Method and system for separating target particles

Abstract

Comprised are: an introduction step for using a particle capturing device (1) to introduce a fluid including target particles (CT) and non-target particles (CA)-(CC) from an inflow passage (18) into a chamber (20); a concentration step for using a filter (12) to concentrate the target particles (CT) and at least some of the non-target particles (CA) by discharging at least some of the fluid body through a first discharge path (8); a separation step for separating the filtered-off non-target particles (CA) from the target particles (CT) via a suctioning means (30); and a storage step for storing the target particles (CT) in a well.

Description

[Technical Field]

[0001] The present invention relates to a separation method and system for separating, capturing, detecting, isolating, etc., target particles such as cells. This application claims priority based on Japanese Patent Application No. 2020-090677, filed on May 25, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Devices that can separate specific particles from particles of various sizes in a liquid have been known for some time. For example, the cellular components of blood are mainly red blood cells, white blood cells, and platelets, but they are known to contain cells with extremely low abundance, such as circulating tumor cells (CTCs) and fetal-derived non-regenerative cells (fNRBCs).

[0003] To separate and obtain such cells with extremely low abundance, there are affinity separation and concentration methods that use antibodies against the surface markers of the target cells, but the separation efficiency depends on the expression level of the surface marker, and cells with low expression levels will be missed. Furthermore, when affinity-based separation and concentration methods are used, molecules such as antibodies bind to the target cells, which can activate or suppress intracellular signaling systems, resulting in a risk that the cells obtained will not reflect their natural state. Therefore, a label-free isolation method that does not affect the target cells is desirable.

[0004] Label-free separation methods include negative selection using antibodies that bind to cells other than the target cells, and methods that rely on differences in physical properties such as cell size, deformability, and density. For example, negative selection involves labeling white blood cells with CD45 antibodies bound to magnetic particles to separate cancer cells from the blood, attracting the labeled white blood cells to a magnet in a column, and identifying cancer cells in the non-attracted cell fraction (Non-Patent Document 1).

[0005] However, the negative selection enrichment method has a low recovery rate because the target cells are also caught up in the large number of white blood cells attracted to the magnet. For example, Non-Patent Document 2 reports a recovery rate of 58%.

[0006] Known methods for separating cells based on differences in physical properties such as size or deformability include separation using porous filters, microfluidic chips with fine comb structures, or stepped structures. Since filters become clogged and separation becomes impossible, the size, shape, and density of the pores must be optimized to capture only the target cells and remove excess cells.

[0007] Cells enriched by this physical separation method are label-free, and to visualize and identify the target cells, immunostaining with antibodies specific to the target cells is performed. However, because the separation filter is flexible, the filter bends during cell identification under a microscope, making it difficult to focus. For this reason, the cells captured on the filter are transferred back to a tube, stained in the tube, and then transferred to a glass slide for observation, but there is a risk of losing cells during the staining process.

[0008] For separation based on size or deformability, there are microchannel chips with microfilter or gap shapes that have been finely processed to provide shape separation functions. In these cases, separation and capture processes are performed within the microchannel, so staining can be performed directly within the chip after processing. In addition, because the chips are more rigid than filters, they can be directly subjected to microscopic observation.

[0009] In separation filter methods and microfluidic chip separation methods, the retained cells can be collected in a tube or the like by reversing the flow direction. The collected cells can then be re-seeded on a glass slide to isolate specific cells. However, if the cells are spread at high density, they will overlap, making observation difficult, and there is also a risk of collecting non-target cells when collecting the cells.

[0010] For this reason, a method for arraying single cells using filters or substrates with compartments with cell-sized partitions is known. For example, a filter with microwells with through-holes at the bottom can simultaneously separate cells by size and deformability and array single cells, which has the advantage of separating adjacent cells by partitions and allowing single cells to be collected and analyzed.

[0011] In the case of a filter with microwells, when observing or recovering cells, the housing or gasket holding the filter is disassembled, and the filter is removed or the flow direction is reversed to recover the retained cells (Non-Patent Document 3).

[0012] However, the number of microwells is finite, and once all the microwells are occupied by cells, further separation and storage becomes impossible. Therefore, separation methods that combine multiple separation techniques are known. For example, a known method involves size separation using a filter, followed by removal of leukocytes by negative selection using a CD45 antibody, and then capturing the cells in the microwells using a dielectrophoresis chip (Non-Patent Document 4). However, this method requires the transfer of cells to the device at each step, which poses the problem of loss of rare cells. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] INTERNATIONAL JOURNAL OF ONCOLOGY 21: 521-530, 2002 521. [Non-patent document 2] Liu, Z. et al. Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients. J. Transl. Med. 9, 70-70 (2011). [Non-patent document 3] S Khetani et adl. Filter-based isolation, enrichment, and characterization of circulating tumor cells. Biotechnol Bioeng. 2018 Oct; 115(10):2504-2529. [Non-patent document 4] Cancer marker-free enrichment and direct mutation detection in rare cancer cells by combining multi-property isolation and microfluidic concentration Lab Chip, 2019,19, 757-766. Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention has been made to solve the above problems, and provides a method and system for separating target particles that can reduce loss of target particles by performing filter separation of target particles, removal of non-target cells, and storage of target particles in wells using a single device. [Means for solving the problem]

