowing through the channel of the channel chip; and a control unit that controls the operation of the solenoid valve based on a signal from the detection unit. A sample solution reservoir is formed in the channel chip, and a positive-pressure constant-pressure air pump for controlling the flow rate of the sample solution is airtightly connected to the gas space above the reservoir via a sample solution reservoir adapter. A channel for introducing the sample solution is connected to the bottom of the sample solution reservoir. The replaceable channel chip includes a pair of sheath fluid introduction channels arranged on either side of the introduction channel; a confluence channel where the pair of sheath fluid introduction channels merge with the sample fluid introduction channel, with sheath fluid flowing on both sides of the sample fluid within the confluence channel; a light irradiation area on the confluence channel; and a pair of opposing branch channels connected to the confluence channel downstream of the light irradiation area. One of the pair of opposing branch channels is airtightly connected to a sorting reservoir via a sorting reservoir adapter, with a normally closed electromagnetic valve and a positive constant-pressure air pump. The other side of the branch channel is connected to a recovery reservoir, with the space above the recovery reservoir being open to atmospheric pressure. A waste reservoir is connected downstream of the confluence channel. The upper gas space of the waste reservoir is airtightly connected to a constant-pressure air pump with a negative pressure lower than atmospheric pressure via a tube via an adapter connecting the waste reservoir to the tube.

[0029] Figure 3(A) shows a channel chip with reservoirs, including a sample fluid reservoir (1), a recovery reservoir (2), a waste fluid reservoir (3), and a sheath fluid reservoir (4). By applying gas pressure to the space above the sample fluid in the sample fluid reservoir for a certain period of time, the sample fluid is pushed out through the sample fluid introduction channel (7) connected to the bottom. Similarly, by applying gas pressure to the space above the sheath fluid in the sheath fluid reservoir for a certain period of time, the sheath fluid is pushed out. The merged streams flow through the main channel (9). Along the main channel, they pass through a light irradiation area (detection area). An optical signal generated when a particle passes through the light irradiation area is detected, and the control unit determines whether the particle is a target particle to be separated based on the optical signal from the detection means. If the particle is determined to be a target particle to be separated (5), a signal is sent to the solenoid valve to open for a short period of time after a delay time until the particle reaches the area where the main channel intersects with the branch channel further downstream. This generates a pulsed flow for a short period of time, and the target particles are taken into the recovery reservoir (2) through the recovery channel (10). Non-target particles, which do not generate a pulsed flow, travel straight through the main channel (9) and flow into the waste liquid reservoir (3) downstream of the channel. The upper gas space of the waste liquid reservoir is airtightly connected via a tube to a negative constant-pressure air pump, and the pressure is adjusted to be lower than atmospheric pressure.

[0030] This section explains how repeated sorting can solve the problems of the Jet in Air method. In the first sorting run, after filling the sample reservoir with sample liquid and processing the entire volume, most of the target particles in the sample liquid are collected in the collection reservoir. However, non-target particles that accidentally received the pulse flow are also collected in the collection reservoir along with the target particles. Therefore, the purity is insufficient, so sorting is performed again. Therefore, the entire particle liquid in the collection reservoir is collected and returned to the upstream sample reservoir for a second sorting run. Before returning the liquid in the collection reservoir to the sample reservoir, there is something that needs to be done. A very large proportion of the particles remaining at the bottom of the sample reservoir are non-target particles from before the sorting process. Therefore, it is important to remove the remaining particles by washing before returning the collected liquid. This procedure is used for the second sorting run. It is also important to perform the third sorting run after washing the particles remaining at the bottom of the sample reservoir. This process is repeated until the desired purity is achieved. Figure 3 illustrates how repeated sorting gradually enriches target cells from non-target cells. Figure 3(C) shows the results of repeated sorting. 8 This is enrichment data for 100 or fewer PC-9 cells intentionally mixed into 100 white blood cells. The number of white blood cells decreases with the number of sorting steps, but the number of target cells remains constant, and the purity of PC9 cells increases by about 100 times with each sorting step (Figure 3(C)). This processing time is within one hour, and the number of target cells in Figure 2 is 100, while the number of non-target cells is 10. 8It can be seen that the values in the column are close to the actual values. The data in Figure 4(A) shows the dependency of purity (=PC-9 count / (PC9 count + white blood cell count) on the number of repeated sorting when the number of spikes of PC-9 in Figure 3 is 10. This shows both the case where the flow channel chip is replaced and the case where the same chip is used continuously. When the flow channel chip is replaced, the purity reaches 90% after two sortings, but with the same chip, it is only about 40% even after the third sorting. This is because the number of white blood cells remaining in the chip deteriorates the purity. Figure 4(B) shows the final purity when the residual white blood cell rate in the chip is changed, simulating the result. This result was derived through simulation, and a zero residual rate corresponds to replacing the flow channel chip, and the purity reaches 90% on the second sort. When using the same chip, a residual rate of 0.7% corresponds to a purity of 40% on the third sort. This residual rate of 40% was obtained by washing the bottom of the sample reservoir, and a washing procedure is preferable for repeated sorting. Furthermore, when the sample fluid is a patient-derived specimen, it is necessary to be able to replace the flow channel chip with a new one to avoid cross-contamination between patients.

