JP6754345B2 - Magnetic labeling method and labeling device for particles - Google Patents

Description

The present disclosure relates to a method for labeling particles with magnetic particles (hereinafter, also referred to as magnetic beads) and a device for labeling. The present disclosure relates, among other things, to a method of magnetically labeling particles trapped in a filter with magnetic beads on the filter and a labeling device used therein.

There are magnetic separation and size separation as a method 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 cell to be separated. On the other hand, size separation is a method that utilizes differences in particle size and deformability.
In the general method 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, in recent years, 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

Sci Transl Med 5, 179ra47 (2013)

Rare cells include medically important cells such as circulating tumor cells / Circulating Tumor Cell (CTC) and immune cells in addition to CTC. An example of CTC is cancer cells released from primary or metastatic tumor tissue. These cancer cells are said to cause metastasis by being transported to other organs or the like through blood or lymph vessels. Further, since CTC in blood is recognized to be useful as a factor for determining the therapeutic effect of metastatic cancer and predicting prognosis, analysis of CTC is being actively carried out.
However, rare cells such as CTC are usually contained in only about 0 to 10 in 10 ml of blood, and in order to perform more accurate analysis, it is necessary to separate or concentrate the rare cells from the sample with high accuracy. It has been demanded.

As described in Patent Document 1, the applicant has developed a method capable of efficiently separating rare cells from blood by using a filter having predetermined characteristics. However, in addition to rare cells, blood contains a large amount of blood cell components such as red blood cells and white blood cells (about tens of billions of red blood cells and about tens of millions of white blood cells), and the size of white blood cells is particularly rare. Since it is about the same or larger than that, it may be captured together 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 rare cells contained in blood (sample) is very small, while the amount of white blood cells is large, so the amount of white blood cells captured by the filter is large compared to the number of rare cells. , Some white blood cells are detected as rare cells, and as a result, false positives may easily occur. Second, when a gene mutation specific to a rare cell is detected, only the genotype of a leukocyte that is not a rare cell is detected, and as a result, false negatives may easily occur.
For these reasons, it is necessary to efficiently separate the leukocytes captured by the filter from the rare cells.

The present disclosure provides a method and a labeling device capable of efficiently magnetically labeling particles trapped in a filter on the filter in one or more embodiments.

The present disclosure is, in one embodiment, a method of labeling particles trapped in a filter having a plurality of through holes with magnetic beads, in which a suspension containing the magnetic beads is supplied to the filter surface on which the particles are trapped. By feeding a part of the suspension from the filter surface toward the other filter surface, the liquid containing the magnetic beads is in contact with both sides of the filter, and the magnetic The present invention relates to a magnetic labeling method comprising back-feeding a liquid containing beads from the other filter surface toward the filter surface in which the particles are captured.

The present disclosure is, in one embodiment, an apparatus for labeling particles with magnetic beads, which comprises a filter portion capable of holding a filter and an introduction flow path capable of introducing a suspension containing magnetic beads into the filter portion. A discharge flow path capable of holding or discharging a liquid that has passed through the filter unit, a liquid feeding means capable of supplying the suspension to the filter unit, and a control unit for controlling the liquid feeding means are provided. The control unit sends a part of the suspension from one filter surface toward the other filter surface so that the liquid containing the magnetic beads is in contact with both sides of the filter, and the control unit supplies the liquid. The present invention relates to a labeling device that controls 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 embodiment, the particles captured by the filter can be efficiently labeled with magnetic beads on the filter.

FIG. 1 shows an example of a cell labeling device that can be used in the magnetic labeling method of the present disclosure. FIG. 2 shows a schematic 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 white blood cells remaining (not magnetically captured) in Example 1 and Comparative Examples 1 and 2. FIG. 4 shows an example of the rare cell capture and recovery device used in Experimental Example 1. FIG. 5 shows an example of the configuration of a cell capture / recovery device that can be used in the cell recovery method of the present disclosure.

Rare cells such as circulating tumor cells / CTC in the blood may be present in the blood. CTCs are cells that have been released from primary or metastatic tumor tissue and infiltrated into blood. Cancer metastasis is thought to occur when cancer cells are carried to other parts of the body through blood vessels and lymph vessels and proliferate, so the number of CTCs in the blood and cancer metastasis It has been reported to be related to the possibility and prognosis of. In addition to CTC, rare cells include medically important cells such as immune cells. Therefore, it is expected that the analysis of rare cells in blood will be carried out more actively in the future.
10 ml of blood usually contains tens of billions of red blood cells and tens of millions of white blood cells, but rare cells such as CTC usually have about 0 to 10. Therefore, in order to perform more accurate analysis, it is necessary to separate and recover rare cells from a sample such as blood with high accuracy.

Rare cells include not only cancer cells larger in size than blood cells, but also small cells and highly deformable cells. 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 the highly deformable cell include human cancer cells (commercially available SNU-1 (cell line name, human cancer cell) has been confirmed as a highly deformable cell).
The applicant has established a method capable of capturing small cells and highly deformable cells with a high capture rate by using a predetermined filter (for example, Patent Document 1).
However, as described above, even when a sample such as blood is separated using the filter, leukocytes contained in a large amount in the sample may be captured together with rare cells. Therefore, a treatment for further separating rare cells and leukocytes is required. In addition, various treatments are required to analyze the rare cells after separation.
On the other hand, since the number of rare cells present in a sample is extremely small as described above, it is necessary to avoid loss during separation by a filter, treatment such as labeling, and recovery as much as possible. For this reason, it has been proposed to perform various treatments for the above-mentioned separation or analysis while the cells are captured by the filter. The treatment on the filter is performed by supplying various treatment liquids to the filter to react with the cells, and then sending the unreacted treatment liquid to remove the liquid. That is, the filter has a function as a reaction field for B / F separation (Bound / Free separation), and can perform various treatments without causing cell loss.
The present inventors have found that in various treatments on this filter, there are a treatment in which a sufficient treatment effect can be obtained and a treatment in which a sufficient treatment effect cannot be obtained. Specifically, although antibody staining can be sufficiently stained, labeling with magnetic particles has a problem that the labeling efficiency is very low and magnetic labeling cannot be sufficiently performed.
In view of the above problems, we tried to collect cells such as rare cells from the filter and label them with magnetic particles, but in this method, the degree of loss of the rare cells captured by the filter is different between workers. Alternatively, there was a problem that it was large between experiments and that the reproducibility was low. In addition, after the labeling treatment, it has occurred that the unreacted surplus magnetic particles and the cells cannot be sufficiently separated. As a result, there is a problem that the rare cells are entrained in a large amount of surplus magnetic particles present at the time of magnetic separation, which is the subsequent processing step, and the separation accuracy is lowered due to magnetic separation of the rare cells.

The present disclosure includes magnetic particles in one aspect so that when labeling with magnetic particles while the cells are trapped in the filter, the entire filter in which the cells are trapped is covered with a suspension containing the magnetic particles. By feeding the suspension, and then feeding the liquid containing the magnetic particles contained in the suspension in the direction opposite to the liquid feeding direction of the suspension (reverse feeding). It is based on a new finding found by the present inventors that the labeling efficiency by magnetic particles can be improved and cells can be separated from a large amount of surplus magnetic particles.

In one aspect of the present disclosure, since the number of cells captured by the filter is larger in leukocytes than in rare cells, if leukocytes, which are cells not intended for recovery, are labeled with magnetic particles, the separation accuracy of rare cells can be improved. It is based on the new findings found by the present inventors that it can be improved. Specifically, it is as follows. That is, in the rare cells CTC, the group expressing epithelial cell antigens such as EpCAM and cytokeratin, and the epithelial-mesenchymal transition in which the expression of these epithelial markers is reduced and the mesenchymal cell antigens such as Vimentin are expressed. It has been clarified that there is a group causing the disease. For this reason, it is difficult to recognize all CTCs with a specific antigen. Therefore, by labeling and 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 the white blood cells regardless of the antigen can be separated. It is based on a new finding found by the present inventors that CTC can be recovered. The labeling target of the present disclosure in the analysis of rare cells such as CTC is not limited to leukocytes, and may be rare cells.

