Particle purification method, single particle dispensing method, cell cluster analysis method, and apparatus 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 method for purifying particles, a method for dispensing single particles, a method for analyzing cell clusters, and an apparatus used therefor.

Background Art

[0002] In order to detect specific cells among a large number of contaminating cells and perform gene analysis or the like, dispensing is required for each individual particle. The present invention relates to a method for purifying or dispensing cells in an amount of about 10 8 to about several hundreds of cells. As an application example, it also includes the separation of circulating tumor cells flowing in the blood of cancer patients. Therefore, the present invention relates to a particle separation and dispensing technique using a disposable exchangeable chip that realizes contamination-free cells between different patients. In this specification, the particles include cells, or particles containing droplets in oil, and the like.

[0003] The prior art and its problems regarding cell sorting and subsequent dispensing techniques will be described.

[0004] (1) Conventional Jet in Air type cell sorter technology As described in Non-Patent Document 1, it is a Jet in Air method in which droplets are formed from a nozzle and cells contained in the droplets are separated in units of droplets.

[0005] (2) Cell sorting technology in a microchannel The sorting speed in sorting by a microchannel is as slow as about 1000 cells / second or less. However, techniques for improving throughput by parallel processing using a plurality of channels, and techniques for returning the cells after processing to the channel and processing them again to improve sorting purity are described in Patent Document 1. Patent Document 2 describes a technique for improving sorting purity by having a flow channel chip with a plurality of sorting units and continuously performing respective processes. Patent Document 3 describes a method of sorting and removing unnecessary cells from a cell group using a micro flow channel chip with a reservoir, returning the remaining cells to the upstream sample reservoir again, and performing a sorting process. Patent Document 3 describes a sorting technique using a pulsed flow in a micro flow channel chip having a reservoir. Further, Patent Document 4 describes a method (repeated negative sorting method) of sorting and removing unnecessary cells in a micro flow channel chip, recovering the remaining cell liquid from a waste liquid reservoir, returning it to the upstream sample reservoir, and repeating negative sorting.

[0006] (3) Single cell dispensing technique If necessary, the sorted cells may be dispensed into a multi-well plate in units of one cell. This technique and problems will be described. As a method for selecting target cells contained in contaminating cells and dispensing the cells into a multi-well plate, a method of directly dispensing droplets collected by a Jet in Air type cell sorter into a multi-well plate is described in Non-Patent Document 2. A method of identifying target cells by image recognition and dispensing them as droplets into a multi-well plate using piezo pressure is described in Patent Document 5. The problem with this method is that when using piezo pressure, there is a limit to the size of the droplets that can be ejected. A technique of sucking up the cells to be dispensed from a suspension with a pipette, photographing the inside of the pipette, and dispensing when it is recognized that one cell is contained is described in Patent Document 6.

[0007] (4) Emulsion droplet dispensing technique There are multiple techniques for dispensing single cells as described above. However, when dispensing emulsion particles in oil, emulsion particles in which droplets settle in the oil can be dispensed in the same way as cells. However, fluorinated oils used in droplet digital PCR or single cell expression analysis have a high specific gravity. Therefore, since emulsion droplets float in the oil, it is 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 by flow cytometry Non-Patent Document 3 describes conventional flow cytometry techniques and analysis methods.

Prior art documents

Patent documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 7

Patent Document 8

Patent Document 6

Patent Document 7

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0011] (1) Problems of Conventional Jet in Air Cell Sorting Technology The sorting speed of a Jet in Air cell sorter is as high as several tens 4 / second. However, the number of cells contained in a droplet must be 1 or less. In the Jet in Air method, droplets formed at high speed in the atmosphere are released. Immediately before the release of the droplets, the droplets containing the target cells are charged, and the direction in which they jump out changes due to an electric field and they are collected. In this method, since the droplets collide with the wall of the collection tube from the atmosphere at high speed, the damage to the cells is large. Therefore, it is necessary to collect cells with sufficient purity in one sorting process. That is, in order to obtain sufficient purity by one treatment, dilution of the cells is necessary. Therefore, as the number of cells increases, the processing time becomes longer. When the sorting speed is 30,000 times / second, the results of obtaining the processing time for a purity of 98% or more after sorting by Poisson distribution analysis are summarized in FIG. 1. From FIG. 1, when the total number of cells is 10 8 cells, the processing time is 30 hours, which is unrealistic. Thus, when the total number of cells is large, there is a problem that dilution is necessary and the processing time becomes long.

[0012] (2) Problems of Conventional Cell Sorting Technology Using Microfluidics In sorting by a microchannel, cells tend to remain in the channel chip after sorting. Therefore, when sorting is repeated, there is a problem that contaminating cells remaining in the channel chip greatly affect the final purity.

[0013] (3) Problems of single-cell dispensing technology In the method of directly dispensing droplets sorted by a Jet in Air type cell sorter into a multi-well plate, in the air, the multi-well plate is moved step and repeat in droplet units and dispensed into different wells. However, when the time between sorting events of target cells is shorter than the movement time of the multi-well plate at the dispensing destination, there is a problem that the target cells are not dispensed. Furthermore, the size of cells that can be dispensed is limited because droplets are formed from the nozzle. Therefore, the size of cells that can be sorted is limited to be equal to or smaller than the size of the droplets to be sorted. Also, in the method of sucking up cells with a pipette from a suspension containing the cells to be dispensed and then dispensing after confirming that only one cell is present by image recognition, there is a problem that the throughput is low because the dispensing time is long.

[0014] (4) Problems of emulsion particle dispensing technology Emulsion droplets in fluorinated oil float because the specific gravity of fluorinated oil is greater than that of water. Moreover, in a resin container, the droplets have the property of moving to the wall surface side of the upper part of the container of the fluorinated oil. Therefore, it is impossible to suck up and then discharge and dispense emulsion droplets one by one with a pipette.

