Patent Application
20220062905
The present invention relates to a method for purifying particles, a method for dispensing a single particle, a method for analyzing cell cluster, and an apparatus using the same.
In order to detect a specific cell in many various cells and analyze gene thereof, it is required to dispense individual particles. The present invention relates to a method for purifying about hundreds of cells out of about 108 total cells, or a dispensing method. In addition, the present invention includes a method for sorting circulating tumor cells flowing in the blood of cancer patients, as an application example. Therefore, the present invention relates to a particle sorting and dispensing technology using a disposable tip wherein contamination of cells between different patients may be prevented. In the present specification, the particle includes a cell and a particle containing a droplet in oil.
Conventional technologies and their problems, regarding cell sorting technologies and dispensing technologies thereafter will be explained.
As disclosed in Non-patent literature 1, in the Jet in Air type, A droplet is formed from the nozzle, and the cells contained in the droplet are separated into individual droplets.
The sorting rate in the sorting by a micro flow path is about 1000/second or less, which is slow. However, Patent literature 1 discloses a technique for improving throughput by parallel processing using a plurality of flow paths, and a technique for returning treated cells to the flow path and treating them again to improve sorting purity.
Patent literature 2 discloses a technique for improving sorting purity by continuously performing each process using a flow path chip having a plurality of sorting portions. Patent literature 2 discloses a method in which non-target cells are removed from a cell cluster using a micro flow-path chip with a reservoir, and the remaining cells are returned to the upstream sample reservoir and sorted.
Patent literature 3 discloses a sorting technique using a pulse flow in a micro flow-path chip with reservoirs. Further, Patent literature 4 discloses a method in which non-target cells are removed by sorting in a micro flow-path chip, the remaining cells are collected from a discharged liquid reservoir, and then returned to an upstream sample reservoir, and the negative sorting is repeated (repeated negative sorting method).
If necessary, the sorted cells may be dispensed individually into a multi-well plate. This technique and problems are described. As a method for selecting the target cells contained in the various cells and dispensing the cells into the multi-well plate, Non-patent literature 2 discloses a method of directly dispensing droplets sorted by an Jet in Air type cell sorter onto a multi-well plate. Patent literature 5 discloses a method of identifying a target cell by image recognition and then dispensing it as a droplet onto a multi-well plate under piezo pressure. This method has the problem that the size of the ejectable droplets is limited when piezo pressure is used. Patent literature 6 discloses a technique in which a cell to be dispensed is sucked up from a suspension with a pipette, the inside of the pipette is photographed, and then when it is recognized that one cell is contained, the cell is dispensed.
As mentioned above, there are multiple techniques for single-cell dispensing. However, when the water in oil droplet in oil is dispensed, the water in oil droplet which is settle in the oil as a droplet can be dispensed in the same way as cells.
However, the fluorocarbon oil used in droplet digital PCR or single-cell expression analysis has a large specific gravity. Therefore, since water in oil droplets float in the oil, it is difficult to dispense the water in oil droplets by dropping them from above. Patent literature 7 discloses a method for dispensing water in oil droplets in a fluorocarbon oil.
Non-patent literature 3 discloses the conventional flow cytometry technique and analysis method.
[Patent literature 1] U.S. Pat. No. 6,976,590 B2
[Patent literature 2] U.S. Pat. No. 8,993,311B2
[Patent literature 3] WO 2012/067259 A1
[Patent literature 4] WO 2016/182034 A1
[Patent literature 5] WO 2011/154042 A1
[Patent literature 7] WO 2018/052137 A1
[Patent literature 8] GB 2566002 A
[Patent literature 6] U.S. Pat. No. 7,268,167 B2
[Patent literature 7] U.S. Pat. No. 9,500,664 B2
[Non-patent literature 1] FACS Aria III pamphlet
[Non-patent literature 2] SONY SH800S pamphlet
[Non-patent literature 3]
https://ls.beckmancoulter.co.jp/files/appli_note/Couter_Practical_Flow_Cytometry.pdf
[Non-patent literature 4] Szczerba, B. M. et al., Nature volume 566, pages 553-557(2019)
The sorting rate in the Jet in Air type cell sorter is about 104/second, which is fast. However, the number of cells contained in the droplet should be 1 or less. In the Jet in Air method, the formed droplet is released at high speed into the atmosphere. Immediately before the droplet is released, the droplet containing a target cell is charged, and the direction of ejection is changed by the electric field, and the droplets are collected. This method causes great cell damage because the droplets collide with the wall surface of the collection tube from the atmosphere at high speed. Therefore, it is necessary to collect cells with sufficient purity in one sorting processing. That is, in order to obtain sufficient purity in one processing, it is necessary to dilute the cells. Therefore, the larger the number of cells, the longer the processing time.