[0031] Next, we will explain how to automate repeated sorting. Specifically, we will explain the necessary technology and the means for solving the problems of conventional cell sorter technology in microchannels below. As shown in the data in Figure 4(B), a mechanism that reduces the number of remaining particles in the channel by washing after each sorting process is preferable. Therefore, in automating repeated sorting, a mechanism that introduces a washing solution into the microchannel chip to wash the remaining particles in the channel is preferable. The technology described in Patent Document 1 does not include this mechanism. In the invention of Patent Document 2, the chip structure does not allow for the required number of repeated sorting operations. Therefore, we will explain how to include a washing step for the required number of times. The following processing means is carried out using a dispensing head equipped with a liquid suction mechanism and a liquid discharge mechanism. 1) After sorting, the pressure inside each reservoir in the channel chip is set to atmospheric pressure. 2) Move the flow channel chip to the side to allow the dispensing head to access the reservoir. 3) Clean any remaining particles in the sample reservoir. 4) The liquid containing particles in the collection reservoir is drawn up by the pipette on the dispensing head. 5) Discharge the liquid in the pipette into the sample liquid reservoir. 6) The channel chip is returned to its original position laterally, and the airtight connection between the channel chip and the external air pressure control system is restored. In step 1), the liquid flow inside the channel chip is controlled by air pressure from outside the channel chip. Therefore, the channel chip must be released to atmospheric pressure after each sorting process. Figures 5(A) and 5(B) show a method for automatically releasing atmospheric pressure. The air pressure in the sample fluid reservoir, sheath fluid reservoir, and waste fluid reservoir inside the channel chip is controlled by connecting them to an air pressure control system, including a constant-pressure air pump, installed on the device via a channel chip adapter (14) and a pressure control unit (15) on the device side, via an airtight connection using deformable rubber (12). The atmospheric pressure inside the channel chip is released by moving the pressure control unit upward. As shown in Figure 6, step 2) involves moving the channel chip laterally so that the dispensing head can access the channel chip reservoirs. In step 3), the dispensing head adds washing solution to the sample fluid reservoir, pipettes it, and then discards the washing solution. This washing procedure of adding washing solution, pipetting it, and discarding it is repeated a predetermined number of times. In step 4), after cleaning, the particles are transferred by sucking up the recovery liquid from the recovery reservoir and discharging it into the sample liquid reservoir. In step 6), airtight connections between the sample liquid reservoir, waste liquid reservoir, and sheath liquid reservoir and the air pressure control system on the device are restored in preparation for the next sorting.

[0032] [2] Single particle dispensing method The single particle dispensing method of the present invention includes the steps of sorting each particle into a collection reservoir connected to a flow path and dispensing the particle from the collection reservoir into another container. Specifically, after sorting one particle, the sorting is temporarily stopped, and the particle collected in the collection reservoir is dispensed into another container (e.g., a well of a multi-well plate). Then, sorting is started, and one particle is sorted and dispensed into the collection reservoir. By repeating these operations, it is possible to reliably dispense one particle into one container (e.g., one well). The number of times the above steps are repeated is not particularly limited, and can be adjusted depending on the number of particles.

