JP2018059931A - Method and device for magnetically labeling particles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of magnetically labeling particles captured by a filter efficiently on the filter.SOLUTION: A method is provided for labeling particles captured by a filter having a plurality of through-holes using magnetic beads, the method comprising steps of; supplying a suspension containing magnetic beads to a filter surface on which particles are captured; moving a portion of the suspension from the filter surface toward the other filter surface to bring both surfaces of the filter into contact with the liquid containing the magnetic beads; moving the liquid containing the magnetic beads backward from the other filter surface toward the filter surface on which the particles were captured.SELECTED DRAWING: None

Description

  The present disclosure relates to a method of labeling particles with magnetic particles (hereinafter also referred to as magnetic beads) and an apparatus for labeling. In particular, the present disclosure relates to a method of magnetically labeling particles trapped on a filter with magnetic beads on the filter and a labeling device used therefor.

Magnetic separation and size separation are methods for separating particles contained in a suspension, particularly rare cells in a biological sample. Magnetic separation is a method that depends on the protein expressed in the cells to be separated. On the other hand, size separation is a method that utilizes differences in particle size and deformability.
In general methods for separating rare cells, only one of the methods is used. As a method using size separation, for example, a method using a filter having a predetermined characteristic has been developed (for example, Patent Document 1).
On the other hand, recently, from the viewpoint of improving the separation accuracy of rare cells, it has been proposed to use separation methods having different principles of magnetic separation and size separation (Patent Document 2 and Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 2015-087382 Special table 2009-511001 gazette

Sci Transl Med 5, 179ra47 (2013)

Rare cells include medically important cells such as circulating cells in the blood / circulating tumor cell (CTC) and immune cells. An example of CTC is cancer cells released from primary tumor tissue or metastatic tumor tissue. These cancer cells are said to cause metastasis by being carried to other organs through blood or lymphatic vessels. Moreover, since CTC in blood is recognized as useful as a factor for determining the therapeutic effect of metastatic cancer and predicting prognosis, analysis of CTC has been actively conducted.
However, rare cells such as CTC usually contain only about 0 to 10 cells in 10 ml of blood, and in order to perform more accurate analysis, it is possible to separate or concentrate rare cells from a specimen with high accuracy. It has been demanded.

As disclosed in Patent Document 1, the present applicant has developed a method capable of efficiently separating rare cells from blood by using a filter having predetermined characteristics. However, blood contains a large amount of blood cell components such as red blood cells and white blood cells in addition to rare cells (red blood cells are about tens of billions and white blood cells are about tens of millions). Since it is about the same or larger, it may be trapped with rare cells even when the above filter is used.
The present inventors have found that when leukocytes are captured together with rare cells, for example, there are the following two problems. The first is that the amount of leukocytes captured by the filter is larger than the number of rare cells because the amount of leukocytes captured by the filter is larger than the amount of leukocytes, while the amount of rare cells contained in the blood (specimen) is very small. Some white blood cells are detected as rare cells, and as a result, false positives are likely to occur. Second, when detecting a gene mutation specific to a rare cell, only the genotype of a leukocyte that is not a rare cell is detected, and as a result, false negatives are likely to occur.
For these reasons, it is necessary to efficiently separate the leukocytes captured by the filter from the rare cells.

  In one or a plurality of embodiments, the present disclosure provides a method and a labeling device capable of efficiently magnetically labeling particles trapped on a filter on the filter.

  In one aspect, the present disclosure is a method for labeling particles captured by a filter having a plurality of through-holes with magnetic beads, and supplying a suspension containing the magnetic beads to the filter surface on which the particles are captured. The liquid containing the magnetic beads is brought into contact with both surfaces of the filter by feeding a part of the suspension from the filter surface to the other filter surface; and The present invention relates to a magnetic labeling method including feeding back a liquid containing beads in the direction of the filter surface where the particles are captured from the other filter surface.

  In one aspect, the present disclosure is an apparatus for labeling particles with magnetic beads, a filter unit capable of holding a filter, and an introduction channel capable of introducing a suspension containing magnetic beads into the filter unit. A discharge passage capable of holding or discharging the liquid that has passed through the filter unit, a liquid feeding unit capable of supplying the suspension to the filter unit, and a control unit for controlling the liquid feeding unit, The control unit sends a part of the suspension from one filter surface to the other filter surface so that the liquid containing the magnetic beads is in contact with both surfaces of the filter, and The present invention relates to a labeling device for controlling the liquid feeding means so as to reversely feed a liquid containing magnetic beads from the other filter surface.

  According to the present disclosure, in one aspect, labeling of particles captured by a filter with magnetic beads can be performed efficiently on the filter.

FIG. 1 shows an example of a cell labeling apparatus that can be used in the magnetic labeling method of the present disclosure. FIG. 2 is a schematic diagram illustrating an example of the procedure of the magnetic labeling method of the present disclosure. FIG. 3 is a graph showing an example of the number of leukocytes remaining (not magnetically captured) in Example 1 and Comparative Examples 1 and 2. FIG. 4 shows an example of a rare cell capture and recovery device used in Experimental Example 1. FIG. 5 shows an example of the configuration of a cell capture and recovery device that can be used in the cell recovery method of the present disclosure.

In the blood, rare cells such as circulating tumor cells / CTC in the blood may be present. CTCs are cells that are released from primary tumor tissue or metastatic tumor tissue and infiltrated into the blood. Cancer metastasis is thought to occur when cancer cells are transported to other parts of the body through blood vessels and lymphatic vessels and proliferate. Therefore, the number of CTCs in the blood and cancer metastasis It has been reported to be related to the likelihood and prognosis. In addition to the CTC, the rare cells include medically important cells such as immune cells. For this reason, the analysis of rare cells in blood is expected to be performed more actively in the future.
10 ml of blood usually contains tens of billions of erythrocytes and tens of millions of leukocytes, but the number of rare cells such as CTC is usually about 0 to 10. For this reason, in order to perform more accurate analysis, it is necessary to separate and collect rare cells from a specimen such as blood with high accuracy.

Rare cells include not only cancer cells larger than blood cells, but also small cells and cells with high deformability. Examples of small cells include human colon cancer cells (commercially available SW620 (cell line name, human colon cancer cells) has been confirmed as small cells). Examples of cells with high deformability include human cancer cells (commercially available SNU-1 (cell line name, human cancer cell) has been confirmed as cells with high deformability).
The present applicant has established a method capable of capturing small cells and cells with high deformability at a high capture rate by using a predetermined filter (for example, Patent Document 1).
However, as described above, even when a specimen such as blood is separated using the filter, white blood cells contained in a large amount in the specimen may be captured together with rare cells. For this reason, a process for further separating rare cells and leukocytes is required. Moreover, in order to analyze the rare cell after isolation | separation, it is necessary to perform various processes.
On the other hand, since rare cells exist in a specimen in an extremely small number as described above, it is necessary to avoid loss during separation and labeling with a filter and recovery as much as possible. For this reason, it has been proposed to perform various processes for the above-described separation or analysis in a state where cells are captured by a filter. The processing on the filter is performed by supplying various processing solutions to the filter and reacting them with cells, and then feeding and removing the unreacted processing solution. That is, the filter has a function as a reaction field for B / F separation (Bound / Free separation) and can perform various treatments without loss of cells.
In various processes on the filter, the present inventors have found a problem that there are a process in which a sufficient processing effect is obtained and a process in which a sufficient processing effect is not obtained. Specifically, antibody staining can be satisfactorily stained, but labeling with magnetic particles is a problem that labeling efficiency is very low and magnetic labeling cannot be sufficiently performed.
In view of the above-mentioned problems, we attempted to collect rare cells and other cells from the filter and label them with magnetic particles. Or there was a problem that it was large between experiments and a problem that reproducibility was low. In addition, after the labeling treatment, it has occurred that the unreacted excess magnetic particles and cells cannot be sufficiently separated. As a result, there has been a problem that the separation accuracy is lowered due to the rare cells being involved in a large amount of excess magnetic particles during magnetic separation, which is a subsequent processing step, and being magnetically separated together.

  In one aspect, the present disclosure includes magnetic particles so that the entire filter in which the cells are captured is covered with a suspension containing the magnetic particles when labeling with the magnetic particles in a state where the cells are captured in the filter. By feeding the suspension, and then feeding the liquid containing the magnetic particles contained in the suspension in the direction opposite to the direction of feeding the suspension (reverse feeding), This is based on the new knowledge found by the present inventors that the efficiency of labeling with magnetic particles can be improved and cells and excessive magnetic particles present in large quantities can be separated.

  In an aspect of the present disclosure, the number of cells captured by the filter is larger in leukocytes than in rare cells. Therefore, if leukocytes, which are not intended for collection, are labeled with magnetic particles, the separation accuracy of rare cells can be improved. Based on the new knowledge found by the present inventors that it can be improved. Specifically, it is as follows. That is, CTCs, which are rare cells, have a group expressing epithelial cell antigens such as EpCAM and cytokeratin, and epithelial-mesenchymal transition in which expression of these epithelial markers is reduced and mesenchymal cell antigens such as Vimentin are expressed. It is clear that there is a group that causes For this reason, it is difficult to recognize all CTCs with a specific antigen. Therefore, by separating leukocytes known to have a specific antigen with magnetic particles instead of CTC, the separation accuracy between CTC and leukocytes can be further improved, and all leukocytes can be used regardless of the antigen. Based on the new findings found by the present inventors that CTCs can be recovered. Note that the labeling target of the present disclosure in the analysis of rare cells such as CTC is not limited to leukocytes, but may be rare cells.

Although the mechanism by which the cells captured by the filter can be efficiently labeled with the magnetic particles on the filter is not clear according to the present disclosure, it is presumed as follows.
When filtering whole blood through a filter with multiple through-holes, the whole blood is sent as a laminar flow, so the flow velocity in the vicinity of the wall of the flow path decreases, resulting in a filter around the wall. In this case, a relatively large amount of red blood cells and white blood cells that are not to be captured remain without being filtered. When staining treatment or labeling treatment is performed in this state, the antibody staining solution, which is a soluble reagent, can be sufficiently stained by diffusion of the reagent itself. The particles cannot be contacted sufficiently and the labeling efficiency is low. The reason for this is considered that the flow velocity around the wall surface where a large number of white blood cells are present is slower than the center of the filter, and the diffusion of magnetic particles is relatively gentle. On the other hand, in the method of the present disclosure, the suspension is fed so that both main surfaces of the filter are in contact with the liquid containing the magnetic particles, and then the solution is fed backward. By immersing both main surfaces of the filter in a liquid containing magnetic particles, the cells captured by the filter are separated from both the cell capturing surface of the filter and the surface of the filter opposite to the cell capturing surface (the upper surface of the filter). And the lower surface of the filter) and in the through-holes, the surface area where the cells come into contact with the suspension (magnetic particles) can be greatly increased. In this state, the cells fitted in the through-holes of the filter are removed from the pores by feeding back from the filter surface opposite to the filter capture surface toward the cell capture surface (from the filter lower surface side to the upper surface side). In addition to being released, the suspension containing the magnetic particles is stirred by the turbulent flow generated by the reverse feeding, so that the contact frequency between the cells and the magnetic particles is further improved. As a result, it is considered that the labeling efficiency by magnetic particles can be improved. However, the present disclosure need not be interpreted as being limited to these mechanisms.

[Labeling method with magnetic particles (magnetic beads)]
In one aspect, the present disclosure relates to a method of labeling particles captured by a filter having a plurality of through holes with a magnetic bead (a labeling method of the present disclosure). In one aspect, the label of the present disclosure supplies a suspension containing magnetic beads to a filter surface on which particles are captured, and a part of the suspension is directed from the filter surface to the other filter surface. The liquid containing the magnetic beads is brought into contact with both surfaces of the filter, and the liquid containing the magnetic beads is brought into contact with the filter surface where the particles are captured from the other filter surface. Including back feeding in the direction. Moreover, the labeling method of the present disclosure is, in one aspect, supplying a suspension containing magnetic beads to the upper surface of the filter in which the particles are captured, and an upper liquid surface and a lower liquid surface of the suspension. A part of the suspension is sent toward the lower surface side of the filter so that the filter is sandwiched between them, and the liquid is fed from the lower surface side of the filter toward the upper surface side of the filter. including. According to the labeling method of the present disclosure, in one or a plurality of embodiments, labeling of particles captured by a filter with magnetic beads can be efficiently performed on the filter without being collected from the filter.

  The labeling method of the present disclosure includes supplying a suspension containing magnetic beads to a filter surface on which particles are captured.

The supply amount of the suspension containing the magnetic beads is not particularly limited, and can be appropriately determined according to the filtration area of the filter. In one or a plurality of embodiments, the supply amount is 50 μL or more or 100 μL or more per filtration area (28 mm ² ) from the viewpoint that magnetic beads can be efficiently and uniformly suspended on the filter, or a small amount of liquid is used for magnetic supply. The amount is 500 μL or less or 300 μL or less because the beads can be efficiently and uniformly suspended.

  In one or a plurality of embodiments, the content (solid content concentration) of the magnetic beads in the suspension containing the magnetic beads is 0.0025% or more or 0 from the viewpoint of efficiently reacting the magnetic beads with the cells. 0.004% or more, and 0.0625% or less or 0.0375% or less from the viewpoint of suppressing non-specific reaction between magnetic beads and cells.

