Target particle separation method and system

Abstract

A particle capture device (1) is used, which has the following steps: an introduction step, in which a fluid containing target particles (CT) and non-target particles (CA) to (CC) is introduced from an inlet path (18) into a chamber (20); a concentration step, in which at least a portion of the fluid is discharged through a first discharge path (8), thereby concentrating the target particles (CT) and at least a portion of the non-target particles (CA) by a filter (12); a separation step, in which the filtered non-target particles (CA) are separated from the target particles (CT) by a suction means (30); and a containing step, in which the target particles (CT) are contained in a pit.

Description

Technical Field

[0001] The present invention relates to a separation method and system for separating, capturing, detecting, or individually separating target particles such as cells.

[0002] This application claims priority based on Japanese Patent Application No. 2020-090677 filed in Japan on May 25, 2020, and the contents are incorporated herein by reference. Background Art

[0003] Equipment for separating specific particles from particles of different sizes mixed in a liquid is known in the past. For example, the cell components of blood are mainly red blood cells, white blood cells, and platelets, and it is known that there are extremely low-occurrence cells such as circulating tumor cells / CTCs (Circulating Tumor Cells) and fetal nucleated red blood cells (fNRBCs) mixed in them.

[0004] In order to separate and obtain such cells with extremely low abundance rates, there are affinity-based separation and concentration methods using antibodies against surface markers of target cells, but the separation efficiency depends on the expression level of the surface marker, and cells with low expression levels tend to escape.

[0005] Furthermore, in the case of affinity-based separation and concentration methods, since molecules such as antibodies bind to target cells, there is a risk that the intracellular signal transduction system is activated or inhibited, and the obtained cells may not reflect the natural state.

[0006] Therefore, a label-free separation method that has no effect on target cells is desired.

[0007] Label-free separation methods include negative selection using antibodies that bind to cells other than target cells and methods based on differences in physical properties of cells such as size, deformability, and density.

[0008] In negative selection, for example, in order to separate cancer cells in the blood, white blood cells are labeled with CD45 antibodies bound to magnetic particles, the labeled white blood cells are attracted to the magnet in a column, and cancer cells are identified in the cell fraction that is not attracted (Non-Patent Document 1).

[0009] However, in the concentration method based on negative selection, when a large number of white blood cells are attracted to the magnet, the target cells are also drawn in, so the recovery rate is low. For example, according to Non-Patent Document 2, the recovery rate is reported to be 58%.

[0010] As a separation method based on differences in physical properties such as cell size or deformability, separation using a filter with holes or a microfluidic chip with a fine comb-shaped structure or a step-difference structure is known. In the case of a filter, if clogging occurs, separation cannot be performed, so the size, shape, and density of the holes must be optimized in a way that only target cells can be captured and excess cells can be removed.

[0011] The cells concentrated by such a separation method based on physical properties are label-free, and in order to visualize and identify the target cells, immunostaining based on antibodies specific to the target cells is performed. However, since the separation filter is soft, when observing and identifying the cells under a microscope, the filter bends and it is difficult to focus. Therefore, after the cells captured in the filter are transferred to a tube again, the staining operation is performed in the tube and transferred to a slide for observation, but there is a risk of cell loss during the staining operation.

[0012] As separation based on size and deformability, there are microfluidic chips formed into microfilter shapes and gap shapes, which are finely processed and have shape separation functions. In this case, the separation and capture process is carried out in the microchannel, so it can be stained directly in the chip after processing. In addition, compared with the filter, it has higher rigidity and can be directly provided for microscopic observation.

[0013] In the separation filter-based method and the microfluidic chip-based separation method, the retained cells can be recovered in a tube or the like by reversing the direction of the flow. The cells recovered in the tube or the like are re-plated on a glass slide, and only specific cells can be isolated individually. However, if the cells are spread at a high density, the cells overlap with each other, making observation difficult, and there is a risk that cells other than the target cells are also recovered when the cells are recovered.

[0014] Therefore, a method of arraying single cells using a filter or substrate having partitions with partitions of single cell size is known. For example, a filter having micro-pits with through holes at the bottom can simultaneously perform separation based on size and deformability and arraying of single cells, thereby achieving separation of partitions from adjacent cells, which has the advantage of being able to recover and analyze single cells.

[0015] In the case of a filter having micro-pits, when observing or recovering cells, the housing or gasket holding the filter is disassembled and the filter is removed, or the direction of flow is reversed to recover the retained cells (Non-Patent Document 3).

[0016] However, the number of micro-pits is limited. If all micro-pits are occupied by cells, further separation and accommodation cannot be performed. Therefore, a separation method combining multiple separation techniques is known. For example, a method is known in which leukocytes are removed by negative selection based on CD45 antibodies after size separation based on a filter, and cells are captured in micro-pits by an electrophoresis chip (non-patent document 4). However, in this method, cells need to be transferred to the device in each step, and there is a problem of loss of rare cells.

[0017] Prior art literature

[0018] Non-patent literature

[0019] Non-patent document 1: INTERNATIONAL JOURNAL OF ONCOLOGY 21: 521-530, 2002 521.

[0020] 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).

[0021] 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.