[0033] The target particle dispensing device of the present invention is a device for dispensing a single target particle and capable of sorting the target particle. The device includes a channel chip for separating particles contained in a sample solution. The channel chip has channels formed in a transparent substrate, and a sample solution reservoir, a sheath solution reservoir, a sorting reservoir, a recovery reservoir, and a waste solution reservoir fluidically connected to the channels. The flow of liquid within the channels is controlled by the air pressure above each reservoir. The channel chip has a confluence channel where an introduction channel from the sample solution reservoir and a pair of sheath solution introduction channels located on either side of the confluence channel converge. A light irradiation area for particle detection is located downstream of the confluence channel. Further downstream, a pair of opposing branch channels are connected to the junction channel from the sides. One of the pair of branch channels is connected to a sorting reservoir, and the other is connected to a recovery reservoir. The top of the recovery reservoir can be released to atmospheric pressure. The target particle dispensing device of the present invention includes a configuration in which, after sorting one target particle into a collection reservoir, sorting is stopped and the one target particle is dispensed from the collection reservoir into another container.

[0034] The single particle dispensing method of the present invention is, for example, a means for dispensing each single cell after sorting. Unintended cells 10 8This section describes a method for dispensing target cells into, for example, a multi-well plate when several hundred specific target cells are contained in a single particle. This section describes a method for dispensing target cells when, for example, a cell suspension purified to approximately 98% purity by repeated sorting in the particle purification method described above is stored in a collection reservoir. As shown in Figure 7, the cell suspension in the collection reservoir is returned to the sample liquid reservoir, and the cells are sorted. As shown in Figure 8(A), dispensing is performed by using a dispensing head to move liquid from the collection reservoir, which is constantly open to atmospheric pressure, to each well in the multi-well plate. For example, when dispensing a single cell, after sorting one cell with one pulse flow, the pressure in the sample liquid reservoir is reduced to stop the flow of sample liquid. Then, a single cell is dispensed from the collection reservoir into each well of the multi-well plate using a pipette on the dispensing head. These operations enable dispensing of individual target cells and prevent loss of target cells due to the target cells passing through the sorting area during dispensing. For example, if 10 cells are dispensed into wells, the flow is stopped after 10 sorting runs, equivalent to 10 cells each. Then, the pipette in the dispensing head dispenses 10 cells from the collection reservoir into each well of the multi-well plate. Then, before the dispensing head returns to the collection reservoir, sorting is resumed. Dispensing of 10 cells can be performed by dispensing all the cells in the collection reservoir after 10 sorting runs. The number of cells dispensed into each well can be set as desired, and dispensing can be performed according to the flowchart shown in Figure 8(B). This flowchart does not show operations to prevent loss of sample liquid from flowing out after each dispensing run. However, if the time interval between target cell detections is shorter than the dispensing run, sample liquid will be lost from flowing out. For example, the time required for this dispensing operation is approximately 5 seconds, so if target cells are detected at intervals shorter than this time, losses will occur. Therefore, it is desirable to set the target cell concentration so that at least one cell or less flows per 5 seconds. Figure 9 is a flowchart of the operation in such a case.

[0035] <Dispensing emulsion droplets> In one embodiment of the method for dispensing a single particle of the present invention, the particle is an emulsion droplet in a fluorine-based oil, and a collection reservoir contains a fluorine-based oil and a mineral oil in advance. The target emulsion droplet is separated based on a fluorescent signal, and the emulsion droplet is taken into the collection reservoir individually. The emulsion droplet is then floated up from the bottom of the collection reservoir, trapped at the dome-shaped interface between the fluorine-based oil and the mineral oil in the collection reservoir, and then sucked up from the top and dispensed into an external container.

[0036] The fluorine-based oil used in the dispensing method of the present invention is not particularly limited, and examples of commercially available products include Fluorinert oil Novec 7500 manufactured by 3M Co., Ltd. The commercially available mineral oil used in the dispensing method of the present invention is not particularly limited, and examples of commercially available mineral oil include mineral oil manufactured by Sigma-Aldrich Co., Ltd. The amount of fluorine-based oil contained in the collection reservoir is not particularly limited, but is 10 μL to 1 mL. The amount of mineral oil contained in the collection reservoir is not particularly limited, but is 10 μL to 1 mL.