[0015] (5) Problems of emulsion formation method In a method of forming emulsion droplets in a fluorinated oil, the problems of a method of recovering emulsion droplets using a reservoir formed on a microchannel chip are as follows. That is, when the amount of emulsion in the recovery reservoir increases and the liquid level of the fluorinated oil rises, a force in the direction of causing the oil to flow backward, which is derived from the mass of the oil, is generated. Therefore, there is a problem that the flow rate decreases as the liquid level rises.

[0016] (6) Problems in cluster cell analysis by flow cytometry In the current analysis by flow cytometry, a distribution derived from a large number of cell data is assumed. Therefore, in the data of one cell, it is impossible to determine whether the cell is a large cell or a mass of multiple cells, and it is also impossible to make a quantitative judgment. According to Non-Patent Document 3, the cell data is an analysis of the relative cell population position on a two-dimensional scatter plot, and there is a problem that there is no judgment criterion based on a quantitative numerical threshold. Specifically, a technique is required to quickly identify and count numerically whether several circulating tumor cells (CTCs) in the blood obtained by the above-mentioned repeated sorting are one large cell or CTCs in a cluster of multiple cells. The reason is that 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 high-concentration particles in a short time. Another object of the present invention is to provide a method or apparatus for reliably dispensing single particles. Furthermore, another object of the present invention is to provide a method for reliably dispensing emulsion droplets in fluorinated oil. Furthermore, another object of the present invention is to provide a method or apparatus capable of determining whether a cell analyzed by a flow cytometer is a single cell or a cell cluster.

Means for Solving the Problems

[0017] As a result of intensive research on a method or apparatus for purifying target particles from high-concentration particles in a short time, the present inventors surprisingly found that the target particles can be purified in a short time by repeatedly sorting the high-concentration particles. Further, as a result of intensive research on a method or apparatus for reliably dispensing single particles, the present inventors surprisingly found that by fractionating into a recovery reservoir connected to a flow path for each sorting of one particle and dispensing the particles from the recovery reservoir into another container, single particles can be reliably dispensed. Furthermore, as a result of intensive research on a method for reliably dispensing emulsion droplets in fluorinated oil, the present inventors surprisingly found that by using fluorinated oil and mineral oil, emulsion droplets in fluorinated oil can be reliably dispensed. Furthermore, as a result of intensive research on a method or apparatus for determining whether cells analyzed in a flow cytometer are single cells or cell clusters, the present inventors surprisingly found that by obtaining the ratio of the forward scattered light signal intensity to the scattered light signal intensity other than forward, single cells and cell clusters can be easily discriminated. The present invention is based on such findings. Accordingly, the present invention provides [1] A method for purifying target particles, comprising a step of sorting the target particles from among high-concentration non-target particles, wherein the sorting step is repeated three or more times. A method for purifying particles, characterized in that [2] A method for purifying target particles, wherein in the step of sorting the target particles from among high-concentration non-target particles, repeated sorting is performed under the condition that the concentration of the first non-target particles is 10 8 particles / mL or more. A method for purifying particles, characterized in that [3] The method for purifying particles according to [1] or [2], wherein the total number of particles including the first target particles is 10 8 particles or more. [4] The method for purifying particles according to any one of [1] to [3], wherein the particles are cells. [5] The method for purifying particles according to any one of [1] to [4], wherein the target particles are fluorescently stained, and after one or more sorting steps, non-target particles are fluorescently stained, and then the subsequent sorting step is performed. [6] The method for purifying particles according to any one of [1] to [5], further including a sorting step for collecting one particle. [7] A method for dispensing single particles, characterized by including a step of sorting one particle into a recovery reservoir connected to a flow path for each sorting of one particle, and then dispensing the particles from the recovery reservoir into another container. [8] An apparatus for purifying target particles capable of repeatedly sorting the target particles. The apparatus includes a flow path chip for separating particles contained in a sample liquid. In the flow path chip, a flow path is 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 that are in fluid connection with the flow path are formed. The flow of the liquid in the flow path is controlled by the air pressure above each reservoir. The flow path chip has a confluence flow path where an introduction flow path from the sample liquid reservoir and a pair of sheath liquid introduction flow paths arranged on both sides thereof converge. Downstream of the confluence flow path, there is a light irradiation region for detecting particles. Further downstream, there are a pair of opposing branch flow paths connected from the side of the confluence flow path. A sorting reservoir is connected to one of the pair of branch flow paths, and a recovery reservoir is connected to the other branch flow path. The 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 movable laterally during repeated sorting, and the liquid movement between each reservoir, the liquid movement from each reservoir to the outside, and the liquid addition from the outside to each reservoir are configured to be performed from above the reservoir. The apparatus for purifying target particles is characterized by this. [9]An apparatus for dispensing a single target particle capable of sorting the target particles, the apparatus including a flow channel chip for separating the particles contained in a sample liquid, the flow channel chip having flow channels 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 formed in fluid connection with the flow channels, and the flow of the liquid in the flow channels being controlled by the air pressure above each reservoir. The flow channel chip has a merging channel where an introduction channel from the sample liquid reservoir and a pair of sheath liquid introduction channels arranged on both sides thereof merge. Downstream of the merging channel, there is a light irradiation region for detecting particles. Further downstream, there is a pair of opposing branch channels connected from the side of the merging channel. A sorting reservoir is connected to one of the pair of branch channels, and a recovery reservoir is connected to the other branch channel. The upper part of the recovery reservoir can be released to atmospheric pressure. After sorting a single target particle into the recovery reservoir, sorting is stopped, and the apparatus includes a configuration for dispensing a single target particle from the recovery reservoir into another container.

[10] In the method for dispensing a single particle, the particle is an emulsion droplet in a fluorinated oil, the recovery reservoir precontains a fluorinated oil and a mineral oil, the target emulsion droplet is fractionated based on a fluorescence signal, the emulsion droplet is taken into the recovery reservoir in units of one, and then floated from the bottom surface of the recovery reservoir, and the emulsion droplet is trapped at the dome-shaped interface between the fluorinated oil and the mineral oil in the recovery reservoir, and the emulsion droplet is sucked up from above and dispensed into an external container. The method for dispensing a single particle according to [7].