When the sorting rate is 30,000 times/second, the processing time for the purity after sorting to be 98% or more was determined by Poisson distribution analysis. The results are shown in
In the sorting by the micro flow path, cells tend to remain in the flow-path chip after sorting. Therefore, when sorting is repeated, there is a problem that the contaminating cells remaining in the flow-path chip have a great influence on the final purity.
In the method of directly dispensing the droplets sorted by the Jet in Air cell sorter onto the multi-well plate, the multi-well plate is moved step-and-repeat in the atmosphere and each droplet is dispensed into a different well. However, if the time between sorting events of the target cells is shorter than the moving time of the multi-well plate to which the target cells are dispensed, there is a problem that the target cells are not dispensed. Furthermore, the size of the cells to be dispensed is limited because the droplets are formed from the nozzles. That is to say, the size of the cells to be sorted is limited to those smaller than the size of the droplets to be sorted. In addition, in the method of sucking up cells from the suspension containing the cells to be dispensed with a pipette and dispensing after confirming that only one cell exists by image recognition, there is a problem that the throughput is low because the dispensing time is long.
Water in oil droplets in fluorocarbon oil float because fluorocarbon oil has a higher specific density than water. Moreover, in a resin container, the droplets easily move to the wall surface of the upper part of the fluorocarbon oil container. Therefore, it is not possible to individually suck up the water in oil droplets with the pipette and then eject and dispense them.
In the method of forming water in oil droplets in a fluorocarbon oil, the problems of the method of collecting water in oil droplets using the reservoirs formed on the microflow-path chip are as follows. That is, when the amount of emulsion in the collection reservoir increases and the upper surface of the fluorocarbon oil becomes higher, a force in the backflow direction of the oil is generated due to the mass of the oil. Therefore, there is a problem that the flow velocity becomes slower as the upper surface becomes higher.
Current flow cytometric analysis is based on a distribution derived from data of a large number of cells. Therefore, with respect to data of cell(s), it is impossible to determine whether the data indicates a large cell or a mass of multiple cells, and it is also impossible to quantitatively determine it. According to Non-patent literature 3, the cell data is an analysis of the relative position of the cell population on a two-dimensional scatter plot, and thus, there is a problem that it does not have a criterion based on a quantitative numerical threshold. Specifically, a following technique is required. That is, it is rapidly determined whether several circulating tumor cells (CTCs) in the blood obtained by the above-mentioned repeated sorting are one large cell or a CTC in a cluster of multiple cells based on numerical values, and counted. This is because it is reported that CTCs in neutrophil clusters shorten the prognosis of cancer patients (Non-Patent literature 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 a single particle. Further, another object of the present invention is to provide a method for reliably dispensing water in oil droplets in fluorocarbon 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.
The present inventors have conducted intensive studies into a method or apparatus for purifying target particles from high-concentration particles in a short time, and as a result, surprisingly found that the target particles can be purified in a short time by repeatedly sorting high-concentration particles. Further, the present inventors have conducted intensive studies into a method or apparatus for reliably dispensing a single particle, and as a result, surprisingly found that a single particle can be reliably dispensed by sorting each one particle into a collection reservoir connected to a flow path and dispensing the one particle from the collection reservoir to another container.
Further, the present inventors have conducted intensive studies into a method for reliably dispensing water in oil droplets in fluorocarbon oil, and as a result, surprisingly found that the water in oil droplets in fluorocarbon oil can be reliably dispensed by using a fluorocarbon oil and mineral oil. Furthermore, the present inventors have conducted intensive studies into a method or apparatus capable of determining whether a cell analyzed by a flow cytometer is a single cell or a cell cluster, and as a result, surprisingly found that the single cell and the cell cluster can be easily determined by calculating a ratio between a forward scattered light signal intensity and a scattered light signal intensity other than forward.