[0015] In order to solve the above problems, each aspect of the present invention has the following configuration. [1] One aspect of the present invention is a method for separating target particles, which separates the target particles from a fluid containing target particles and non-target particles, a particle capture device comprising a chamber, an inlet path to the chamber, an outlet path from the chamber, a filter provided in the outlet path that does not allow the target particles to pass through but allows the fluid to pass through, and a target particle capture membrane provided within the chamber, the target particle capture membrane being formed facing the inside of the chamber and having a plurality of wells therein that can hold the particles, an introducing step of introducing the fluid containing the target particles and the non-target particles into the chamber through the inlet channel; a concentrating step of concentrating the target particles and at least a portion of the non-target particles in the filter by discharging at least a portion of the fluid through the first discharge path; a separation step of separating the non-target particles filtered out by the filter from the target particles by a suction means that sucks only the non-target particles; and storing the target particles in the wells within the chamber.

[0016] According to the target particle separation method, a fluid containing target particles and non-target particles is introduced into the chamber from the inlet channel of the particle capture device, and at least a portion of the fluid is discharged through the first outlet channel, thereby concentrating the target particles and at least a portion of the non-target particles with the filter. Next, the non-target particles filtered out by the filter are separated from the target particles by a suction means that sucks in only the non-target particles, and the target particles are stored in the well within the chamber.

[0017] This method allows a single particle capture device to filter target particles, remove non-target cells filtered out along with the target particles, and store the target particles in wells, thereby reducing loss of target particles.

[0018] [2] The method according to the aspect [1] further comprises a labeling step of labeling the non-target particles with a magnetic antibody prior to the introducing step, and in the separating step, magnet The labeled non-target particles may be attracted by a magnetic force of 0.1 to 0.25, and the labeled non-target particles may be separated from the target particles.

[0019] According to this method, the non-target particles are labeled with a magnetic antibody prior to the introduction step, and in the separation step, magnet A magnetic force is generated by the magnetic field, which attracts the labeled non-target particles and separates them from the target particles. Therefore, the attraction force of the non-target particles can be easily controlled by controlling the magnetic force, making it easy to achieve optimal separation conditions.

[0020] [3] In the above aspect [1], a negative selection marker that selectively binds to the non-target particles may be fixed to the filter, and in the separation process, the non-target particles may be separated from the target particles by binding to the negative selection marker fixed to the filter.

[0021] According to this method, a negative selection marker is fixed to the filter, and this negative selection marker binds to the non-target particles filtered out by the filter, thereby separating the non-target particles from the target particles, making it easy to separate the non-target particles with high selectivity.

[0022] [4] In the above aspect [1], a suction section to which a negative selection marker that selectively binds to the non-target particles is fixed may be provided within the chamber, and in the separation process, the non-target particles may be separated from the target particles by the negative selection marker fixed to the suction section binding to the non-target particles.

[0023] According to this method, a negative selection marker is fixed at a specific position within the chamber, and this negative selection marker binds to the non-target particles filtered out by the filter, thereby separating the non-target particles from the target particles, making it easy to separate the non-target particles with high selectivity and at a position away from the target particles.

[0024] [5] In the above embodiments [3] and [4], the non-target particles may be leukocytes, and the negative selection marker may be at least one of a CD34 antibody and a CD45 antibody. In this case, at least one of the CD34 antibody and the CD45 antibody binds to the leukocytes filtered out by the filter, thereby enabling the leukocytes to be separated from the target particles with high selectivity.

[0025] [6] In the above aspects [1] to [5], a staining step may be further provided in which the target particles are stained before or after being stored in the wells. In this case, the target particles are stained in the staining step, so that the target particles can be easily detected after being captured in the wells.

[0026] [7] In the above aspect [6], a detection step may be further provided in which, after the staining step, the inside of the well is observed to identify the stained target particles. In this case, the target particles stained in the staining step are detected in the detection step, so that the target particles captured in the well can be easily inspected and collected.

[0027] [8] In the above aspect [7], a recovery step of recovering the identified target particles may be further provided after the detection step. In this case, the recovery can be easily performed because the target particles are recovered after their positions are identified.

[0028] [9] Another aspect of the present invention is a system for separating target particles from a fluid containing target particles and non-target particles, comprising: a particle capture device comprising: a chamber; an inlet path to the chamber; an outlet path from the chamber; a filter provided in the outlet path that does not allow the target particles to pass through but allows the fluid to pass through; and a target particle capture membrane provided within the chamber, the target particle capture membrane being formed facing the inside of the chamber and having a plurality of wells therein that can contain the particles; The apparatus includes a suction means for sucking only the non-target particles and separating them from the target particles.