[0022] 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

[0023] Problems to be solved by the invention

[0024] The present invention is completed to solve the above-mentioned problems, and provides a method and system for separating target particles, which can suppress the loss of target particles by filtering target particles, removing non-target cells, and accommodating target particles in pits in one device.

[0025] Means for solving problems

[0026] In order to solve the above-mentioned problems, each aspect of the present invention has the following configurations.

[0027] [1] One embodiment of the present invention is a method for separating target particles from a fluid containing target particles and non-target particles, using a particle capturing device, the particle capturing device comprising:

[0028] a chamber; an inflow path into the chamber; an exhaust path out of the chamber; a filter disposed in the exhaust path and through which the target particles are not permeable but the fluid is permeable; and a target particle capture membrane disposed in the chamber, wherein the target particle capture membrane has a plurality of pits formed toward the inner side of the chamber and capable of accommodating the particles therein,

[0029] The separation method comprises the following steps:

[0030] An introduction step of introducing the fluid containing the target particles and the non-target particles into the chamber from the inlet passage;

[0031] a concentration step of discharging at least a portion of the fluid through the first discharge path, thereby concentrating the target particles and at least a portion of the non-target particles by the filter;

[0032] a separation step of separating the non-target particles filtered out by the filter from the target particles by using an attraction means for attracting only the non-target particles; and

[0033] The accommodating step is to accommodate the target particle in the recess in the chamber.

[0034] According to the aforementioned target particle separation method, a fluid containing target particles and non-target particles is introduced into the aforementioned chamber from the aforementioned inlet path of the aforementioned particle capture device, and at least a portion of the aforementioned fluid is discharged through the aforementioned first discharge path, thereby concentrating the aforementioned target particles and at least a portion of the aforementioned non-target particles by the aforementioned filter. Next, the non-target particles filtered out by the aforementioned filter are separated from the target particles by a suction means that only attracts the non-target particles, and the aforementioned target particles are contained in the aforementioned pit in the aforementioned chamber.

[0035] According to this method, filter separation of target particles, removal of non-target cells filtered out together with the target particles, and storage of target particles in the pits can be performed in one particle capturing device, thereby suppressing the loss of target particles.

[0036] [2] Alternatively, in the aforementioned method [1], it also includes a labeling step of labeling the aforementioned non-target particles with a magnetic antibody before the aforementioned introduction step, and in the aforementioned separation step, the aforementioned non-target particles that have been labeled are attracted by the magnetic force of the aforementioned attraction means to separate the aforementioned non-target particles that have been labeled from the aforementioned target particles.

[0037] According to this method, non-target particles are labeled with magnetic antibodies before the introduction process. In the separation process, the magnetic force is generated by the attraction means, and the magnetic force is used to attract the labeled non-target particles and separate them from the target particles. Therefore, the attraction force of the non-target particles can be easily controlled by controlling the magnetic force, and the optimal separation conditions can be easily achieved.

[0038] [3] Alternatively, in the aforementioned method [1], a negative selection marker that selectively binds to the aforementioned non-target particles is fixed in the aforementioned filter, and in the aforementioned separation step, the aforementioned negative selection marker fixed on the aforementioned filter binds to the aforementioned non-target particles, thereby separating the aforementioned non-target particles from the aforementioned target particles.

[0039] According to this method, a negative selection marker is immobilized on the filter, and the negative selection marker binds to non-target particles filtered out by the filter to separate the non-target particles from the target particles, so that the non-target particles can be easily separated with high selectivity.

[0040] [4] Alternatively, in the aforementioned method [1], an attraction portion is provided in the aforementioned chamber, and the attraction portion is fixed with a negative selection marker that selectively binds to the aforementioned non-target particles. In the aforementioned separation step, the aforementioned negative selection marker fixed to the aforementioned attraction portion binds to the aforementioned non-target particles, thereby separating the aforementioned non-target particles from the aforementioned target particles.

[0041] According to this method, a negative selection marker is fixed at a specific position in the aforementioned chamber, and the negative selection marker binds to the non-target particles filtered out by the filter, thereby separating the non-target particles from the target particles. Therefore, it is easy to separate the non-target particles with high selectivity and at a position far away from the target particles.

[0042] [5] Alternatively, in the above-mentioned aspects [3] and [4], the non-target particles are leukocytes, and the negative selection marker is at least one of CD34 antibody and CD45 antibody. In this case, at least one of CD34 antibody and CD45 antibody binds to the leukocytes filtered out by the filter, and the leukocytes can be separated from the target particles with high selectivity.

[0043] [6] Alternatively, in the above-mentioned methods [1] to [5], a dyeing step of dyeing the target particle before or after the target particle is contained in the above-mentioned pit may be further provided. In this case, the target particle is dyed by the dyeing step, and then the target particle is easily detected after being captured by the pit.

[0044] [7] Alternatively, in the above-mentioned method [6], after the above-mentioned dyeing step, there is also a detection step of observing the inside of the above-mentioned pit to determine the above-mentioned dyed target particles. In this case, the target particles dyed in the dyeing step are detected in the detection step, and then the target particles captured by the pit can be easily inspected and recovered.

[0045] [8] In the above-mentioned aspect [7], a recovery step of recovering the identified target particles may be further provided after the detection step. In this case, the recovery is performed after the position of the target particles is determined, so the recovery can be easily performed.