The present invention is based on the above findings.
Accordingly, the present invention relates to
[1] a method for purifying target particles, characterized in that the method comprises a step of sorting the target particles from a high concentration of non-target particles, wherein the sorting step is repeated for three times or more,
[2] a method for purifying target particles, characterized in that the method comprises a step of sorting the target particles from a high concentration of non-target particles, wherein a primary concentration of non-target particles is 108 particles/mL or more, and the sorting is repeated,
[3] the method for purifying target particles of item [1] or [2], wherein primary numbers of total particles containing the target particles is 108 or more,
[4] the method for purifying target particles of items [1] to [3], wherein the particles are cells,
[5] the method for purifying target particles of items [1] to [4], wherein the target particles are fluorescently-stained, the non-target particles are fluorescently-stained after one or more sorting step, and then the sorting step is carried
[6] the method for purifying target particles of items [1] to [5], further comprising a sorting step for sorting one particle,
[7] a method for dispensing a single particle, characterized in that the method comprises a step of sorting each one particle into a collection reservoir connected to a flow path and dispensing the one particle from the collection reservoir to another container,
[8] an apparatus for purifying target particles, capable of repeatedly sorting target particles, characterized in that the apparatus comprises a flow-path chip for separating particles contained in a sample liquid,
wherein the flow-path chip has flow paths formed in a transparent substrate, and has a sample liquid reservoir, a sheath liquid reservoir, a sorting reservoir, a collection reservoir, and a discharged liquid reservoir which are fluidically-connected to the flow paths, and wherein a liquid flow in the flow paths is controlled by an air pressure above each reservoir,
wherein the flow-path chip has an introduction flow path from the sample liquid reservoir, a confluence flow path in which a pair of sheath liquid introduction flow paths arranged on both sides are joined to the introduction flow path, an illumination region for detecting the particle at downstream of the confluence flow path, and a pair of branched flow paths that are connected to the side of the confluence flow path so as to face each other at further downstream of the confluence flow path, and wherein the sorting reservoir is connected to one of the pair of branched flow paths, the collection reservoir is connected to the other of the branched flow paths, an upper part of the collection reservoir can be opened to atmospheric pressure, and the sample liquid reservoir can be opened to atmospheric pressure, and
wherein the flow-path chip can be moved laterally when sorting is repeated, and liquid movement between each reservoir, liquid movement from each reservoir to the outside, and liquid addition from the outside to each reservoir are configured to perform from the above of the reservoirs,
[9] an apparatus for singly dispensing a target particle, capable of sorting target particles, wherein the apparatus comprises a flow-path chip for separating particles contained in a sample liquid,
wherein the flow-path chip has flow paths formed in a transparent substrate, and has a sample liquid reservoir, a sheath liquid reservoir, a sorting reservoir, a collection reservoir, and a discharged liquid reservoir which are fluidically-connected to the flow paths, and wherein a liquid flow in the flow paths is controlled by an air pressure above each reservoir,
wherein the flow-path chip has an introduction flow path from the sample liquid reservoir, a confluence flow path in which a pair of sheath liquid introduction flow paths arranged on both sides are joined to the introduction flow path, an illumination region for detecting the particle at downstream of the confluence flow path, and a pair of branched flow paths that are connected to the side of the confluence flow path so as to face each other at further downstream of the confluence flow path, and wherein the sorting reservoir is connected to one of the pair of branched flow paths, the collection reservoir is connected to the other of the branched flow paths, and an upper part of the collection reservoir can be opened to atmospheric pressure, and
wherein the apparatus comprises a configuration in which sorting is stopped after sorting one target particle into the collection reservoir and one target particle is dispensed from the collection reservoir into another container,
[10] the method for dispensing a single particle of item [7], wherein the particle is a water in oil droplet in a fluorocarbon oil, the fluorocarbon oil and a mineral oil are contained in the collection reservoir in advance, a target water in oil droplet is sorted based on a fluorescence signal, the water in oil droplet is taken up in the collection reservoir one by one, and the water in oil droplet is trapped at the dome-shaped interface between the fluorocarbon oil and mineral oil in the collection reservoir, and the water in oil droplet is sucked up from up above and dispensed into an external container,
[11] a method for analyzing cell cluster by analyzing flow cytometry data, characterized in that a ratio between a forward scattered light signal intensity and a scattered light signal intensity other than forward detected in each cell is calculated, and by the obtained value of the ratio, whether individual cells are single cells or cell cluster are identified,
[12] the A method for analyzing cell cluster of item [11], wherein the target cells are circulating tumor cells, the circulating tumor cells are fluorescently-stained, and wherein whether the circulating tumor cells are single cells or cell cluster are analyzed by a combination with a fluorescence signal, and
[13] an apparatus for analyzing cell cluster using a flow cytometer, wherein ratios between plural scattered light signals are calculated, and by using values thereof, whether single cell or cell cluster is identified.