[11] A method for analyzing data of a flow cytometer, characterized by obtaining a ratio of the forward scattered light signal intensity detected by each cell to the scattered light signal intensity other than the forward direction, and identifying whether each cell is a single cell or a cell cluster based on the numerical value.

[12] The target cell is a circulating tumor cell in blood, the circulating tumor cell in blood is fluorescently stained, and the circulating tumor cell in a cluster or as a single cell is analyzed in combination with a fluorescence signal. The cluster analysis method according to

[11] , and

[13] A flow cytometer device, a cell cluster analysis device that obtains the ratio of scattered light signals in a plurality of directions and uses the numerical value to identify whether it is a single cell or a cell cluster, relates to.

Effect of the Invention

[0018] According to the particle purification method and apparatus of the present invention, the problems of the conventional Jet in Air type cell sorting technology can be solved. That is, it is possible to purify target particles from high-concentration particles in a short time. For example, it is possible to separate target cells from 10 8 non-target cells within several hours. Further, according to another aspect, the problems of cell sorting technology in a conventional microchannel can be solved. That is, in an automatic operation when performing repeated sorting, removal and washing of cells remaining in the flow channel chip can be incorporated any number of times. According to the single particle dispensing method and apparatus of the present invention, the problems of single cell dispensing technology can be solved. That is, according to the single particle dispensing method and apparatus of the present invention, single particles can be reliably dispensed. Specifically, when the time between sorting events of target cells is shorter than the moving time of the multi-well plate at the dispensing destination, the problem that the target cells are not dispensed can be solved. According to the single particle dispensing method and apparatus of the present invention, the problems of the dispensing technology of emulsion particles in fluorine-based oil can be solved. That is, emulsion droplets in fluorine oil can be reliably dispensed. Specifically, since the specific gravity of fluorine-based oil is greater than that of water, the droplets float, and it is difficult to suck them up with a pipette due to the adsorption phenomenon on the resin wall surface after floating. Further, when the amount of emulsion in the recovery reservoir increases and the liquid level of the fluorine-based oil rises, the problem that a force in the direction of backflow of the oil derived from the mass of the oil is generated can be solved. According to the data analysis method of the present invention, the problems of cluster cell analysis in flow cytometry can be solved. That is, for each detected individual cell or cell mass, it is possible to determine whether the cell is a large single cell or a mass of multiple cells by converting it back to a quantitative value.

Brief Description of the Drawings

[0019]

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[0020] [1] Method for purifying particles The method for purifying particles of the present invention is a method for purifying target particles. The method 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, the "particles" may be emulsion droplets. The emulsion droplets may contain cells. Also, the target particles are the particles to be purified, and the non-target particles are the particles that are different from the target particles and are removed by sorting.

[0021] 《Sorting Step》 The sorting step may be performed three or more times, and the number of times is not limited. Preferably, it is three or more times, more preferably four or more times. The upper limit is not particularly limited, but it is six or less times, more preferably five or less times. The number of times required for repeated sorting varies depending on 1) the number of non-target cells in the initial sample solution, 2) the number of target cells, 3) the residual rate of non-target cells after sorting, 4) the recovery rate of target cells after sorting, and 5) the purity of the final target cells. For example, if the number of non-target cells is 10 8 cells, 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 times is calculated to be 4 times. When the number of target cells is 10, the required number of times is 5 times.

[0022] In the method for purifying the particles of the present invention, the concentration of the first non-target particles (or the concentration of the total particles) is not particularly limited. For example, it is 10 6 cells / mL or more, preferably 10 7 cells / mL or more, more preferably 2x10 7 cells / mL or more, still more preferably 5x10 7 cells / mL or more, still more preferably 7x10 7 cells / mL or more, still more preferably 1x10 8 cells / mL or more, still more preferably 2x10 8 cells / mL or more. In the sorting step, since non-target particles are removed each time it is repeated, the concentration of non-target particles (or the concentration of total particles) decreases.

[0023] The total number of particles in the method for purifying particles of the present invention is not particularly limited. For example, it is 10 6 or more, preferably 10 7 or more, more preferably 2×10 7 or more, still more preferably 5×10 7 or more, still more preferably 7×10 7 or more, still more preferably 1×10 8 or more, still more preferably 2×10 8 or more.

[0024] In the method for purifying particles of the present invention, although not limited, the target particles are fluorescently stained. After one or more sorting steps, non-target particles can be fluorescently stained, and subsequent sorting steps can be performed. Before the sorting step, the target particles and non-target particles may be specifically stained by fluorescent staining for sorting. However, only the target particles can be fluorescently stained and the sorting step can be performed. In the purification method of the present invention, relatively high-concentration non-target particles are used. Therefore, when fluorescently staining non-target particles before the first sorting, a large amount of antibody needs to be used. However, if non-target particles are fluorescently stained after one or more sorting steps, since the number of non-target particles has decreased, the amount of antibody used for fluorescently staining non-target particles can be reduced.

[0025] The method for purifying particles of the present invention, although not limited, can further include a sorting step for separating one particle. "Separation of one particle" can be carried out according to the "single particle dispensing method" described below.