[0046] [9] Another aspect of the present invention is a target particle separation system for separating the target particles from a fluid containing target particles and non-target particles, the system comprising a particle capture device and an attraction means for attracting only the non-target particles and separating them from the target particles, wherein the particle capture device comprises:

[0047] A chamber; an inflow path into the aforementioned chamber; an exhaust path out of the aforementioned chamber; a filter arranged in the aforementioned exhaust path and through which the aforementioned target particles do not pass but through which the aforementioned fluid passes; and a target particle capture membrane arranged in the aforementioned chamber, wherein the aforementioned target particle capture membrane has a plurality of pits formed toward the inner side of the aforementioned chamber and capable of accommodating the aforementioned particles therein.

[0048] According to such a target particle separation system, a fluid containing target particles and non-target particles is introduced from the inlet path of the particle capture device into the chamber, and at least a portion of the fluid is discharged through the first discharge path, so that the target particles and at least a portion of the non-target particles can be concentrated by the filter. Next, the non-target particles filtered out by the filter are separated from the target particles by a suction means that only attracts the non-target particles, and the target particles are contained in the pit in the chamber. According to this system, filter separation of target particles, removal of non-target cells filtered out together with target particles, and containment of target particles in pits can be performed in one particle capture device, and the loss of target particles can be suppressed.

[0049]

[10] Alternatively, in the above-mentioned methods [1] to [9], the area of ​​the target particle capture membrane of the above-mentioned particle capture device is smaller than the area of ​​the above-mentioned filter. In this case, the target particles can be captured with a small area, which has the advantage of facilitating the inspection and recovery of the target particles.

[0050] Effects of the Invention

[0051] 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 together with target particles, and accommodation of target particles in pits can be performed in one particle capturing device, thereby suppressing the loss of target particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] [ Figure 1 ] is a flow chart showing a first embodiment of the target particle separation method of the present invention.

[0053] [ Figure 2 ] is a longitudinal cross-sectional view of a particle capturing device showing the process of introducing a fluid containing target particles and non-target particles into a chamber in the first embodiment.

[0054] [ Figure 3 ] is a longitudinal cross-sectional view of a particle capturing device showing the process of concentrating target particles by a filter in the first embodiment.

[0055] [ Figure 4 ] is a longitudinal cross-sectional view of the particle capturing device showing the state after the target particles are concentrated by the filter in the first embodiment.

[0056] [ Figure 5 ] is a longitudinal cross-sectional view of a particle capturing device showing the process of attracting non-target particles and separating them from target particles in the first embodiment.

[0057] [ Figure 6] is a longitudinal cross-sectional view of a particle capturing device showing the process of accommodating target particles in a pit in the first embodiment.

[0058] [ Figure 7 ] is a longitudinal cross-sectional view of a particle capturing device showing the process of recovering target particles in the first embodiment.

[0059] [ Figure 8 ] is a three-dimensional enlarged view showing an example of a target particle capture membrane.

[0060] [ Fig. 9 ] is a longitudinal cross-sectional view of a particle capturing device showing the process of attracting non-target particles and separating them from target particles in the second embodiment of the target particle separation method of the present invention.

[0061] [ Fig.10 ] is a longitudinal cross-sectional view of a particle capturing device showing the process of attracting non-target particles and separating them from target particles in the third embodiment of the target particle separation method of the present invention.

[0062] [ Fig.11 ] is a longitudinal cross-sectional view of a particle capturing device used in the seventh embodiment of the target particle separation method of the present invention. DETAILED DESCRIPTION

[0063] Hereinafter, embodiments of the target particle separation method and system of the present invention will be described in detail with reference to the drawings. Figure 1 This is a flowchart showing a first embodiment of the present invention. Figure 2 to Figure 7 To illustrate a longitudinal cross-sectional view of the particle capture device in each step of the first embodiment, first use Figure 2 The structure of the particle capture device 1 described above will be described.

[0064] The particle capture device 1 has a device body 2 in the shape of a container with an open upper surface, a target particle capture membrane 6 is arranged in the device body 2, and the space below the target particle capture membrane 6 is a first exhaust path 8. The shape of the device body 2 is not limited, and can be any shape, for example, it can have a flat bottom with a planar shape such as a rectangular, elliptical or circular shape, and a shape with a peripheral wall portion vertically rising from the bottom. The target particle capture membrane 6 is preferably arranged horizontally and flatly in the use state of the device body 2, but it does not have to be horizontal and flat, for example, it can also be slightly inclined, or it can be curved as needed.

[0065] The device body 2 is provided with a discharge port 4 at any position, and the fluid in the first discharge path 8 can be discharged through the discharge port 4. The fluid passes through the target particle capture membrane 6 and flows from top to bottom as it is discharged. In the example shown in the figure, a discharge port 4 is formed in the center of the bottom of the device body 2, but it is not limited to this structure, and it can be formed in the peripheral wall portion of the device body 2, other parts, or multiple portions, or the entire lower end of the device body 2 can be opened to serve as the discharge port 4.