[0029] According to this target particle separation system, a fluid containing target particles and non-target particles is introduced into the chamber through the inlet channel of the particle capture device, and at least a portion of the fluid is discharged through the first outlet channel, thereby concentrating the target particles and at least a portion of the non-target particles using the filter. Next, the non-target particles filtered out by the filter are separated from the target particles using a suction means that sucks in only the non-target particles, and the target particles are stored in the wells within the chamber. This system allows the filter separation of target particles, the removal of non-target cells filtered out along with the target particles, and the storage of the target particles in the wells to be performed using a single particle capture device, thereby reducing loss of target particles.

[0030]

[10] In the above aspects [1] to [9], the area of ​​the target particle capture film of the particle capture device may be smaller than the area of ​​the filter. In this case, since the target particles can be captured in a small area, there is an advantage that the target particles can be easily inspected and collected. [Effects of the Invention]

[0031] As described above, according to the target particle separation method and system of the present invention, filter separation of target particles, removal of non-target cells filtered out along with the target particles, and storage of the target particles in wells can be performed using a single particle capture device, thereby reducing loss of target particles. [Brief explanation of the drawings]

[0032] [Figure 1] 1 is a flowchart showing a first embodiment of a method for separating target particles according to the present invention. [Figure 2] FIG. 10 is a longitudinal cross-sectional view of the particle capturing device showing a step of introducing a fluid containing target particles and non-target particles into a chamber in the same embodiment. [Figure 3] FIG. 10 is a longitudinal cross-sectional view of the particle capturing device showing a process of concentrating target particles using a filter in the same embodiment. [Figure 4] FIG. 2 is a longitudinal cross-sectional view of the particle capturing device in the same embodiment, showing a state in which target particles are concentrated by a filter. [Figure 5] FIG. 10 is a longitudinal cross-sectional view of the particle capturing device showing a process of suctioning non-target particles and separating them from target particles in the same embodiment. [Figure 6] FIG. 10 is a longitudinal cross-sectional view of the particle capturing device showing the step of storing target particles in wells in the same embodiment. [Figure 7] FIG. 10 is a longitudinal cross-sectional view of the particle capturing device showing a step of collecting target particles in the same embodiment. [Figure 8] FIG. 2 is an enlarged perspective view showing an example of a target particle trapping film. [Figure 9] 10 is a longitudinal sectional view of a particle capturing device showing a step of sucking in non-target particles and separating them from target particles in a second embodiment of the target particle separation method according to the present invention. FIG. [Figure 10] 10 is a longitudinal sectional view of a particle capturing device showing a step of sucking in non-target particles and separating them from target particles in a third embodiment of the target particle separation method according to the present invention. FIG. [Figure 11] FIG. 13 is a longitudinal sectional view showing a particle capturing device used in a seventh embodiment of the method for separating target particles according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0033] Hereinafter, embodiments of a target particle separation method and system according to the present invention will be described in detail with reference to the drawings. Fig. 1 is a flowchart showing a first embodiment of the present invention, and Figs. 2 to 7 are vertical cross-sectional views of a particle capture device in each step of the embodiment. First, the structure of the particle capture device 1 will be described with reference to Fig. 2.

[0034] The particle capture device 1 has a device body 2 in the shape of a container with an open top. A target particle capture film 6 is disposed within the device body 2, and the space below the target particle capture film 6 serves as a first discharge channel 8. The shape of the device body 2 is not limited and may be any shape, including, for example, a shape having a flat bottom with a planar shape such as a rectangle, ellipse, or circle, and a peripheral wall portion standing vertically from the bottom. The target particle capture film 6 is preferably disposed horizontally and flat when the device body 2 is in use, but does not necessarily have to be horizontal and flat; for example, it may be slightly inclined or, if necessary, curved.

[0035] An outlet 4 is formed somewhere in the device body 2, and the fluid in the first outlet channel 8 can be discharged through the outlet 4, and as the fluid is discharged, it flows from top to bottom through the target particle capture film 6. In the example shown, one outlet 4 is formed in the center of the bottom of the device body 2, but this configuration is not limited to this, and outlets 4 may be formed on the peripheral wall or other parts of the device body 2, or multiple outlets may be formed, or the entire bottom end of the device body 2 may be open as the outlet 4.

[0036] A filter holding part 10 is arranged above the target particle capture film 6 of the device body 2, at a distance from the target particle capture film 6. A filter 12 is arranged at the lower end of the filter holding part 10, and a second discharge path 14 is formed above the filter 12 inside the filter holding part 10, with a discharge port 16 leading from the second discharge path 14 to the outside. In this example, the filter 12 is preferably arranged horizontally and flat when the device body 2 is in use, but it does not necessarily have to be horizontal and flat; for example, it may be slightly inclined or, if necessary, curved.

[0037] The filter holding part 10 may be fixed to the device body 2, but it is more preferable that it is detachable from the device body 2. In this case, when the filter holding part 10 is removed from the device body 2, the upper surface of the target particle capture film 6 can be exposed, and as shown in Figure 7, for example, the target particles CT captured on the upper surface of the target particle capture film 6 can be easily manipulated directly using an operating means 21 such as a micropipette.