  The magnetic beads (magnetic particles) are not particularly limited, and can be appropriately determined according to the type of particles to be labeled. As the magnetic beads, in one or a plurality of embodiments, known magnetic beads used for magnetic labeling of particles such as cells or magnetic beads that can be developed in the future can be used. In one or a plurality of embodiments, the magnetic beads include magnetic particles (beads) in which a substance that specifically reacts with a binding molecule of a particle to be labeled is immobilized on the surface. Examples of the substance include avidin, streptavidin, neutravidin, an antibody, and an antigen in one or a plurality of embodiments. The size of the magnetic beads is not particularly limited, and can be appropriately determined according to the size of the particles and / or the size of the opening (hole) of the filter used for magnetic labeling. When the size of the particles to be labeled is 5 μm to 20 μm in diameter, such as white blood cells or CTCs, in one or a plurality of embodiments, 1 μm or less, 0.8 μm or less, or 0. 5 μm or less. From the viewpoint of improving the magnetic separation efficiency, it is 0.05 μm or more, or 0.1 μm or more. From the viewpoint that excess magnetic beads can be removed by washing after magnetic labeling, the size of the magnetic beads is smaller than the size of the opening (hole) of the filter used for magnetic labeling.

  In the labeling method of the present disclosure, a part of a suspension supplied to a filter surface (particle capturing surface or filter upper surface) on which particles are captured is transferred to the other filter surface (the filter surface on the side opposite to the particle capturing surface ( Liquid flow toward the opposite surface or the lower surface of the filter), thereby bringing the liquid containing the magnetic beads into contact with both surfaces of the filter (both the particle capturing surface and the opposite surface). In addition, in one or a plurality of embodiments, a part of the suspension is supplied to the lower surface of the filter so that the upper liquid surface and the lower liquid surface of the liquid sandwich the filter. It may include feeding toward the side. In one or a plurality of embodiments, the liquid feeding is performed so that the liquid containing the magnetic beads is held in the vicinity of the opposite filter surface. In one or more embodiments, the liquid feeding may be performed by pumping from the opposite side so that the suspension passes through the filter and the liquid containing the magnetic beads contacts both sides of the filter. it can. That is, according to the labeling method of the present disclosure, a part of the suspension is actively fed toward the filter surface opposite to the particle capturing surface, so that the liquid unintentionally penetrates or diffuses into the filter. This is different from the phenomenon that the liquid reaches the vicinity of the lower surface of the filter.

  The liquid containing magnetic beads is not particularly limited as long as the liquid contains magnetic beads. In one or a plurality of embodiments, the suspension containing the magnetic beads supplied to the filter surface, and the suspension are provided. Examples include a liquid in which a turbid liquid is diluted with another liquid. The concentration (solid content concentration) of the magnetic beads in the liquid containing the magnetic beads is 0.00009% or more, 0.00012% or more, or 0.00021% or more, or 0.0581% in one or more embodiments. Hereinafter, it is 0.0415% or less or 0.00249% or less. Other liquids include, in one or more embodiments, phosphate buffered saline (PBS), PBS-BSA (eg, 0.2%), PBS-EDTA (eg, 1 mg / ml) and buffer, isotonic. Examples thereof include aqueous solvents such as liquid and water. Examples of the buffer solution include Tris buffer solution such as Tris-HCl buffer solution, phosphate buffer solution, HEPES buffer solution, citrate-phosphate buffer solution and the like in one or a plurality of embodiments. Examples of the isotonic solution include physiological saline, sucrose isotonic solution, glucose isotonic solution, mannose isotonic solution, and the like.

  In one or a plurality of embodiments, the ratio of the suspension to be fed to the opposite surface side is the suspension supplied to the particle trapping surface from the viewpoint that both surfaces of the filter are sufficiently in contact with the liquid containing magnetic beads. 10% or more, 20% or more, or 40% or more of the liquid, and 90% or less from the point of efficiently sending all of the suspension when feeding from the opposite surface side to the particle capturing surface side 80% or less, or 60% or less.

  In one or a plurality of embodiments, the flow rate of the suspension is 0.07 mm / second or more and 0.1 mm / second per opening area of the filter from the viewpoint of improving the reaction efficiency between the particles captured by the filter and the magnetic beads. Or 0.3 mm / second or more, and from the viewpoint of improving the reaction efficiency between the particles trapped in the filter and the magnetic beads, 14 mm / second or less, 1.4 mm / second or less or 0. 7 mm / second or less. The flow rate of the suspension is 0.05 μL / min or more, 0.1 μL / min or more, or 0.2 μL / min or more from the viewpoint of improving the reaction efficiency between the particles captured by the filter and the magnetic beads. From the viewpoint of improving the reaction efficiency between the captured particles and the magnetic beads, it is 10000 μL / min or less, 1000 μL / min or less, or 500 μL / min or less. The “flow rate per opening area of the filter” in the present disclosure refers to the velocity of the liquid passing through the opening (hole) of the filter. In one or a plurality of embodiments, the flow velocity per opening area can be calculated by dividing the flow rate of the entire system by the total area of the openings (holes). The “flow rate” in the present disclosure refers to the amount (volume) of liquid that flows per unit time. Examples of the flow rate include the amount (volume) of the liquid that passes through the filter in a unit time in one or a plurality of embodiments.

  The labeling method of the present disclosure is directed to the filter surface side (from the filter lower surface side to the upper surface side) on which the particles are captured from the filter surface side opposite to the particle capturing surface with respect to the filter having both surfaces immersed in the suspension. It includes feeding (returning liquid). A suspension containing magnetic beads is sent to the particle capture surface side from the opposite surface side to a filter in which both main surfaces are in contact with the liquid containing magnetic beads (immersed state). By allowing the particles to flow and releasing the particles captured by the filter from the filter, the reactivity between the magnetic beads and the particles to be labeled can be improved and the labeling efficiency can be improved.

  In one or a plurality of embodiments, the backflow may feed only the suspension located on the filter surface side (filter lower surface side) opposite to the particle capturing surface, or may be a liquid different from the suspension. May be fed. The liquid different from the suspension is, in one or more embodiments, phosphate buffered saline (PBS), PBS-BSA (eg 0.2%), PBS-EDTA (eg 1 mg / ml) and buffer. And aqueous solvents such as isotonic solutions and water. Examples of the buffer solution include Tris buffer solution such as Tris-HCl buffer solution, phosphate buffer solution, HEPES buffer solution, citrate-phosphate buffer solution and the like in one or a plurality of embodiments. Examples of the isotonic solution include physiological saline, sucrose isotonic solution, glucose isotonic solution, mannose isotonic solution, and the like.

  In one or a plurality of embodiments, the liquid feeding flow rate from the opposite surface side to the particle capturing surface side (reverse flow velocity) may be appropriately determined according to the flow rate of the specimen when the particles are captured by the filter. In one or a plurality of embodiments, the reverse flow velocity may be higher than the flow velocity of the specimen, or may be slower than the flow speed of the specimen. In one or a plurality of embodiments, the reverse flow rate is 1/10 or more times the sample flow rate, 1/5 or more flow rate, 1/2 or more flow rate, 1 or more times flow rate, 2 or more times flow rate. Examples include a flow rate, a flow rate of 3 times or more, or a flow rate of 5 times or more. In one or a plurality of embodiments, the reverse flow rate may be higher than the flow rate of the specimen from the viewpoint of improving the labeling efficiency with the magnetic beads.

  In one or a plurality of embodiments, the flow rate of liquid feeding from the opposite surface side to the particle capturing surface side is 0.01 mm / second or more per filter opening area, in order to further improve the labeling efficiency with the magnetic beads. It is 1 mm / second or more or 1 mm / second or more, and is 150,000 mm / second or less, 100000 mm / second or less, or 50000 mm / second or less per opening area of the filter from the viewpoint of suppressing damage to cells. The back flow rate is 10 μL / min or more, 100 μL / min or more, or 1000 μL / min or more from the viewpoint of improving the labeling efficiency by magnetic beads, and 100 million μL / min or less, 50 million from the viewpoint of suppressing damage to cells. μL / min or less or 10 million μL / min or less.

In one or a plurality of embodiments, the amount of the liquid to be reversed is 50 μL or more or 100 μL or more per filtration area (28 mm ² ) from the viewpoint of efficiently feeding the liquid containing the magnetic beads to the particle capturing surface, or From the viewpoint of maintaining the concentration of the suspension, it is 1000 μL or less, 500 μL or less, or 300 μL or less.

  The number of times of immersing the filter in the suspension and sending the liquid from the opposite surface side to the particle capturing surface side (back flow) is not particularly limited, and may be one or two or more times. It may be.

  In one or a plurality of embodiments, the labeling method of the present disclosure further stirs the liquid containing the magnetic beads on the particle capturing surface and the particles to be labeled after the above-described reverse flow, in order to further improve the efficiency of labeling with magnetic beads. It may include. Stirring includes pipetting in one or more embodiments.

  The labeling method of the present disclosure may include standing after stirring in one or a plurality of embodiments from the viewpoint of further improving the efficiency of labeling with magnetic beads. In one or a plurality of embodiments, the standing time is 1 minute or more, 5 minutes or more, or 10 minutes or more from the point of sufficiently reacting the particles with the magnetic beads, and the non-specific reaction between the particles and the magnetic beads is performed. From the point to prevent, it is 60 minutes or less, 45 minutes or less, or 30 minutes or less.

  In one or more embodiments, the labeling method of the present disclosure further improves the efficiency of labeling with magnetic beads. In one or a plurality of embodiments, after pipetting, the liquid containing the magnetic beads and the particles to be labeled is filtered and then filtered. It may include standing still. Thereby, the particles previously captured by the filter containing the particles to be labeled can be captured again by the filter, and the particles and the magnetic beads can be contacted again. In one or a plurality of embodiments, the standing is preferably performed in a state where both surfaces of the filter are covered with a liquid containing magnetic beads and particles to be labeled.

  In one or a plurality of embodiments, the flow rate of the filter filtration is 0.07 mm / second or more, 0.1 mm / second per opening area of the filter from the viewpoint of improving the reaction efficiency between the particles captured by the filter and the magnetic beads. From the point of improving the reaction efficiency between the particles trapped in the filter and the magnetic beads, it is 14 mm / second or less, 1.4 mm / second or less, or 0. 0.7 mm / second or less. The flow rate of the suspension is 0.05 μL / min or more, 0.1 μL / min or more, or 0.2 μL / min or more from the viewpoint of improving the reaction efficiency between the particles captured by the filter and the magnetic beads. From the viewpoint of improving the reaction efficiency between the captured particles and the magnetic beads, it is 10000 μL / min or less, 1000 μL / min or less, or 500 μL / min or less.

  In one or a plurality of embodiments, the standing time is 1 minute or more, 5 minutes or more, or 10 minutes or more from the point of sufficiently reacting the particles with the magnetic beads, and the non-specific reaction between the particles and the magnetic beads is performed. From the point to prevent, it is 60 minutes or less, 45 minutes or less, or 30 minutes or less.

  In one or a plurality of embodiments, the labeling method of the present disclosure may include feeding a cleaning solution in order to wash excess magnetic beads that have not been used for labeling. In one or more embodiments, the washing solution includes phosphate buffered saline (PBS), PBS-BSA (eg, 0.2%), PBS-EDTA (eg, 1 mg / ml) and buffer, isotonic solution, and An aqueous solvent such as water may be mentioned. Examples of the buffer solution include Tris buffer solution such as Tris-HCl buffer solution, phosphate buffer solution, HEPES buffer solution, citrate-phosphate buffer solution and the like in one or a plurality of embodiments. Examples of the isotonic solution include physiological saline, sucrose isotonic solution, glucose isotonic solution, mannose isotonic solution, and the like.

  In one or more embodiments of the labeling method of the present disclosure, particles may be captured in advance on a filter, or particles may be captured using a filter prior to supplying a suspension containing magnetic beads. May be included. In one or a plurality of embodiments, capture of particles by a filter can be performed by supplying a sample containing particles to be labeled to the filter and filtering the sample.

  In one or a plurality of embodiments, the flow rate of the specimen improves the capture rate of particles in the specimen. In one or more embodiments, the specimen flow rate is 0.01 mm / second or more, 0.05 mm / second or more, or 0.1 mm / second. From the same point, it is 100 mm / second or less, 50 mm / second or less, 25 mm / second or less, 10 mm / second or less, 5 mm / second or less, or 2 mm / second or less. The flow rate of the specimen is 0.01 ml / min or more, 0.05 ml / min or more, or 0.1 ml / min or more in one or more embodiments from the viewpoint of improving the capture rate of particles in the specimen, From the same point, it is 100 ml / min or less, 50 ml / min or less, 25 ml / min or less, or 10 ml / min or less.

  In one or more embodiments of the labeling method of the present disclosure, prior to the supply of the suspension containing magnetic beads, the liquid is filled in the flow path on the filter surface side opposite to the filter surface on which the particles are captured. May include. In one or a plurality of embodiments, the liquid may be filled so that a filter in which particles are trapped is immersed in the liquid. Examples of the liquid include a liquid that can be used as the above-described cleaning liquid in one or a plurality of embodiments.

  In one or a plurality of embodiments, the labeling method of the present disclosure can be performed using a device having a separation unit including a filter having a plurality of through holes. In one or a plurality of embodiments, the labeling method of the present disclosure can improve operability and can efficiently label even a small amount of magnetic bead suspension. It is preferable to carry out with this apparatus.

  In the labeling method of the present disclosure, the above-described processing such as supply of a suspension, liquid feeding, and stirring may be performed automatically or manually in one or a plurality of embodiments. Alternatively, some processes may be performed manually and the remaining processes may be performed automatically.