[0066] A filter holder 10 is disposed on the upper side of the device body 2 and away from the target particle capture membrane 6. A filter 12 is disposed at the lower end of the filter holder 10, and a second discharge path 14 is formed on the filter 12 inside the filter holder 10, and a discharge port 16 is formed from the second discharge path 14 to the outside. The filter 12 of this example is preferably disposed horizontally and flatly in the use state of the device body 2, but it is not necessarily horizontal and flat, and for example, it may be slightly inclined or curved as required.

[0067] The filter holding portion 10 may be fixed to the device body 2, but is preferably detachable from the device body 2. In this case, if the filter holding portion 10 is removed from the device body 2, the upper surface of the target particle capture membrane 6 can be exposed, for example, Figure 7 As shown, it becomes easy to directly manipulate the target particles CT captured on the upper surface of the target particle capturing film 6 using a manipulation means 21 such as a micropipette.

[0068] The filter holding part 10 has a bottom formed by the filter 12, a second exhaust passage 14 formed inside, and an exhaust port 16 leading to the outside from the second exhaust passage 14. In this example, a single exhaust port 16 is formed in the center of the top plate of the filter holding part 10, but the present invention is not limited to this configuration, and the exhaust port 16 may be formed on the side of the filter holding part 10, or a pair of exhaust ports 16 communicating with both ends of the second exhaust passage 14 may be formed.

[0069] A chamber 20 is formed between the target particle capture membrane 6 of the device body 2 and the filter 12 of the filter holding part 10. In addition, an inflow path 18 communicating with the chamber 20 is formed at least partially between the device body 2 and the filter holding part 10. The entire upper surface of the device body 2 may be sealed by the filter holding part 10, and the inflow path 18 may be formed on the side of the device body 2.

[0070] like Figure 8As shown, the target particle capture film 6 of this example is regularly formed with a large number of fine pits 22 that are concave toward the upper surface. In this example, each pit 22 is a regular hexagonal shape when viewed from above, and has a hexagonal pit bottom 28 and a regular hexagonal cylindrical pit wall 24 rising from each pit bottom 28, and the pit wall 24 has a honeycomb structure as a whole. The shape of the pit 22 is not limited to such a hexagonal shape, and can be circular or quadrilateral.

[0071] Each pit 22 has two pit outlets 26 formed at the center of the pit bottom 28, which are connected vertically, and the fluid in the pit 22 is discharged to the lower surface side of the target particle capture membrane 6 through the pit outlets 26. In this example, each pit 22 has two pit outlets 26, but the number of pit outlets 26 of each pit 22 can be 1 or 3 or more. In the case of more than 2, there is an advantage that the pit outlet 26 is not easily completely blocked even if the target particle CT enters the pit 22.

[0072] The size and depth of the pit 22 can be selected according to the size of the target particle CT to be captured. Specifically, the size and depth of the pit 22 are set to be slightly larger than the target particle CT to be captured, and a target particle CT is stored in the pit 22 with a small gap between it and the pit wall 24. As needed, the size can be set to accommodate a predetermined number of target particles CT, such as 2, 3 or more, in the pit 22.

[0073] The target particle capture film 6 can be directly mounted on the device body 2, or alternatively, can be fixed to a frame (not shown) that is detachably mounted on the device body 2. In this case, there is an advantage that the size of the recess 22 can be selected by replacing the target particle capture film 6 together with the frame. There is an advantage that the device body 2 can be reused even if the target particle capture film 6 becomes unusable.

[0074] The filter 12 is a porous membrane having a large number of through-holes of almost constant size, and the size of the through-holes is larger than the non-target particles ( Figure 2 to Figure 7 CB and CC in the figure are smaller than the target particles CT and the non-target particles ( Figure 2 to Figure 7 Thus, non-target particles CB and CC pass through the filter 12, while target particles CT and non-target particles CA cannot pass through the filter 12. The filter holder 10 is individually provided according to the size of each mesh of the filter 12, and the size of the through hole can be changed by replacing the filter holder 10.

[0075] The spacing L between the filter 12 and the target particle capturing membrane 6 ( Figure 2) is preferably almost constant over the entire range of the target particle capture membrane 6. In this case, the target particles CT falling from the lower surface of the filter 12 sway in the fluid while landing, and arrive at the target particle capture membrane 6 with almost the same dispersion area, so that the probability of the pits 22 capturing the target particles CT over the entire range of the target particle capture membrane 6 can be almost equal.

[0076] In this embodiment, the filter 12 and the target particle capturing membrane 6 have almost the same area, but as described below, Fig.11 As in the embodiment shown, the area of ​​the target particle capture membrane 6 is made smaller than the area of ​​the filter 12, and the target particles CT are concentrated and landed on a small area, thereby making it easy to inspect the target particles CT and recover them based on the operating means 21. In addition, the area of ​​the target particle capture membrane 6 can also be made larger than the filter 12 according to certain needs.

[0077] Next, follow Figure 1 The flow chart of the first embodiment is used to illustrate the target particle separation method. The following description is equivalent to, for example, using human or animal blood, or a dilution or component extract of blood as a sample to capture specific target cells from a variety of cells present in the blood, but the present invention is not limited to blood samples, and any biological sample or non-biological sample can be used, and any particles contained therein can also be used as target particles. As a fluid, various buffers suitable for storing target particles CT can be used.