According to the method for purifying particles and the apparatus for purifying particles of the present invention, the problems with conventional Jet in Air type cell sorter technology can be solved. That is to say, target particles can be purified from high-concentration particles in a short time. For example, target cells can be purified within a few hours from non-target cells with a total number of 108 cells. Further, according to another embodiment of the present invention, the problems with conventional cell sorting technology in micro flow path can be solved. That is to say, in an automatic operation during repeated sorting, the cells remaining in the flow path chip can be washed and removed as many times as necessary.
According to the method for dispensing a single particle and the apparatus for dispensing a single particle of the present invention, the problems with single cell dispensing technology can be solved. That is to say, according to the method for dispensing a single particle and the apparatus for dispensing a single particle of the present invention, the single cell can be reliably dispensed. Specifically, the problem that the target cells are not dispensed, if the time between sorting events of the target cells is shorter than the moving time of the multi-well plate to which the target cells are dispensed, can be solved.
According to the method for dispensing a single particle and the apparatus for dispensing a single particle of the present invention, the problems with dispensing technique of a water in oil droplet in fluorocarbon oil can be solved. That is to say, the water in oil droplet in fluorocarbon oil can be reliably dispensed. Specifically, the following problems can be solved. The problem is that the droplets float because the fluorocarbon oil has a higher specific density than water, and the floated droplets adsorb on the resin wall surface after floating, and thus it is difficult to suck up with a pipette. Furthermore, when the amount of emulsion in the collection reservoir increases and the upper surface of the fluorocarbon oil becomes higher, the problem that a force in the backflow direction of the oil is generated due to the mass of the oil can be solved.
According to the method for analyzing data of the present invention, the problems with cluster cell analysis technology by flow cytometry can be solved. That is to say, for each detected cell or cell mass, it is possible to determine whether the cell is a large single cell or a mass of multiple cells based on a quantitative numerical value.
The sorting rate of Jet in Air sorting is 30,000/second, and the sorting rate of the repeated sorting of the present invention is 1000/second.
In addition to the lateral movement of the flow path tip, the vertical movement and the horizontal movement of the dispensing pipette is required.
The method for purifying particles of the present invention is for purifying target particles. The method is characterized in that it comprises a step of sorting the target particles from a high concentration of non-target particles, wherein the sorting step is repeated for three times or more.
In the present specification, the term “particle” includes a cell. Further, the “particle” may be a water in oil droplet. Furthermore, the water in oil droplet may contain a cell. In addition, the target particle means a particle to be purified, and the non-target particle means a particle different from the target particle, which is removed by sorting.
The number of the sorting step is not limited, so long as it is carried out three times or more. The number of the sorting step is preferably 3 or more, more preferably 4 or more. The upper limit thereof is not particularly limited, but is 6 or less, more preferably 5 or less.
The required number of the repeated sorting varies depending on 1) the number of non-target cells in the initial sample liquid, 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 final purity of the target cells. For example, the required number of repeated sorting is calculated to be 4 times. when the number of non-target cells is 108, 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 purity of target cells is 98%. Further, when the number of target cells is 10, the required number of repeated sorting is 5 times.
The initial concentration of the non-target particles in the method for purifying particles of the present invention is not particularly limited, but is, for example, 106/mL or more, preferably 107/mL or more, more preferably 2×107/mL or more, further preferably 5×107/mL or more, further preferably 7×107/mL or more, further preferably 1×108/mL or more, further preferably 2×108/mL or more.
In the sorting step, the concentration of the non-target particles (or the concentration of the total particles) decreases because the non-target particles are removed each time the step is repeated.