  Examples of the filter used in the labeling method of the present disclosure include a filter capable of capturing rare cells in a specimen in one or a plurality of embodiments. In one or a plurality of embodiments, the filter is preferably a filter capable of capturing at least small cancer cells, highly deformable cancer cells, or both of these cells. In one or a plurality of embodiments, the filter is preferably a filter capable of capturing at least SNU-1, SW620, or both, and more preferably a filter capable of capturing CTC in a specimen. As the filter, in one or a plurality of embodiments, a conventionally known filter or a filter capable of capturing rare cells that will be developed in the future can be used. Examples of the filter include a filter described in Patent Document 1 and a filter described in Japanese Patent Application No. 2006-057313 in one or more embodiments. Moreover, for example, a sheet-like single membrane including a large number of through-holes such as an ellipse, a perfect circle or a rectangle having a minor axis diameter of 1 μm to 50 μm can be mentioned, and a material other than a nonwoven fabric is preferable.

  In one or a plurality of embodiments, the material of the filter may be a metal such as nickel, SUS, gold, silver, barrel, aluminum, tungsten, or chromium.

The filtration area is not particularly limited, and in one or more embodiments, it is 5 mm ² or more, or 10 mm ² or more, or 2,000 mm ² or less, 1,000 mm ² or less, 500 mm ² or less, 200 mm ^2. Hereinafter, it is 150 mm ² or less, 100 mm ² or less, 80 mm ² or less, 50 mm ² or less, 40 mm ² or less, 30 mm ² or less, or 25 mm ² or less. The “filtration area” in the present disclosure refers to the area of the entire area of the filter where the suspension or specimen containing the magnetic beads contacts (sends).

  In one or a plurality of embodiments, the particles labeled by the labeling method of the present disclosure can be separated from the target particles by a known magnetic separation device.

In one or a plurality of embodiments, the labeling method of the present disclosure performs labeling according to the characteristics of the particles and the protein expressed in the particles even when a plurality of types of particles are captured by the filter. The particles of interest can be specifically labeled. In one or a plurality of embodiments, even when rare cells and leukocytes are captured by a filter in the analysis of rare cells such as CTC, according to the labeling method of the present disclosure, leukocytes can be magnetized with high labeling efficiency. Can be labeled. For this reason, after labeling by the labeling method of the present disclosure, the collected suspension can be magnetically separated, whereby the rare cells and the magnetically labeled leukocytes can be separated with high accuracy, and thereby the rare cells can be collected with high accuracy. it can. In one or a plurality of embodiments in which rare cells and leukocytes are captured by the filter, the rare cells may be magnetically labeled instead of leukocytes.
In one or a plurality of embodiments, recovery after labeling can be performed by feeding the recovery liquid in a direction opposite to the filtration direction at the time of particle capture by the filter. In one or a plurality of embodiments, the collection liquid is collected from the direction opposite to the flow direction of the specimen for cell capture on the filter in which the cells are captured, as in the cell collection method described later. Pass the liquid so that the ratio of the liquid flow rate to the sample flow rate ([collected liquid flow rate] / [specimen flow rate]) is 25 or more, or a flow rate of 1 mm / second or more per opening area. This can be done by passing the solution through. Specific conditions and the like are the same as in the cell collection method described later.

  The particles in the present disclosure are not particularly limited, and examples thereof include human or non-human animal cells. Examples of cells that are not particularly limited include rare cells, leukocytes, erythrocytes, platelets, and their undifferentiated cells. Rare cells refer to cells other than blood cell components (red blood cells, white blood cells, and platelets) that can be contained in the blood of humans or non-human animals. In one or more embodiments, rare cells include cancer cells, circulating tumor cells, vascular endothelial cells, vascular endothelial progenitor cells, cancer stem cells, epithelial cells, hematopoietic stem cells, mesenchymal stem cells, fetal cells, stem cells, Examples include cells selected from the group consisting of undifferentiated leukocytes, undifferentiated red blood cells, and combinations thereof.

  Examples of the specimen in the present disclosure include a sample that may contain particle cells that can be applied to the labeling method of the present disclosure. Examples of the specimen include a blood specimen in one or a plurality of embodiments. In one or a plurality of embodiments that are not limited, the blood sample includes blood or a diluted product thereof from the viewpoint of simple and rapid processing and the suppression of damage to rare cells in blood.

[Marking device]
In one aspect, the present disclosure relates to an apparatus for labeling particles with magnetic beads (a labeling apparatus of the present disclosure). The labeling device of the present disclosure includes a filter unit that can hold a filter, an introduction channel that can introduce a suspension containing magnetic beads in the filter unit, and a discharge channel that can hold or discharge the liquid that has passed through the filter unit. And a liquid feeding means capable of supplying the suspension to the filter unit, and a control unit for controlling the liquid feeding means. In the labeling device of the present disclosure, the control unit feeds the suspension so that at least a part of the suspension supplied to the filter unit is held through the filter unit, and passes the magnetic field that has passed therethrough. The liquid feeding means is controlled so as to reversely feed the liquid containing the beads. According to the labeling device of the present disclosure, the labeling method of the present disclosure can be easily performed.

  In one or a plurality of embodiments, the labeling device of the present disclosure may include a stirring unit that stirs the liquid on the surface of the filter or in the vicinity thereof.

  In one or a plurality of embodiments, the control unit may control the liquid feeding unit so as to supply the specimen including the particles to be labeled to the filter unit. The control unit may control the liquid feeding means so as to feed the specimen at a flow rate of 0.01 mm / second to 100 mm / second per opening area of the filter. The control unit may control the liquid feeding means so that the flow rate of the reverse feeding liquid is faster than 1/10 of the flow speed of the specimen. The control unit may control the liquid feeding unit so that the liquid feeding flow rate of the reverse feeding is 0.01 mm / second or more per opening area of the filter.

  Hereinafter, a non-limiting embodiment of the labeling method of the present disclosure will be described.

  Description will be made using the rare cell labeling apparatus shown in FIG.

An example of a rare cell labeling apparatus that can be used in the labeling method of the present disclosure is shown in FIG.
A rare cell labeling apparatus shown in FIG. 1 includes a supply port 1 for supplying a suspension containing magnetic beads, a cleaning solution and a staining solution, a discharge port 2 for discharging a waste solution such as a cleaning solution, an upper part 3 of the device, It has the device lower part 4, the filter part 5, and the liquid feeding mechanism (not shown) which can be fed in the arrow A and B direction. The filter unit 5 is disposed between the device upper part 3 and the device lower part 4. The device upper part 3 includes a flow path 6 and a support member 7 for fixing the filter unit 5, and the flow path 6 communicates with the supply port 1. The device lower part 4 includes a flow path 8 and a support member 9 for fixing the filter unit 5, and the flow path 8 communicates with the discharge port 2. The filter unit 5 includes a filter 10 and bonding materials (for example, double-sided tape) 11 and 12 for fixing the filter 10. The filter 10 is fixed to the device upper part 3 and the device lower part 4 by bonding materials 11 and 12. In the flow path 6 that connects the supply port 1 and the filter unit 5, a connection unit (not shown) and a three-way cock valve (not shown) capable of switching the liquid may be arranged on the upper part of the filter unit 5. . Further, in the flow path 6 connecting the supply port 1 and the filter unit 5, as a mechanism capable of supplying liquid at a constant pressure or a mechanism capable of supplying liquid at a constant flow rate so that the pressure applied to filtration is constant. A) pressurizing means (not shown) may be arranged. Moreover, you may pull with a syringe pump so that the pressure concerning filtration may become fixed level or more.

  Next, an example of a particle labeling method using the rare cell labeling apparatus of FIG. 1 will be described with reference to FIG. In this embodiment, a filter capable of capturing rare cells is used as a filter, and the whole blood containing the rare cells is filtered with the filter, whereby the leukocytes captured by the filter are labeled with magnetic beads as an example. I will explain to you. In FIG. 2, the gray portion schematically shows a portion including magnetic beads. Note that the present disclosure is not limited to the embodiment.

  First, the blood sample is sent to the separation unit through the flow path 6 and filtered by the filter 10. As a result, rare cells and leukocytes are captured by the filter 10.

  Next, from the viewpoint of improving the reactivity of the reagent with respect to leukocytes, the red blood cells remaining on the filter 10 may be removed by supplying a hemolytic agent and processing.

  Next, a buffer solution is supplied from the supply port 1 to fill the channel 8 with the buffer solution. The buffer solution is supplied so that the cell capturing surface (upper surface) 10a of the filter 10 is immersed. Next, a suspension 21 containing magnetic particles is supplied to the cell capturing surface 10a of the filter 10 (FIG. 2 (a)). Prior to the supply of the suspension 21, the white blood cells may be immunostained with an antibody in order to improve the reactivity between the magnetic particles and the white blood cells. Examples of the antibody include an antibody having a binding molecule bound thereto. Examples of the binding molecule include biotin and the like in one or a plurality of embodiments. Moreover, as the magnetic particles, for example, magnetic particles on which a substance that specifically reacts with the binding molecule is immobilized may be used. Examples of the substance that specifically reacts include, in one or more embodiments, proteins such as streptavidin and neutravidin.

  The suspension 21 is passed through the cells of the filter 10 so that a part of the suspension 21 containing magnetic particles moves to the filter surface (lower surface) 10b side (channel 8 side) opposite to the cell capture surface 10a. Liquid is fed from the capturing surface 10a side toward the filter surface 10b side (in the direction of arrow A in FIGS. 1 and 2) (FIG. 2B).

  Next, the liquid is fed (reversely fed) from the filter surface 10b side toward the cell capture surface 10a side (in the direction of arrow B in FIGS. 1 and 2) (FIG. 2 (c)). As a result, the cells released from the filter 10 are agitated on the cell capturing surface (upper surface) 10a of the filter 10, and the liquid containing the magnetic particles on the cell capturing surface 10a of the filter 10 is agitated. The frequency of contact with magnetic particles can be improved, and the labeling efficiency can be improved. Only the suspension located on the filter surface 10b side may be fed back, or a different liquid may be fed back according to the suspension. The different liquids may contain magnetic particles or may not contain magnetic particles.

  Next, on the cell capturing surface 10a of the filter 10, the suspension containing the magnetic particles and the cells is stirred with a pipette or the like, and then allowed to stand for a predetermined time. Thereby, the contact frequency of leukocytes and magnetic particles can be further improved, and the labeling efficiency can be improved.

  Next, the suspension 23 containing cells and magnetic particles on the cell capturing surface 10a of the filter 10 is directed from the cell capturing surface 10a side to the opposite filter surface 10b side (in the direction of arrow A in FIGS. 1 and 2). ) The liquid is fed again and then left for a predetermined time (FIG. 2 (d)). Thereby, the cells contained in the suspension are captured by the filter 10. In addition, the magnetic particles pass through the filter 10, but when passing through the filter 10, the magnetic particles can be brought into contact with the cells captured by the filter 10 again, thereby further improving the contact frequency between the white blood cells and the magnetic particles. And the labeling efficiency can be improved. The liquid feeding is preferably performed such that the cell capturing surface 10a and the opposite filter surface 10b of the filter 10 are sandwiched by a liquid containing magnetic particles.

  Then, after the liquid 23 containing magnetic particles is lowered to the vicinity of the cell capturing surface 10a of the filter 10, the cleaning liquid 24 is supplied from the cell capturing surface 10a side of the filter 10 (in the direction of arrow A in FIGS. 1 and 2) (FIG. 2 ( e)) The liquid is fed so that the liquid level of the cleaning liquid 24 is close to the cell capturing surface 10a of the filter 10 (FIG. 2 (f)). Thereby, unreacted magnetic particles remaining on the filter 10 are removed. The liquid feeding of the cleaning liquid 24 may be repeated a plurality of times.

  After labeling leukocytes with the magnetic particles, rare cells may be immunostained with antibodies. Thereby, the detection of rare cells after separation and collection can be performed efficiently. Examples of the antibody include an antibody recognizing an antigen expressed in CTC cells such as Cytokeratin. From the viewpoint of reacting an antibody with an antigen in a rare cell, an immobilization treatment and a membrane permeation treatment may be performed prior to labeling with the antibody.

  Finally, the collected liquid containing cells (white blood cells and rare cells) captured by the filter 10 is sent onto the cell capturing surface 10a by sending the collected liquid from the filter surface 10b side (in the direction of arrow B in FIGS. 1 and 2). And collect it using a pipette or the like. By separately performing magnetic separation on the collected recovered liquid, leukocytes labeled with magnetic particles and rare cells can be separated.

The present disclosure may relate to one or more of the following embodiments.
[1] A method of labeling particles captured by a filter having a plurality of through holes with magnetic particles,
Supplying a suspension containing magnetic particles to the filter surface on which the particles are trapped;
By sending a part of the suspension from the filter surface in the direction of the other filter surface so that the liquid containing the magnetic particles is in contact with both surfaces of the filter; and
A magnetic labeling method comprising: feeding back a liquid containing the magnetic particles from the other filter surface in the direction of the filter surface where the particles are captured.
[2] The method according to [1], wherein the liquid containing the magnetic particles is stirred on the filter surface on which the particles are captured after liquid feeding from the other filter surface to the filter surface on which the particles are captured. Labeling method.
[3] The labeling method according to [1] or [2], comprising feeding the specimen containing the particles to the filter so that the particles are captured by the filter.
[4] The labeling method according to [3], wherein the flow rate of the specimen is 0.01 mm / second to 100 mm / second per opening area of the filter.
[5] The labeling method according to [3] or [4], wherein the reverse feeding flow rate is faster than 1/10 of the flow rate of the specimen.
[6] The labeling method according to any one of [1] to [5], wherein the reverse feeding flow rate is 0.01 mm / second or more per opening area of the filter.