[0078] In step S1, Figure 2 As shown, a fluid containing target particles CT and non-target particles CA, CB, and CC is introduced into the chamber 20 from the inlet path 18. In the example of the figure, there are three types of non-target particles, but this is only an example, and the non-target particles can be one or two, or more than four. The target particle CT can also contain multiple particles.

[0079] In step S2, Figure 3 As shown, while the fluid is appropriately supplied from the inlet 18, the fluid in the second outlet 14 is sucked from the outlet 16 by a suction means (not shown). As a result, the non-target particles CB and CC of a size that pass through the filter 12 enter the second outlet 14 and are discharged from the outlet 16. Figure 4 As shown, the filter 12 can be used to concentrate almost only the target particles CT and the non-target particles CA in the chamber 20 .

[0080] In step S3, Figure 5As shown, among 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 suction means (not shown), and are separated from the target particles CT remaining in the filter 12 .

[0081] In the first embodiment, as a suction means, a balance is used among the inflow and outflow of the fluid from the discharge port 4, the inflow and outflow of the fluid from the discharge port 16, and the discharge of the fluid from the inflow path 18. That is, by controlling the three flow rates of the inflow and outflow of the fluid from the discharge port 4, the inflow and outflow of the fluid from the discharge port 16, and the discharge of the fluid from the inflow path 18, the flow rate of the fluid flowing through the filter 12 is adjusted, and the target particles CT remain on the lower surface of the filter 12, while the non-target particles CA fall off from the filter 12, and the non-target particles CA that fall off from the filter 12 are discharged from the inflow path 18.

[0082] Such an attraction means can be used because there is a difference in specific gravity, size, and / or adhesion to the filter 12 (strength of physical adhesion, surface affinity, particle shape difference, etc.) between the target particles CT and the non-target particles CA, and the difference in the flow rate of the fluid flowing through the filter 12 is used to produce a difference in the amount of particles remaining on the lower surface of the filter 12 or falling off the filter 12. If such control of the remaining / falling off based on the fluid flow rate cannot be performed, separation based on magnetic force, separation based on a negative selection marker, or separation based on electrostatic force, etc., as described in the second to fourth embodiments below, can be used.

[0083] In step S4, Figure 6 As shown, in the chamber 20, the target particles CT are caused to fall off from the filter 12, fall from the fluid, and are contained in the pit 22 of the target particle capture membrane 6. At this time, the flow of fluid from the discharge port 4, the discharge port 16, and the inflow path 18 may be stopped. Alternatively, in order to promote the capture of the target particles CT, the fluid may be discharged from the discharge port 4 as needed, the target particles CT may be sucked into the pit 22, and the fluid may be supplied to the chamber 20 from the inflow path 18.

[0084] In step S5, Figure 7 As shown, the filter holding part 10 can be removed from the device body 2, and the position of the target particle CT captured by any recess 22 of the target particle capture membrane 6 can be determined using a microscope or an inspection device (not shown), and the target particles CT can be recovered one by one from the recess 22 using an operating means 21 such as a micropipette. Alternatively, the target particles CT can be subjected to various inspections while being captured in the recess 22 of the target particle capture membrane 6 without recovering the target particles CT.

[0085] In order to facilitate the determination of the target particle CT, a dyeing step may be provided for pre-dyeing the target particle CT with various dyes. The dyeing step may be performed before step S1, i.e., at the time of preparing the sample, or the target particle CT may be dyed by adding a dye to the fluid in any of steps S2 to S4. The type of dye is not limited, and may be any dye that has been used for the same purpose in the past.

[0086] As a detection process for observing the inside of the pit to determine the dyed target particle, based on the emission wavelength or absorption wavelength of the dye, observation based on a microscope, observation using a fluorescence microscope, or these and image processing using a computer can be used to determine at which position of the target particle capture film 6 the pit 22 captures the target particle CT. When the coordinates are determined by the computer, the target particle CT in the pit 22 at that position can be automatically recovered or inspected.

[0087] According to the above first embodiment, target particles CT can be separated by the filter 12, non-target cells CA filtered out together with the target particles CT can be separated and removed, and target particles CT can be accommodated in the pits 22 in one particle capturing device 1, thereby suppressing the loss of target particles CT.

[0088] [Second embodiment using magnetic force]

[0089] Fig. 9 Step S3 (non-target particle removal process) in the second embodiment of the present invention is shown. Unless otherwise specified, the other steps S1, S2, S4, and S5 are the same as those in the first embodiment, but in the second embodiment, before step S1 (introduction process), there is also a labeling process of pre-labeling the non-target particles CA that do not pass through the filter 12 with magnetic antibodies. As the magnetic antibody, for example, magnetic beads with antibodies pre-bound to the surface can be used, and the antibody will bind to the antigen present on the surface of the non-target particles CA that do not pass through the filter 12.

[0090] For example, the following materials can be used as magnetic beads: spinel ferrite (AFe2O4 (A is Mn, Co, Ni, Cu, Zn, etc.), hexagonal ferrite (AFe 12 O 19 A core of a ferrite such as Ba (A is Ba, Sr, Pb, etc.), the surface of the core is coated with a hydrophilic polymer or lipid to impart functional groups, and the aforementioned functional groups are further imparted with antibodies or proteins such as avidin (Japanese: アジビン), albumin, protein A, protein G, etc.; or, a core of a high molecular polymer in which the aforementioned ferrite or other magnetizable substance is dispersed, and the core is imparted with antibodies in the same manner as the aforementioned; etc.