[0026] Using numerical simulation results, it is explained that the problems in the Jet in Air type cell sorter can be solved by the method for purifying particles of the present invention. The processing time of the method of the present invention for repeatedly sorting a sample solution containing high-concentration cells until the required purity, and the processing time for diluting a sample solution containing high-concentration cells to a low concentration in which only one cell is contained in the droplet to be sorted and achieving the required purity in a single sorting process were compared by numerical simulation. FIG. 2 shows the comparison result of the processing times between Jet in Air sorting (JS) and the repeated sorting (RS) of the present invention. The processing time until obtaining a purity of 98% or more was determined by Poisson distribution analysis as in FIG. 1. The speed of Jet in Air sorting is 30,000 cells / second, while the repeated sorting of the present invention is 1,000 cells / second. When the number of unwanted cells is 10 8 pieces, the processing time of Jet in Air sorting becomes 30 hours due to the increase in the volume of the sample solution by dilution. On the other hand, the processing time of repeated sorting ends in a short time because there is no need to dilute the sample solution and the volume is small. In a method for shortening the processing time of sorting for collecting target cells when the number of unwanted cells is very large, it is not necessary to collect a single target cell in a single sorting, and even under cell concentration conditions where a plurality of unwanted cells are also accidentally collected, the target cells can be purified to the required purity by repeating the sorting.

[0027] The target particle purification device of the present invention is a device for purifying target particles that enables repeated sorting of the target particles. The target particle purification device includes a flow channel chip for separating particles contained in a sample liquid. In the flow channel chip, flow channels are 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 that are in fluid connection with the flow channels are formed. The flow of the liquid in the flow channel is controlled by the air pressure above each reservoir. The flow 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 thereof converge. Downstream of the confluence channel, there is a light irradiation region for detecting particles. Further downstream, there are a pair of opposing branch channels connected from the side of the confluence channel. A sorting reservoir is connected to one of the pair of branch channels, and a recovery reservoir is connected to the other branch channel. The upper part of the recovery reservoir can be released to atmospheric pressure, and the sample liquid reservoir can be released to atmospheric pressure. During repeated sorting, the flow channel chip is movable laterally and is configured to perform liquid transfer between each reservoir, liquid transfer from each reservoir to the outside, and liquid addition from the outside to each reservoir from above the reservoir.

[0028] The means for the repeated sorting will be specifically described. A replacement type flow path chip in which a flow path is formed in a flat substrate, a light irradiation means for irradiating light on particles in a sample liquid flowing through the flow path, a scattered light or fluorescence generated from the particles when the light is irradiated, and detecting and identifying the particles based on their signal intensities, and a detection means for detecting target particles, and as means for applying a pressure pulse to the particles in the sample liquid flowing through the flow path of the flow path chip, a constant pressure air pump (such as an electro-pneumatic regulator or a cylinder pump) and an electromagnetic valve connected thereto, and a control unit for controlling the operation of the electromagnetic valve based on a signal from the detection means are used. In the flow path chip, a sample liquid reservoir is formed, and in the gas space above the reservoir, a positive pressure constant pressure air pump for controlling the flow rate of the sample liquid is airtightly connected via an adapter for the sample liquid reservoir. A flow path for introducing the sample liquid is connected to the bottom surface of the sample liquid reservoir. The replacement type flow path chip includes a pair of sheath liquid introduction flow paths arranged on both sides of the introduction flow path, and a confluence flow path in which the pair of sheath liquid introduction flow paths merge into the introduction flow path of the sample liquid, and a sheath liquid flows on both sides sandwiching the sample liquid in the confluence flow path, a light irradiation region on the confluence flow path, and a pair of opposing branch flow paths connected to the confluence flow path downstream of the light irradiation region. An electromagnetic valve in a normally closed state and a positive pressure constant pressure air pump are airtightly connected to one of the pair of opposing branch flow paths to a sorting reservoir via an adapter for the sorting reservoir. The other side of the branch flow path is connected to a recovery reservoir. However, the space above the recovery reservoir can be released to atmospheric pressure. A waste liquid reservoir is connected downstream of the confluence flow path. In the gas space above the waste liquid reservoir, a negative pressure constant pressure air pump having a pressure lower than atmospheric pressure is airtightly connected through a tube via an adapter connecting the waste liquid reservoir and the tube.

[0029] FIG. 3(A) shows a flow channel chip having reservoirs such as a sample liquid reservoir (1), a recovery reservoir (2), a waste liquid reservoir (3), and a sheath liquid reservoir (4). By applying gas pressure to the upper space of the sample liquid in the sample liquid reservoir for a certain period of time, the sample liquid is pushed out from the sample liquid introduction flow channel (7) connected to the bottom. Similarly, by applying gas pressure to the upper space of the sheath liquid in the sheath liquid reservoir for a certain period of time, the sheath liquid is pushed out. The combined flow flows through the main flow channel (9). In the middle of the main flow channel, it passes through the light irradiation region (detection region). When particles pass through the light irradiation region, the generated optical signal is detected, and the control unit determines whether the particles are the target particles to be separated based on the optical signal from the detection means. When it is determined that the particles are the target particles (5) to be separated, after a delay time until the particles reach the region where they intersect with the branch flow channel downstream of the main flow channel, a signal is given to the electromagnetic valve to keep it open for only a short time. As a result, a pulse flow is generated for only a short time, and the target particles are taken into the recovery reservoir (2) through the recovery flow channel (10). Particles other than the target particles flow straight through the main flow channel (9) and flow into the waste liquid reservoir (3) downstream of the flow channel because no pulse flow is generated. The upper gas space of the waste liquid reservoir is airtightly connected to a constant negative pressure air pump via a tube and is adjusted to be lower than the atmospheric pressure.