In addition to the above method, the present disclosure provides the following apparatus. It goes without saying that the following apparatus is clearly and fully disclosed by those skilled in the art in the above description.
(A1) An apparatus for labeling particles captured by a filter having a plurality of through holes with magnetic particles,
Means for supplying a suspension containing magnetic particles to the upper surface of the filter on which the particles are trapped;
Means for feeding a part of the suspension toward the lower surface side of the filter so that the upper liquid surface and the lower liquid surface of the suspension sandwich the filter; Means for feeding liquid from the lower surface side toward the upper surface side.
(A2) The labeling device according to (A1), including means for stirring the suspension on the upper surface of the filter after feeding from the lower surface side to the upper surface side of the filter.
(A3) The labeling apparatus according to (A1) or (A2), including means for feeding the specimen containing the particles to the upper surface of the filter.
(A4) The labeling apparatus according to (A3), wherein the specimen liquid feeding means feeds the specimen so that the flow rate of the specimen is 0.01 mm / second to 100 mm / second per opening area of the filter.
(A5) The means for sending the liquid from the lower surface side to the upper surface side of the filter sends the liquid flow rate from the lower surface side to the upper surface side of the filter so that it is faster than 1/10 of the flow rate of the sample. The labeling device according to (A3) or (A4).
(A6) The means for feeding liquid from the lower surface side to the upper surface side of the filter is such that the liquid flow rate from the lower surface side to the upper surface side of the filter is 0.01 mm / second or more per opening area of the filter. The labeling device according to any one of (A1) to (A5), which sends liquid.
[B1] An apparatus for labeling particles with magnetic particles,
A filter section capable of holding a filter;
An introduction flow path capable of introducing a suspension containing magnetic particles into the filter section;
A discharge channel capable of holding or discharging the liquid that has passed through the filter unit;
Liquid feeding means capable of supplying the suspension to the filter unit;
A controller for controlling the liquid feeding means,
The control unit sends a part of the suspension from one filter surface to the other filter surface so that the liquid containing the magnetic particles is in contact with both surfaces of the filter, and A labeling device for controlling the liquid feeding means so as to reversely feed a liquid containing magnetic particles from the other filter surface.
[B2] The labeling device according to [B1], including means for stirring the liquid.
[B3] The labeling device according to [B1] or [B2], including means for stirring the liquid on or near the filter surface.
[B4] The labeling device according to any one of [B1] to [B3], wherein the control unit controls the liquid feeding unit so as to supply a specimen including the particles to be labeled to the filter unit.
[B5] The labeling apparatus according to [B4], wherein the control unit controls the liquid feeding unit so that the specimen is fed at a flow rate of 0.01 mm / second to 100 mm / second per opening area of the filter.
[B6] The labeling apparatus according to [B4] or [B5], wherein the control unit controls the liquid feeding unit so that the flow rate of the reverse feeding solution is faster than 1/10 of the flow rate of the specimen.
[B7] The control unit controls the liquid feeding unit so that a liquid feeding flow rate of the reverse feeding is 0.01 mm / second or more per opening area of the filter. [B1] to [B6] The labeling device according to any one of the above.

[Method for recovering cells in specimen]
In order to analyze rare cells with high accuracy, it is necessary to collect cells captured by the filter with high accuracy. Thus, in another aspect, the present disclosure relates to a method capable of recovering cells captured by a filter from the filter with a high recovery rate.

  As a method for separating or recovering a specific component from blood, for example, Patent Document 1 discloses a method in which blood is treated with a filter having predetermined characteristics, and rare cells in the blood are captured by the filter and analyzed. It is disclosed.

  The collection of cells from body fluids is also performed for uses other than inspection. For example, Japanese Patent Laid-Open No. 10-201470 (Patent No. 4412621) discloses a method of recovering target cells from a body fluid such as bone marrow for hematopoietic stem cell transplantation. In the examples of JP-A-10-2014470, bone marrow or peripheral blood is introduced into a nonwoven fabric, and after white blood cells or CD34 positive cells are captured with the nonwoven fabric, a viscosity of 17.2 mPa · s to 31.8 mPa · s is obtained. Disclosed is a method for recovering leukocytes or CD34-positive cells trapped in a nonwoven fabric by reversing the liquid that it has.

However, in the method disclosed in JP-A-10-201470, the viscosity of the recovered liquid used for recovery needs to be 5 mPa · s or more and 31.8 mPa · s or less, and there is a problem that the composition used for the recovered liquid is limited. is there. Further, as described above, since JP-A-10-201470 is a method for separating and collecting cells for hematopoietic stem cell transplantation and the like, the cells captured by the capturing means can be collected with a large amount of collected liquid. Therefore, there is a problem that the recovered cells are not suitable for use in analysis.
Patent Document 1 does not disclose a technique for collecting rare cells captured by a filter.

By using a predetermined filter, the present applicant has established a method capable of capturing small cells such as SW620 and cells having high deformability such as SNU-1 at a high capture rate as described above. (For example, Patent Document 1).
On the other hand, the analysis of rare cells is performed, for example, by observing rare cells, counting the number, and measuring nucleic acids extracted from the rare cells. In order to perform these analyzes with high accuracy, it is necessary to separate and collect the rare cells captured by the filter from the filter.

  In collecting the rare cells from the filter in which the rare cells are captured in the process of earnest research, the present inventors have made a ratio of the flow rate of the collected liquid to the flow rate of the collected liquid when the rare cells are captured ( It has been found that cells can be recovered with a high recovery rate and a small amount of recovery liquid by allowing the recovery liquid to flow backward so that [flow rate of recovery liquid] / [flow rate of specimen] is 25 or more. In addition, the present inventors captured the rare cells in a filter while suppressing damage to the cells by causing the collected liquid to flow backward at a flow rate of 1 mm / second or more per filter opening area. It has been found that cells can be recovered at a high recovery rate.

  As one of the reasons why the rare cells captured by the filter cannot be collected with a high recovery rate and a small amount of the collected liquid, the present inventors have some rare cells and other mixed cells at the time of filtration (capture) of the rare cells. The collected filter contains both closed and non-closed holes. As a result, the rare cells trapped in the closed holes have sufficient force to push the recovered liquid through. Found that it does not act on. Then, the collected liquid is passed through from the direction opposite to the specimen flow direction for cell capture, and the flow rate is changed to the filter surface through which the collected liquid is passed (opposite to the cell capture surface of the filter). It was found that the rare cells captured by the filter can be recovered with a high recovery rate and a small amount of recovery liquid by setting the flow rate at which turbulent flow is generated on the filter surface on the side.

In JP-A-10-201470, 50 ml of peripheral whole blood is passed through a filter made of polyester nonwoven fabric at a flow rate of about 5 ml / min, and then 30 ml of a recovered solution having a viscosity exceeding 17 mPa · s is passed at 100 ml / min. It is disclosed that the cells captured by the filter are collected by doing (Examples 1 to 5). On the other hand, the document also discloses that when a recovered liquid (physiological saline) having a viscosity of 1.0 mPa · s is used instead of the recovered liquid having the above viscosity, the recovery rate is 31% or less. (Comparative Examples 1 and 2).
In contrast, according to the method of the present disclosure, the ratio of the flow rate of the collected liquid to the flow rate of the sample at the time of cell capture or the flow rate of the collected liquid is set to a specific range, and a specific filter (multiple filters) By using a filter having a through-hole, the cells captured by the filter can be recovered at a high recovery rate regardless of the viscosity of the recovery solution. Furthermore, the method of the present disclosure can realize that even a recovered liquid having a viscosity that cannot be sufficiently recovered in Japanese Patent Laid-Open No. 10-201470 can be recovered with a high recovery rate. That is, the method of the present disclosure is a technology that is completely different from the technical idea of Japanese Patent Laid-Open No. 10-201470.
The method disclosed in JP-A-10-201470 is a method for selectively recovering leukocytes selectively from a mixture of leukocytes, platelets and erythrocytes for hematopoietic stem cell transplantation. As a means for capturing cells (white blood cells), those having a large surface area per volume such as a nonwoven fabric are preferably exemplified. On the other hand, the method of the present disclosure is a technique that can be suitably used for collecting rare cells captured by a filter having a plurality of through holes for analysis and diagnosis. That is, the purpose of the method disclosed in JP-A-10-2014470 is completely different from that of the method disclosed herein. Further, the nonwoven fabric used in JP-A-10-201470 and the filter having a plurality of through-holes used in the present disclosure are greatly different in the mechanism of capturing cells and the captured state. The method and the Japanese Patent Laid-Open No. 10-201470 are completely different technical ideas.

  In the present disclosure, the “fluid flow rate” refers to the amount (volume) of the liquid that flows per unit time. Examples of the flow rate of liquid flow include the amount (volume) of the liquid that passes through the filter in a unit time in one or a plurality of embodiments.

  In the present disclosure, the “flow rate per opening area of the filter” refers to the velocity of the liquid passing through the opening (hole) of the filter. In one or a plurality of embodiments, the flow rate per opening area can be calculated by dividing the liquid flow rate by the total area of the openings (holes).

[Cell recovery method]
In one aspect, the present disclosure relates to a method of recovering cells captured by a filter having a plurality of through holes from the filter (a recovery method of the present disclosure). In one aspect of the collection method of the present disclosure, the flow rate of the collected liquid and the flow rate of the sample are passed through the filter in which the cells are captured from the direction opposite to the direction of flow of the sample for capturing the cells. And the ratio ([flow rate of recovered liquid] / [flow rate of specimen]) is 25 or more, and the recovered liquid collected on the filter is recovered. Further, in one aspect of the collection method of the present disclosure, the cell is captured at a flow rate of 1 mm / second or more per opening area from the direction opposite to the direction in which the specimen is passed for capturing the collection liquid. And passing through the filter, and collecting the collected liquid collected on the filter.

  According to the present disclosure, in one or a plurality of embodiments, cells captured by a filter can be recovered at a high recovery rate. According to the present disclosure, in one or a plurality of embodiments, cells captured by the filter can be recovered at a high recovery rate while suppressing damage to the cells. In addition, according to the present disclosure, in one or a plurality of embodiments, cells can be collected with a small amount of collection liquid (for example, a level that can be stored in a microtube).

  The collection method of the present disclosure includes passing the collected liquid through a filter having a plurality of through-holes in which cells are captured from a direction opposite to the direction in which the specimen is captured for capturing the cells. . In one or a plurality of embodiments, the ratio of the flow rate of the collected liquid to the flow rate of the sample ([flow rate of the collected liquid] / [flow rate of the sample]) is 25 or more. Or by passing the solution at a flow rate of 1 mm / second or more per opening area. In one or a plurality of embodiments, the cells captured by the filter can be collected at a high recovery rate while suppressing the damage to the cells in one or a plurality of embodiments by causing the collected liquid to flow back to the filter having a plurality of through holes. It can be recovered. In the recovery method of the present disclosure, “flowing in the reverse direction (reverse flow)” means introducing the recovery liquid from the filter surface on the side opposite to the filter surface on which the sample is introduced in order to capture cells. Alternatively, it can also be said that the recovery liquid is caused to flow from the side (downstream side) from which the specimen flows out toward the side (upstream side) from which the specimen flows in when the specimen is passed.

  The ratio of the flow rate of the collected liquid to the flow rate of the sample ([flow rate of the collected liquid] / [flow rate of the sample]) is 25 or more in one or more embodiments, and the cells are collected from the filter. From the point which improves a rate, they are 100 or more, 150 or more, 200 or more, 300 or more, or 400 or more. From the viewpoint of suppressing damage to cells, it is 100,000 or less, 50,000 or less, or 25,000 or less. Moreover, from the point which further improves the collection | recovery rate of a cell, it is 1,000 or more, 1,200 or more, 1,500 or more, 2,000 or more, 2,200 or more, or 4,000 or more, From the same point, 15 , 1,000 or less, 12,000 or less, or 10,000 or less.

  In one or more embodiments, the flow rate of the collected liquid is 25 ml / min or more, 50 ml / min or more, 100 ml / min or more, 110 ml / min or more, or 120 ml / min in one or a plurality of embodiments. That's it. From the viewpoint of suppressing damage to cells, it is 100,000 ml / min or less, 50,000 ml / min or less, or 10,000 ml / min or less. From the point of further improving the cell recovery rate, it is 300 ml / min or more, 500 ml / min or more, or 550 ml / min or more, and from the same point, it is 7,000 ml / min or less, 6,000 ml / min or less, 5, 000 ml / min or less, 4,000 ml / min or less, 3,000 ml / min or less, 2,000 ml / min or less, or 1,000 ml / min or less.

  In one or a plurality of embodiments, the flow rate of the collected liquid is such that turbulence is generated on the filter surface opposite to the filter surface on which the specimen is introduced in order to capture the cells, and the cell recovery rate from the filter 1 mm / second or more, 5 mm / second or more, 10 mm / second or more, 15 mm / second or more, 20 mm / second or more, 25 mm / second or more, 30 mm / second or more, or 35 mm / second or more per opening area It is. From the viewpoint of suppressing damage to cells, it is 100,000 mm / second or less, 50,000 mm / second or less, 30,000 mm / second or less, 10,000 mm / second or less, or 5,000 mm / second or less. From the point of further improving the cell recovery rate, it is 100 mm / second or more, 150 mm / second or more, or 160 mm / second or more. From the same point, it is 4,000 mm / second or less, 3,500 mm / second or less, 3,000 mm / second. Second or less, 2,000 mm / second or less, or 1,500 mm / second or less.

  The ratio of the flow rate of the collected liquid and the flow rate of the specimen ([volume of collected liquid] / [volume of specimen passed through the filter]) is less than 0.6 in one or more embodiments, From the point of improving the recovery rate of cells from the filter, it is 0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, or 0.005 or more. From the viewpoint of facilitating analysis of the collected cells, it is less than 0.6, 0.5 or less, or 0.4 or less. Moreover, from the point which further improves the collection | recovery rate of a cell, it is 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, or 0.01 or more, From the same point, 0.3 or less, 0 .2 or less or 0.1 or less.