[0091] Magnetic beads can be micron (μm) sized or nanometer (nm) sized. In the case of micron size, the magnetic force of each particle is strong, so the capture effect brought by magnetism is high, and the amount of active substances that can be bound per unit mass of beads decreases corresponding to the amount of specific surface area reduction. On the other hand, nanometer-sized beads have a large specific surface area, so the amount of active substances that can be bound per unit mass of beads is large, which is easy to improve the sensitivity of detecting target particles CT. It can be used according to the purpose.

[0092] In the method of the second embodiment, Figure 3 In step S2 shown, while the fluid is appropriately supplied from the inlet path 18, the fluid in the second outlet path 14 is sucked from the outlet 16 by a suction means (not shown). As a result, the non-target particles CB and CC of a size that can pass through the filter 12 are discharged from the outlet 16 through the second outlet path 14, while on the other hand, the target particles CT and non-target particles CA of a size larger than the through hole of the filter 12 fail to pass through the filter 12 and remain in the chamber 20.

[0093] At this time, or freely Figure 3 From the beginning of the filtering of step S2 shown in FIG. Fig. 9 As shown, a magnet 30 is pre-arranged on the side of the filter 12 opposite to the particle capturing surface, that is, on the upper side of the filter 12. As a result, the non-target particles CA having magnetic beads pre-attached to the surface are attracted by the magnetism generated by the magnet 30 and pressed against the filter 12, thereby maintaining the state of being captured by the filter 12. In contrast, the target particles CT have no magnetic beads attached to them, and therefore are not affected by the magnetic force generated by the magnet 30, so they are easily detached from the filter 12, fall in the fluid, reach the target particle capturing membrane 6, and are captured in the pit 22.

[0094] The magnet 30 can be a permanent magnet or an electromagnet. When an electromagnet is used, it has the advantages of being able to switch the attraction / non-attraction of non-target particles CA by turning the current on / off, and being able to control the magnitude of the attraction force by adjusting the current. In the illustrated example, the magnet 30 is arranged on the filter holding portion 10, but is not limited to this structure. The magnet 30 can also be arranged inside the filter holding portion 10, and the magnet 30 can be divided into a plurality of small magnets so that they can turn on / off the magnetic force together; or, it can also be configured to be held on a common support body and can move up and down at the same time.

[0095] After the target particle CT is captured in the pit 22, the magnet 30 and the filter holder 10 are removed together, and the non-target particles CA and the filter 12 are removed together to expose the target particle capture membrane 6. The target particle CT in the pit 22 is inspected or recovered using an operating means 21 such as a micropipette.

[0096] According to this method, before step S1, non-target particles CT are labeled with magnetic antibodies in advance. In step S3, the magnetic force of the magnet 30 is used to attract the labeled non-target particles CA to separate them from the target particles CT. Therefore, the attraction of the non-target particles CA can be easily controlled by controlling the magnetic force, and the optimal separation conditions can be easily achieved.

[0097] [Third Embodiment]

[0098] Fig.10 Step S3 (non-target particle removal process) in the third embodiment of the present invention is shown. Unless otherwise specified, the other steps S1, S2, S4, and S5 are the same as those in the first embodiment, but the third embodiment also has a labeling process of pre-labeling the non-target particles CA that do not pass through the filter 12 with magnetic antibodies before step S1 (introduction process) as in the second embodiment. The magnetic antibodies can be the same as in the second embodiment.

[0099] In the method of the third embodiment, Figure 3 In step S2 shown, the fluid is appropriately supplied from the inlet path 18, and at the same time, the fluid in the second outlet path 14 is sucked from the outlet 16. As a result, the non-target particles CB and CC of a size that can pass through the filter 12 are discharged from the outlet 16 through the second outlet path 14, while on the other hand, the target particles CT and non-target particles CA of a size larger than the through hole of the filter 12 fail to pass through the filter 12 and remain in the chamber 20.

[0100] After filtering, if Fig.10 As shown, the magnet 32 ​​is inserted into the chamber 20 from the inflow path 18. As a result, the non-target particles CA with magnetic beads attached to the surface in advance are attracted by the magnetism generated by the magnet 32, away from the filter 12 and adsorbed to the magnet 32. In contrast, the target particles CT are not attached with magnetic beads, so they are not affected by the magnetic force generated by the magnet 32. Once they fall off the filter 12, they fall into the fluid and reach the target particle capture membrane 6, and are captured in the pit 22.

[0101] After the target particles CT are captured in the pits 22, the non-target particles CA are removed together with the magnet 32, the filter holder 10 is removed to expose the target particle capture membrane 6, and the target particles CT in the pits 22 are inspected or recovered using an operating means 21 such as a micropipette.

[0102] According to this method, the non-target particles CA labeled with the magnetic antibody can be attracted to a position away from the target particles CT by the magnet 32 ​​placed away from the filter 12, so there is an advantage that the non-target particles CA can be easily separated.