The total particle numbers in the method for purifying particles of the present invention is not particularly limited, but is, for example, 106 or more, preferably 107 or more, more preferably 2×107 or more, further preferably 5×107 or more, further preferably 7×107 or more, further preferably 1×108 or more, further preferably 2×108 or more.
In the method for purifying particles of the present invention, the target particles are fluorescently-stained, and the non-target particles are fluorescently-stained after one or more sorting step, and then the sorting step can be carried, but is not limited thereto.
The target particles and non-target particles may be specifically stained by the fluorescence staining before the sorting step, and then may be sorted. However, only the target particles are fluorescently-stained and then the sorting step may be carried out. In the purifying method of the present invention, a comparatively high concentration of non-target particles is used. Therefore, when the non-target particles are fluorescently-stained before a first sorting, it is necessary to use a large amount of antibody. However, when the non-target particles are fluorescently-stained after one or more sorting step, the amount of antibody used for fluorescent staining of non-target particles can be reduced since the number of non-target particles is reduced.
The method for purifying particles of the present invention is not limited, but may further comprise a sorting step for sorting one particle. The “sorting one particle” can be performed according to the “the method for dispensing a single particle” described later.
It will be explained by using the results of numerical simulation that the problem with 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, in which the sample liquid containing high-concentration cells is repeatedly sorted to the required purity, and the processing time of the method, in which a sample liquid containing high-concentration cells is diluted to a low concentration sample liquid wherein only one cell is contained in the sorted droplets to obtain the desired purity in one sorting processing, were compared by the numerical simulation.
The apparatus for purifying target particles of the present invention can repeatedly sort target particles. The apparatus for purifying target particles comprises a flow-path chip for separating particles contained in a sample liquid, wherein the flow-path chip has flow paths formed in a transparent substrate, and has a sample liquid reservoir, a sheath liquid reservoir, a sorting reservoir, a collection reservoir, and a discharged liquid reservoir which are fluidically-connected to the flow paths, and wherein a liquid flow in the flow paths is controlled by an air pressure above each reservoir. The flow-path chip has an introduction flow path from the sample liquid reservoir, and a confluence flow path in which a pair of sheath liquid introduction flow paths arranged on both sides are joined to the introduction flow path. It has an illumination region for detecting the particle at downstream of the confluence flow path, and a pair of branched flow paths that are connected to the side of the confluence flow path so as to face each other at further downstream of the confluence flow path. Further, the sorting reservoir is connected to one of the pair of branched flow paths, the collection reservoir is connected to the other of the branched flow paths, an upper part of the collection reservoir can be opened to atmospheric pressure, and the sample liquid reservoir can be opened to atmospheric pressure.
The flow-path chip can be moved laterally when sorting is repeated, and liquid movement between each reservoir, liquid movement from each reservoir to the outside, and liquid addition from the outside to each reservoir are configured to perform from the above of the reservoirs.
A method of the repeated sorting will be specifically explained. An apparatus for separating particles comprising a disposable flow-path chip wherein a flow path is formed in a flat substrate, an illumination unit configured to illuminate particles in a sample liquid flowing through the flow path, a detection unit configured to detect target particles by detecting a scattered light or fluorescence generated from the particle when the particle is illuminated, and identifying the particle based on its signal intensity, a constant air pump (such as an electropneumatic regulator or a cylinder pump) which applies a pressure pulse to the particles in the sample liquid flowing through the flow path in the flow-path chip, and an electromagnetic valve connected thereto, and a control unit configured to control a movement of the electromagnetic valve based on the signal from the detection unit, is used. A sample liquid reservoir is formed in the flow path tip, and a constant positive pressure air pump for controlling the flow rate of the sample liquid is airtightly connected to a gas space above the reservoir via an adapter for the sample liquid reservoir. The introduction flow path for the sample liquid is connected to a bottom of the sample liquid reservoir. The disposable flow-path chip comprises a pair of sheath liquid introduction flow path, arranged on both sides of the introduction flow path for the sample liquid; a confluence flow path for joining the introduction flow path for the sample liquid and the pair of the sheath liquid introduction flow path, in which the sheath liquid flows along either side of the sample liquid in the confluence flow path; an illumination region on the confluence flow path, and a pair of oppositely-branched flow paths connected to the confluence flow path, downstream of the illumination region. In one of the pair of oppositely-branched flow paths, an electromagnetic valve, which is normally closed, and a constant positive pressure pump are airtightly connected to the sorting reservoir via an adapter for the sorting reservoir. A collection reservoir is connected to the other of the branched flow paths. However, an upper space of the collection reservoir can be opened to atmospheric pressure. A discharged liquid reservoir is connected at downstream of the confluence flow path. In the upper gas space of the discharged liquid reservoir, a constant negative pressure air pump with a pressure lower than atmospheric pressure is airtightly connected to the discharged liquid reservoir via an adapter that connects the tubes.