According to the present disclosure, the mechanism by which cells trapped in the filter can be efficiently labeled with magnetic particles on the filter is not clear, but it is presumed as follows.
When whole blood is sent to a filter having a plurality of through holes and filtered, the whole blood is sent as a laminar flow, so that the flow velocity near the wall surface of the flow path decreases, and as a result, the filter around the wall surface. Red blood cells and white blood cells that are not the targets of capture are not filtered and remain in a relatively large amount. When staining or labeling is performed in this state, the antibody staining solution, which is a soluble reagent, enables sufficient staining by diffusing the reagent itself, but labeling with magnetic particles magnetizes the leukocytes around the wall surface. The particles cannot be sufficiently contacted and the labeling efficiency is low. The reason for this is considered to be that the flow velocity around the wall surface where a large number of leukocytes are present is slower than that in the central part 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 magnetic particles, and then the suspension is fed back. By immersing both main surfaces of the filter in a liquid containing magnetic particles, the cells captured by the filter can be transferred to both the cell capture surface of the filter and the filter surface opposite to the cell capture surface (the upper surface of the filter). And the underside of the filter) and in the through holes, the cells can come into contact with the suspension (magnetic particles), greatly increasing the surface area. In this state, by back-feeding the liquid from the filter surface opposite to the filter capture surface toward the cell capture surface (from the lower surface side to the upper surface side of the filter), the cells fitted in the through holes of the filter are released from the pores. The suspension containing the magnetic particles is agitated by the turbulence generated by the back-delivery liquid as well as being liberated, so that the frequency of contact 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 may not be construed as limiting to these mechanisms.

[Labeling method with magnetic particles (magnetic beads)]
The present disclosure relates to, in one aspect, a method of labeling particles trapped in a filter having a plurality of through holes with magnetic beads (labeling method of the present disclosure). In one aspect, the labeling of the present disclosure is to supply a suspension containing magnetic beads to a filter surface on which particles are captured, in which a portion of the suspension is directed from the filter surface to the other filter surface. By sending the liquid to the filter surface, the liquid containing the magnetic beads is brought into contact with both sides of the filter, and the liquid containing the magnetic beads is brought into contact with the filter surface on which the particles are captured from the other filter surface. Includes reverse feeding in the direction. Further, in one embodiment, the labeling method of the present disclosure is to supply a suspension containing magnetic beads to the upper surface of a filter in which the particles are captured, and to obtain an upper liquid level and a lower liquid level of the suspension. A part of the suspension is sent toward the lower surface side of the filter so as to sandwich the filter, and the liquid is sent to the filter from the lower surface side to the upper surface side of the filter. including. According to the labeling method of the present disclosure, in one or more embodiments, labeling of particles captured by the filter with magnetic beads can be efficiently performed on the filter without being recovered from the filter.

The labeling method of the present disclosure comprises supplying a suspension containing magnetic beads to the filter surface on which the 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 and the like. The feed amount is 50 μL or more or 100 μL or more per filtration area (28 mm ² ), or magnetic with a small amount of liquid, in that the magnetic beads can be efficiently and uniformly suspended on the filter in one or more embodiments. It is 500 μL or less or 300 μL or less from the viewpoint that beads can be suspended efficiently and uniformly.

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 in one or more embodiments. It is .004% or more, and 0.0625% or less or 0.0375% or less from the viewpoint of suppressing the non-specific reaction between the magnetic beads and the cells.

The magnetic beads (magnetic particles) are not particularly limited, and can be appropriately determined according to the type of particles to be labeled and the like. As the magnetic beads, in one or more embodiments, known magnetic beads used for magnetically labeling particles such as cells or magnetic beads that can be developed in the future can be used. Examples of the magnetic beads include magnetic particles (beads) in which a substance that specifically reacts with a binding molecule of the particles to be labeled is fixed on the surface in one or more embodiments. Examples of the substance include avidin, streptavidin, neutravidin, antibodies, antigens and the like in one or more embodiments. The size of the magnetic beads is not particularly limited, and can be appropriately determined depending on the size of the particles and / or the size of the opening (hole) of the filter used for the magnetic labeling. When the size of the particles to be labeled has a diameter of 5 μm to 20 μm, such as leukocytes or CTC, in one or more embodiments, from the viewpoint of improving labeling efficiency, 1 μm or less, 0.8 μm or less, or 0. It is 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. The size of the magnetic beads is smaller than the size of the opening (hole) of the filter used for the magnetic labeling from the viewpoint that excess magnetic beads can be removed by cleaning after the magnetic labeling.

In the labeling method of the present disclosure, a part of the suspension supplied to the filter surface (particle trapping surface or the upper surface of the filter) in which the particles are captured is subjected to the other filter surface (the filter surface opposite to the particle trapping surface (the filter surface opposite to the particle capturing surface) This includes sending the liquid toward the opposite surface (or the lower surface of the filter)) so that the liquid containing the magnetic beads is in contact with both surfaces of the filter (both surfaces of the particle trapping surface and the opposite surface). Further, in one or more embodiments, a part of the suspension is sent from the lower surface of the filter so that the upper liquid surface and the lower liquid surface of the liquid containing the magnetic beads sandwich the filter. It may include sending liquid toward the side. In one or more embodiments, the liquid feeding is carried out so that the liquid containing the magnetic beads is held in the vicinity of the filter surface on the opposite side. The liquid delivery may be carried out in one or more embodiments by pumping from the opposite side so that the suspension passes through the filter and the liquid containing the magnetic beads is in contact with both sides of the filter. it can. That is, in the labeling method of the present disclosure, a part of the suspension is positively sent toward the filter surface side opposite to the particle trapping surface, so that the liquid unintentionally permeates or diffuses into the filter. This is different from the phenomenon in which the liquid reaches the vicinity of the lower surface of the filter.

The liquid containing the magnetic beads is not particularly limited as long as it is a liquid containing the magnetic beads, and in one or a plurality of embodiments, a suspension containing the magnetic beads supplied to the filter surface and the suspension thereof. Examples thereof include a liquid obtained by mixing a turbid liquid with another liquid and diluting the 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, 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. Aqueous solvents such as liquid and water can be mentioned. Examples of the buffer solution include, in one or more embodiments, a Tris buffer solution such as Tris-HCl buffer solution, a phosphate buffer solution, a HEPES buffer solution, a citric acid-phosphate buffer solution, and the like. Examples of the isotonic solution include physiological saline, sucrose isotonic solution, glucose isotonic solution, mannose isotonic solution and the like.

The proportion of the suspension delivered to the opposite side is the suspension supplied to the particle trapping surface from the point of ensuring that both sides of the filter are in good contact with the liquid containing the magnetic beads in one or more embodiments. It is 10% or more, 20% or more, or 40% or more of the liquid, and 90% or less from the point of efficiently sending the entire suspension when the liquid is sent from the opposite surface side toward the particle trapping surface side. , 80% or less, or 60% or less.

In one or more embodiments, the flow velocity of the suspension is 0.07 mm / sec or more and 0.1 mm / sec 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. More than or equal to 0.3 mm / sec or more, and similarly, from the viewpoint of improving the reaction efficiency between the particles captured by the filter and the magnetic beads, 14 mm / sec or less, 1.4 mm / sec or less, or 0. It is 7 mm / sec 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 10,000 μL / min or less, 1000 μL / min or less, or 500 μL / min or less. The "flow velocity per opening area of the filter" in the present disclosure means the velocity of the liquid passing through the opening (hole) of the filter. 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) in one or more embodiments. The "flow rate" in the present disclosure refers to the amount (volume) of liquid flowing in a unit time. The flow rate includes, in one or more embodiments, the amount (volume) of liquid that passes through the filter in a unit time.

In the labeling method of the present disclosure, with respect to a filter whose both sides are immersed in a suspension, the particles are captured from the filter surface side opposite to the particle trapping surface toward the filter surface side (from the lower surface side to the upper surface side of the filter). Includes sending (backflowing) liquid. To the filter in which both main surfaces are in contact with the liquid containing magnetic beads (immersed), the liquid is sent from the opposite side toward the particle trapping surface side, and the suspension containing the magnetic beads is sent. 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.

For backflow, in one or more embodiments, only the suspension located on the filter surface side (filter lower surface side) opposite to the particle trapping surface may be fed, or a liquid different from the suspension. May be sent. Liquids different from suspensions, in one or more embodiments, are phosphate buffered saline (PBS), PBS-BSA (eg 0.2%), PBS-EDTA (eg 1 mg / ml) and buffer. , Isotonic solution and aqueous solvent such as water. Examples of the buffer solution include, in one or more embodiments, a Tris buffer solution such as Tris-HCl buffer solution, a phosphate buffer solution, a HEPES buffer solution, a citric acid-phosphate buffer solution, and the like. Examples of the isotonic solution include physiological saline, sucrose isotonic solution, glucose isotonic solution, mannose isotonic solution and the like.