  The liquid passing pressure of the recovered liquid is 30 kPa or more and 150 kPa or less. From the point of improving the recovery rate of rare cells from the filter, the flow rate of the collected liquid is 35 kPa or higher, 40 kPa or higher, 45 kPa or higher, 50 kPa or higher, or 55 kPa or higher in one or more embodiments. To 130 kPa or less, or 120 kPa or less.

  In one or a plurality of embodiments, the recovery liquid includes phosphate buffered saline (PBS), PBS-BSA (for example, 0.2%), PBS-EDTA (for example, 1 mg / ml), and buffer, isotonic liquid. And aqueous solvents such as water. Examples of the buffer solution include Tris buffer solution such as Tris-HCl buffer solution, phosphate buffer solution, HEPES buffer solution, citrate-phosphate buffer solution and the like in one or a plurality of embodiments. Examples of the isotonic solution include physiological saline, sucrose isotonic solution, glucose isotonic solution, mannose isotonic solution, and the like.

  In the recovery method of the present disclosure, the viscosity of the recovered liquid is not limited to a specific range defined in Japanese Patent Laid-Open No. 10-201470, and may be a viscosity in other ranges. The viscosity of the recovered liquid is not particularly limited, and in one or more embodiments, the viscosity at 25 ° C. is less than 5 mPa · s, or the viscosity at 25 ° C. is 0, in order to further improve the cell recovery rate. It is 5 mPa · s or more.

  In one or a plurality of embodiments, the recovery liquid can be passed using a driving force capable of passing the recovery liquid so as to satisfy the liquid passing pressure or flow rate. In one or a plurality of embodiments, the recovery liquid may be introduced by a method using pressurization from the outlet direction of the flow path (side from which the specimen flows out (downstream side)), or from the inlet direction of the flow path (from which the specimen flows in) (Upstream side)), a method using reduced pressure, a syringe pump, a tube pump (also referred to as a roller pump or a peristaltic pump) or a plunger pump, or a method using mechanical drive.

  According to the recovery method of the present disclosure, in one or a plurality of embodiments, cells can be recovered from the filter with a high recovery rate with a small amount of recovery liquid. In one or a plurality of embodiments, the flow rate of the collected liquid is 10 ml or less, 8 ml or less, 6 ml or less, 5 ml or less, 4 ml or less, 2 ml or less, or 1 ml or less, or 0.05 ml or more, 0.1 ml or more. Or it is 0.2 ml or more.

  In one or a plurality of embodiments, the collection method of the present disclosure may include passing a specimen containing cells through a filter. By passing the specimen through the filter, rare cells that can be contained in the specimen can be captured by the filter.

  The flow rate of the specimen is 0.01 ml / min or more, 0.05 ml / min or more, or 0.1 ml / min or more in one or more embodiments from the viewpoint of improving the cell capture rate. From the point, it is 100 ml / min or less, 50 ml / min or less, 25 ml / min or less, or 10 ml / min or less.

  In one or more embodiments, the flow rate of the specimen is 0.01 mm / second or more, 0.05 mm / second or more, or 0.1 mm / second or more, in one or more embodiments, in terms of improving the cell capture rate. From the same point, it is 100 mm / second or less, 50 mm / second or less, 25 mm / second or less, 10 mm / second or less, 5 mm / second or less, or 2 mm / second or less.

Liquid passing amount of the specimen (the amount of the blood sample to pass through the filter), for example, with respect to the filtration area of 5mm ² ~10,000mm ^2, or 0.07 ml, 0.25 ml or more, or 0.5 ml, or 1ml More than 1 ml, 2 ml or more, 3 ml or more, or 4 ml or more. Furthermore, liquid passing amount of the specimen, in one or more embodiments, from the viewpoint of maintaining the retention rates of rare cells, relative to the filtration area of 5mm ² ~10,000mm ^2, 6L or less, 4L below, 2L or less 400 ml or less, 200 ml or less, 100 ml or less, 80 ml or less, 50 ml or less, 30 ml or less, 25 ml or less, 12 ml or less, 11 ml or less, 10 ml or less, 9 ml or less, or 8 ml or less. Alternatively, filter capacity, in one or more embodiments, the viewpoint of processing many samples, from the viewpoint of maintaining the retention rates of aspects and rare cells to capture deformable cells of 15 mm ² to 200 mm ² at a time More than 0.2 ml or less than 130 ml or 130 ml or less with respect to the filtration area, and from the same viewpoint, it is 0.8 ml to 90 ml, 0.8 ml to 60 ml, 2 ml to 50 ml, or 2 ml to 40 ml.

  In one or a plurality of embodiments, the fluid passage pressure (ΔP1) of the specimen is 0.1 kPa or more or 0.2 kPa or more, or 2.6 kPa or less, 1.5 kPa or less in terms of improving the cell capture rate. 1.3 kPa or less, 1.0 kPa or 0.5 kPa or less. In the present disclosure, “specimen flow pressure” refers to the pressure difference of the entire system from the inlet to the outlet of the system, for example, the pressure difference of the system including the channel including the tank from which the specimen is disposed to the waste liquid tank. Say. For this reason, the pressure more than the description may be sufficient depending on the channel structure. The liquid passing pressure (system pressure difference) can be measured by a known method using a commercially available pressure gauge, or can be calculated from the relationship between the flow rate and the flow path structure.

  The filter used in the recovery method of the present disclosure is a filter having a plurality of through holes. Since the cells are captured by the filter having the through-hole, in one or a plurality of embodiments, the cells are removed from the filter with a small amount of a recovery liquid and a high recovery rate while reducing damage to the cells, as compared with the nonwoven fabric. It can be recovered. Examples of the filter include a slit-shaped filter having a plurality of through holes in one or a plurality of embodiments. As a shape of a through-hole, in one or some embodiment, a rounded rectangle or an ellipse is mentioned.

  Examples of the filter used in the collection method of the present disclosure include a filter capable of capturing rare cells in a specimen in one or a plurality of embodiments. As the filter, a filter that can be used in the labeling method of the present disclosure can be used.

  By passing the collected liquid through the filter after passing the sample in the direction opposite to the direction of passing the sample, the rare cells are separated from the filter, and the collected liquid containing the separated rare cells is put on the filter. Exists. In one or a plurality of embodiments, the recovery method of the present disclosure may include recovering the recovery liquid present on the filter after passing the recovery liquid. In one or a plurality of embodiments, the recovery liquid can be recovered using a pipette or the like. Further, during the recovery, the recovered liquid may or may not be stirred.

  In one or a plurality of embodiments, the collection method of the present disclosure may include performing treatment on a filter on cells captured by the filter. Examples of the treatment include fixing treatment, cell membrane permeation treatment, and staining treatment in one or a plurality of embodiments.

[Method of analyzing rare cells]
The present disclosure, as another aspect, analyzes rare cells in a blood sample, including collecting rare cells by the collection method of the present disclosure, and analyzing the cells by observing the dynamics or measuring the activity of the cells. It relates to a method (analysis method of the present disclosure). According to the analysis method of the present disclosure, since rare cells are collected by the collection method of the present disclosure, it is possible to collect rare cells with a high recovery rate. For this reason, according to the analysis method of the present disclosure, it is possible to measure or analyze rare cells with high accuracy.

[Cell recovery device]
This indication is related with a cell recovery device (cell recovery device of this indication) as other modes. According to the cell collection device of the present disclosure, cells can be collected at a high collection rate by the collection method of the present disclosure. In one or a plurality of embodiments, the cell collection device of the present disclosure can collect cells captured by a filter from the filter, capture cells using the filter, and the like.

  As an aspect, the cell collection device of the present disclosure introduces a separation unit including a filter having a plurality of through holes for capturing cells and a holding unit for holding the filter, and a specimen containing cells to the separation unit. At least one introduction channel for supplying the sample to the separation unit through the introduction channel, a discharge channel for discharging the sample that has passed through the separation unit to the outside, and the separation unit A container for storing a recovery liquid for recovering the captured cells, a reverse liquid feeding means for supplying the recovery liquid from the container to the separation section through the outlet channel, and a cell by the cell recovery method of the present disclosure A controller for controlling the reverse liquid feeding means. In one or a plurality of embodiments, the control unit has a ratio of the flow rate of the collected liquid to the flow rate of the sample ([flow rate of the collected liquid] / [flow rate of the sample]) of 25 or more and / or The reverse liquid feeding means is controlled so that the flow rate of the recovered liquid is 1 mm / second or more per opening area.

  In one or a plurality of embodiments, the separation unit includes at least a filter and is detachably attached to the introduction channel and the discharge channel. As a filter, in one or some embodiment, said filter is mentioned.

  In one or a plurality of embodiments, the liquid feeding unit and the reverse liquid feeding unit include a pressurizing unit, a depressurizing unit, a syringe pump, a tube pump (also referred to as a roller pump or a peristaltic pump), a plunger pump, or a mechanical drive. Is mentioned.

  In one or a plurality of embodiments, the liquid feeding means may be capable of introducing a treatment liquid for treating cells in the specimen into the separation unit through the introduction channel. Examples of the treatment liquid include treatment liquids used in fixation treatment, cell membrane permeation treatment, staining treatment, and the like in one or a plurality of embodiments. In one or a plurality of embodiments, the cell collection device of the present disclosure may include a container for storing a treatment liquid for treating cells in a specimen.

  In one or a plurality of embodiments, the control unit can record the filter and filtration conditions used at the time of capturing cells. In one or a plurality of embodiments, the control unit can be realized by a processor included in the computer of the collection apparatus executing a predetermined program.

  In one or a plurality of embodiments, the cell collection device of the present disclosure includes a collection unit that collects the collection liquid supplied to the separation unit. Examples of the recovery unit include a nozzle mechanism in one or a plurality of embodiments. Cells can be collected by aspirating the collected liquid accumulated in the upper part of the filter with a nozzle or pipette.

  As another aspect, the cell collection device of the present disclosure includes a control program capable of collecting cells by the collection method of the present disclosure.

[Cell recovery system]
This indication is related with a cell recovery system (cell recovery system of this indication) as other modes. According to the cell collection system of the present disclosure, cells can be collected at a high collection rate by the collection method of the present disclosure. In one or a plurality of embodiments, the cell collection system of the present disclosure can collect cells captured by a filter from the filter, capture cells using the filter, and the like.

  In one or a plurality of embodiments, a cell collection system according to the present disclosure includes a filter having a plurality of through holes for capturing cells, a holding unit that holds the filter, and the filter unit. A separation unit comprising a possible flow path part, and a liquid feeding means capable of passing a specimen containing cells through the flow path part to the filter, a flow path part in which the filter unit can be disposed, and a passage of the separation unit. A recovery unit comprising a reverse liquid feeding means capable of flowing the collected liquid through the filter from the direction opposite to the liquid direction, and the reverse feeding for recovering cells by the cell recovery method of the present disclosure. And a control unit for controlling the liquid means. In one or a plurality of embodiments, the control unit has a ratio of the flow rate of the collected liquid to the flow rate of the sample ([flow rate of the collected liquid] / [flow rate of the sample]) of 25 or more and / or the collected liquid. The liquid feeding means of the separation unit and the liquid feeding means of the recovery unit are controlled so that the flow rate of the liquid becomes 1 mm / second or more per opening area. In one or a plurality of embodiments, the filter unit can be attached to and detached from the flow path portions of the separation unit and the recovery unit. According to the cell collection system of the present disclosure, in one or a plurality of embodiments, after the cells are captured in the separation unit, the filter unit is transferred to the collection unit, and the cells captured by the filter are collected in the collection unit. be able to.

  In one or a plurality of embodiments, the cell collection system of the present disclosure separates a specimen containing cells from a separation unit including a filter having a plurality of through holes for capturing cells and a holding unit for holding the filter. At least one introduction channel for introducing into the unit, at least one liquid feeding means for supplying the sample to the separation unit through the introduction channel, a discharge channel for discharging the sample that has passed through the separation unit to the outside, A container for storing a recovery liquid for recovering cells captured by the separation unit, a reverse liquid feeding means for supplying the recovery liquid from the container to the separation unit through a discharge channel, and a cell recovery method of the present disclosure And a control means for controlling the reverse liquid feeding means in order to collect cells. In one or a plurality of embodiments, the control means has a ratio between the flow rate of the collected liquid and the flow rate of the sample ([flow rate of the collected liquid] / [flow rate of the sample]) of 25 or more and / or The reverse liquid feeding means is controlled so that the flow rate of the recovered liquid is 1 mm / second or more per opening area.

  An example of a rare cell capture and recovery device that can be used in the recovery method or analysis method of the present disclosure is shown in FIG. The rare cell capture and recovery device shown in FIG. 4 includes a supply port 41 and a discharge port 42 for supplying a specimen, a washing solution, a staining solution, and the like to a filter, a flow path 43 that connects the supply port 41 and the discharge port 42, and a flow It has a separation part arranged at a position corresponding to a part of the path 43. The separation unit includes a filter 44 and a filter holder 45 that holds the filter 44. The filter holder 45 includes a support portion (base) 46 on which the filter 44 can be installed, an O-ring 47 that fixes the filter 44, and a cover 48. A filter 44 is installed on the support 46, an O-ring 47 and a cover 48 are placed on the upper surface of the filter 44, and the filter 44 is fixed to form a separation unit (filter device) having a filter having holes. The flow path 43 connecting the supply port 41 and the separation unit and the separation unit and the discharge port 42 is formed by connecting a Saffy (trademark) tube (made by Terumo Corporation) to both ends of the separation unit. In the flow path 43 connecting the supply port 41 and the separation part, a connection part (not shown) and a three-way cock (not shown) capable of switching the liquid may be arranged on the upper part of the separation part. Further, in the flow path 43 connecting the supply port 41 and the separation unit, a pressurizing means 49 (as a mechanism capable of feeding a liquid at a constant pressure or a mechanism capable of feeding a liquid at a constant flow rate) is arranged to perform filtration. The pressure may be kept constant, or the solution may be sent so that the pressure applied for filtration by pulling with a syringe pump becomes a certain value or more.