[0030] It is explained that repeating sorting can solve the problems of the Jet in Air method. In the first sorting, after putting the sample liquid into the sample liquid reservoir and processing the entire amount, most of the target particles in the sample liquid are recovered into the recovery reservoir. However, in the recovery reservoir, non-target particles that accidentally received a pulse flow along with the target particles are also recovered. Therefore, since the purity is insufficient, sorting is performed again. Thus, the entire particle liquid in the recovery reservoir is recovered, returned to the upstream sample liquid reservoir, and the second sorting is performed. Before returning the liquid in the recovery reservoir to the sample liquid reservoir, something is necessary. The particles remaining at the bottom of the sample liquid reservoir have a very high proportion of non-target particles before the sorting process. Therefore, it is important to wash and remove the remaining particles before returning the recovered liquid. The second sorting is performed according to this procedure. It is also important to perform the third sorting after washing the particles remaining at the bottom of the sample liquid reservoir. Repeat until the desired purity is reached. Figure 3 illustrates how the target cells are gradually concentrated from non-target cells by repeating sorting. Figure 3(C) is the enrichment data of less than 100 PC-9 cells intentionally mixed in 10 8 white blood cells. The number of white blood cells decreases with the number of sortings, but the number of target cells is constant, and the purity of PC9 cells increases by about 100 times for each sorting (Figure 3(C)). This processing time is within 1 hour, the number of target cells in Figure 2 is 100, and the number of non-target cells is 10 8It can be seen that the values in each column are close to the actual values. The data in Fig. 4(A) shows the dependence of the purity (= number of PC-9 cells / (number of PC-9 cells + number of white blood cells)) on the number of repeated sorting operations when the number of spikes of PC-9 is 10 in the data of Fig. 3. It shows both the case of replacing the flow channel chip and the case of continuously using the same chip. When the chip is replaced, the purity reaches 90% after two sorts, but with the same chip, it is only about 40% even after the third sort. The reason for this is that the remaining number of white blood cells in the chip deteriorates the purity. Fig. 4(B) shows the result of simulating the final purity when the remaining rate of white blood cells in the chip is changed. When the remaining rate is zero, it is reproduced that the purity reaches 90% at the second time corresponding to the case of replacing the flow channel chip. When the same chip is used, the case with a remaining rate of 0.7% corresponds to a purity of 40% at the third sorting operation. This remaining rate of 40% is the result obtained by cleaning the bottom of the sample liquid reservoir, and a cleaning operation is preferred for repeated sorting. Also, when the sample liquid is a specimen from a patient, it is necessary to be able to replace the flow channel chip with a new one to avoid cross-contamination between patients.

[0031] Next, means for automating repeated sorting will be described. Specifically, the necessary technologies and means for solving the problems of the cell sorter technology in a conventional microchannel will be described below. As shown by the data in Fig. 4(B), a mechanism for reducing the number of remaining particles in the channel by cleaning for each sorting process is preferred. Therefore, in automating repeated sorting, a mechanism for introducing a cleaning liquid into the microchannel chip and cleaning the remaining particles in the channel is preferred. The technology described in Patent Document 1 does not include this mechanism. In the invention of Patent Document 2, due to the chip structure, it is not possible to perform the required number of repeated sorting operations. Therefore, means including the required number of cleaning steps will be described below. Using a dispensing head having a liquid suction mechanism and a discharge mechanism, the following processing means are implemented. 1) After sorting, set the pressure in each reservoir in the flow channel chip to atmospheric pressure. 2) Move the flow channel chip horizontally to make the dispensing head accessible to the reservoir. 3) Wash the residual particles in the sample liquid reservoir. 4) Aspirate the liquid containing particles in the recovery reservoir with the pipette of the dispensing head. 5) Dispense the liquid in the pipette into the sample liquid reservoir. 6) Return the position of the flow path chip to its original position in the horizontal direction and restore the airtight connection between the flow path chip and the external air pressure control system. In the above step 1), since the flow of the liquid inside the flow path chip is controlled by air pressure from outside the flow path chip, the inside of the flow path chip must be opened to atmospheric pressure for each sorting process. Methods for automatically releasing the atmospheric pressure are shown in FIGS. 5(A) and (B). In the air pressure control of the sample liquid reservoir, sheath liquid reservoir, and waste liquid reservoir inside the flow path chip, an airtight connection using a deformable material rubber (12) is made via a flow path chip adapter (14) and a pressure control unit (15) on the device side, and it is connected to an air pressure control system including a constant pressure air pump installed on the device side. The release of the atmospheric pressure inside the flow path chip is performed by moving to the upper part of the pressure control unit. The above step 2) is an operation of moving the flow path chip in the horizontal direction so that the dispensing head can access the reservoir of the flow path chip, as shown in FIG. 6. In step 3), a cleaning liquid is added to the sample liquid reservoir by the dispensing head, and the cleaning liquid is discarded after pipetting. The cleaning by the procedures of adding the cleaning liquid, pipetting, and discarding is repeated a predetermined number of times. In step 4), after cleaning, the recovered liquid in the recovery reservoir is aspirated and dispensed into the sample liquid reservoir to transfer the particles. Step 6) is an operation of restoring the airtight connection between the sample liquid reservoir, waste liquid reservoir, sheath liquid reservoir, and the air pressure control system on the device side in preparation for the next sorting.

[0032] [2] Single particle dispensing method The single-particle dispensing method of the present invention includes a step of sorting one particle into a recovery reservoir connected to a flow path and then dispensing the particle from the recovery reservoir into another container. Specifically, after sorting one particle, the sorting is temporarily stopped, and the single particle recovered in the recovery reservoir is dispensed into another container (for example, a well of a multi-well plate). Then, the sorting is started, one particle is sorted, and collected in the recovery reservoir. By repeating these operations, one particle can be reliably dispensed into one container (for example, one well). The number of repetitions of the above steps is not particularly limited and can be adjusted according to the number of particles.

[0033] The target particle dispensing device of the present invention is a device for singly dispensing target particles capable of sorting the target particles, and the device includes a flow path chip for separating the particles contained in the sample liquid. In the flow path chip, flow paths are 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 that are in fluid connection with the flow paths are formed, and the flow of the liquid in the flow path is controlled by the air pressure above each reservoir. The flow path chip has a confluence flow path where an introduction flow path from the sample liquid reservoir and a pair of sheath liquid introduction flow paths arranged on both sides thereof converge. Downstream of the confluence flow path, there is a light irradiation region for detecting particles. Further downstream, there are a pair of opposing branch flow paths connected from the side of the confluence flow path. A sorting reservoir is connected to one of the pair of branch flow paths, and a recovery reservoir is connected to the other branch flow path. The upper part 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 the recovery reservoir, the sorting is stopped, and one target particle is dispensed from the recovery reservoir into another container.