[0037] Regarding emulsion droplets in fluorinated oil, the specific gravity of the fluorinated oil is greater than that of water, so the emulsion droplets float in the fluorinated oil. Then, as shown in the photograph in Figure 11(A), they are adsorbed to the plastic resin wall on the surface of the fluorinated oil. Therefore, it is difficult to suck up the emulsion droplets individually from above the fluorinated oil with a pipette. To solve this problem, mineral oil is used as a cover for the fluorinated oil, as shown in Figure 11(B). In this case, the interface between the mineral oil and the fluorinated oil forms a dome shape, and the emulsion droplets in the fluorinated oil are trapped at the top of the dome. In the photograph in Figure 11(B), it can be seen that the emulsion droplets are distributed in a dome shape because they contain a dye. Therefore, by filling the collection reservoir with fluorine-based oil and mineral oil in advance, the sorted emulsion droplets are trapped at the center of the reservoir. This makes it easy to suck up the emulsion droplets from the collection reservoir with a pipette. This will be explained below with reference to Figure 10.

[0038] When emulsion droplets are formed in oil, they do not necessarily contain cells. That is, emulsion droplets containing one cell or less account for approximately 1 / 10 of the total number of emulsions, according to the Poisson distribution. Therefore, sorting of emulsion droplets is necessary to select emulsion droplets containing cells. The selection of emulsions containing cells is performed based on signals such as side-scattered light signals and cell autofluorescence signals generated when the emulsions pass through the laser light irradiation area (20). In this sorting method, fluorinated oil is used as the sheath flow. When the emulsion droplets containing cells reach the sorting area, a pulsed flow is generated. The emulsion droplets are then drawn into the collection channel, and only the emulsion droplets containing cells are accumulated in the collection reservoir. As mentioned above, the proportion of emulsion droplets containing cells is approximately 1 / 10, so a single sorting run results in a purity of approximately 98%. After sorting a single emulsion droplet, it is trapped in the upper center of the dome-shaped interface at the top of the collection reservoir. This makes it easy to pick up the emulsion droplets with a pipette and dispense them into a multi-well plate.

[0039] Next, to demonstrate the feasibility of dispensing individual emulsion droplets, we performed the following procedure. A water-soluble fluorescent reagent (FITC) was added to the liquid forming the emulsion droplets, making the droplets identifiable by fluorescence after formation. After sorting a single fluorescent emulsion droplet, we observed the emulsion droplet in the collection reservoir before dispensing. We also observed the emulsion droplet after dispensing it into a well of a 384-well plate using a pipette. The results are shown in Figure 12. Figure 12(A) is a micrograph showing an emulsion droplet (40 μm in diameter) trapped at the top inside of the dome-shaped interface between the fluorinated oil and mineral oil from the bottom of the collection reservoir before dispensing. Figure 12(B) is a micrograph of the emulsion droplet after being sucked up by the pipette of the dispensing head and dispensed into a well of a 384-well plate together with the mineral oil and fluorinated oil. In mineral oil, a small amount of fluorinated oil forms spheres. Emulsion droplets are observed inside the spheres, demonstrating that the oil is dispensed individually.

[0040] The structure of a channel chip that forms emulsion droplets in fluorinated oil is described below. The following structure can solve the problem. Specifically, a structure without a collection reservoir is sufficient. As shown in Figure 13, an oil reservoir and a sample liquid reservoir are formed in the channel chip. Emulsion droplets are formed at the intersection of the oil channel and the sample liquid channel. Furthermore, emulsion is collected downstream of the channel by a tube connected downward. In the structure shown in Figure 14, an oil reservoir and a sample liquid reservoir are formed in the channel chip. Emulsion droplets are formed at the intersection of the oil channel and the sample liquid channel, and the emulsion is collected by a tube connected horizontally. Figure 15(A) is a photograph of the droplet formation channel region when emulsion droplets are formed using the emulsion formation channel chip with the vertically downward tube collection method shown in Figure 13. Figure 15(B) is a photograph of the wide channel region downstream of the droplet formation channel region.