  FIG. 5 shows another example of a rare cell capture and recovery device that can be used in the recovery method or analysis method of the present disclosure. The rare cell capture and recovery apparatus shown in FIG. 5 discharges from the introduction flow path 56 for introducing the specimen in the container 52 into the separation unit 51, the separation unit 51 having a filter, the container 55 for storing the collected liquid, and the separation unit 51. A discharge passage 57 for discharging the waste liquid to be discharged is provided. The container 55 that stores the recovered liquid is connected to the discharge flow path 57 through the flow path so that the recovered liquid can be fed back to the separation unit 51 through the discharge flow path 57. The container 55 is connected to liquid feeding means such as a pump for feeding the recovered liquid back to the separation unit 51. In addition to the container 52 containing the specimen, for example, the container 13 containing the cleaning liquid and the container 54 containing the processing liquid for processing the cells in the specimen can be connected to the introduction channel 56 through the flow path. A container 58 for collecting waste liquid can be disposed at one end of the discharge channel 57.

  In the present disclosure, the “cell” is not particularly limited, and examples thereof include human or non-human animal cells.

In the present disclosure, “rare cells” refer to cells other than blood cell components (red blood cells, white blood cells, and platelets) that can be contained in the blood of a human or non-human animal. The rare cells include tumor cells or cancer cells in one or more embodiments. In general, tumor cells and cancer cells circulating in the blood are called CTCs. As the number of rare cells in the blood, depending on the specimen, there are cases in which several to several tens or at most several hundred to 1,000 cells are contained in 10 ml of blood. In one or a plurality of embodiments, the “rare cell” is a cancer cell, circulating tumor cell, vascular endothelial cell, vascular endothelial progenitor cell, cancer stem cell, epithelial cell, hematopoietic stem cell, mesenchymal stem cell, fetal cell, stem cell. And a cell selected from the group consisting of these and combinations thereof.
In one or a plurality of embodiments that are not particularly limited, the rare cells include human colon cancer cells, human stomach cancer cells, human colon cancer cells, human lung cancer cells, human lung cancer cells, human lung cancer cells, human lung cancer cells, and human lung cancer cells. Is mentioned.

  In the present disclosure, the “specimen” includes a sample that can contain cells that can be applied to the recovery method of the present disclosure. Examples of the specimen include a blood specimen in one or a plurality of embodiments. The blood sample is a sample containing a component constituting blood in one or a plurality of embodiments, and in one or a plurality of embodiments not particularly limited, blood, a blood-derived product containing blood cell components, blood or blood-derived Examples include body fluids and urine mixed with substances, and samples prepared from these. Examples of the blood include blood collected from a living body, and examples of the living body include humans or animals other than humans (for example, mammals). Examples of blood-derived substances containing blood cell components include those isolated or prepared from blood and containing blood cell components or their dilution / concentration, for example, blood cell fractions from which plasma has been removed, blood cells Concentrates, lyophilized products of blood or blood cells, samples or hemolyzed samples obtained by lysing whole blood and removing red blood cell components, centrifuged blood, spontaneously precipitated blood, washed blood cells, specific fractions, and the like. Among these, in one or a plurality of embodiments that are not limited, from the viewpoint of simple and rapid processing and the suppression of damage to rare cells in blood, the sample containing blood is derived from blood containing blood or blood cell components Are preferred.

The present disclosure may relate to one or more of the following embodiments.
[C1] A method of recovering cells captured by a filter having a plurality of through-holes from the filter,
Passing the collected liquid through the filter in which the cells are captured from the direction opposite to the direction in which the specimen is passed for capturing the cells,
The flow of the recovered liquid is performed so that the ratio of the flow rate of the recovered liquid and the flow rate of the sample ([flow rate of recovered liquid] / [flow rate of sample]) is 25 or more. Collection method.
[C2] A method for recovering cells captured by a filter having a plurality of through-holes from the filter,
The collected liquid is passed through the filter in which the cells have been captured from a direction opposite to the direction in which the specimen is passed for capturing the cells at a flow rate of 1 mm / second or more per opening area of the filter. A method for recovering cells.
[C3] The recovery method according to [C1], including passing the recovery liquid through the filter at a flow rate of 1 mm / second or more per opening area of the filter.
[C4] including passing the specimen containing cells through the filter at a flow rate of 0.01 mm / second to 100 mm / second per opening area, thereby capturing the cells on the filter; C3].
[C5] The collection method according to any one of [C1] to [C4], wherein the filter is a filter capable of capturing rare cells.
[C6] The collection method according to any one of [C1] to [C5], wherein the material of the filter is selected from the group consisting of nickel, SUS, gold, silver, cylinder, aluminum, tungsten, and chromium.
[C7] The collection method according to any one of [C1] to [C7], wherein the cells are rare cells. [C8] In a blood sample, comprising collecting rare cells by the collection method according to any one of [C1] to [C7], and analyzing the cells by observing the dynamics or measuring the activity of the cells. Rare cell analysis method.
[C9] a separation unit including a filter having a plurality of through holes for capturing cells, and a holding unit for holding the filter;
At least one introduction channel for introducing a specimen containing cells into the separation unit;
At least one liquid feeding means for supplying the specimen to the separation section through the introduction channel;
A discharge channel for discharging the specimen that has passed through the separation unit to the outside;
A container for storing a collection liquid for collecting the cells captured by the separation unit;
Reverse liquid feeding means for supplying the recovered liquid from the container to the separation section through the discharge flow path;
A control unit for controlling the reverse feeding means so that a ratio of the flow rate of the collected liquid to the flow rate of the sample ([flow rate of the collected liquid] / [flow rate of the sample]) is 25 or more; A cell recovery apparatus.
[C10] a separation unit comprising a filter having a plurality of through holes for capturing cells, and a holding unit for holding the filter;
At least one introduction channel for introducing a specimen containing cells into the separation unit;
At least one liquid feeding means for supplying the specimen to the separation section through the introduction channel;
A discharge channel for discharging the specimen that has passed through the separation unit to the outside;
A container for storing a collection liquid for collecting the cells captured by the separation unit;
Reverse liquid feeding means for supplying the recovered liquid from the container to the separation section through the discharge flow path;
A cell recovery apparatus comprising: a control unit that controls the reverse feeding means so that the recovery liquid is passed at a flow rate of 1 mm / second or more per opening area.
[C11] A cell recovery apparatus comprising the control program for the recovery method according to any one of [C1] to [C7].
[C12] The cell recovery device according to [C9] or [C10], comprising the control program for the recovery method according to any one of [C1] to [C7].
[C13] The cell recovery apparatus according to any one of [C9] to [C12], further including a recovery unit that recovers the recovery liquid supplied to the separation unit.
[C14] a filter unit comprising a filter having a plurality of through holes for capturing cells, and a holding unit for holding the filter;
A separation unit comprising a flow path section in which the filter unit can be disposed, and a liquid feeding means capable of passing a specimen containing cells through the flow path section to the filter;
A recovery unit comprising: a flow path portion in which the filter unit can be disposed; and a reverse liquid feeding means capable of flowing a recovery liquid through the flow path portion from the direction opposite to the liquid flow direction of the separation unit. ,
The liquid feeding means of the separation unit and the recovery unit so that the ratio of the flow rate of the collected liquid and the flow rate of the sample ([flow rate of collected liquid] / [flow rate of sample]) is 25 or more. And a control unit for controlling the liquid feeding means.
[C15] a filter unit comprising a filter having a plurality of through holes for capturing cells, and a holding unit for holding the filter;
A separation unit comprising a flow path section in which the filter unit can be disposed, and a liquid feeding means capable of passing a specimen containing cells through the flow path section to the filter;
A recovery unit comprising: a flow path portion in which the filter unit can be disposed; and a reverse liquid feeding means capable of flowing a recovery liquid through the flow path portion from the direction opposite to the liquid flow direction of the separation unit. ,
A cell recovery system comprising a control unit for controlling the liquid supply means of the separation unit and the liquid supply means of the recovery unit so that the recovery liquid is passed at a flow rate of 1 mm / second or more per opening area .

  Hereinafter, the present disclosure will be further described using examples. However, the present disclosure is not construed as being limited to the following examples. The following cell capturing / labeling device and cell capturing device may be configured by combining a plurality of units.

1. Confirmation of magnetic labeling efficiency (Example 1)
Using the cell capturing / labeling apparatus shown in FIG. 1, cells (leukocytes) were captured and labeled with magnetic particles to confirm the labeling efficiency.

[Cell labeling device]
A cell labeling apparatus shown in FIG. 1 was prepared. The cell labeling apparatus of FIG. 1 includes a device upper part 3, a filter part 5 including a filter 10 and a device lower part 4, and a liquid feeding mechanism (not shown) capable of feeding in the arrow A direction and the arrow B direction. The filter 10 is disposed between the device upper part 3 and the device lower part 4 and is fixed by double-sided tapes 11 and 12. As the filter 10, a filter having a plurality of through holes and having the following characteristics was used.
<Filter characteristics>
・ Hole Short axis diameter: 6.5 μm, Long axis diameter: 88 μm, Area: 572 μm ² , Shape: Pitch between slit and hole centers: 14 μm × 100 μm (short axis diameter side × major axis diameter side)
・ Pore density: 714 / mm ²
・ Opening ratio: 41%
・ Film thickness: 5μm
・ Material: Nickel ・ Filtration area: 79mm ²

[Magnetic particle labeling]
Using the cell labeling apparatus of FIG. 1, cells (white blood cells) contained in blood were labeled with magnetic particles in the following steps 1 to 8.
Step 1: 8 mL of blood containing rare cells and leukocytes was introduced into the cell labeling apparatus, and the blood was filtered to capture cells such as rare cells and leukocytes in a size-selective manner (filtration flow rate: 100 μL / min, flow rate : 0.144 mm / sec).
Step 2: An antibody solution containing an antibody against CD45 and CD50 and an antibody solution containing a biotinylated antibody that recognizes the antibody are supplied to the filter in this order to react with cells, and CD45 and CD50-expressing cells (for example, leukocytes) Biotin labeling was performed. The antibody solution stays not only at the upper part of the filter but also at the lower part of the filter so as to contact both sides of the filter.
Step 3: Neutravidin-labeled magnetic particle suspension is supplied to the upper surface of the filter, and a part of the suspension is sent in the direction of arrow A in FIG. 1 so that the lower surface of the suspension is positioned below the lower surface of the filter. did.
Step 4: Back flow was performed from the bottom of the filter in the direction of arrow B in FIG. 1 (back flow rate: 10,000 μL / min, flow rate: 14.441 mm / sec).
Step 5: Pipet the suspension above the filter.
Step 6: The suspension above the filter (mixed solution of cells and magnetic particles) was fed again (250 μL / min) from the direction of arrow A in FIG.
Step 7: A cleaning liquid containing no magnetic particles was fed from the direction of arrow A in FIG. Thereby, unreacted magnetic particles were removed.
Step 8: The collected liquid is sent from the lower part of the filter in the direction of arrow B in FIG. 1, and the collected liquid collected on the upper part of the filter is sucked with a pipette, and cells (white blood cells) and rare cells labeled with magnetic particles together with the collected liquid Was recovered.

[Evaluation of labeling efficiency]
The cells collected in step 8 were magnetically separated using a neodymium magnet. The number of leukocytes collected without being captured by the magnet was counted. The result is shown in FIG.

(Comparative Example 1)
It carried out like Example 1 except not having performed process 4-6. The result is shown in FIG.

(Comparative Example 2)
The same procedure as in Example 1 was performed except that Step 4 was not performed. The result is shown in FIG.

  As shown in FIG. 3, according to the method of Example 1, the number of remaining white blood cells, that is, the number of white blood cells that were not captured by the magnet by magnetic separation, could be greatly reduced. Therefore, according to the method of Example 1, it can be said that leukocytes could be magnetically labeled with high labeling efficiency on the filter.

(Example 2)
[Preparation of sample]
Fluorescent and Biotin-labeled leukocytes were prepared by the following procedure.
First, blood was collected from a human using a vacuum blood collection tube (EDTA 2K).
Then, according to the package insert of HetaSep (STEMCELL), leukocytes were separated from the collected whole blood.
1 × 10 ⁶ leukocytes separated by HetaSep were reacted at 23 ° C. for 10 minutes in a blocking solution (10% goat serum, 0.000001% Avidin and 0.2% BSA dissolved in Dulbecco's PBS (−)). After centrifuging and removing the supernatant, it was resuspended using Dulbecco's PBS (-). It was centrifuged again and the supernatant was removed.
Next, a primary antibody reaction solution was prepared by adding anti-CD45 antibody and anti-CD50 antibody to Dulbecco's PBS (-) in which 10% goat serum, 0.01% Biotin and 0.2% BSA were dissolved, respectively, according to the package insert. Cells (white blood cells) were suspended in the prepared primary antibody reaction solution and reacted at 23 ° C. for 15 minutes. After centrifuging and removing the supernatant, it was suspended using Dulbecco's PBS (-). Thereafter, the step of removing the supernatant by centrifugation was performed three times in total, and the cells were washed thoroughly.
Add 2 μg / ml Hoechst33342, 10% goat serum and 0.2% BSA dissolved Dulbecco's PBS (-), and add Alexa594-labeled anti-mouse IgG (Fab) and Biotin-labeled anti-mouse IgG (Fc) respectively according to the package insert. An antibody reaction solution was prepared. Cells washed with the prepared secondary antibody reaction solution were suspended and reacted at 23 ° C. for 15 minutes. After centrifuging and removing the supernatant, it was suspended using Dulbecco's PBS (-). The step of centrifuging and removing the supernatant was performed three times in total, and the cells (leukocytes) were thoroughly washed.
Thus, a sample containing fluorescent and Biotin-labeled leukocytes was prepared.