The flow rate of liquid feeding (backflow flow rate) from the opposite surface side to the particle capture surface side may be appropriately determined depending on the flow rate of the sample when the particles are captured by the filter in one or more embodiments. The backflow flow rate may be faster than or slower than the flow rate of the sample in one or more embodiments. As the backflow flow velocity, in one or more embodiments, a flow velocity of 1/10 times or more, a flow rate of 1/5 times or more, a flow rate of 1/2 times or more, a flow rate of 1 time or more, and a flow rate of 2 times or more The flow velocity is 3 times or more, or 5 times or more. The backflow flow rate may be higher than the flow rate of the sample in one or more embodiments from the viewpoint of improving the labeling efficiency by the magnetic beads.

The flow velocity of the liquid to be sent from the opposite surface side to the particle trapping surface side is 0.01 mm / sec or more per opening area of the filter, from the viewpoint of further improving the labeling efficiency by the magnetic beads in one or more embodiments. It is 1 mm / sec or more or 1 mm / sec or more, and is 150,000 mm / sec or less, 100,000 mm / sec or less, or 50,000 mm / sec or less per opening area of the filter from the viewpoint of suppressing damage to cells. The backflow 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 and 50 million from the viewpoint of suppressing damage to cells. It is μL / min or less or 10 million μL / min or less.

The amount of liquid to be regurgitated is 50 μL or more or 100 μL or more per filtration area (28 mm ² ), or 100 μL or more, from the viewpoint of efficiently delivering the liquid containing magnetic beads to the particle trapping surface in one or more embodiments. 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 the filter is immersed in the suspension and the liquid is sent (backflow) from the opposite surface side to the particle trapping surface side is not particularly limited, and may be once or twice or more. It may be.

In the labeling method of the present disclosure, in one or more embodiments, the magnetic beads on the particle trapping surface and the liquid containing the particles to be labeled are stirred in one or more embodiments from the viewpoint of further improving the labeling efficiency with the magnetic beads. It may include that. Stirring may include pipetting in one or more embodiments.

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

In the labeling method of the present disclosure, from the viewpoint of further improving the labeling efficiency with magnetic beads, in one or more embodiments, after pipetting, the liquid containing the magnetic beads and the particles to be labeled is filtered. It may include standing still. As a result, the particles previously captured by the filter containing the particles to be labeled can be captured by the filter again, and the particles and the magnetic beads can be brought into contact with each other again. In one or more embodiments, the standing is preferably carried out with both sides of the filter covered with a liquid containing magnetic beads and particles to be labeled.

In one or more embodiments, the flow rate of the filter filtration is 0.07 mm / sec or more, 0.1 mm / sec, 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. It is 12 mm / sec or more or 0.3 mm / sec or more, and similarly, from the viewpoint of improving the reaction efficiency between the particles captured by the filter and the magnetic beads, 14 mm / sec or less, 1.4 mm / sec or less or 0 per filter opening area. It is 0.7 mm / sec 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 10,000 μL / min or less, 1000 μL / min or less, or 500 μL / min or less.

The standing time is 1 minute or more, 5 minutes or more, or 10 minutes or more from the point of sufficient reaction between the particles and the magnetic beads in one or more embodiments, and the non-specific reaction between the particles and the magnetic beads is caused. From the point of prevention, it is 60 minutes or less, 45 minutes or less, or 30 minutes or less.

The labeling method of the present disclosure may include, in one or more embodiments, feeding a cleaning solution to clean the excess magnetic beads that have not been used for labeling. The cleaning solution includes, in one or more embodiments, 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 can be mentioned. Examples of the buffer solution include, in one or more embodiments, a Tris buffer solution such as Tris-HCl buffer solution, a phosphate buffer solution, a HEPES buffer solution, a citric acid-phosphate buffer solution, and the like. Examples of the isotonic solution include physiological saline, sucrose isotonic solution, glucose isotonic solution, mannose isotonic solution and the like.

In the labeling method of the present disclosure, in one or more embodiments, the particles may be captured in advance by the filter, or the particles may be captured by the filter prior to the supply of the suspension containing the magnetic beads. May include. Capturing particles by a filter can be performed in one or more embodiments by feeding a sample containing the particles to be labeled to the filter and filtering.

The flow velocity of the sample is 0.01 mm / sec or more, 0.05 mm / sec or more, or 0.1 mm / sec per opening area in one or more embodiments from the viewpoint of improving the capture rate of particles in the sample. From the same point, it is 100 mm / sec or less, 50 mm / sec or less, 25 mm / sec or less, 10 mm / sec or less, 5 mm / sec or less, or 2 mm / sec or less. The flow rate of the sample 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 sample. 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 present disclosure, the flow path on the filter surface side opposite to the filter surface on which the particles are captured is filled with a liquid prior to the supply of the suspension containing the magnetic beads. May include doing. The filling of the liquid may be carried out in one or more embodiments so that the filter in which the particles are captured is immersed in the liquid. Examples of the liquid include liquids that can be used as the above-mentioned cleaning liquid in one or more embodiments.

The labeling method of the present disclosure can be carried out in one or more embodiments by using an apparatus or the like having a separation portion including a filter having a plurality of through holes. In one or more embodiments, the labeling method of the present disclosure is an open system in which the upper part of the filter is open, because operability can be improved and even a small amount of magnetic bead suspension can be efficiently labeled. It is preferable to carry out with the device of.

The above-mentioned treatments such as suspension supply, liquid feeding and stirring in the labeling method of the present disclosure may be performed automatically or manually in one or more embodiments. Further, a part of the processing may be performed manually and the remaining processing may be performed automatically.

Examples of the filter used in the labeling method of the present disclosure include filters capable of capturing rare cells in a sample in one or more embodiments. In one or more embodiments, the filter is preferably a filter capable of capturing at least small cancer cells, highly deformable cancer cells, or both of these cells. Further, in one or more embodiments, the filter is preferably a filter capable of capturing at least SNU-1, SW620, or both of them, and more preferably a filter capable of capturing CTC in a sample. As the filter, in one or more embodiments, a filter capable of capturing rare cells conventionally known or developed in the future can be used. Examples of the filter include the filter described in Patent Document 1 and the filter described in Japanese Patent Application No. 2016-057313 in one or more embodiments. Further, for example, a single sheet-shaped membrane having 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 those other than non-woven fabrics are preferable.

The material of the filter may, in one or more embodiments, a metal such as nickel, SUS, gold, silver, body, 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 Below, 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 "filtered area" in the present disclosure refers to the area of the portion of the total area of the filter that the suspension containing the magnetic beads or the sample comes into contact with (delivered).

The particles labeled by the labeling method of the present disclosure can separate the particles of interest by a known magnetic separation device in one or more embodiments.

In one or more 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 target particle can be specifically labeled. In one or more embodiments, in the analysis of rare cells such as CTC, even when rare cells and leukocytes are captured by the filter, according to the labeling method of the present disclosure, leukocytes are magnetically labeled with high labeling efficiency. Can be labeled. Therefore, after labeling by the labeling method of the present disclosure, the rare cells and the magnetically labeled leukocytes can be accurately separated by magnetically separating the collected suspension, whereby the rare cells can be recovered with high accuracy. it can. In one or more embodiments where the filter captures the rare cells and the leukocytes, the rare cells may be magnetically labeled instead of the leukocytes.
Recovery after labeling can be performed in one or more embodiments by feeding the recovery solution in a direction opposite to the filtration direction when the particles are captured by the filter. In one or more embodiments, the collected liquid is collected through the filter in which the cells are captured from the direction opposite to the flow rate of the sample for capturing the cells, as in the method for collecting cells described later. Pass the liquid so that the ratio of the flow rate of the liquid to the flow rate of the sample ([flow rate of the recovered liquid] / [flow rate of the sample]) is 25 or more, or flow rate of 1 mm / sec or more per opening area. It can be done by passing the liquid through. The specific conditions and the like are the same as the cell recovery 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 without particular limitation include rare cells, leukocytes, erythrocytes, platelets and their undifferentiated cells. Rare cells refer to cells other than blood cell components (erythrocytes, white blood cells, and platelets) that can be contained in the blood of humans or non-human animals. Rare cells include, in one or more embodiments, cancer cells, circulating tumor cells, vascular endothelial cells, vascular endothelial precursor cells, cancer stem cells, epithelial cells, hematopoietic stem cells, mesenchymal stem cells, fetal cells, stem cells, Examples thereof include undifferentiated leukocytes, undifferentiated erythrocytes, and cells selected from the group consisting of combinations thereof.