[0038] The filter holding part 10 has a bottom formed by a filter 12, a second discharge path 14 formed inside, and a discharge port 16 formed leading from the second discharge path 14 to the outside. In this example, one discharge port 16 is formed in the center of the top plate part of the filter holding part 10, but this configuration is not limited thereto, and the discharge port 16 may be formed on the side surface of the filter holding part 10, or a pair of discharge ports 16 leading to both ends of the second discharge path 14 may be formed.

[0039] A chamber 20 is formed between the target particle capturing film 6 of the device body 2 and the filter 12 of the filter holding part 10. An inlet channel 18 that communicates with the chamber 20 is also formed in at least a portion between the device body 2 and the filter holding part 10. It is also possible to configure the filter holding part 10 to seal the entire top surface of the device body 2, and to form the inlet channel 18 on the side surface of the device body 2.

[0040] 8, the target particle trapping membrane 6 of this example has a large number of fine wells 22 formed regularly, each concave toward the upper surface. In this example, each well 22 has a regular hexagonal shape in plan view, a hexagonal well bottom 28, and a well wall 24 of a regular hexagonal cylinder standing up from each well bottom 28, with the well wall 24 having an overall honeycomb structure. The shape of the wells 22 is not limited to this hexagonal shape, and may be circular or rectangular.

[0041] Two well outlets 26 are formed in the center of the well bottom 28 for each well 22, penetrating vertically, and the fluid in the well 22 is discharged through the well outlets 26 to the underside of the target particle capture membrane 6. In this example, two well outlets 26 are formed for each well 22, but the number of well outlets 26 for each well 22 may be one, or three or more. If there are two or more well outlets 26, this has the advantage that the well outlets 26 are less likely to be completely blocked even if target particles CT get into the well 22.

[0042] The dimensions and depth of the well 22 can be selected depending on the dimensions of the target particles CT to be trapped. Specifically, the dimensions and depth of the well 22 are set to be slightly larger than the target particles CT to be trapped so that one target particle CT can fit inside the well 22 with a slight gap between it and the well wall 24. If necessary, the wells 22 may be sized to accommodate a predetermined number of target particles CT, for example, two, three, or more.

[0043] The target particle trapping film 6 may be attached directly to the device body 2, or alternatively, may be fixed to a frame (not shown), which may be detachably attached to the device body 2. In this case, the size of the well 22 can be selected by replacing the target particle trapping film 6 together with the frame. Another advantage is that the device body 2 can be reused even if the target particle trapping film 6 becomes unusable.

[0044] The filter 12 is a porous membrane having a large number of pores of approximately uniform size, and the pore size is larger than the size of non-target particles to be passed through (CB and CC in FIGS. 2 to 7) and smaller than the size of target particles CT and non-target particles that should not be passed through (CA in FIGS. 2 to 7). This allows the non-target particles CB and CC to pass through the filter 12, but the target particles CT and non-target particles CA cannot pass through the filter 12. A separate filter holder 10 may be provided for each mesh size of the filter 12, so that the size of the pores can be changed by replacing the filter holder 10.

[0045] It is preferable that the distance L (FIG. 2) between the filter 12 and the target particle capture film 6 is approximately constant over the entire area of ​​the target particle capture film 6. In this case, the target particles CT that have fallen from the underside of the filter 12 sway as they descend through the fluid and reach the target particle capture film 6 with approximately the same distribution area, making it possible to make the probability that the wells 22 will capture the target particles CT approximately equal over the entire area of ​​the target particle capture film 6.

[0046] In this embodiment, the areas of the filter 12 and the target particle trapping film 6 are approximately the same, but as in the embodiment shown in Fig. 11 described later, the area of ​​the target particle trapping film 6 may be made smaller than the area of ​​the filter 12 so that the target particles CT fall in a concentrated manner over a small area, facilitating inspection of the target particles CT and recovery by the operating means 21. Furthermore, if necessary, the area of ​​the target particle trapping film 6 may be made larger than that of the filter 12.

[0047] Next, a target particle separation method according to the first embodiment will be described with reference to the flowchart in Figure 1. The following description corresponds to a case in which, for example, human or animal blood, or a diluted or component extract of blood, is used as a sample to capture specific target cells from among multiple types of cells present in the blood. However, the present invention is not limited to blood samples, and any biological or non-biological sample may be used, and any particle contained therein may be used as the target particle. As the fluid, various buffers suitable for storing target particle CT can be used.

[0048] In step S1, as shown in Fig. 2, a fluid containing target particles CT and non-target particles CA, CB, and CC is introduced into chamber 20 through inlet 18. In the example shown in the figure, three types of non-target particles are used, but this is merely an example, and the number of non-target particles may be one, two, or four or more. The target particles CT may also contain multiple types of particles.

[0049] 3, while fluid is appropriately supplied from the inlet channel 18, the fluid in the second outlet channel 14 is sucked from the outlet 16 by a suction means (not shown). As a result, non-target particles CB and CC having a size that can pass through the filter 12 enter the second outlet channel 14 and are discharged from the outlet 16. Eventually, as shown in FIG. 4, the filter 12 can concentrate almost only the target particles CT and non-target particles CA in the chamber 20.