The optical signal generated when passing through the illumination region is detected, and the control unit determines whether or not the particle is a target particle to be separated based on the optical signal from the detecting unit. When it is determined that the target particle (5) should be separated, a signal to open the electromagnetic valve for a short time is provided, after a delay time for the particle to reach the region that intersects the branched flow path at downstream of the main flow path. As a result, a pulse flow is generated for a short time, and the target particles are taken into the collection reservoir (2) through the collection flow path (10). No pulse flow is generated for the non-target particles, and thus the non-target particles go straight through the main flow path (9) and flow into the discharged liquid reservoir (3) at downstream of the flow path. The upper gas space of the discharged liquid reservoir is airtightly connected to a constant negative pressure air pump via a tube, and is adjusted below atmospheric pressure.
It will be explained that the problem with Jet in Air type cell sorter can be solved by repeating sorting. In the first sorting, when the sample solution is placed in the sample liquid reservoir and the entire amount is processed, most of the target particles in the sample liquid are recovered in the collection reservoir. However, in addition to the target particles, the non-target particles that are accidentally pulsed are also recovered in the collection reservoir. Therefore, since the purity thereof is insufficient, the sorting is performed again. That is, the entire particle liquid in the collection reservoir is collected and then returned to the upstream sample liquid reservoir for the second sorting. Before returning the liquid from the collection reservoir to the sample liquid reservoir, the following should be done. A very large proportion of unprocessed non-target particles remain at the bottom of the sample fluid reservoir. Therefore, it is important to return the recovered liquid after removing the remaining particles in the sample liquid reservoir by washing. The second sorting is performed by the above procedure. It is also important that the third sorting is performed after washing the remaining particles on the bottom of the sample liquid reservoir. The sorting is repeated until the desired purity is obtained.
Next, the method for automating the repeated sorting will be explained. Specifically, the necessary technology and method for solving the problems the conventional cell sorting technology in micro flow path, will be explained. As shown in the data of
1) After sorting, each reservoir in the flow path chip opens to atmospheric pressure.
2) The flow path tip is moved laterally to make the dispensing head accessible in the reservoir.
3) Remaining particles in the sample liquid reservoir are washed.
4) The liquid containing the particles in the collection reservoir is sucked up by the pipette of the dispensing head.
5) The liquid in the pipette is discharged into the sample liquid reservoir.
6) The flow path tip is laterally moved to return it to its original position, and the flow path chip and the external air pressure control system are airtightly connected restoratively.
In the above step 1), the flow of the liquid inside the flow path chip is controlled by air pressure from the outside of the flow path chip, and thus the inside of the flow path chip must be opened to atmospheric pressure after each sorting process. The method of automatically opening to atmospheric pressure is shown in
The method for dispensing a single particle of the present invention comprises a step of sorting each one particle into a collection reservoir connected to a flow path and dispensing the one particle from the collection reservoir to another container. Specifically, after sorting one particle, the sorting is temporarily stopped. Then, one particle collected in the collection 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 dispensed into the collection reservoir. By repeating these procedures, one particle can be reliably dispensed into one container (such as one well).
The number of repetitions of the above steps is not particularly limited and can be adjusted according to the number of particles.
The apparatus for dispensing target particles of the present invention can sort the target particles, and singly dispense the target particle. The apparatus comprises a flow-path chip for separating particles contained in a sample liquid. The flow-path chip has flow paths formed in a transparent substrate, and has a sample liquid reservoir, a sheath liquid reservoir, a sorting reservoir, a collection reservoir, and a discharged liquid reservoir which are fluidically-connected to the flow paths. Further, a liquid flow in the flow paths is controlled by an air pressure above each reservoir.