Examples of the sample in the present disclosure include a sample that may contain particle cells that can be applied to the labeling method of the present disclosure. Samples include, in one or more embodiments, blood samples. Examples of the blood sample include blood or a diluted product thereof in one or more embodiments without limitation, from the viewpoint of simple and rapid processing and suppression of damage to rare cells in the blood.

[Signing device]
The present disclosure relates to, in one aspect, a device for labeling particles with magnetic beads (the labeling device of the present disclosure). The labeling device of the present disclosure includes a filter unit capable of holding a filter, an introduction flow path capable of introducing a suspension containing magnetic beads into the filter unit, and a discharge flow path capable of holding or discharging a liquid passing through the filter unit. A liquid feeding means capable of supplying the suspension to the filter unit, and a control unit for controlling the liquid feeding means are provided. In the labeling device of the present disclosure, the control unit feeds and magnetically passes the suspension so that at least a part of the suspension supplied to the filter unit passes through the filter unit and is held. The liquid feeding means is controlled so that the liquid containing the beads is fed back. According to the labeling device of the present disclosure, the labeling method of the present disclosure can be easily performed.

The labeling device of the present disclosure may include, in one or more embodiments, a stirring means for stirring the liquid at or near the surface of the filter.

In one or more embodiments, the control unit may control the liquid feeding means to supply the filter unit with a sample containing the particles to be labeled. The control unit may control the liquid feeding means so that the sample is fed at a flow rate of 0.01 mm / sec to 100 mm / sec per opening area of the filter. The control unit may control the liquid feeding means so that the flow rate of the reverse liquid is faster than 1/10 of the flow rate of the sample. The control unit may control the liquid feeding means so that the liquid feeding flow rate of the reverse liquid is 0.01 mm / sec or more per the opening area of the filter.

Hereinafter, an embodiment, which is not limited to the labeling method of the present disclosure, will be described.

This will be described using the rare cell labeling device shown in FIG.

An example of a rare cell labeling device that can be used in the labeling method of the present disclosure is shown in FIG.
The rare cell labeling device shown in FIG. 1 includes a supply port 1 for supplying a suspension containing magnetic beads, a washing liquid, a staining liquid, and the like, a discharge port 2 for discharging waste liquid such as the washing liquid, and an upper part 3 of the device. It has a lower part 4 of the device, a filter part 5, and a liquid feeding mechanism (not shown) capable of feeding liquid in the directions of arrows A and B. The filter unit 5 is arranged between the upper part 3 of the device and the lower part 4 of the device. The device upper portion 3 has a flow path 6 and a support member 7 for fixing the filter portion 5, and the flow path 6 communicates with the supply port 1. The lower part 4 of the device has a flow path 8 and a support member 9 for fixing the filter portion 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 or the like) 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 the joining materials 11 and 12. In the flow path 6 connecting the supply port 1 and the filter portion 5, a connection portion (not shown) and a three-way stopcock valve (not shown) capable of switching the liquid may be arranged above the filter portion 5. .. Further, in the flow path 6 connecting the supply port 1 and the filter unit 5, the pressure applied to the filtration is constant (as a mechanism capable of supplying liquid at a constant pressure or a mechanism capable of supplying liquid at a constant flow rate). ) Pressurizing means (not shown) may be arranged. Further, it may be pulled by a syringe pump so that the pressure applied to the filtration becomes above a certain level.

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

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

Then, from the viewpoint of improving the reactivity of the reagent with white blood cells, the red blood cells remaining in the filter 10 may be removed by supplying and treating the hemolytic agent.

Next, the buffer solution is supplied from the supply port 1 to fill the flow path 8 with the buffer solution. The buffer solution is supplied so that the cell capture surface (upper surface) 10a of the filter 10 is immersed. Then, the suspension 21 containing the magnetic particles is supplied to the cell capture surface 10a of the filter 10 (FIG. 2A). From the viewpoint of improving the reactivity between the magnetic particles and the leukocytes, the leukocytes may be immunostained with an antibody prior to the supply of the suspension 21. Examples of the antibody include an antibody to which a binding molecule is bound. Examples of the binding molecule include biotin and the like in one or more embodiments. Further, as the magnetic particles, for example, magnetic particles in which a substance that specifically reacts with the binding molecule is immobilized may be used. Specific reactive substances include, in one or more embodiments, proteins such as streptavidin and neutravidin.

The cells of the filter 10 are moved to the filter surface (lower surface) 10b side (flow path 8 side) opposite to the cell capture surface 10a so that a part of the suspension 21 containing the magnetic particles moves. Liquid is sent from the trapping 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 sent (reversely fed) from the filter surface 10b side toward the cell capture surface 10a side (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 capture surface (upper surface) 10a of the filter 10, and the liquid containing the magnetic particles on the cell capture surface 10a of the filter 10 is agitated, resulting in leukocytes and leukocytes. The frequency of contact with magnetic particles can be improved, and labeling efficiency can be improved. Only the suspension located on the filter surface 10b side may be back-delivered, or different liquids may be back-delivered according to the suspension. The different liquids may or may not contain magnetic particles.

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

Next, the suspension 23 containing the cells on the cell capture surface 10a of the filter 10 and the magnetic particles is moved from the cell capture surface 10a side toward the opposite filter surface 10b side (directions of arrows A in FIGS. 1 and 2). ) The liquid is sent again and then allowed to stand for a predetermined time (FIG. 2 (d)). As a result, the cells contained in the suspension are captured by the filter 10. Further, although the magnetic particles pass through the filter 10, when the magnetic particles pass through the filter 10, the magnetic particles can be re-contacted with the cells captured by the filter 10, so that the contact frequency between the leukocytes and the magnetic particles is further improved. It is possible to improve the labeling efficiency. It is preferable that the liquid feeding is performed so that the cell capture surface 10a of the filter 10 and the filter surface 10b on the opposite side are sandwiched by the liquid containing the magnetic particles.

Then, after lowering the liquid 23 containing the magnetic particles to near the cell capture surface 10a of the filter 10, the cleaning liquid 24 is supplied from the cell capture surface 10a side of the filter 10 (in the direction of arrow A in FIGS. 1 and 2) (FIG. 2 (FIG. 2). e)), the liquid is sent so that the liquid level of the cleaning liquid 24 is close to the cell capture surface 10a of the filter 10 (FIG. 2 (f)). As a result, the unreacted magnetic particles remaining on the filter 10 are removed. The cleaning liquid 24 may be repeatedly fed a plurality of times.

After labeling leukocytes with the above magnetic particles, rare cells may be immunostained with an antibody. This makes it possible to efficiently detect rare cells after separation and recovery. Examples of the antibody include an antibody that recognizes an antigen expressed in cells of CTC, such as Cytokeratin. From the viewpoint of reacting the antibody with the antigen in the rare cells, immobilization treatment and membrane permeation treatment may be performed prior to labeling with the antibody.

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

The present disclosure may relate to one or more embodiments below.
[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 captured,
By sending a part of the suspension from the filter surface toward the other filter surface, the liquid containing the magnetic particles is brought into contact with both sides of the filter, and
A magnetic labeling method comprising back-feeding a liquid containing the magnetic particles from the other filter surface toward the filter surface on which the particles are captured.
[2] The method according to [1], which comprises sending a liquid from the other filter surface to the filter surface on which the particles are captured, and then stirring the liquid containing the magnetic particles on the filter surface on which the particles are captured. Marking method.
[3] The labeling method according to [1] or [2], which comprises feeding the sample containing the particles to the filter to capture the particles in the filter.
[4] The labeling method according to [3], wherein the flow velocity of the sample is 0.01 mm / sec to 100 mm / sec per the opening area of the filter.
[5] The labeling method according to [3] or [4], wherein the reverse liquid flow rate is made faster than 1/10 of the flow rate of the sample.
[6] The labeling method according to any one of [1] to [5], wherein the reverse liquid flow rate is 0.01 mm / sec or more per the opening area of the filter.