[0034] The single-particle dispensing method of the present invention is, for example, a means for performing dispensing for each sorting of single cells. Non-target cell 10 8When, among about several hundreds, about several tens of specific target cells are included, means for dispensing the target cells into, for example, a multi-well plate will be described. Although not limited, for example, when a cell suspension in which the target cells are purified to about 98% purity is stored in a recovery reservoir by repeated sorting in the above-described method for purifying the particles, a method for dispensing the target cells will be described. As shown in FIG. 7, the cell suspension in the recovery reservoir is returned to the sample liquid reservoir, and the cells are sorted. As shown in FIG. 8(A), for dispensing, the liquid is moved from the recovery reservoir, which is always open to atmospheric pressure, to each well in the multi-well plate using a dispensing head. For example, when dispensing one cell, after sorting one cell in one pulse flow, the pressure of the sample liquid reservoir is decreased to stop the flow of the sample liquid. Then, one cell is dispensed from the recovery reservoir to each well of the multi-well plate by the pipette of the dispensing head. By these operations, it becomes possible to dispense the target cells unit by unit, and it becomes possible to prevent the loss of dispensing in which the target cells pass through the sorting region during the dispensing operation. For example, when dispensing 10 cells into each well, after sorting 10 times corresponding to 10 cells, the flow is stopped. Then, 10 cells are dispensed from the recovery reservoir to each well of the multi-well plate by the pipette of the dispensing head. Then, sorting is restarted before the dispensing head returns to the recovery reservoir. By dispensing all the cells in the recovery reservoir after a total of 10 times of sorting, it is possible to dispense the cells in units of 10. The number of dispensed cells to each well can be set as appropriate, and dispensing can be carried out according to the flowchart shown in FIG. 8(B). This flowchart does not illustrate the operation for preventing the outflow loss of the sample liquid for each number of dispensing times. However, when the detection time interval of the target cells is shorter than the dispensing operation, an outflow loss of the sample liquid occurs. That is, for example, since the time required for this dispensing operation is about 5 seconds, loss occurs when the target cells are detected at intervals shorter than this time. Therefore, it is desirable to set the target cell concentration to a concentration such that at least less than 1 cell flows per 5 seconds. FIG. 9 is a flowchart of the operation in such a case.

[0035] "Dispensing of Emulsion Droplets" In one embodiment of the method for dispensing single particles of the present invention, the particles are emulsion droplets in a fluorinated oil, and the recovery reservoir is pre-filled with fluorinated oil and mineral oil. The target emulsion droplets are sorted based on a fluorescence signal, the emulsion droplets are taken into the recovery reservoir one by one, and then lifted from the bottom surface of the recovery reservoir. The emulsion droplets are trapped at the dome-shaped interface between the fluorinated oil and the mineral oil in the recovery reservoir, and the emulsion droplets are sucked up from above and dispensed into an external container.

[0036] The fluorinated oil used in the dispensing method of the present invention is not particularly limited. For example, among commercially available products, Novec 7500 of Fluorinert oil manufactured by 3M can be mentioned. The commercially available product of the mineral oil used in the dispensing method of the present invention is not particularly limited, but the mineral oil manufactured by Sigma-Aldrich can be mentioned. The amount of fluorinated oil contained in the recovery reservoir is not particularly limited, but is 10 μL to 1 mL. The amount of mineral oil contained in the recovery reservoir is not particularly limited, but is 10 μL to 1 mL.

[0037] Regarding the emulsion droplets in the fluorinated oil, since the specific gravity of the fluorinated oil is greater than that of water, the emulsion droplets float in the fluorinated oil. And on the surface of the fluorinated oil, as shown in the photograph of Fig. 11(A), they adsorb to the wall surface of the plastic resin. Therefore, it is difficult to suck up the emulsion droplets one by one with a pipette from above the fluorinated oil. As a method for solving this problem, as shown in Fig. 11(B), mineral oil is used as a cover for the fluorinated oil. 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 upper part of the dome shape. In the photograph of Fig. 11(B), since the emulsion droplets contain a dye, it can be seen that the emulsion droplets are distributed in a dome shape. Therefore, by pre - filling the recovery reservoir with fluorinated oil and mineral oil, the sorted emulsion droplets are trapped at the central position of the reservoir. Therefore, it becomes easy to suck up the emulsion droplets from the recovery reservoir with a pipette. Hereinafter, it will be described with reference to FIG. 10.

[0038] When forming emulsion droplets in oil, the formed droplets do not necessarily contain cells. That is, the emulsion droplets with one or fewer cells are about 1 / 10 of the total number of emulsions according to the Poisson distribution. Therefore, in order to select the emulsion droplets containing cells, sorting of the emulsion droplets is necessary. The selection of the emulsion containing cells is performed based on signals such as the side - scattered light signal and the autofluorescence signal of the cells generated when passing through the laser light irradiation region (20). In this sorting, fluorinated oil is used as the sheath flow. When the emulsion droplet containing cells reaches the sorting region, a pulsed flow is generated. Then, the emulsion droplet is taken into the recovery flow path, and only the emulsion droplets containing cells are accumulated in the recovery reservoir. As described above, since the proportion of cells is about 1 / 10, the purity becomes about 98% by one - time sorting. After sorting the emulsion droplets in units of one, the emulsion droplets are trapped at the upper - central part of the domed interface at the top of the recovery reservoir. Therefore, it becomes easy to suck up the emulsion droplets with a pipette and dispense them into a multi - well plate.