The flow-path chip has an introduction flow path from the sample liquid reservoir, and a confluence flow path in which a pair of sheath liquid introduction flow paths arranged on both sides are joined to the introduction flow path. Further, the flow-path chip has an illumination region for detecting the particle at downstream of the confluence flow path, and a pair of branched flow paths that are connected to the side of the confluence flow path so as to face each other at further downstream of the confluence flow path. The sorting reservoir is connected to one of the pair of branched flow paths, and the collection reservoir is connected to the other of the branched flow paths. Furthermore, an upper part of the collection reservoir can be opened to atmospheric pressure. The apparatus for dispensing target particles of the present invention comprises a configuration in which sorting is stopped after sorting one target particle into the collection reservoir and one target particle is dispensed from the collection reservoir into another container.
In the method for dispensing a single particle of the present invention, for example, a single cell is dispensed with each single cell sorting.
For example, when about hundreds of specific target cells are contained in about 108 of non-target cells, a method for dispensing the target cells into a multi-well plate will be explained. For example, in the case that a cell suspension containing the target cells purified to about 98% purity by repeated sorting of the method for purifying particles is stored in the collection reservoir, a method of dispensing the target cells will be explained, but it is not limited thereto. As shown in
In one embodiment of the method for dispensing a single particle of the present invention, the particle is a water in oil droplet in a fluorocarbon oil, the fluorocarbon oil and a mineral oil are contained in the collection reservoir in advance, a target water in oil droplet is sorted based on a fluorescence signal, the water in oil droplet is taken up in the collection reservoir one by one, and floats from a bottom of the collection reservoir, and the water in oil droplet is trapped at the dome-shaped interface between the fluorocarbon oil and mineral oil in the collection reservoir, and the water in oil droplet is sucked up from up above and dispensed into an external container.
The fluorocarbon oil used in the dispensing method of the present invention, is not particularly limited, but there may be mentioned Novec7500 which is 3M Fluorocarbonrt oil, as a commercial product. The mineral oil used in the dispensing method of the present invention, is not particularly limited, but there may be mentioned Sigma-Aldrich's mineral oil, as a commercial product.
The amount of fluorocarbon 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.
As to the water in oil droplet in the fluorocarbon oil, water in oil droplets float in fluorocarbon oil because fluorocarbon oil has a higher specific density than water. Further, on the surface of the fluorocarbon oil, the water in oil droplets are adsorbed to the wall surface of the plastic resin, as shown in the photograph of
Accordingly, by pre-adding the fluorocarbon oil and the mineral oil to the collection reservoir, the sorted water in oil droplets are trapped at the center of the reservoir. Therefore, the water in oil droplets are easily sucked up from the collection reservoir with the pipette. This will be explained below using
When water in oil droplets are formed in oil, the formed droplets do not always contain a cell. That is, based on the Poisson distribution, there are about 1/10 of the total number of water in oil droplets with one or less cells. Therefore, it is necessary to sort the water in oil droplets in order to separate the water in oil droplets containing a cell. Emulsions containing cells are sorted based on signals such as sideward scattering light signals generated when passing through the laser illumination region (20) and autofluorescence signals of cells.
In this sorting, a fluorocarbon oil is used as a sheath flow. When the water in oil droplet containing the cell reaches the sorting region, a pulse flow is generated. Then, the water in oil droplets are taken into a collection flow path, and only the water in oil droplets containing the cell are accumulated in the collection reservoir. As mentioned above, the droplets containing the cell are about 1/10, and thus a single sorting gives about 98% purity. Then, after sorting the water in oil droplets one by one, the water in oil droplets are trapped in the upper center of the dome-shaped interface at the top of the collection reservoir. Therefore, the water in oil droplets can be easily sucked up with a pipette and dispensed into a multi-well plate.
Next, in order to demonstrate that it is possible to dispense the water in oil droplets one by one, the following operation was performed. A water-soluble fluorescent reagent (FITC) was added to the liquid that forms the water in oil droplets so that the formed droplets can be identified by fluorescence. After sorting one water in oil droplet with fluorescence, one water in oil droplet before dispensing was observed in the collection reservoir. In addition, the water in oil droplet was observed after dispensing into a well of a 384-well plate with a pipette. The results are shown in
The configuration of the flow path chip that forms water in oil droplets in fluorocarbon oil will be explained below. The problem can be solved by the following configuration. Specifically, the configuration without a collection reservoir is appropriate. As shown in
In the method for analyzing cell cluster of the present invention, a flow cytometry data is analyzed. A ratio between a forward scattered light signal intensity and a scattered light signal intensity other than forward detected in each cell is calculated, and whether individual cells are single cells or cell cluster are identified by the obtained value of the ratio. The scattered light signal intensity other than forward detected is not particularly limited, but is preferably a side scattered light signal intensity, or a back scattered light signal intensity.