The present disclosure also provides the following devices in addition to the above methods. It goes without saying that the following devices are clearly and sufficiently disclosed to those skilled in the art in the above description.
(A1) A device for labeling particles captured by a filter having a plurality of through holes with magnetic particles.
A means for supplying a suspension containing magnetic particles to the upper surface of a filter in which the particles are captured,
A means for sending a part of the suspension toward the lower surface side of the filter so that the upper liquid level and the lower liquid level of the suspension sandwich the filter, and the filter with respect to the filter. A magnetic labeling device, including means for delivering liquid from the lower surface side to the upper surface side of the device.
(A2) The labeling apparatus according to (A1), comprising means for stirring the suspension on the upper surface of the filter after the liquid is fed from the lower surface side to the upper surface side of the filter.
(A3) The labeling apparatus according to (A1) or (A2), which comprises means for sending a sample containing the particles to the upper surface of the filter.
(A4) The labeling apparatus according to (A3), wherein the sample liquid feeding means feeds the sample so that the flow velocity of the sample is 0.01 mm / sec to 100 mm / sec per the opening area of the filter.
(A5) The means for delivering the liquid from the lower surface side to the upper surface side of the filter sends the liquid so that the flow velocity of the liquid from the lower surface side to the upper surface side of the filter is faster than 1/10 of the flow velocity of the sample. The labeling device according to (A3) or (A4), which is liquid.
(A6) The means for feeding the liquid from the lower surface side to the upper surface side of the filter so that the liquid flow velocity from the lower surface side to the upper surface side of the filter is 0.01 mm / sec or more per the opening area of the filter. , The labeling device according to any one of (A1) to (A5).
[B1] A device for labeling particles with magnetic particles.
The filter part that can hold the filter and
An introduction flow path into which a suspension containing magnetic particles can be introduced into the filter portion,
A discharge flow path capable of holding or discharging the liquid that has passed through the filter portion, and
A liquid feeding means capable of supplying the suspension to the filter unit, and
A control unit for controlling the liquid feeding means is provided.
The control unit sends a part of the suspension from one filter surface toward the other filter surface so that the liquid containing the magnetic particles is in contact with both surfaces of the filter. A labeling device that controls 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], which comprises means for stirring a liquid.
[B3] The labeling device according to [B1] or [B2], which comprises means for stirring a liquid at 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 means so as to supply a sample containing the labeled particles to the filter unit.
[B5] The labeling device according to [B4], wherein the control unit controls the liquid feeding means so as to feed the sample at a flow rate of 0.01 mm / sec to 100 mm / sec per opening area of the filter.
[B6] The labeling device according to [B4] or [B5], wherein the control unit controls the liquid feeding means so that the flow rate of the reverse liquid is faster than 1/10 of the flow rate of the sample.
[B7] The control unit controls the liquid feeding means so that the liquid feeding flow rate of the reverse liquid is 0.01 mm / sec or more per the opening area of the filter, according to [B1] to [B6]. The marking device according to any one.

[Method of collecting cells in a sample]
In order to analyze rare cells with high accuracy, it is required to collect the cells captured by the filter with high accuracy. Therefore, the present disclosure relates to a method in which cells trapped in a filter can be recovered from the filter with a high recovery rate in another aspect.

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

Recovery of cells from body fluids is also performed for purposes other than testing. For example, Japanese Patent Application Laid-Open No. 10-2014770 (Patent 441,621) discloses a method for recovering a target cell from a body fluid such as bone marrow for hematopoietic stem cell transplantation or the like. In the examples of JP-A-10-201470, bone marrow or peripheral blood is introduced into a non-woven fabric, and leukocytes or CD34-positive cells are captured by the non-woven fabric, and then the viscosity is 17.2 mPa · s or more and 31.8 mPa · s or less. Disclosed is a method for recovering leukocytes or CD34-positive cells trapped in a non-woven fabric by regurgitating the liquid having.

However, in the method of JP-A-10-201470, the viscosity of the recovery 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 recovery liquid is limited. is there. Further, since Japanese Patent Application Laid-Open No. 10-201470 is a method for separating and recovering cells for hematopoietic stem cell transplantation and the like as described above, the cells captured by the capture means can be recovered with a large amount of recovery liquid. Therefore, there is a problem that the recovered cells are not suitable for analysis.
Patent Document 1 does not disclose a technique for recovering rare cells trapped in a filter.

Applicants have established a method capable of capturing small cells such as SW620 and highly deformable cells such as SNU-1 with a high capture rate by using a predetermined filter as described above. (For example, Patent Document 1).
On the other hand, the analysis of rare cells is performed, for example, by observing the rare cells, measuring the number of the rare cells, and measuring the nucleic acid extracted from the rare cells. In order to perform these analyzes accurately, it is necessary to separate and collect the rare cells trapped in the filter from the filter.

In the process of intensive research, the present inventors, in collecting rare cells from a filter in which rare cells were captured, the ratio of the flow rate of the collected liquid to the flow rate of the sample at the time of capturing the rare cells ( It has been found that cells can be recovered with a high recovery rate and a small amount of recovery liquid by backflowing the recovery liquid so that [flow rate of recovery liquid] / [flow rate of sample]) is 25 or more. In addition, the present inventors regurgitated the collected solution to the filter that captured the rare cells at a flow rate of 1 mm / sec or more per opening area of the filter, thereby suppressing damage to the cells and capturing the cells. We have found that cells can be recovered with a high recovery rate.

The present inventors have one of the reasons why the rare cells captured by the filter cannot be recovered with a high recovery rate and a small amount of recovery solution, and some of the rare cells and other mixed cells are mixed during filtration (capture) of the rare cells. The pores of the cells are blocked, and the filter at the time of collection contains both blocked and unoccluded holes. As a result, the rare cells trapped in the blocked holes have sufficient force to push out the recovered fluid by passing it through. It was found that it does not act on. Then, the collected liquid is passed from the direction opposite to the flowing direction of the sample for cell capture, and the flow velocity is set to the filter surface (opposite to the cell capture surface of the filter) through which the collected liquid is passed. It was found that the rare cells trapped in the filter can be recovered with a high recovery rate and a small amount of recovery liquid by setting the flow velocity at which turbulence occurs on the side filter surface).

According to JP-A-10-201470, 50 ml of peripheral whole blood is passed through a filter made of polyester non-woven 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 the above (Examples 1 to 5). On the other hand, the same document also discloses that when a recovery solution having a viscosity of 1.0 mPa · s (physiological saline) is used instead of the recovery solution having the above viscosity, the recovery rate is 31% or less. (Comparative Examples 1 and 2).
On the other hand, according to the method of the present disclosure, the ratio of the flow rate of the recovered solution to the flow rate of the sample at the time of cell capture or the flow rate of the recovered solution is set in a specific range, and a specific filter (plurality). By using a filter having through-holes in the filter), it is possible to recover cells trapped in the filter at a high recovery rate regardless of the viscosity of the recovery liquid. Further, the method of the present disclosure has realized that even a recovery liquid having a viscosity for which a sufficient recovery rate cannot be obtained in Japanese Patent Application Laid-Open No. 10-201470 can be recovered with a high recovery rate. That is, the method of the present disclosure is a technique having a completely different technical idea from Japanese Patent Application Laid-Open No. 10-201470.
Further, the method of JP-A-10-201470 is a method of selectively and efficiently recovering leukocytes from a mixture of leukocytes, platelets and erythrocytes for hematopoietic stem cell transplantation and the like. As a means for capturing cells (white blood cells), those having a large surface area per volume, such as non-woven fabrics, 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, diagnosis, and the like. That is, the purposes of Japanese Patent Application Laid-Open No. 10-201470 and the method of the present disclosure are completely different. Further, the non-woven fabric used in Japanese Patent Application Laid-Open No. 10-201470 and the filter having a plurality of through holes used in the present disclosure differ greatly in the mechanism by which cells are captured and the state in which they are captured. The method and JP-A-10-201470 are completely different technical ideas.

In the present disclosure, the "flow rate of liquid" means the amount (volume) of liquid flowing in a unit time. The flow rate of the liquid includes, in one or more embodiments, the amount (volume) of the liquid passing through the filter in a unit time.