[0039] Next, in order to demonstrate that it is possible to dispense a single unit of the emulsion droplets, the following operations were performed. A water-soluble fluorescent reagent (FITC) was added to the liquid forming the emulsion droplets so that the formed droplets could be identified by fluorescence. After sorting one fluorescent emulsion droplet, one emulsion droplet before dispensing in the recovery reservoir was observed. Also, after dispensing the emulsion droplets into the wells of a 384-well plate using a pipette, observation was carried out. The results are shown in Fig. 12. Fig. 12(A) is a micrograph showing an emulsion droplet (diameter 40 μm) trapped inside the upper part of the domed interface between the fluorinated oil and the mineral oil from the bottom of the recovery reservoir before dispensing. Fig. 12(B) is a micrograph of an emulsion droplet that was sucked up by the pipette of the dispensing head and dispensed into the well in the 384-well plate together with the mineral oil and the fluorinated oil. In the mineral oil, a small amount of the fluorinated oil forms a sphere. An emulsion droplet is observed inside the sphere, indicating that it can be dispensed in single units.

[0040] The structure of the flow channel chip for forming emulsion droplets in the fluorinated oil will be described below. With the following structure, the problem can be solved. Specifically, a structure without a recovery reservoir is sufficient. As shown in Fig. 13, an oil reservoir and a sample liquid reservoir are formed in the flow channel chip, and emulsion droplets are formed in the intersection region of the oil flow channel and the sample liquid flow channel. Further downstream of the flow channel, the emulsion is recovered by a tube connected downward. In the structure shown in Fig. 14, an oil reservoir and a sample liquid reservoir are formed in the flow channel chip. Emulsion droplets are formed in the intersection region of the oil flow channel and the sample liquid flow channel, and the emulsion is recovered by a tube connected horizontally. Fig. 15(A) is a photograph of the droplet formation flow channel region when emulsion droplets are formed using the emulsion formation flow channel chip with the vertical tube recovery method of Fig. 13. Fig. 15(B) is a photograph of the wide flow channel region downstream of this droplet formation flow 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 obtains the ratio of the forward scattered light signal intensity detected in each individual cell to the scattered light signal intensity other than the forward direction, and based on the numerical value, identifies whether each individual cell is a single cell or a cell cluster. The scattered light signal intensity other than the forward direction is not particularly limited, but is preferably the side scattered signal intensity or the back scattered signal intensity.

[0042] The cell cluster analysis apparatus of the present invention is a flow cytometer apparatus, which obtains the ratio of scattered light signals in a plurality of directions and uses the numerical value to identify whether it is a single cell or a cell cluster.

[0043] The cell cluster analysis method of the present invention is a means for quantitatively analyzing a single unit of cell cluster. The forward scattered light signal (FSC) is the intensity of the low-angle scattered light component in the light irradiation direction and mainly reflects the scatterer size. In contrast, the side scattered signal component (SSC) is the intensity of the high-angle scattered light component from the light irradiation direction and is said to mainly reflect the fine structure in the scatterer. This is the same result based on the Mie light scattering theory. Therefore, a method of identifying a single cell and a cell cluster based on the numerical value of the ratio of the signal intensities of FSC and SSC will be described below using actual data. FIG. 17(A) is a histogram distribution of the SSC / FSC values of the cell line PC-9. FIG. 17(B) is a granulocyte of the white blood cell component, and FIG. 17(C) is a histogram distribution of the SSC / FSC values of the white blood cell cluster. PC-9 is a single cell, and granulocytes are single cells having a fine structure inside the cell. The white blood cell cluster is a component confirmed to be a cluster composed of a plurality of cells according to the nuclear staining amount. The above three types of cell states are distinguished by the flow cytometry data of the mixture of white blood cells, white blood cell clusters, and cell line PC-9 cells in FIG. 16. The horizontal axis of FIG. 16 is hematoxylin staining, and the vertical axis is cytokeratin staining. PC9, an epithelial cell, is distributed in the portion enclosed by the gate marked P25, and has a high cytokeratin expression level. In contrast, granulocytes are distributed in the area enclosed by the gate marked P23, have a large nuclear staining amount among leukocyte components, and are characterized by weak positive cytokeratin. In contrast, the leukocyte cluster is distributed in the area enclosed by the gate marked P26, and its characteristic is a distribution in which the nuclear staining amount and the cytokeratin staining amount are correlated. This is considered to be a granulocyte cluster, but since neutrophils are also one of the granulocyte components, the counting of this leukocyte cluster of P26 is important. After CTC sorting, it is important to determine whether there is something determined to be this leukocyte cluster. According to the histogram distributions of FIGS. 17(A), (B), and (C), the discrimination of the leukocyte cluster is in the distribution from about 0.2 to 1, but since it overlaps with granulocytes from 0.2 to 0.3, it can be said that it is desirable to confirm by microscopic observation. In the case of CTC sorting, it can be said that discrimination is easy by combining conditions with other fluorescent stains, but generally, a technique for discriminating only by scattered light signals is important. FIG. 18 shows the flow cytometry data of a sample in which cytokeratin-positive cells are actually sorted and concentrated from the blood of cancer patients, in a two-dimensional scatter plot of the cytokeratin fluorescence signal intensity and the SSC / FSC value. From this graph, cytokeratin-positive cells forming clusters can be discriminated. That is, the CTC cluster can be counted based on the number of data in the quantitative numerical range of the SSC / FSC value and the cytokeratin signal value.

Example

[0044] Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

[0045] 《Example 1》 In this example, circulating tumor cells (CTCs) in the blood of cancer patients were sorted by repeated sorting. 8 mL of blood was collected from a cancer patient and placed in a blood storage tube. The following pretreatment was performed within 2 days. In 8 mL of blood, the white blood cell count was about 108 It contains about that many. The containers used in the following pretreatment were washed and coated with 0.5% BSA / On-chip T-buffer in advance. This is to prevent cell loss due to cell adsorption to resin containers. As the buffer reagents used in the pretreatment, commercially available reagents such as red blood cell lysis buffer, fixing reagent, cell permeabilization reagent, and FCR blocker were used. These protocols followed the attached documents of the reagents.