[0050] In step S3, as shown in FIG. 5, of the target particles CT and non-target particles CA captured on the lower surface of the filter 12, only the non-target particles CA are dropped from the filter 12 by a suction means (not shown) and separated from the target particles CT remaining on the filter 12.

[0051] In this first embodiment, the suction means utilizes a balance between the amount of fluid flowing in and out from the outlet 4, the amount of fluid flowing in and out from the outlet 16, and the amount of fluid discharged from the inlet channel 18. That is, by controlling the three flow rates of the amount of fluid flowing in and out from the outlet 4, the amount of fluid flowing in and out from the outlet 16, and the amount of fluid discharged from the inlet channel 18, the flow rate of the fluid flowing through the filter 12 is adjusted so that the target particles CT remain on the lower surface of the filter 12, while the non-target particles CA drop off from the filter 12, and the non-target particles CA that have dropped off from the filter 12 are discharged from the inlet channel 18.

[0052] Such suction means can be used when there is a difference in specific gravity, size, and / or adhesion to the filter 12 (strength of physical adhesion, surface affinity, particle shape, etc.) between the target particles CT and the non-target particles CA, and the flow rate of the fluid flowing through the filter 12 determines whether the particles remain on the lower surface of the filter 12 or fall off from the filter 12. If it is not possible to control whether the particles remain or fall off by changing the fluid flow rate, separation by magnetic force, separation by negative selection marker, separation by electrostatic force, or the like can be used, as in the second to fourth embodiments described below.

[0053] 6, in step S4, the target particles CT are dropped from the filter 12 in the chamber 20, allowed to descend through the fluid, and stored in the wells 22 of the target particle capture membrane 6. At this time, the flow of fluid through the outlet 4, outlet 16, and inlet channel 18 may be stopped. Alternatively, if necessary to promote the capture of the target particles CT, the fluid may be discharged from the outlet 4 to suck the target particles CT into the wells 22, while the fluid is supplied from the inlet channel 18 into the chamber 20.

[0054] 7, in step S5, the filter holder 10 may be removed from the device body 2, and the positions of the target particles CT captured in any of the wells 22 of the target particle capture film 6 may be identified using a microscope or inspection device (not shown), and the target particles CT may be collected one by one from the wells 22 using an operating means 21 such as a micropipette. Alternatively, various tests may be performed on the identified target particles CT while they are still captured in the wells 22 of the target particle capture film 6, without collecting the target particles CT.

[0055] To facilitate identification of the target particle CT, a staining step may be provided in which the target particle CT is stained in advance with various staining agents. The staining step may be performed before step S1, i.e., at the time of sample preparation, or the target particle CT may be stained by adding a dye to the fluid in any of steps S2 to S4. The type of staining agent is not limited, and any staining agent conventionally used for the same purpose may be used.

[0056] In the detection process of observing the inside of the well and identifying the stained target particle, it is possible to identify the position of the well 22 in the target particle capture membrane 6 in which the target particle CT is captured by observation with a microscope, observation with a fluorescence microscope, or image processing using a computer in conjunction with these, in accordance with the emission wavelength or absorption wavelength of the dye. When the coordinates are identified by a computer, it becomes possible to automatically recover or inspect the target particle CT in the well 22 at that position.

[0057] According to the first embodiment described above, the separation of the target particles CT by the filter 12, the separation and removal of the non-target cells CA filtered out together with the target particles CT, and the storage of the target particles CT in the wells 22 can be performed by a single particle capturing device 1, thereby making it possible to suppress loss of the target particles CT.

[0058] [Second embodiment using magnetic force] 9 shows step S3 (non-target particle removal step) in the second embodiment of the present invention. The other steps S1, S2, S4, and S5 may be the same as those in the first embodiment unless otherwise specified, but this second embodiment further includes a labeling step in which non-target particles CA that do not pass through the filter 12 are pre-labeled with a magnetic antibody prior to step S1 (introduction step). As the magnetic antibody, for example, magnetic beads having pre-bound antibodies on their surface that bind to antigens present on the surface of non-target particles CA that do not pass through the filter 12 can be used.

[0059] The magnetic beads are, for example, spinel ferrite (AFe2O4 (A is Mn, Co, Ni, Cu, Zn, etc.)) or hexagonal ferrite (AFe 12 O 19 The core may have a core such as ferrite (A is Ba, Sr, Pb, etc.), the surface of which is coated with a hydrophilic polymer or lipid to provide functional groups, and antibodies or proteins such as avidin, albumin, protein A, or protein G to which the functional groups are further provided; or the core may have a core of a high molecular weight polymer with a magnetizable substance such as the ferrite dispersed therein, to which an antibody is provided in the same manner as above.

[0060] Magnetic beads can be either micron (μm) or nano (nm) sized. Micron-sized beads have a strong magnetic force per particle, resulting in a high magnetic collection effect, but their small specific surface area reduces the amount of active substance that can be bound per unit mass of beads. On the other hand, nano-sized beads have a large specific surface area, meaning that a large amount of active substance can be bound per unit mass of beads, making it easier to increase the sensitivity of detecting target particle CT. They can be used according to the purpose.