[Capture cells]
A sample containing fluorescent and Biotin-labeled leukocytes (1 × 10 ⁵ ) was introduced into the cell labeling apparatus of FIG. 1 from the direction of arrow 1 in the same manner as in Example 1, and a flow rate of 100 μL / min (flow rate: 0.144 mm / min). Second).
As the filter 10, a filter having a plurality of through holes and having the following characteristics was used. <Filter characteristics>
・ Hole Short axis diameter: 6.5 μm, Long axis diameter: 88 μm, Area: 572 μm ² , Shape: Pitch between slit and hole centers: 14 μm × 100 μm (short axis diameter side × major axis diameter side)
・ Pore density: 714 / mm ²
・ Opening ratio: 41%
・ Film thickness: 5μm
Material: Nickel filtration area: 28mm ^2.

[Magnetic particle labeling]
A solution of 0.2% BSA dissolved in Dulbecco's PBS (−) was sent to the apparatus shown in FIG. 200 μL of magnetic particle liquid (Neutravidin-labeled magnetic particle suspension, solid content concentration: 0.0125%) is added to the upper surface of the filter 10, and the lower surface of the magnetic particle liquid is added so as to be located below the filter 10. A part (100 μL) of the magnetic particle solution was filtered. 200 μL of a solution of 0.2% BSA dissolved in Dulbecco's PBS (−) was fed back at a flow rate of 10000 μL / min using a back feeding mechanism (in the direction of arrow B in FIG. 1). The solution on the filter 10 (solid content concentration: 0.0062%) was pipetted and reacted at 23 ° C. for 15 minutes. Next, 300 μL of a magnetic particle solution containing leukocytes on the upper surface of the filter 10 was fed at a flow rate of 250 μL / min (in the direction of arrow A in FIG. 1), and reacted at 23 ° C. for another 15 minutes. Thereby, leukocytes were magnetically labeled with magnetic particles.

[Cleaning of magnetic particles]
Thereafter, 1000 μL of Dulbecco's PBS (−) in which 1 mg / mL EDTA was dissolved was added to the upper surface of the filter 10 and then fed at a flow rate of 1000 μL / min (in the direction of arrow A in FIG. 1). This process was performed a total of four times to wash away excess magnetic particles on the filter 10.

[Recovery of cells]
1000 μL of a solution obtained by dissolving 0.2% BSA in Dulbecco's PBS (−) was fed back at a flow rate (14 mm / sec) of 10,000 μL / min (in the direction of arrow B in FIG. 1) using a back feeding mechanism. Then, 1100 μL of the solution on the filter 10 was recovered by pipetting.

[Evaluation of labeling efficiency]
The collected suspension is magnetically separated, and leukocytes that have not been magnetically separated (not captured by a magnet) are judged not to be magnetically labeled (or the labeling is insufficient) to evaluate the labeling efficiency. went.
That is, the collected cells were magnetically separated using a neodymium magnet. The number of leukocytes collected without being captured by the magnet was counted.
Specifically, among the cells contained in 1000 μL of the collected suspension, cells positive for both Alexa594 and Hoechst33342 were counted using a fluorescence microscope (the number of leukocytes after magnetic separation (LEUK-a)).
On the other hand, among the suspensions collected after labeling, leukocytes in 100 μL of suspension not used for magnetic separation were counted using a hemocytometer (Moxi-Z), and the white blood cell count in 1000 μL (White blood cell count before magnetic separation (LEUK-b)).
Using the obtained white blood cell counts (LEUK-a and LEUK-b), the non-labeling rate was calculated from the following formula. The results are shown in Table 1 below.
[Unlabeled rate] = {1-([LEUK-b]-[LEUK-a] / [LEUK-b])} × 100

(Examples 3 to 5)
The same procedure as in Example 2 was performed, except that a sample newly prepared by the same preparation method as in Example 2 was used, and the flow rate during reverse flow of the magnetic particle suspension was changed to the flow rate shown in Table 1 below. . The results are shown in Table 1 below.

(Comparative Example 3)
The same procedure as in Example 2 was performed, except that a sample newly prepared by the same preparation method as in Example 2 was used and no reverse flow was performed in magnetic particle labeling. That is, a part (100 μL) of the magnetic particle liquid (200 μL) added to the upper surface of the filter is filtered and then the solution on the filter is pipetted and reacted at 23 ° C. for 30 minutes for magnetic particle labeling. It was. The results are shown in Table 1 below.

  As shown in Table 1, by labeling by the methods of Examples 2 to 5, the unlabeled rate, that is, the proportion of leukocytes not magnetically labeled (or insufficiently labeled) is reduced to less than 1%. I was able to. That is, according to the methods of Examples 2 to 5, it can be said that leukocytes could be magnetically labeled with high labeling efficiency on the filter.

(Example 6)
A sample newly prepared by the same method as in Example 2 was used, and the reverse flow after a part of the magnetic particle liquid was filtered was divided into two 100 μL portions (total liquid supply amount: 200 μL). ) Was carried out in the same manner as in Example 2. The results are shown in Table 2 below.

(Example 7)
The same procedure as in Example 2 was performed except that a sample newly prepared by the same method as in Example 2 was used. The results are shown in Table 2 below.

As shown in Table 2, in both cases of Examples 6 and 7, leukocytes could be magnetically labeled on the filter with high labeling efficiency. It has also been found that the desired effect can be obtained by performing the back flow after the liquid particle filtration of the magnetic particle liquid at least once.
The reason why the unlabeling rate differs between Example 7 and Example 2 where the processing conditions are the same is considered to be the difference in the sample used, specifically, the difference in the white blood cells contained in the sample. In the above examples, CD particles expressed on the surface of leukocytes are used to label magnetic particles. However, CD antigen expression is subject to individual-to-individual variation (individual differences) and intra-individual variation. It is known. It is considered that the unlabeled rate is different due to the difference between the samples. In the method of the present disclosure, it has been confirmed that sufficient reproducibility can be obtained for the labeling rate for the same specimen.

(Example 8)
The same procedure as in Example 2 was performed except that a sample newly prepared by the same method as in Example 2 was used. The results are shown in Table 3 below.

Example 9
The same procedure as in Example 8 was performed except that a sample newly prepared by the same method as in Example 2 was used and pipetting was not performed. The results are shown in Table 3 below.

  As shown in Table 3, leukocytes could be magnetically labeled with high labeling efficiency on the filter by the methods of Examples 8 and 9. In particular, the efficiency of magnetic labeling could be further improved by performing pipette agitation after the reverse flow.

(Comparative Example 4)
Similar to Example 8 except that a sample newly prepared by the same method as in Example 2 was used and a part of the magnetic particle liquid (FIG. 2B) was not sent before the reverse flow. Went to. The results are shown in Table 4 below together with the results of Example 8.

  As shown in Table 4, the leukocytes captured by the filter could be magnetically labeled with high labeling efficiency by feeding and filtering a part of the magnetic particle solution added to the upper surface of the filter prior to the reverse flow solution. That is, it can be seen that the labeling efficiency is improved by reversely flowing after immersing both sides of the filter in the suspension.

2. Cell capture and recovery test with filter (Experimental example 1)
Using the rare cell trap shown in FIG. 4, the rare cells in the blood sample were collected.

As the filter, a filter having the following characteristics was used. A filter having the following characteristics can be captured by both SNU-1 and SW620.
<Filter characteristics>
Hole Short axis diameter: 6.5 μm, Long axis diameter: 88 μm, Area: 572 μm ² , Shape: Pitch between centers of slit holes (short axis diameter side × major axis diameter side): 14 μm × 100 μm
Pore density: 714 / mm ²
Opening ratio: 41%
Film thickness: 5μm
Material: Nickel filtration area: 79mm ²

  The rare cell capturing / recovering apparatus shown in FIG. 4 including the above-described filter and a liquid feeding unit including a reverse liquid feeding mechanism was configured. The member support part 46 which fixes a filter used the member made from aluminum. Using the rare cell capture / recovery apparatus, the rare cells were captured by filtration under the conditions shown in Table 5 below, and the captured cells were collected from the filter. Specifically, it was as follows.

First, cells were captured by filtration. A blood sample containing SNU-1 stained with Celltracker green (7.5 ml) was passed at a constant pressure from the direction of arrow A in FIG. 4 over about 30 minutes (ΔP1 = 0.75 kPa: filtration flow rate of 0.25 ml / min) ( The liquid flow at this low pressure may use the water head pressure, or may use the same or a separate pump as the above-mentioned reverse flow mechanism. This captured SNU-1 on the filter. Next, the filter was washed by passing 4 mL of PBS (−) twice, and then 900 μL of ammonium chloride solution (ACS) was passed 3 times, and hemolysis was performed at room temperature for 10 minutes. PBS (−) 4 mL was passed through and washed to replace the solution in the device. After removing the flow path connecting the supply port 41 and the separation part and sealing with a cover glass, the number of SNU-1 captured on the filter was counted by microscopic observation.
After counting, the cover glass was taken out, and an acrylic plate provided with a recovery well designed to match the position and size of the flow path above the filter was set on the cover 48.
Thereafter, the cells captured by the filter were collected. PBS (−) (recovered liquid, viscosity: 0.95 mPa · s) using a reverse flow mechanism from the direction opposite to the direction in which the cells were trapped on the filter (the direction of arrow B in FIG. 4). ) 1 ml was passed (flow rate 120 ml / min). The collected liquid collected on the upper part of the filter was sucked with a pipette to collect the whole amount. The number of SNU-1 in the collected liquid was counted by microscopic observation. The recovery rate was determined by dividing the number of SNU-1 in the recovered liquid by the number of SNU-1 captured on the filter. The results are shown in Table 5 below.

(Experimental example 2)
Cells were captured and collected in the same manner as in Experimental Example 1 except that the volume of the collected liquid was 0.25 ml and the flow rate was 6,000 ml / min. The results are shown in Table 5 below.

(Experimental example 3)
Cells were captured and collected in the same manner as in Experimental Example 1 except that the volume of the collected liquid was 0.5 ml and the flow rate was 6,000 ml / min. The results are shown in Table 5 below.

(Experimental example 4)
Cells were captured and collected in the same manner as in Experimental Example 1 except that the flow rate of the collected liquid was 6,000 ml / min. The results are shown in Table 5 below.

(Experimental example 5)
Cells were captured and collected in the same manner as in Experimental Example 1 except that the amount of the collected liquid was 0.25 ml and the flow rate was 600 ml / min. The results are shown in Table 5 below.

(Experimental example 6)
Cells were captured and collected in the same manner as in Experimental Example 1 except that the flow rate of the collected liquid was 600 ml / min. The results are shown in Table 5 below.

(Experimental example 7)
Cell capture and recovery were carried out in the same manner as in Experimental Example 1, except that the filtration area was 20 mm ² , ΔP1 was 0.4 kPa (liquid feeding over about 60 to 120 minutes), and the flow rate of the recovery liquid was 600 ml / min. went. The results are shown in Table 5 below.

(Experimental example 8)
Cells were captured and collected in the same manner as in Experimental Example 1 except that the filtration area was 20 mm ² and ΔP1 was 0.4 kPa (liquid feeding over about 60 to 120 minutes). The results are shown in Table 5 below.

(Experimental example 9)
Cells were captured and collected in the same manner as in Experimental Example 1, except that the filtration area was 20 mm ² , ΔP1 was 0.4 kPa (liquid was transferred over about 30 minutes), and the amount of blood sample was 4 ml. The results are shown in Table 5 below.

(Experimental example 10)
Cells were captured and collected in the same manner as in Experimental Example 1 except that the filtration area was 20 mm ² , ΔP1 was 0.75 kPa (delivered over about 10 minutes), and the amount of blood sample was 4 ml. The results are shown in Table 5 below.

(Experimental example 11)
First, cells were captured by filter filtration. A blood sample 2 ml containing SW620 stained with Celltracker green was passed at a constant pressure in the same manner as in Experimental Example 1 over about 8 minutes from the direction of arrow A in FIG. 4 (ΔP1 = 0.4 kPa: filtration flow rate 0.25 ml) / Min). This captured SW620 on the following filter. Hemolysis was performed in the same manner as in Experimental Example 1, and after washing with PBS (−), immobilization and cell membrane permeation were performed on the filter. The immobilization treatment was carried out by passing 4% paraformamide: PFA (PBS (-) solution) to fill the vicinity of the filter and then reacting at room temperature for 15 minutes. The cell membrane permeabilization treatment was performed by passing 0.2% Triton-X100 (PBS (-) solution) to fill the vicinity of the filter and then reacting at room temperature for 15 minutes. The device was washed with PBS (−), and the solution in the device was replaced. After removing the flow path connecting the supply port 41 and the separation part and sealing with a cover glass, the number of SW620 captured on the filter was counted by microscopic observation.
After counting, the cover glass was taken out, and an acrylic plate provided with a recovery well designed to match the position and size of the flow path above the filter was set on the cover 48.
Thereafter, the cells captured by the filter were collected. 1 ml of PBS (-) (collected liquid) was passed through the filter using a reverse flow mechanism from the direction opposite to the direction in which the cells were captured (the direction of arrow B in FIG. 4) (flow rate 6). , 1,000 ml / min). The collected liquid collected on the upper part of the filter was sucked with a pipette to collect the whole amount. The number of SW620 in the collected liquid was counted by microscopic observation. The recovery rate was determined by dividing the number of SW620 in the recovered liquid by the number of SW620 captured on the filter. The results are shown in Table 5 below.
<Filter characteristics>
Hole Short axis diameter: 6.5 μm, Long axis diameter: 9.8 μm, Area: 50 μm ² , Shape: Pitch between centers of elliptical holes (short axis diameter side × major axis diameter side): 19 μm × 14 μm
Pore density: 3,759 holes / mm ²
Opening ratio: 19%
Film thickness: 5μm
Material: Nickel filtration area: 20mm ²

(Experimental example 12)
The immobilization treatment was performed by reacting with 1% PFA (PBS (−) solution) at room temperature for 10 minutes, and the cell membrane permeabilization treatment was only performed by passing 0.2% Triton-X100 (PBS (−) solution) through the filter. The cells were captured and collected in the same manner as in Experimental Example 11 except that the reaction time in the state where the vicinity of the filter was filled was not taken, but the reaction was carried out only during the passage of liquid. The results are shown in Table 5 below.