[0046] Pretreatment (I) The collected blood was processed in the order of hemolysis, removal and washing of red blood cell fragments by centrifugation, fixation, permeabilization treatment, and fluorescence staining (only cytokeratin antibody labeling). By these treatments, red blood cells were removed from the sample solution, and it is considered that several or more CTCs are contained in 10 8 white blood cells. Since the sample solution before treatment was adjusted to 0.3 mL, the cell concentration is about 3.3x10 8 cells / mL.

[0047] CTC sorting treatment (I) Only CTCs derived from cancer cells that are epithelial cells are cytokeratin positive, and it is considered that granulocytes in the white blood cell component are weakly cytokeratin positive. Therefore, as the condition for identifying and sorting CTCs, the cytokeratin positive signal level was set to be equal to or higher than the cytokeratin fluorescence signal level of granulocytes, and cytokeratin positive cells were sorted. When sorting was repeated twice under this condition, the number of white blood cells decreased to about 10 3 cells. The amount of decrease in the number of white blood cells due to repeated sorting was shown in Figure 3. This data is a sample in which the cell line cell PC-9 derived from lung cancer was spiked into 4 mL of blood, and the number of white blood cells and the number of PC-9s for each number of repeated sorting were graphed.

[0048] Pretreatment (II) At the stage of reducing white blood cells, Hoechst nuclear staining, additional cytokeratin fluorescence labeling for staining, CD45 fluorescence antibody labeling, vimentin fluorescence antibody labeling, PD-L1 fluorescence antibody labeling, (or HER2 fluorescence antibody labeling, EGFR fluorescence antibody labeling, AXL fluorescence antibody labeling) were performed. After staining, washing by centrifugation was performed with 0.5% BSA / On-chip T-buffer buffer.

[0049] CTC sorting process (II) For CTC identification, sorting for collecting CTCs was repeated twice under the conditions of Hoechst positive, CD45 negative, and cytokeratin positive. According to the CTC concentration method, in an experiment where a known number of PC-9 cells of a cell line were added to blood, the recovery rate > 70% and the purity > 80%.

Industrial Applicability

[0050] The method for separating a small number of target cells from a cell group with a total cell number of about 10 8 or more, as described in the examples, can be used for the purpose of separating and analyzing CTCs from the blood of cancer patients. In addition, it can be used for antibody screening to select cells that produce antibodies specifically binding to a target antigen from a large number of antibody-producing cells in antibody drug development. Furthermore, the method for dispensing single particles of the present invention, when the particles are emulsion droplets, can be used for the purpose of separating cells that secrete specific substances because the cell secretions do not diffuse but accumulate in the emulsion droplets. Moreover, since the lysate does not diffuse even when cells are lysed in the emulsion droplets, it can be used for cell expression analysis.

Explanation of Signs

[0051] 1 ··· Sample liquid reservoir formed on the flow channel chip; 2 ··· Recovery reservoir formed on the flow channel chip; 3 ··· Waste liquid reservoir formed on the flow channel chip; 4 ··· Sheath liquid reservoir formed on the flow channel chip; 5 ··· Target cells or target particles; 6 ··· Non-target cells or non-target particles; 7 ··· Sample liquid introduction flow path; 8 ··· Sheath flow path; 9 ··· Main flow path; 10 ··· Recovery flow path; 11 ··· Flow path chip; 12 ··· Elastic deformation part for airtight retention (rubber); 13 ··· Reservoir formed in the flow path chip; 14 ··· Flow path chip adapter; 15 ··· Pressure control unit (adapter for airtight connection on the device side); 16 ··· Air piping tube; 17 ··· Pipette of the dispensing head; 18 ··· Constant pressure air pump such as an electro-pneumatic regulator or a cylinder pump; 19 ··· Pipette at the tip of the dispensing head; 20 ··· Light irradiation region (detection region); 21 ··· Fluorine-based oil; 22 ··· Mineral oil; 23 ··· Droplets in fluorine-based oil; 24 ··· Multi-well plate; 25 ··· Sample liquid reservoir; 26 ··· Oil reservoir; 27 ··· Emulsion droplet formation region (intersection region of the oil flow path and the sample liquid flow path); 28 ··· Tube for recovering emulsion droplets; 29 ··· Resin container; 30 ··· Wells in a 384-well plate;

Claims

1. A method for dispensing a single particle, comprising the steps of: collecting particles taken into a flow path by a pulsed flow in individual particles into a collection reservoir connected to the flow path; and dispensing each particle from the collection reservoir to each well of another multiwell plate after sorting each particle.

2. A method for dispensing single particles, the method comprising the steps of: the particles are emulsion droplets in a fluorinated oil; a collection reservoir previously containing fluorinated oil having a higher specific gravity than the droplets; separating the target emulsion droplets based on a fluorescent signal; separating the emulsion droplets one by one into a branched flow path and collecting them in a collection reservoir connected to the flow path; and, for each sorting of a particle, floating the droplets in the oil from the bottom of the collection reservoir and then dispensing them into another container.

3. 3. The method for dispensing a single particle according to claim 2, 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, 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 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, and the emulsion droplets are sucked up from the top and dispensed into an external container.

4. 2. A method for dispensing a single particle as described in claim 1, wherein the process of sorting each particle into a collection reservoir connected to a flow path is started after confirming the completion of the process of dispensing the particles from the collection reservoir into another container, and the order of dispensing follows the order of sorting.

5. A device for dispensing a single target particle capable of sorting the target particle, comprising: The device includes a flow channel chip for separating particles contained in a sample liquid, The flow channel chip has a flow 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 flow channel, and the flow of liquid in the flow channel is controlled by the air pressure above each reservoir, the flow channel chip has a confluence flow channel where an introduction flow channel from the sample liquid reservoir and a pair of sheath liquid introduction flow channels arranged on both sides of the confluence flow channel join together, a light irradiation area for detecting particles is provided downstream of the confluence flow channel, and a pair of opposing branch flow channels connected from the sides of the confluence flow channel are provided downstream thereof, a sorting reservoir is connected to one of the pair of branch flow channels and a recovery reservoir is connected to the other of the branch flow channels, 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.

Images