In the present disclosure, the "velocity rate per opening area of the filter" means the speed of the liquid passing through the opening (hole) of the filter. The flow velocity per opening area can be calculated by dividing the flow rate of the liquid by the total area of the openings (holes) in one or more embodiments.

[Cell recovery method]
The present disclosure relates to, in one embodiment, a method of recovering cells trapped in a filter having a plurality of through holes from the filter (recovery method of the present disclosure). In one embodiment, the collection method of the present disclosure allows the flow rate of the collected liquid and the flow rate of the sample to pass through the filter in which the cells are captured from the direction opposite to the flow direction of the sample for capturing the cells. It includes passing the recovered liquid through the filter so that the ratio to and ([flow rate of the recovered liquid] / [flow rate of the sample]) is 25 or more, and recovering the recovered liquid collected on the filter. Further, in one aspect of the collection method of the present disclosure, cells are captured by collecting the collected solution from a direction opposite to the flow direction of the sample for capturing cells at a flow rate of 1 mm / sec or more per opening area. This includes passing the liquid through the filter and collecting the recovered liquid accumulated on the filter.

According to the present disclosure, in one or more embodiments, the cells trapped in the filter can be recovered with a high recovery rate. According to the present disclosure, in one or more embodiments, the cells captured by the filter can be recovered at a high recovery rate while suppressing damage to the cells. Also, according to the present disclosure, in one or more embodiments, cells can be recovered with a small amount of recovery solution (eg, at a level that can be contained in a microtube).

The recovery method of the present disclosure includes passing a recovery solution through a filter having a plurality of through holes in which cells are captured from a direction opposite to the flow direction of the sample for capturing the cells. .. In one or more embodiments, the flow rate of the recovered liquid is such that the ratio of the flow rate of the recovered liquid to the flow rate of the sample ([flow rate of the recovered liquid] / [flow rate of the sample]) is 25 or more. It can be carried out by passing the liquid at a flow rate of 1 mm / sec or more per opening area. By regurgitating the recovered solution under the above-mentioned liquid passing conditions to the filter having a plurality of through holes, in one or more embodiments, the cells captured by the filter can be recovered at a high recovery rate while suppressing damage to the cells. Can be recovered. In the recovery method of the present disclosure, "flowing from the reverse direction (backflow)" means that the recovery liquid is introduced 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 recovered liquid flows from the side where the sample flows out (downstream side) to the side where the sample flows in (upstream side) when the sample is passed.

The ratio of the flow rate of the collected liquid to the flow rate of the sample ([flow rate of the recovered 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 viewpoint of improving the rate, it is 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. Further, from the viewpoint of further improving the cell recovery rate, the values are 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, and 15 from the same point. It is 000 or less, 12,000 or less, or 10,000 or less.

The flow rate of the recovered solution 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 more embodiments from the viewpoint of improving the recovery rate of cells from the filter. That is all. 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. Further, from the viewpoint 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, 7,000 ml / min or less, 6,000 ml / min or less, 5, It is 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.

As for the flow velocity of the recovery liquid, in one or more embodiments, turbulence is generated on the filter surface on the side opposite to the filter surface on the side where the sample is introduced to capture the cells, and the recovery rate of the cells from the filter is determined. From the viewpoint of improving, 1 mm / sec or more, 5 mm / sec or more, 10 mm / sec or more, 15 mm / sec or more, 20 mm / sec or more, 25 mm / sec or more, 30 mm / sec or more, or 35 mm / sec or more per opening area. Is. From the viewpoint of suppressing damage to cells, it is 100,000 mm / sec or less, 50,000 mm / sec or less, 30,000 mm / sec or less, 10,000 mm / sec or less, or 5,000 mm / sec or less. From the point of further improving the cell recovery rate, it is 100 mm / sec or more, 150 mm / sec or more or 160 mm / sec or more, and from the same point, 4,000 mm / sec or less, 3,500 mm / sec or less, 3,000 mm / sec. Seconds or less, 2,000 mm / sec or less, or 1,500 mm / sec or less.

The ratio of the amount of the recovered liquid to the amount of the sample to be passed ([volume of the recovered liquid] / [volume of the sample to be passed through the filter]) is less than 0.6 in one or more embodiments. From the viewpoint 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. It is less than 0.6, 0.5 or less, or 0.4 or less from the viewpoint of facilitating the analysis of the collected cells. Further, from the viewpoint of further improving the cell recovery rate, it is 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more or 0.01 or more, and from the same point, 0.3 or less, 0. .2 or less or 0.1 or less.

The flow pressure of the recovered liquid is 30 kPa or more and 150 kPa or less. The flow pressure of the recovered solution 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 from the viewpoint of improving the recovery rate of rare cells from the filter. Therefore, it is 130 kPa or less, or 120 kPa or less.

As the recovery liquid, in one or more embodiments, 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. Examples of the buffer solution include, in one or more embodiments, a Tris buffer solution such as Tris-HCl buffer solution, a phosphate buffer solution, a HEPES buffer solution, a citric acid-phosphate buffer solution, and the like. 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 recovery liquid is not limited to the specific range specified in Japanese Patent Application Laid-Open No. 10-201470, and may be a viscosity in another range. The viscosity of the recovered solution is not particularly limited, and the viscosity at 25 ° C. is less than 5 mPa · s, or the viscosity at 25 ° C. is 0 in one or more embodiments from the viewpoint of further improving the recovery rate of cells. It is 5.5 mPa · s or more.

In one or more embodiments, the recovered liquid can be passed by using a driving force capable of passing the recovered liquid so as to satisfy the above-mentioned liquid passing pressure or flow rate. As the introduction of the recovered liquid, in one or more embodiments, a method using pressurization from the outlet direction of the flow path (the side where the sample flows out (downstream side)) and the inlet direction of the flow path (the side where the sample flows in). A method using decompression from (upstream side)), a method using a syringe pump, a tube pump (also referred to as a roller pump or a peristaltic pump) or a plunger pump, a method using mechanical drive, and the like can be mentioned.

According to the recovery method of the present disclosure, in one or more embodiments, cells can be recovered from the filter at a high recovery rate with a small amount of recovery solution. The flow rate of the recovered 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 and 0.1 ml or more in one or more embodiments. Or 0.2 ml or more.

The recovery method of the present disclosure may include, in one or more embodiments, passing a sample containing cells through a filter. By passing the sample through the filter, rare cells that can be contained in the sample can be captured by the filter.

The flow rate of the sample 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, and is the same. From the point of view, it is 100 ml / min or less, 50 ml / min or less, 25 ml / min or less, or 10 ml / min or less.

The flow rate of the sample is 0.01 mm / sec or more, 0.05 mm / sec or more, or 0.1 mm / sec or more per opening area in one or more embodiments from the viewpoint of improving the cell capture rate. From the same point, it is 100 mm / sec or less, 50 mm / sec or less, 25 mm / sec or less, 10 mm / sec or less, 5 mm / sec or less, or 2 mm / sec 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 An amount greater 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 It is more than 0.2 ml, less than 130 ml or less than 130 ml 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.

The fluid flow pressure (ΔP1) of the sample is 0.1 kPa or more or 0.2 kPa or more, or 2.6 kPa or less, 1.5 kPa or less in one or more embodiments from the viewpoint of improving the cell capture rate. , 1.3 kPa or less, 1.0 kPa or 0.5 kPa or less. The "liquid flow pressure of a sample" in the present disclosure means 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 flow path including the tank in which the sample is arranged to the waste liquid tank. To say. Therefore, depending on the flow path structure, the pressure may be higher than described. The flow pressure (pressure difference of the system) 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 cells are captured by a filter with through holes, in one or more embodiments, cells can be removed from the filter with a small amount of recovery fluid and a high recovery rate while reducing damage to the cells as compared to non-woven fabrics. Can be recovered. Examples of the filter include, in one or more embodiments, a slit-shaped filter having a plurality of through holes. The shape of the through hole may be a rounded rectangle or an ellipse in one or more embodiments.

Examples of the filter used in the recovery method of the present disclosure include filters capable of capturing rare cells in a sample in one or more embodiments. As the filter, a filter that can be used in the labeling method of the present disclosure can be used.

By passing the recovered solution through the filter after passing the sample from the direction opposite to the direction in which the sample was passed, the rare cells are separated from the filter, and the recovered solution containing the separated rare cells is placed on the filter. Exists. The recovery method of the present disclosure may include, in one or more embodiments, recovering the recovery liquid present on the filter after passing the recovery liquid. The recovered liquid can be recovered by using a pipette or the like in one or more embodiments. Further, at the time of the recovery, the recovered liquid may or may not be stirred.