The apparatus for analyzing cell cluster of the present invention is a flow cytometer, and ratios between plural scattered light signals are calculated, and whether single cell or cell cluster is identified by using values thereof.
In the method for analyzing cell cluster of the present invention, individual cell clusters are quantitatively analyzed.
The forward scattered light signal (FSC) is an intensity of the scattered light component at a low angle in the light irradiation direction and mainly means the scatterer size. On the other hand, the sideward scattering signal component (SSC) is an intensity of the scattered light component at a high angle in the light irradiation direction, and mainly means the microstructure in the scatterer. These are consistent with the results based on the Mie's theory of light scattering. Here, a method of distinguishing a single cell from a cell cluster based on the numerical value of the signal intensity ratio between FSC and SSC will be explained below using actual data.
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The present invention now will be further illustrated by, but is by no means limited to, the following Examples.
In this example, circulating tumor cells (CTC) of a cancer patient were sorted by the 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. 8 mL of blood contains about 108 white blood cells. The containers used in the following pretreatments were previously washed and coated with 0.5% BSA/On-chip T-buffer, in order to prevent a loss of cells due to adsorption of cells to the resin containers. Commercially available reagents such as an erythrocyte hemolysis buffer, a fixation reagent, a cell permeability reagent, and a FCR blocker were used as the buffers and reagents used in the pretreatment. The protocols thereof were in accordance with the attached documents of the reagents.
The collected blood was treated in the order of hemolysis, removal and washing of erythrocyte debris by centrifugation, fixation, permeation treatment, and fluorescent staining (cytokeratin antibody only). Erythrocytes were removed from the sample liquid by these treatments, and it was considered that 108 leukocytes contained several or more CTCs. Since the sample liquid before treatment was adjusted to 0.3 mL, the cell concentration was about 3.3×108 cells/mL.
Only CTCs derived from cancer cells, which are epithelial cells, are cytokeratin-positive, and granulocytes, which are leukocyte components, are weakly positive for cytokeratin. Therefore, the condition for identifying and sorting CTCs is to set the cytokeratin-positive signal level higher than the cytokeratin fluorescence signal level of granulocytes, whereby the cytokeratin-positive cells were sorted. When the sorting was repeated twice under these conditions, the number of leukocytes decreased to about 103.
The samples with reduced leukocytes were stained with Hoechst nuclear, fluorescent labeled cytokeratin antibody for additional staining, fluorescent labeled CD45 antibody, fluorescent labeled vimentin antibody, fluorescent labeled PD-L1 antibody, (or fluorescent labeled HER2 antibody, fluorescent labeled EGFR antibody, fluorescent labeled AXL antibody). After staining, the cells were washed by centrifugation with 0.5% BSA/On-chip T-buffer buffer.
CTCs were identified by repeated sorting for separating CTCs under a condition of Hoechst-positive, CD45-negative, and cytokeratin-positive. In the experiment in which the known number of PC-9 cells (cell lines) was added to blood, the recovery rate was >70% and the purity was >80% according to the above CTC concentration method.
The method for purifying particles of the present invention, i.e., a method for separating a small number of target cells from a cell population having a total number of cells of about 108 or more, can be used for collecting and analyzing CTC from the blood of cancer patients, as shown in Examples. In addition, it can be used for antibody screening to select cells that produce antibodies that specifically bind to the antigen of interest from a large number of antibody-producing cells in antibody drug development. Furthermore, in the method for dispensing a single particle of the present invention, when the particles are water in oil droplets, cell secretions are accumulated in the water in oil droplets without diffusing, and therefore, it can be used for sorting cells that secrete specific substances. Furthermore, since the lysate of cells does not diffuse even if the cells are lysed in the water in oil droplets, it can be used for an expression analysis of cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-141895 | Aug 2020 | JP | national |