[0103] [Fourth Embodiment]

[0104] The fourth embodiment of the present invention is characterized in that a negative selection marker that selectively binds to the non-target particles CA that do not pass through the filter 12 is immobilized in advance on the filter 12 .

[0105] As the negative selection marker, for example, an antibody that selectively binds to an antigen present on the surface of the non-target particle CA that does not pass through the filter 12 can be used. Figure 3 In step S3, the negative selection marker fixed in the filter 12 binds to the non-target particle CA, thereby maintaining the state in which the non-target particle CA is attached to the filter 12. On the other hand, the target particle CT is not bound to the negative selection marker of the filter 12, and therefore falls off from the filter 12 and falls in the fluid, reaches the target particle capture membrane 6, and is captured in the pit 22.

[0106] According to this method, the negative selection marker is immobilized on the filter 12 and binds to the labeled non-target particles CA filtered out by the filter 12 to separate the non-target particles CA from the target particles CT, so that the non-target particles CA can be separated with high selectivity.

[0107] [Fifth Embodiment]

[0108] 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 fixed in advance to a portion other than the filter 12. Specifically, a suction portion is provided in the chamber 20, and the suction portion is fixed with a negative selection marker that selectively binds to the non-target particles CA. In the separation process, the negative selection marker fixed to the aforementioned suction portion 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, and therefore fall off the filter 12 and fall into the fluid, reaching the target particle capture membrane 6 and being captured in the pit 22.

[0109] The position of the suction part is not limited as long as it is in the chamber 20. For example, the negative selection marker may be fixed to the inner wall surface of the chamber 20 of the device body 2 in advance, or the member having the suction part may be inserted into the chamber 20 through the inflow path 18 or a specially provided insertion port through the wall surface of the device body 2. Alternatively, the member 32 provided with the suction part may be inserted into the chamber 20 instead of Fig.10 The magnet 32.

[0110] According to this method, a negative selection marker is fixed at a specific position within the chamber 20, and the negative selection marker combines with the non-target particles CA filtered out by the filter 12, thereby separating the non-target particles CA from the target particles CT, making it easy to separate the non-target particles CA with high selectivity and at a position away from the target particles CT.

[0111] [Sixth Embodiment]

[0112] The sixth embodiment of the present invention is characterized in that 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 the CD34 antibody and the CD45 antibody. In this case, the target particles CT are not limited, and may be, for example, circulating tumor cells / CTCs (Circulating Tumor Cells) in the blood, fetal nucleated red blood cells (fNRBCs), etc.

[0113] The CD34 antibody selectively binds to the CD34 antigen present on the surface of the hematopoietic stem cells that produce the white blood cells in the blood. The CD45 antibody selectively binds to the CD45 antigen, which is a common antigen of white blood cells. At least one of these CD34 antibodies and CD45 antibodies is used as a negative selection marker in the method of the fourth embodiment or the fifth embodiment, so that the white blood cells CA, which are non-target particles, are bound to the CD34 antibodies and the CD45 antibodies, and the white blood cells CA are separated from the target particles CT. On the other hand, the target particles CT do not bind to the CD34 antibodies and the CD45 antibodies, so they fall off from the filter 12 and fall into the fluid, reaching the target particle capture membrane 6 and being captured in the pit 22.

[0114] This method has the advantage that the leukocyte CA, which is difficult to distinguish from the target particle CT, can be removed from the target particle CT with high selectivity.

[0115] [Seventh Embodiment]

[0116] Fig.11The longitudinal sectional view of the seventh embodiment of the particle capture device 1 that can be used in the present invention is shown. In the seventh embodiment, the device body 2 has a bottom 2A, a cone 2B and an enlarged portion 2C. The bottom 2A has a bottom plate and a peripheral wall rising from the periphery thereof. In this example, a discharge port 4 is formed in the center of the bottom plate of the bottom 2A. The discharge port 4 may not be at this position. A cone 2B is formed from the upper end of the peripheral wall of the bottom 2A toward the upper side, and the cone 2B is enlarged in the horizontal direction as it moves upward to form a cone shape. The target particle capture membrane 6 is horizontally arranged along the boundary between the bottom 2A and the cone 2B in the use form, and the frame 7 supporting the target particle capture membrane 6 is arranged in a manner that can be removed along the inner side of the peripheral wall of the bottom 2A. The planar shape of the device body 2 is not limited, and may also be rectangular, circular, elliptical, or have semicircular portions on both sides of the rectangle, etc.

[0117] A cylindrical expansion portion 2C is formed upward from the upper end of the tapered portion 2B, and the filter holder 10 is detachably mounted on the inner side of the expansion portion 2C in a horizontal and detachable manner in the use state. In order to make the landing density of the target particles CT to the target particle capture membrane 6 uniform, it is preferred to arrange the filter 12 of the filter holder 10 in a manner that the center position of the filter 12 is roughly consistent with the center position of the target particle capture membrane 6, but the two do not necessarily have to be consistent.

[0118] An opening 19 is formed in a part of the peripheral wall of the tapered portion 2B and is connected to the inflow path 18. Fig.10 or insert the magnet 30 into the filter holding portion 10 as above; Fig. 9 The magnet 30 is arranged as in the embodiment of FIG. The other structures can be the same as those of the other embodiments described above, so the description is omitted.