[0041] [3] Cell cluster analysis method The cell cluster analysis method of the present invention is a data analysis method for a flow cytometer, which calculates the ratio of the forward scattered light signal intensity to the non-forward scattered light signal intensity detected for each cell, and identifies each cell as a single cell or a cell cluster based on this ratio. The non-forward scattered light signal intensity is not particularly limited, but is preferably side scattered light signal intensity or back scattered light signal intensity.

[0042] The cell cluster analysis device of the present invention is a flow cytometer device that determines the ratio of scattered light signals in multiple directions and uses the resulting value to distinguish between single cells and cell clusters.

[0043] The cell cluster analysis method of the present invention is a means for quantitatively analyzing individual cell clusters. The forward scatter signal (FSC) is the intensity of the scattered light component at low angles relative to the direction of light irradiation and primarily reflects the size of the scatterer. In contrast, the side scatter signal (SSC) is the intensity of the scattered light component at high angles relative to the direction of light irradiation and is said to primarily reflect the fine structure within the scatterer. This is consistent with the results based on Mie light scattering theory. Therefore, we will explain below using actual data a method for distinguishing single cells from cell clusters based on the numerical ratio of FSC to SSC signal intensities. Figure 17(A) shows the histogram distribution of the SSC / FSC values of a cell line called PC-9. Figure 17(B) shows the histogram distribution of the SSC / FSC values of granulocytes, a white blood cell component, and Figure 17(C) shows the histogram distribution of the SSC / FSC values of white blood cell clusters. PC-9 is a single cell, and granulocytes are single cells with fine structures within the cell. White blood cell clusters are components confirmed to be clusters consisting of multiple cells based on the amount of nuclear staining. The above three types of cell states are distinguished from each other in the flow cytometry data of leukocytes, leukocyte clusters, and mixtures with cell line PC-9 cells in FIG. The horizontal axis of Figure 16 represents Hoechst staining, and the vertical axis represents cytokeratin staining. PC9 epithelial cells are distributed within the area enclosed by the gate labeled P25 and exhibit high levels of cytokeratin expression. Granulocytes, on the other hand, are distributed within the gate labeled P23. These cells are characterized by high levels of nuclear staining and weak cytokeratin positivity. Leukocyte clusters, on the other hand, are distributed within the gate labeled P26. These clusters are characterized by a correlation between the amount of nuclear staining and the amount of cytokeratin staining. While these clusters are considered granulocyte clusters, since neutrophils are also granulocyte components, counting these P26 leukocyte clusters is crucial. After CTC sorting, it is important to determine whether any of these clusters are identifiable. According to the histogram distributions in Figures 17(A), (B), and (C), leukocyte clusters can be identified within a range of approximately 0.2 to 1. However, because the range of 0.2 to 0.3 overlaps with granulocytes, microscopic observation is recommended. In the case of CTC sorting, identification can be facilitated by combining conditions with other fluorescent stains, but for general purposes, a technique that can identify them using only scattered light signals is important. Figure 18 shows flow cytometry data from a sample of cytokeratin-positive cells sorted and enriched from the blood of a cancer patient, plotted as a two-dimensional scatter plot of cytokeratin fluorescent signal intensity and SSC / FSC values. This graph allows identification of clustered cytokeratin-positive cells. In other words, CTC clusters can be counted based on the number of data points within the quantitative range of SSC / FSC values and cytokeratin signal values. [Example]

[0044] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0045] Example 1 In this example, circulating tumor cells (CTCs) from the blood of a cancer patient were separated by repeated sorting. Eight milliliters of blood was collected from a cancer patient and placed in a blood storage tube. The following pretreatment was performed within two days. The 8 milliliters of blood contained approximately 10 white blood cells.8 The containers used in the following pretreatment were washed and coated with 0.5% BSA / On-chip T-buffer in advance. This was to prevent cell loss due to cell adsorption to the resin container. The buffer reagents used in the pretreatment were commercially available reagents such as red blood cell hemolysis buffer, fixation reagent, cell permeability reagent, and FCR blocker agent. These protocols were performed according to the package inserts of the reagents.