The recovery method of the present disclosure may include, in one or more embodiments, treating cells trapped in the filter on the filter. Examples of the treatment include fixation treatment, cell membrane permeation treatment, staining treatment and the like in one or more embodiments.

[Analysis method for rare cells]
The present disclosure also comprises the analysis of rare cells in a blood sample, comprising recovering the rare cells by the recovery methods of the present disclosure and analyzing by methods including observation of the dynamics of the cells or measurement of activity. Regarding the method (analytical method of the present disclosure). According to the analysis method of the present disclosure, since the rare cells are recovered by the recovery method of the present disclosure, the rare cells can be recovered with a high recovery rate. Therefore, according to the analysis method of the present disclosure, rare cells can be measured or analyzed with high accuracy.

[Cell recovery device]
The present disclosure relates to a cell recovery device (cell recovery device of the present disclosure) as another aspect. According to the cell recovery device of the present disclosure, cells can be recovered at a high recovery rate by the recovery method of the present disclosure. In one or more embodiments, the cell recovery device of the present disclosure can recover cells captured by a filter from the filter, capture cells using the filter, and the like.

As one aspect of the cell recovery device of the present disclosure, 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 sample containing cells are introduced into the separation unit. At least one introduction flow path for the purpose, at least one liquid feeding means for supplying the sample to the separation part through the introduction flow path, a discharge flow path for discharging the sample passing through the separation part to the outside, and a separation part. Cells by the container for storing the recovery liquid for collecting the captured cells, the reverse liquid feeding means for supplying the recovery liquid from the container to the separation part through the above-mentioned flow path, and the cell recovery method of the present disclosure. It is provided with a control unit that controls the reverse liquid feeding means in order to collect the liquid. In one or more embodiments, the control unit has a ratio of the flow rate of the recovered liquid to the flow rate of the sample ([flow rate of the recovered liquid] / [flow rate of the sample]) of 25 or more, and / or Control the reverse liquid feeding means in which the flow rate of the recovered liquid is 1 mm / sec or more per opening area.

In one or more embodiments, the separator comprises at least a filter and is detachably attached to the inlet and outlet channels. Examples of the filter include the above-mentioned filters in one or more embodiments.

The liquid feeding means and the reverse liquid feeding means include, in one or more embodiments, a pressurizing means, a depressurizing means, a syringe pump, a tube pump (also referred to as a roller pump or a peristaltic pump), a plunger pump, a mechanical drive, or the like. Can be mentioned.

In one or more embodiments, the liquid feeding means may be capable of introducing a treatment liquid for treating cells in a sample into a separation unit through an introduction flow path. Examples of the treatment liquid include treatment liquids used for fixation treatment, cell membrane permeation treatment, staining treatment and the like in one or more embodiments. The cell recovery device of the present disclosure may include, in one or more embodiments, a container for containing a treatment solution for treating cells in a sample.

The control unit can record the filter, filtration conditions, etc. used at the time of cell capture in one or more embodiments. In one or more embodiments, the control unit can be realized by a processor included in the computer of the recovery device executing a predetermined program.

The cell recovery device of the present disclosure includes, in one or more embodiments, a recovery means for recovering the recovery liquid supplied to the separation unit. Examples of the collecting means include a nozzle mechanism and the like in one or more embodiments. The cells can be collected by sucking the collected liquid collected on the upper part of the filter with a nozzle or a pipette.

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

[Cell recovery system]
The present disclosure relates to a cell recovery system (cell recovery system of the present disclosure) as another aspect. According to the cell recovery system of the present disclosure, cells can be recovered at a high recovery rate by the recovery method of the present disclosure. In one or more embodiments, the cell recovery system of the present disclosure can recover cells trapped in a filter from the filter, capture cells using the filter, and the like.

In one or more embodiments, the cell recovery system of the present disclosure comprises a filter unit having a plurality of through holes for capturing cells, a holding portion for holding the filter, and the filter unit. A separation unit including a possible flow path portion and a liquid feeding means capable of passing a sample containing cells through the flow path portion to the filter, a flow path portion in which the filter unit can be arranged, and a passage of the separation unit. A recovery unit including a reverse liquid feeding means capable of passing a recovered liquid through the filter from a direction opposite to the liquid direction through a flow path portion, and the reverse feeding for collecting cells by the cell recovery method of the present disclosure. A control unit for controlling the liquid means is provided. In one or more embodiments, the control unit has a ratio of the flow rate of the recovered liquid to the flow rate of the sample ([flow rate of the recovered liquid] / [flow rate of the sample]) of 25 or more, and / or the recovered liquid. The liquid feeding means of the separation unit and the liquid feeding means of the recovery unit are controlled so that the flow velocity of the above is 1 mm / sec or more per opening area. The filter unit can be attached to and detached from the flow path portion of the separation unit and the recovery unit in one or more embodiments. According to the cell recovery system of the present disclosure, in one or more embodiments, after the cells are captured in the separation unit, the filter unit is transferred to the recovery unit, and the cells captured by the filter are recovered in the recovery unit. be able to.

In one or more embodiments, the cell recovery system of the present disclosure separates a cell containing a filter having a plurality of through holes for capturing cells and a holding section holding the filter, and a sample containing cells. At least one introduction flow path for introducing into the unit, at least one liquid feeding means for supplying the sample to the separation unit through the introduction flow path, and a discharge flow path for discharging the sample passing through the separation unit to the outside. A container for storing a recovery solution for collecting cells captured in the separation section, a reverse liquid feeding means for supplying the recovery solution from the container through a discharge channel, and a cell recovery method of the present disclosure. It is provided with a control means for controlling the reverse liquid feeding means in order to collect cells. In one or more embodiments, the control means has a ratio of the flow rate of the recovered liquid to the flow rate of the sample ([flow rate of the recovered 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 / sec or more per opening area.

FIG. 4 shows an example of a rare cell capture / recovery device that can be used in the recovery method or analysis method of the present disclosure. The rare cell capture / recovery device shown in FIG. 4 has a supply port 41, a discharge port 42, a flow path 43 communicating the supply port 41 and the discharge port 42, and a flow for supplying a sample, a washing liquid, a staining liquid, etc. to the filter. It has a separation portion arranged at a position corresponding to a part of the road 43. The separation unit includes a filter 44 and a filter holder 45 that holds the filter 44. The filter holder 45 is composed of a support portion (base) 46 on which the filter 44 can be installed, an O-ring 47 for fixing the filter 44, and a cover 48. By installing the filter 44 on the support portion 46, placing the O-ring 47 and the cover 48 on the upper surface of the filter 44, and fixing the filter 44, a separation portion (filter device) having a filter having holes is formed. The flow path 43 connecting the supply port 41 and the separation portion and the separation portion and the discharge port 42 is formed by connecting Safeed (trademark) tubes (manufactured by Terumo Corporation) to both ends of the separation portion. In the flow path 43 connecting the supply port 41 and the separation portion, a connection portion (not shown) and a three-way stopcock (not shown) capable of switching the liquid may be arranged above the separation portion. Further, a pressurizing means 49 (as a mechanism capable of sending liquid at a constant pressure or a mechanism capable of sending liquid at a constant flow rate) is arranged in the flow path 43 connecting the supply port 41 and the separation portion to perform filtration. The pressure may be constant, or the liquid may be sent so that the pressure applied to the filtration is above a certain level by pulling with a syringe pump.