[0061] 3, fluid is supplied appropriately from the inlet channel 18, while the fluid in the second outlet channel 14 is sucked from the outlet 16 by a suction means (not shown). As a result, non-target particles CB and CC having a size that can pass through the filter 12 are discharged from the outlet 16 via the second outlet channel 14, while target particles CT and non-target particles CA having a size larger than the pores of the filter 12 cannot pass through the filter 12 and remain in the chamber 20.

[0062] At this time, or from the start of filtering in step S2 shown in Fig. 3, a magnet 30 is placed on the side opposite the particle capture surface of filter 12, i.e., above filter 12, as shown in Fig. 9. As a result, non-target particles CA, which have magnetic beads already attached to their surfaces, are attracted by the magnetic force generated by magnet 30 and pressed against filter 12, thereby maintaining their capture by filter 12. In contrast, target particles CT, which do not have magnetic beads attached, are not affected by the magnetic force generated by magnet 30 and therefore easily fall off filter 12, descend through the fluid, reach target particle capture membrane 6, and are captured in well 22.

[0063] The magnet 30 may be a permanent magnet or an electromagnet. When an electromagnet is used, it is possible to switch between attracting and not attracting non-target particles CA by turning the current on and off, and it has the advantage of being able to control the strength of the attractive force by adjusting the current. In the illustrated example, the magnet 30 is placed on top of the filter holder 10, but this structure is not limiting; the magnet 30 can also be placed inside the filter holder 10, or the magnet 30 can be divided into multiple small magnets whose magnetic force can be turned on and off simultaneously, or they can be held by a common support and moved up and down simultaneously.

[0064] Once the target particles CT are captured in the well 22, the magnet 30 is removed along with the filter holder 10, the non-target particles CA are removed along with the filter 12, the target particle capture membrane 6 is exposed, and the target particles CT in the well 22 are inspected or recovered by an operating means 21 such as a micropipette.

[0065] According to this method, the non-target particles CT are labeled with magnetic antibodies prior to step S1, and in step S3, the labeled non-target particles CA are attracted by the magnetic force of the magnet 30 and separated from the target particles CT. This makes it easy to control the attraction force of the non-target particles CA by controlling the magnetic force, making it easy to achieve optimal separation conditions.

[0066] [Third embodiment] 10 shows step S3 (non-target particle removal step) in the third embodiment of the present invention. The other steps S1, S2, S4, and S5 may be the same as those in the first embodiment unless otherwise specified. However, like the second embodiment, this third embodiment also includes a labeling step of labeling non-target particles CA that do not pass through the filter 12 with a magnetic antibody before step S1 (introduction step). The magnetic antibody may be the same as that in the second embodiment.

[0067] 3, fluid is supplied appropriately from the inlet channel 18 while fluid in the second outlet channel 14 is sucked from the outlet 16. As a result, non-target particles CB and CC having a size that can pass through the filter 12 are discharged from the outlet 16 via the second outlet channel 14, while target particles CT and non-target particles CA having a size larger than the pores of the filter 12 cannot pass through the filter 12 and remain in the chamber 20.

[0068] Once filtering is complete, as shown in Figure 10, a magnet 32 ​​is inserted into the chamber 20 through the inlet channel 18. As a result, non-target particles CA, which have magnetic beads already attached to their surfaces, are attracted by the magnetic force generated by the magnet 32, and are separated from the filter 12 and adsorbed to the magnet 32. In contrast, since the target particles CT do not have magnetic beads attached, they are not affected by the magnetic force generated by the magnet 32. When they fall off the filter 12, they descend through the fluid to the target particle capture membrane 6 and are captured in the well 22.

[0069] Once the target particles CT are captured in the well 22, the non-target particles CA are removed together with the magnet 32, the filter holder 10 is removed, the target particle capture membrane 6 is exposed, and the target particles CT in the well 22 are inspected or recovered by an operating means 21 such as a micropipette.

[0070] According to this method, the non-target particles CA labeled with magnetic antibodies can be attracted to a position away from the target particles CT by the magnet 32 ​​placed at a position away from the filter 12, which has the advantage of making it easy to separate the non-target particles CA.

[0071] [Fourth embodiment] The fourth embodiment of the present invention is characterized in that a negative selection marker that selectively binds to non-target particles CA that do not pass through the filter 12 is immobilized on the filter 12 in advance.

[0072] The negative selection marker can be, for example, an antibody that selectively binds to an antigen present on the surface of non-target particles CA that do not pass through the filter 12. As a result, in step S3 shown in Figure 3, the negative selection marker fixed to the filter 12 binds to the non-target particles CA, thereby maintaining the non-target particles CA attached to the filter 12. On the other hand, the target particles CT do not bind to the negative selection marker of the filter 12, so they drop off the filter 12 and descend in the fluid, reach the target particle capture membrane 6, and are captured in the well 22.