(Experimental example 13)
Immobilization treatment was performed with 5 mg / ml dimethylsuberimidate: DMS (PBS (−) solution) at room temperature for 10 minutes, and cell membrane permeation treatment was only passed through 0.2% Triton-X100 (PBS (−) solution). Cells were captured and collected in the same manner as in Experimental Example 11 except that the changes were made. The results are shown in Table 5 below.

(Experimental example 14)
First, cells were captured by filter filtration. 2 ml of blood sample containing unstained SW620 was passed at a constant pressure in the same manner as in Experimental Example 1 over about 8 minutes from the direction of arrow A in FIG. 4 (ΔP1 = 0.4 kPa: filtration flow rate 0.25 ml / min) . Thereby, SW620 was captured on the filter (the same filter as in Experimental Example 1 except that the filtration area was 20 mm ² ). The sample was hemolyzed in the same manner as in Experimental Example 1, washed with PBS-EDTA, fixed on a filter with 1% PFA (PBS (−) solution) at room temperature for 10 minutes, and 0.2% Triton-X100 (PBS The (−) solution) was passed through to perform cell membrane permeabilization treatment. Thereafter, FcR blocking treatment was performed, and staining was performed with a fluorescently labeled anti-cytokeratin antibody. The device was washed with PBS-EDTA, and the solution in the device was replaced. After removing the flow path connecting the supply port 41 and the separation part and sealing with a cover glass, the number of SW620 captured on the filter was counted by microscopic observation.
After counting, the cover glass was taken out, and an acrylic plate provided with a recovery well designed to match the position and size of the flow path above the filter was set on the cover 48.
Thereafter, the cells captured by the filter were collected. 1 ml of PBS-EDTA (recovered solution) was passed through the filter from the direction opposite to the direction in which the cells were captured (in the direction of arrow B in FIG. 4) using a reverse flow mechanism (flow rate 6, 000 ml / min). The collected liquid collected on the upper part of the filter was sucked with a pipette to collect the whole amount. The number of SW620 in the collected liquid was counted by microscopic observation. The recovery rate was determined by dividing the number of SW620 in the collected liquid by the number of SW620 captured on the filter. The results are shown in Table 5 below.

(Experimental example 15)
First, cells were captured by filter filtration. A resin member is used as a member for fixing the filter, and an 8 ml blood sample containing SUN-1 stained with Celltracker green and SW620 stained with Celltracker orange is determined over about 32 minutes from the direction of arrow A in FIG. The liquid was passed at a flow rate (filtration flow rate 0.25 ml / min). Thereby, SNU-1 and SW620 were captured on the filter (the same filter as Experimental Example 1). Next, the filter was washed 4 times with 2 mL of PBS-EDTA, and then 900 μL of ACS was passed 3 times, and hemolysis was performed at room temperature in the same manner as in Experimental Example 1 (in order to increase the accuracy of counting after collection). The plate was washed 4 times with 2 mL of PBS-BSA, and the solution in the device was replaced.
Subsequently, the cells captured by the filter were collected. 1 ml of PBS-BSA (recovered solution, viscosity: 1.50 mPa · s) was passed from the direction opposite to the direction in which the cells were trapped (in the direction of arrow B in FIG. 4) using a reverse flow mechanism. (Flow rate 600 ml / min). Thereafter, the collected liquid at the top of the filter was stirred twice with a pipette, and then 500 μl of the collected liquid collected at the top of the filter was collected by sucking with a pipette, and then further stirred twice to collect the remaining amount in the same manner. The number of SNU-1 and SW620 in the collected liquid was counted by microscopic observation. The recovery rate of each cell was determined by dividing the number of each recovered cell by the number of each added cell. The results are shown in Table 5 below.

(Experimental example 16)
The cells were captured and recovered in the same manner as in Experimental Example 15 except that the flow rate of the recovered liquid was 316 ml / min (liquid passing pressure: 58 kPa). The results are shown in Table 5 below.

(Experimental example 17)
Cell capture and recovery were carried out in the same manner as in Experimental Example 15, except that the filtration area was 28 mm ² , the filtration flow rate was 0.1 ml / min, and the cells used were NCI-H1650 (human lung cancer) stained with Celltracker green. went. The results are shown in Table 5 below.

(Experiment 18)
First, cells were captured by filter filtration. A resin member was used as a member for fixing the filter, and 8 ml of blood sample containing unstained NCI-H1650 was passed at a constant flow rate from the direction of arrow A in FIG. / Min). Thereby, NCI-H1650 was captured on the filter (the same filter as in Experimental Example 1 except that the filtration area was 28 mm ² ). Next, cell staining such as hemolysis, immobilization, cell membrane permeabilization and antibody staining is performed on the captured NCI-H1650, and finally washed with an isotonic solution based on sucrose / glucose. Liquid replacement was performed.
Subsequently, the cells captured by the filter were collected. 1 ml of the isotonic solution (recovered solution, viscosity: 1.75 mPa · s) is passed through a reverse flow mechanism from the direction opposite to the direction in which the cells have been captured (the direction of arrow B in FIG. 4). Liquid (flow rate 600 ml / min). Thereafter, the collected liquid at the top of the filter was stirred twice with a pipette, and then 250 μl of the collected liquid collected on the filter was collected. Then, after stirring twice more, the remaining total amount was collected in the same manner. The number of NCI-H1650 in the collected liquid was counted by microscopic observation. The recovery rate of each cell was determined by dividing the number of each recovered cell by the number of each added cell. The results are shown in Table 5 below.

(Experimental example 19)
Cells were captured and collected in the same manner as in Experimental Example 18 except that the volume of the collected liquid was changed to 0.5 ml. The results are shown in Table 5 below.

(Experiment 20)
Cells were captured and collected in the same manner as in Experimental Example 18 except that the volume of the collected liquid was 0.5 ml and the filtration flow rate was 1 ml / min. The results are shown in Table 5 below.

(Experimental example 21)
The filtration area was 28 mm ² , the filtration flow rate was 1 ml / min, the volume of the recovered liquid was 0.5 ml, the flow rate was 100 ml / min, and the cells used were NCI-H1975 (human lung cancer) stained with Celltracker green. In the same manner as in Experimental Example 15, cells were captured and recovered. The results are shown in Table 5 below.

(Experimental example 22)
The cells were captured and collected in the same manner as in Experimental Example 21, except that the cells used were SW620 that had been stained with Celltracker orange. The results are shown in Table 5 below.

(Experimental example 23)
Cells were captured and collected in the same manner as in Experimental Example 21, except that the cells used were NCI-H1650 that had been stained with Celltracker green. The results are shown in Table 5 below.

(Experimental example 24)
Cells were captured and collected in the same manner as in Experimental Example 21, except that NCI-H1650 stained with Celltracker green was used, and immobilization was added after hemolysis. The results are shown in Table 5 below.

(Experimental example 25)
Cells were captured and collected in the same manner as in Experimental Example 21 except that the flow rate of the collected liquid was 50 ml / min. The results are shown in Table 5 below.

(Experimental example 26)
Cells were captured and collected in the same manner as in Experimental Example 22 except that the flow rate of the collected liquid was 50 ml / min. The results are shown in Table 5 below.

(Experiment 27)
Cells were captured and collected in the same manner as in Experimental Example 23, except that the flow rate of the collected liquid was 50 ml / min. The results are shown in Table 5 below.

(Experimental example 28)
Cells were captured and collected in the same manner as in Experimental Example 21, except that the flow rate of the collected liquid was 25 ml / min. The results are shown in Table 5 below.

(Experimental example 29)
Cells were captured and collected in the same manner as in Experimental Example 22 except that the flow rate of the collected liquid was 25 ml / min. The results are shown in Table 5 below.

(Experiment 30)
Cells were captured and collected in the same manner as in Experimental Example 23, except that the flow rate of the collected liquid was 25 ml / min. The results are shown in Table 5 below.

(Experimental example 31)
Cells were captured and recovered in the same manner as in Experimental Example 24 except that the flow rate of the recovery solution was 25 ml / min. The results are shown in Table 5 below.

(Comparative Experimental Example 1)
Cells were captured and collected in the same manner as in Experimental Example 21 except that the flow rate of the collected liquid was 10 ml / min and the cells used were SNU-1 that had been stained with Celltracker green. The results are shown in Table 5 below.

(Comparative Experimental Example 2)
Cells were captured and collected in the same manner as in Comparative Experimental Example 1 except that the flow rate of the collected liquid was changed to 5 ml / min. The results are shown in Table 5 below.

(Comparative Experiment 3)
Cells were captured and collected in the same manner as in Comparative Experimental Example 1, except that the flow rate of the collected liquid was 2.5 ml / min. The results are shown in Table 5 below.

(Comparative Experimental Example 4)
Cells were captured and collected in the same manner as in Comparative Experimental Example 1, except that the flow rate of the collected liquid was 1 ml / min. The results are shown in Table 5 below.

(Comparative Experimental Example 5)
The cells were captured and collected in the same manner as in Comparative Example 3 except that the cells used were SW620 stained with Celltracker orange. The results are shown in Table 5 below.

  As shown in Table 5, cells could be recovered from the filter at a recovery rate exceeding 70% in any of the experimental examples. Moreover, a high recovery rate could be realized with a small amount of recovered liquid.

  In the above Experimental Examples 1 to 30, after the collection of the collected liquid using the reverse flow mechanism, the entire collected liquid collected on the upper part of the filter was collected, but after collected so as to leave a small amount of collected liquid on the filter. In some cases, the recovery rate may be further improved by passing the recovered liquid again using the backflow mechanism.

  As shown in Table 5, cells are recovered from the filter at a high recovery rate exceeding 70% by setting the flow rate (per opening area) of the recovery liquid to at least 15 mm / second or more, preferably 36 mm / second or more. I was able to.

Claims (14)

A method of labeling particles captured by a filter having a plurality of through holes with magnetic beads,
Supplying a suspension containing magnetic beads to the filter surface on which the particles are trapped;
By sending a part of the suspension from the filter surface toward the other filter surface so that the liquid containing the magnetic beads is in contact with both surfaces of the filter; and
A magnetic labeling method comprising: feeding back the liquid containing the magnetic beads from the other filter surface in the direction of the filter surface where the particles are captured.   The labeling method according to claim 1, further comprising stirring the liquid containing the magnetic beads after the reverse feeding on a filter surface on which the particles are captured.   Passing the collected liquid from the direction opposite to the flow direction of the specimen for capturing the cells to the filter in which the cells have been captured, and aspirating and collecting the collected liquid collected on the filter The labeling method according to claim 1 or 2.   The labeling method according to any one of claims 1 to 3, further comprising capturing the particles on the filter by feeding a specimen containing the particles to the filter.   The labeling method according to claim 4, wherein the flow rate of the specimen is 0.01 mm / second to 100 mm / second per opening area of the filter.   6. The labeling method according to claim 4 or 5, wherein the flow rate of the reverse feeding liquid is higher than 1/10 of the flow rate of the specimen.   The labeling method according to any one of claims 1 to 6, wherein a flow rate of the reverse feeding liquid is 0.01 mm / second or more per opening area of the filter. An apparatus for labeling particles with magnetic beads,
A filter section capable of holding a filter;
An introduction flow path capable of introducing a suspension containing magnetic beads into the filter section;
A discharge channel capable of holding or discharging the liquid that has passed through the filter unit;
Liquid feeding means capable of supplying the suspension to the filter unit;
A controller for controlling the liquid feeding means,
The control unit sends a part of the suspension from one filter surface to the other filter surface so that a liquid containing the magnetic beads is in contact with both surfaces of the filter, and A labeling device for controlling the liquid feeding means so as to reversely feed a liquid containing magnetic beads from the other filter surface.   9. The marking device according to claim 8, comprising means for stirring the liquid at or near the surface of the filter. It further comprises a recovery means for recovering the particles captured by the filter unit,
The control means passes the collected liquid through the filter in which the cells are captured from a direction opposite to the direction in which the specimen is passed for capturing the cells, and sucks the collected liquid collected on the filter. 10. The labeling device according to claim 8 or 9, wherein the collecting means is controlled so as to collect.   The labeling device according to any one of claims 8 to 10, wherein the control unit controls the liquid feeding unit so as to supply a specimen containing particles to be labeled to the filter unit.   12. The labeling apparatus according to claim 11, wherein the control unit controls the liquid feeding unit to feed the sample at a flow rate of 0.01 mm / second to 100 mm / second per opening area of the filter.   The labeling device according to claim 11 or 12, wherein the control unit controls the liquid feeding unit so that a flow rate of the reverse feeding solution is faster than 1/10 of a flow rate of the specimen.   The labeling device according to any one of claims 8 to 13, wherein the control unit controls the liquid feeding unit such that a flow rate of the reverse liquid feeding is 0.01 mm / second or more per opening area of the filter. .