[0119] according to Fig.11 In the seventh embodiment, the device body 2 is in an upwardly enlarged shape, and the area of ​​the target particle capture membrane 6 is smaller than the area of ​​the filter 12. The target particles CT are captured in this small area, which has the advantage that the target particles CT can be easily inspected and recovered after the filter holder 10 is removed. For the inclination angle of the cone 2B relative to the plumb line, an angle is selected to the extent that the target particles CT will not adhere to the inner wall surface of the cone 2B and stop. In order to set the angle as close to the horizontal as possible, a vibration device can also be provided in the device body 2 to cause the device body 2 to vibrate slightly to promote the target particles CT to fall toward the target particle capture membrane 6.

[0120] Various embodiments of the present invention are described above, but the present invention is not limited to the aforementioned embodiments and can be broadly interpreted according to the scope of the claims. The various structures of the aforementioned embodiments can be changed to other known structures, and some structures can be omitted, and the embodiments can be appropriately reorganized with each other.

[0121] Industrial Applicability

[0122] According to the target particle separation method and system of the present invention, it is possible to filter and separate target particles, remove non-target cells filtered out together with target particles, and accommodate target particles in pits in one particle capture device, thereby suppressing the loss of target particles. Therefore, it can be used in production.

[0123] Description of Reference Numerals

[0124] 1: Particle capture device, 2: Device body, 2A: Bottom, 2B: Cone, 2C: Enlarged part, 4: Exhaust port, 6: Target particle capture membrane, 7: Frame,

[0125] 8: first discharge path, 10: filter holding portion, 12: filter, 14: second discharge path, 16: discharge port, 18: inflow path, 19: opening, 20: chamber,

[0126] 21: operating means, 22: pit, 24: pit wall, 26: pit outlet,

[0127] 28: pit bottom, 30: magnet, 32: magnet,

[0128] CA~CC: non-target particles, CT: target particles.

Claims

1. A method for separating target particles, which is a method for separating target particles from a fluid containing target particles and non-target particles, characterized in that: Use a particle capture device that has: Chamber; the inflow path into the chamber; A filter through which the target particles do not pass but through which the fluid passes; A target particle capture membrane is disposed in the chamber, and a spacing between the target particle capture membrane and the filter is constant; a first discharge path, which is disposed below the target particle capture membrane; a second discharge path, which is disposed above the filter; a discharge outlet provided at the first discharge path; another discharge outlet provided in the second discharge path; and a suction means for sucking the fluid in the inflow path, the first discharge path, or the second discharge path, respectively; The filter is arranged in the chamber closer to the first discharge path than the target particle capture membrane, and the target particle capture membrane has a plurality of pits formed toward the inside of the chamber and capable of accommodating the particles therein. The separation method comprises the following steps: An introducing step of introducing the fluid containing the target particles and the non-target particles from the inlet path into the chamber; a concentrating step of causing the fluid supplied from the inlet path to flow through the filter by sucking the fluid in the second outlet path, thereby discharging at least a portion of the fluid; 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 flowing through the filter so as to use the suction means for suctioning only the non-target particles; and The step of accommodating the target particle is performed in the recess in the chamber.

2. The method for separating target particles according to claim 1, further comprising a labeling step of labeling the non-target particles with a magnetic antibody before the introduction step. In the separation step, the labeled non-target particles are attracted by the magnetic force of the attracting means, so that the labeled non-target particles are separated from the target particles.

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

4. The method for separating target particles according to claim 1, wherein: The chamber is provided with an attraction portion, the attraction portion having fixed thereon a negative selection marker that selectively binds to the non-target particles. In the separation step, the negative selection marker immobilized on the suction section is bound to the non-target particles, thereby separating the non-target particles from the target particles.

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 . The method for separating target particles according to claim 1 , further comprising a dyeing step of dyeing the target particles before or after accommodating the target particles in the recesses. 7 . The target particle separation method according to claim 6 , further comprising a detection step of observing the inside of the pit to identify the stained target particle after the staining step. 8 . The target particle separation method according to claim 7 , further comprising a recovery step of recovering the identified target particles after the detection step.

9. A target particle separation system, which is a target particle separation system for separating the target particle from a fluid containing the target particle and non-target particles, characterized in that: A particle capturing device and an attracting means for attracting only the non-target particles and separating them from the target particles are provided. The particle capture device comprises: Chamber; the inflow path into the chamber; A filter through which the target particles do not pass but through which the fluid passes; A target particle capture membrane is disposed in the chamber, and a spacing between the target particle capture membrane and the filter is constant; a first discharge path, which is disposed below the target particle capture membrane; a second discharge path, which is disposed above the filter; a discharge outlet provided at the first discharge path; another discharge outlet provided in the second discharge path; and a suction means for sucking the fluid in the inflow path, the first discharge path, or the second discharge path, respectively; The filter is disposed in the chamber and is closer to the first discharge path than the target particle capture membrane. The target particle capturing film has a plurality of recesses formed toward the inside of the chamber and capable of accommodating the particles therein.

10. The target particle separation system according to claim 9, characterized in that: The target particle capture membrane of the particle capture device has an area smaller than that of the filter.

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