[0073] According to this method, a negative selection marker is fixed to the filter 12, and this negative selection marker binds to the labeled non-target particles CA filtered out by the filter 12, thereby separating the non-target particles CA from the target particles CT, thereby enabling the separation of the non-target particles CA with high selectivity.

[0074] [Fifth embodiment] The fifth embodiment of the present invention is characterized in that a negative selection marker that selectively binds to non-target particles CA that do not pass through the filter 12 is immobilized at a location other than the filter 12. Specifically, a suction section to which a negative selection marker that selectively binds to non-target particles CA is immobilized is provided in the chamber 20, and in the separation process, the negative selection marker immobilized at the suction section binds to the non-target particles CA, thereby separating the non-target particles CA from the target particles CT. On the other hand, the target particles CT do not bind to the negative selection marker of the filter 12, so they drop off the filter 12, descend in the fluid, reach the target particle capture membrane 6, and are captured in the well 22.

[0075] The position of the attraction part is not limited as long as it is inside the chamber 20, but for example, a negative selection marker may be fixed to the inner wall surface of the chamber 20 of the device main body 2, or a member having an attraction part may be inserted into the chamber 20 by penetrating the wall surface of the device main body 2 from the inflow channel 18 or a specially provided insertion port. A structure in which a member 32 having an attraction part is inserted into the chamber 20 may be used instead of the magnet 32 ​​in FIG.

[0076] According to this method, a negative selection marker is fixed at a specific position within the chamber 20, and this negative selection marker binds to the non-target particles CA filtered out by the filter 12, thereby separating the non-target particles CA from the target particles CT. This makes it easy to separate the non-target particles CA with high selectivity and at a position away from the target particles CT.

[0077] [Sixth embodiment] In a sixth embodiment of the present invention, the non-target particles CA that do not pass through the filter 12 are white blood cells, and the negative selection marker is at least one of a CD34 antibody and a CD45 antibody. In this case, the target particles CT are not limited to, but may be, for example, circulating tumor cells (CTCs) or fetal NRBCs.

[0078] The CD34 antibody selectively binds to the CD34 antigen present on the surface of hematopoietic stem cells that produce white blood cells in the blood. The CD45 antibody selectively binds to the CD45 antigen, a leukocyte common antigen. By using at least one of these CD34 and CD45 antibodies as a negative selection marker in the method of the fourth or fifth embodiment, the non-target particles, leukocyte CA, are bound to the CD34 and CD45 antibodies, and the leukocyte CA is separated from the target particles CT. Meanwhile, the target particles CT do not bind to the CD34 and CD45 antibodies, so they fall off the filter 12, descend in the fluid, reach the target particle capture membrane 6, and are captured in the well 22.

[0079] This method has the advantage that leukocytes CA, which are difficult to separate from target particles CT, can be removed from the target particles CT with high selectivity.

[0080] [Seventh embodiment] FIG. 11 is a longitudinal cross-sectional view showing a seventh embodiment of a particle capture device 1 usable in the present invention. In this seventh embodiment, the device body 2 has a bottom 2A, a tapered portion 2B, and an expanded portion 2C. The bottom 2A has a bottom plate and a peripheral wall rising from its periphery. In this example, the outlet 4 is formed in the center of the bottom plate of the bottom 2A. However, the outlet 4 does not have to be located at this position. A tapered portion 2B extends obliquely upward from the upper end of the peripheral wall of the bottom 2A, expanding horizontally as it extends upward to form a tapered shape. A target particle capture film 6 is horizontally disposed along the boundary between the bottom 2A and the tapered portion 2B in the usage configuration, and a frame 7 supporting the target particle capture film 6 is removably disposed along the inside of the peripheral wall of the bottom 2A. The planar shape of the device body 2 is not limited and may be rectangular, circular, elliptical, or a shape with semicircular portions on both sides of a rectangle.

[0081] A cylindrical expansion portion 2C is formed upward from the upper end of the tapered portion 2B, and a filter holding portion 10 is detachably attached inside this expansion portion 2C so that it is horizontal and detachable in use. It is preferable that the center position of the filter 12 of the filter holding portion 10 and the center position of the target particle trapping film 6 are arranged so that they approximately coincide with each other in order to make the falling density of the target particles CT onto the target particle trapping film 6 uniform, but the two do not necessarily have to coincide with each other.

[0082] An opening 19 is formed in a part of the peripheral wall of the tapered portion 2B, and is connected to an inflow channel 18. It is also possible to insert a magnet 30 along the inflow channel 18 as in the embodiment of Figure 10, or to place the magnet 30 above the filter holding portion 10 as in the embodiment of Figure 9. The other configurations may be the same as those of the other embodiments described so far, and therefore description thereof will be omitted.

[0083] According to the seventh embodiment of Fig. 11, the device body 2 has a shape that expands upward, and the area of ​​the target particle capture film 6 is smaller than the area of ​​the filter 12. This means that the target particles CT are captured in this small area, which has the advantage of making it easy to inspect and collect the target particles CT after removing the filter holder 10. The inclination angle of the tapered portion 2B with respect to the vertical line is selected to be an angle that prevents the target particles CT from adhering to and stopping on the inner wall surface of the tapered portion 2B. In order to set this angle as close to horizontal as possible, a vibrating device may be provided in the device body 2 to slightly vibrate the device body 2 and promote the target particles CT falling onto the target particle capture film 6.