[0046] Pretreatment (I) The collected blood was processed in the following order: hemolysis, centrifugation to remove red blood cell debris, washing, fixation, permeabilization, and fluorescent staining (cytokeratin antibody label only). These processes removed red blood cells from the sample solution, and the blood was collected in 10 8 It is believed that several or more CTCs are contained in each leukocyte. The sample solution before treatment was adjusted to 0.3 mL, so the cell concentration was 3.3 x 10 8 It is about 1 / mL.

[0047] CTC sorting process (I) It is thought that only CTCs derived from cancer cells, which are epithelial cells, are cytokeratin positive, and granulocytes, which are white blood cell components, are weakly cytokeratin positive. Therefore, the conditions for identifying and separating CTCs were set so that the cytokeratin positive signal level was higher than the cytokeratin fluorescent signal level of granulocytes, and cytokeratin positive cells were separated. When sorting was repeated twice under these conditions, white blood cells were separated at 10 3 The reduction in white blood cell count due to repeated sorting is shown in Figure 3. This data is a graph of the white blood cell count and PC-9 count after each repeated sorting step, using a sample in which PC-9 cells derived from lung cancer were spiked into 4 mL of blood.

[0048] Pretreatment (II) After leukocyte reduction, Hoechst nuclear staining was performed, followed by additional staining with cytokeratin fluorescent labels, CD45 fluorescent antibody labels, vimentin fluorescent antibody labels, PD-L1 fluorescent antibody labels (or HER2 fluorescent antibody labels, EGFR fluorescent antibody labels, and AXL fluorescent antibody labels). After staining, the cells were washed by centrifugation with 0.5% BSA / On-chip T-buffer.

[0049] CTC sorting process (II) CTCs were identified by Hoechst-positive, CD45-negative, and cytokeratin-positive sorting, and were sorted twice. The CTC enrichment method demonstrated a recovery rate of >70% and purity of >80% in experiments in which a known number of PC-9 cells was spiked into blood. [Industrial Applicability]

[0050] The particle purification method of the present invention allows for a total cell count of 10 8 As explained in the Examples, this method for isolating a small number of target cells from a cell population of approximately 100 or more can be used to separate and analyze CTCs from the blood of cancer patients. In addition, it can be used for antibody screening, which selects cells that produce antibodies that specifically bind to a target antigen from a large number of antibody-producing cells in antibody drug development. Furthermore, when the particles are emulsion droplets, the single particle dispensing method of the present invention can be used to separate cells that secrete specific substances, because cell secretions accumulate in the emulsion droplets without diffusing. Furthermore, because lysing cells within emulsion droplets does not result in diffusion of the lysate, this method can be used for cell expression analysis. [Explanation of symbols]

[0051] 1···Sample solution reservoir formed on the channel chip; 2···Collection reservoir formed on the channel chip; 3···Waste reservoir formed on the channel chip; 4···Sheath fluid reservoir formed on the channel chip; 5···Target cells or particles; 6···Non-target cells or particles; 7···Sample solution introduction channel; 8···Sheath flow channel; 9...Main flow path; 10···recovery channel; 11···Flow channel chip; 12···Elastically deformable parts (rubber) for maintaining airtightness; 13···Reservoir formed on the channel chip; 14···Fluid chip adapter; 15···Pressure control unit (adapter for airtight connection on the device side); 16···Air piping tube; 17···Dispensing head pipette; 18···Constant-pressure air pumps such as electropneumatic regulators and cylinder pumps; 19···Pipette with dispensing head; 20···Light irradiation area (detection area); 21···Fluorinated oil; 22···Mineral oil; 23···Droplets in fluorinated oil; 24···multiwell plate; 25···Sample fluid reservoir; 26···Oil reservoir; 27···Emulsion droplet formation region (intersection region of the oil channel and the sample liquid channel); 28···Tube for collecting emulsion droplets; 29···Resin container; 30···wells in a 384-well plate;

Claims

1. A method for purifying target particles, comprising a step of sorting the target particles from among non-target particles at a high concentration, the step being repeated three or more times.

2. A method for purifying target particles, comprising: a step of sorting target particles from non-target particles with a high concentration, wherein the initial concentration of non-target particles is 10 ８ A method for purifying particles, characterized by repeatedly sorting under conditions of particles / mL or more.