[0084] Although various embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, but should be broadly interpreted based on the scope of the claims. Each configuration of the above-described embodiments may be changed to another well-known configuration, some configurations may be omitted, and configurations of the embodiments may be appropriately rearranged. [Industrial Applicability]

[0085] According to the target particle separation method and system of the present invention, filter separation of target particles, removal of non-target cells filtered out along with the target particles, and storage of the target particles in wells can be performed using a single particle capture device, making it possible to reduce loss of target particles and therefore making it suitable for industrial use. [Explanation of symbols]

[0086] 1: particle capture device; 2: device body; 2A: bottom portion; 2B: tapered portion; 2C: Enlarged portion, 4: Discharge port, 6: Target particle capture membrane, 7: Frame body, 8: First discharge passage, 10: Filter holding portion, 12: Filter, 14: Second discharge passage, 16: Discharge port, 18: Inlet passage, 19: Opening, 20: Chamber, 21: Operating means, 22: Well, 24: Well wall, 26: Well outlet, 28: well bottom; 30: magnet; 32: magnet; CA~CC: non-target particles, CT: target particles.

Claims

1. 1. A method for separating target particles, comprising: separating the target particles from a fluid containing target particles and non-target particles; A chamber; an inlet to the chamber; a filter disposed within the chamber, the filter blocking the target particles but allowing the fluid to pass through; a target particle capturing membrane provided in the chamber; a first discharge path capable of sucking the fluid in the chamber through the target particle capture membrane; a second discharge path capable of sucking the fluid in the chamber through the filter; a particle capture device in which the target particle capture membrane has a plurality of wells formed facing the inside of the chamber and capable of accommodating the particles therein, and a well outlet penetrating the bottom of each well and not allowing the target particles to pass through; an introducing step of introducing the fluid containing the target particles and the non-target particles into the chamber through the inlet channel; a concentrating step of concentrating the target particles and at least a portion of the non-target particles in the filter by discharging at least a portion of the fluid through the second discharge path; a separation step of separating the non-target particles filtered out by the filter from the target particles by adjusting the flow rate of the fluid passing through the filter using a suction means that sucks the inside of the second discharge path; and storing the target particles separated from the non-target particles in the chamber in the well by sucking them through the first discharge path.

2. The method further comprises a labeling step of labeling the non-target particles with a magnetic antibody in advance prior to the introducing step, 2. The method for separating target particles according to claim 1, wherein the separating step comprises attracting the labeled non-target particles by the magnetic force of a magnet, and separating the labeled non-target particles from the target particles.

3. a negative selection marker that selectively binds to the non-target particles is immobilized on the filter; 2. The method for separating target particles according to claim 1, wherein in the separation step, the negative selection marker fixed to the filter binds to the non-target particles, thereby separating the non-target particles from the target particles.

4. a suction section having a negative selection marker immobilized thereon that selectively binds to the non-target particles; 2. The method for separating target particles according to claim 1, wherein in the separation process, the negative selection marker fixed to the suction section binds to the non-target particles filtered out by the filter, thereby separating the non-target particles from the target particles.

5. 5. The method for separating target particles according to claim 3, wherein the non-target particles are leukocytes, and the negative selection marker is at least one of CD34 and CD45.

6. 6. The method for separating target particles according to claim 1, further comprising a staining step of staining the target particles before or after they are stored in the wells.

7. 7. The method for separating target particles according to claim 6, further comprising a detection step of observing the inside of the well after the staining step to identify the stained target particles.

8. The method for separating target particles according to claim 7 , further comprising a recovery step of recovering the identified target particles after the detection step.

9. 1. A target particle separation system for separating target particles from a fluid containing target particles and non-target particles, comprising: A chamber; an inlet to the chamber; a filter disposed within the chamber, the filter blocking the target particles but allowing the fluid to pass through; a target particle capturing membrane provided in the chamber; a first discharge path capable of sucking the fluid in the chamber through the target particle capture membrane; a second discharge path capable of sucking the fluid in the chamber through the filter; a particle capture device in which the target particle capture membrane is formed facing the inside of the chamber and has a plurality of wells therein capable of accommodating the particles, and a well outlet penetrating the bottom of each well and through which the target particles cannot pass; A target particle separation system characterized by comprising a suction means for separating the non-target particles filtered out by the filter from the target particles by adjusting the flow rate of the fluid passing through the filter by suctioning through the second discharge path.

10. 10. The system for separating target particles according to claim 9, wherein the area of ​​the target particle capturing membrane of the particle capturing device is smaller than the area of ​​the filter.

11. A target particle separation system as described in claim 9 or 10, characterized in that the distance between the filter and the target particle capture film is approximately constant over the entire area of ​​the target particle capture film, and the target particle capture film is positioned below the filter.

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