3. The total number of particles, including the first target particle, is 10 ８ The method for purifying particles according to claim 1 or 2, wherein the number of particles is equal to or more than 100.

4. The method for purifying particles according to any one of claims 1 to 3, wherein the particles are cells.

5. A particle purification method according to any one of claims 1 to 4, wherein the target particles are fluorescently dyed, and after one or more sorting steps, non-target particles are fluorescently dyed and a subsequent sorting step is performed.

6. The method for purifying particles according to any one of claims 1 to 5, further comprising a sorting step for separating a single particle.

7. A method for dispensing a single particle, comprising the steps of collecting the particle into a collection reservoir connected to a flow path for each sorting step, and dispensing the particle from the collection reservoir into another container.

8. An apparatus for purifying target particles, capable of repeatedly sorting target particles, comprising: the device includes a channel chip for separating particles contained in a sample liquid; The channel chip has a channel formed in a transparent substrate, and a sample liquid reservoir, a sheath liquid reservoir, a sorting reservoir, a recovery reservoir, and a waste liquid reservoir fluidically connected to the channel, and the flow of liquid in the channel is controlled by the air pressure above each reservoir; the channel chip has a confluence channel where an introduction channel from the sample liquid reservoir and a pair of sheath liquid introduction channels arranged on both sides of the confluence channel join together, a light irradiation area for detecting particles is provided downstream of the confluence channel, and a pair of opposing branch channels connected to the sides of the confluence channel are provided further downstream thereof, one of the pair of branch channels is connected to a sorting reservoir and the other of the branch channel is connected to a recovery reservoir, an upper part of the recovery reservoir can be released to atmospheric pressure, and the sample liquid reservoir can be released to atmospheric pressure, The flow path chip is capable of moving laterally during repeated sorting, and is configured to move liquid between each reservoir, move liquid from each reservoir to the outside, and add liquid to each reservoir from the outside from the top of the reservoir, thereby achieving a target particle purification device.

9. A device for dispensing a single target particle, capable of sorting the target particle, the device includes a channel chip for separating particles contained in a sample liquid; The channel chip has a channel formed in a transparent substrate, and a sample liquid reservoir, a sheath liquid reservoir, a sorting reservoir, a recovery reservoir, and a waste liquid reservoir fluidically connected to the channel, and the flow of liquid in the channel is controlled by the air pressure above each reservoir; the channel chip has a confluence channel where an introduction channel from the sample liquid reservoir and a pair of sheath liquid introduction channels arranged on both sides of the confluence channel join together, a light irradiation area for detecting particles is provided downstream of the confluence channel, and a pair of opposing branch channels connected to the confluence channel from the sides are provided further downstream thereof, one of the pair of branch channels is connected to a sorting reservoir and the other of the branch channel is connected to a recovery reservoir, and an upper part of the recovery reservoir can be released to atmospheric pressure; A target particle dispensing device including a configuration for stopping sorting after sorting one target particle into a collection reservoir and dispensing the one target particle from the collection reservoir into another container.

10. 8. The method for dispensing a single particle according to claim 7, wherein the particles are emulsion droplets in a fluorine-based oil, the collection reservoir contains the fluorine-based oil and the mineral oil in advance, the target emulsion droplets are separated based on the fluorescent signal, the emulsion droplets are taken into the collection reservoir one by one, and are caused to float up from the bottom of the collection reservoir, the emulsion droplets are trapped at the dome-shaped interface between the fluorine-based oil and the mineral oil in the collection reservoir, the emulsion droplets are sucked up from above, and dispensed into an external container.

11. A cell cluster analysis method that is a data analysis method for a flow cytometer, characterized by calculating the ratio of the forward scattered light signal intensity to the non-forward scattered light signal intensity detected for each cell, and identifying each cell as a single cell or a cell cluster based on the ratio.

12. The cluster analysis method according to claim 11, wherein the target cells are circulating tumor cells, the circulating tumor cells are fluorescently stained, and clusters or single circulating tumor cells are analyzed in combination with the fluorescent signal.

13. A cell cluster analysis device that is a flow cytometer device that determines the ratio of scattered light signals in multiple directions and uses this value to distinguish between single cells and cell clusters.

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