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 / recovery device shown in FIG. 5 discharges the sample in the container 52 from the introduction flow path 56 for introducing the sample into the separation section 51, the separation section 51 provided with a filter, the container 55 for accommodating the recovery solution, and the separation section 51. A discharge flow path 57 for discharging the waste liquid to be discharged is provided. The container 55 for accommodating the recovered liquid is connected to the discharge flow path 57 through the flow path so that the recovered liquid can be back-fed to the separation portion 51 through the discharge flow path 57. A liquid feeding means such as a pump for back-feeding the recovered liquid to the separating portion 51 is connected to the container 55. In addition to the container 52 for accommodating the sample, for example, a container 13 for accommodating the washing liquid and a container 54 for accommodating the treatment liquid for treating cells in the sample can be connected to the introduction flow path 56 through the flow path. A container 58 for collecting the waste liquid can be arranged at one end of the discharge flow path 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, the "rare cell" refers to a cell other than blood cell components (erythrocytes, white blood cells, and platelets) that can be contained in the blood of a human or a non-human animal. Rare cells include tumor cells or cancer cells in one or more embodiments. Tumor cells and cancer cells that circulate in the blood are generally called CTCs. The number of rare cells in the blood depends on the sample, but may be contained in 10 ml of blood from several to several tens, and at most several hundreds to 1,000. "Rare cells" are, in one or more embodiments, cancer cells, circulating tumor cells, vascular endothelial cells, vascular endothelial precursor cells, cancer stem cells, epithelial cells, hematopoietic stem cells, mesenchymal stem cells, fetal cells, stem cells. And cells selected from the group consisting of combinations thereof.
Rare cells include, in one or more embodiments, human colon cancer cells, human gastric cancer cells, human colon cancer cells, human lung cancer cells, human lung cancer cells, human lung cancer cells, human lung cancer cells, human lung cancer cells, and the like. Can be mentioned.

In the present disclosure, the “sample” includes a sample that may contain cells that can be applied to the recovery method of the present disclosure. Samples include, in one or more embodiments, blood samples. The blood sample is a sample containing components constituting blood in one or more embodiments, and in one or more embodiments without particular limitation, blood, a blood-derived substance containing a blood cell component, blood or blood-derived. Examples thereof include body fluids and urine in which substances are mixed, and samples prepared from these. Examples of the blood include blood collected from a living body, and examples of the living body include humans and non-human animals (for example, mammals). Examples of blood-derived products containing blood cell components include those separated or prepared from blood and containing blood cell components or dilutions / concentrates thereof, for example, blood cell fractions from which plasma has been removed, and blood cells. Includes concentrates, lyophilized blood or blood cells, samples or hemolytic samples in which whole blood is hemolyzed to remove erythrocyte components, centrifuged blood, naturally precipitated blood, washed blood cells, specific fractions, and the like. Among these, in one or more embodiments without limitation, the sample containing blood is derived from blood or blood containing a blood cell component from the viewpoint of simple and rapid treatment and suppression of damage to rare cells in blood. Specimens are preferred.

The present disclosure may relate to one or more embodiments below.
[C1] A method of collecting cells trapped in a filter having a plurality of through holes from the filter.
This includes passing the collected liquid through the filter in which the cells have been captured from the direction opposite to the flow direction of the sample for capturing the cells.
The flow rate of the recovered solution is carried out so that the ratio of the flow rate of the recovered solution to the flow rate of the sample ([flow rate of the recovered solution] / [flow rate of the sample]) is 25 or more. Collection method.
[C2] A method of collecting cells trapped in 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 the direction opposite to the flow direction of the sample for capturing the cells at a flow rate of 1 mm / sec or more per opening area of the filter. Methods of collecting cells, including.
[C3] The recovery method according to [C1], which comprises passing the recovery liquid through the filter at a flow rate of 1 mm / sec or more per opening area of the filter.
[C4] From [C1] to [C1], which comprises capturing the cells in the filter by passing a sample containing the cells through the filter at a flow rate of 0.01 mm / sec to 100 mm / sec per opening area. The collection method according to any one of C3].
[C5] The recovery method according to any one of [C1] to [C4], wherein the filter is a filter capable of capturing rare cells.
[C6] The recovery 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, body, aluminum, tungsten, and chromium.
[C7] The recovery method according to any one of [C1] to [C7], wherein the cell is a rare cell. [C8] In a blood sample, which comprises collecting rare cells by the recovery method according to any one of [C1] to [C7], and analyzing by a method including observation of the dynamics of the cells or measurement of activity. Method for analyzing rare cells.
[C9] A separation unit including a filter having a plurality of through holes for capturing cells, a holding unit for holding the filter, and a separation unit.
At least one introduction channel for introducing a sample containing cells into the separation part, and
At least one liquid feeding means for supplying the sample to the separation portion through the introduction flow path, and
A discharge flow path for discharging the sample that has passed through the separation part to the outside,
A container for storing the recovery liquid for collecting the cells captured in the separation part, and
A reverse liquid feeding means for supplying the recovered liquid from the container to the separating portion through the discharge flow path, and
A control unit that controls the reverse liquid feeding means so that the ratio of the flow rate of the recovered liquid to the flow rate of the sample ([flow rate of the recovered liquid] / [flow rate of the sample]) is 25 or more. A cell recovery device.
[C10] A separation unit including a filter having a plurality of through holes for capturing cells, a holding unit for holding the filter, and a separation unit.
At least one introduction channel for introducing a sample containing cells into the separation part, and
At least one liquid feeding means for supplying the sample to the separation portion through the introduction flow path, and
A discharge flow path for discharging the sample that has passed through the separation part to the outside,
A container for storing the recovery liquid for collecting the cells captured in the separation part, and
A reverse liquid feeding means for supplying the recovered liquid from the container to the separating portion through the discharge flow path, and
A cell recovery device including a control unit that controls the back-feeding means so that the recovery liquid is passed at a flow rate of 1 mm / sec or more per opening area.
[C11] A cell recovery device 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 device according to any one of [C9] to [C12], comprising a recovery means for recovering the recovery liquid supplied to the separation unit.
[C14] A filter unit including a filter having a plurality of through holes for capturing cells, and a holding portion for holding the filter.
A separation unit including a flow path portion in which the filter unit can be arranged and a liquid feeding means capable of passing a sample containing cells through the flow path portion to the filter.
A recovery unit including a flow path portion in which the filter unit can be arranged and a reverse liquid feeding means capable of passing the recovery liquid through the flow path portion from a 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 recovered liquid to the flow rate of the sample ([flow rate of the recovered liquid] / [flow rate of the sample]) is 25 or more. A cell recovery system including a control unit that controls a liquid feeding means.
[C15] A filter unit including a filter having a plurality of through holes for capturing cells, and a holding portion for holding the filter.
A separation unit including a flow path portion in which the filter unit can be arranged and a liquid feeding means capable of passing a sample containing cells through the flow path portion to the filter.
A recovery unit including a flow path portion in which the filter unit can be arranged and a reverse liquid feeding means capable of passing the recovery liquid through the flow path portion from a direction opposite to the liquid flow direction of the separation unit. ,
A cell recovery system including a control unit that controls a liquid feeding means of the separation unit and a liquid feeding means of the recovery unit so that the recovered liquid is passed at a flow rate of 1 mm / sec or more per opening area. ..

The present disclosure will be further described below with reference to examples. However, this disclosure is not construed as limited to the following examples. The cell capture / labeling device and the cell capture device described below may be configured by combining a plurality of units.

1. Confirmation of magnetic labeling efficiency (Example 1)
Using the cell capture / labeling device shown in FIG. 1, cells (leukocytes) were captured and labeled with magnetic particles, and the labeling efficiency was confirmed.

[Cell labeling device]
The cell labeling device shown in FIG. 1 was prepared. The cell labeling device of FIG. 1 includes a device upper part 3, a filter unit 5 including a filter 10, a device lower part 4, and a liquid feeding mechanism (not shown) capable of feeding liquid in the directions of arrow A and arrow B. The filter 10 is arranged between the upper part 3 of the device and the lower part 4 of the device, and is fixed by the 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 minor axis diameter: 6.5 μm, major axis diameter: 88 μm, area: 572 μm ² , shape: pitch between slit and hole center: 14 μm × 100 μm (minor axis diameter side × major axis diameter side)
・ Pore density: 714 pieces / mm ²
・ Aperture ratio: 41%
・ Film thickness: 5 μm
・ Material: Nickel ・ Filtration area: 79mm ²

[Magnetic particle label]
Using the cell labeling device 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 a cell labeling device, 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 velocity). : 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 a filter in this order and reacted with cells to cause CD45 and CD50 expressing cells (for example, leukocytes). Biotin labeling was performed on the cells. The antibody solution stays not only in the upper part of the filter but also in the lower part of the filter so as to be in contact with 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 fed from the upper part of the filter in the direction of arrow A in FIG. 1 so that the lower surface of the suspension is below the lower surface of the filter. did.
Step 4: Backflow was performed from the lower part of the filter in the direction of arrow B in FIG. 1 (backflow flow rate: 10,000 μL / min, flow rate: 14.441 mm / sec).
Step 5: The suspension on top of the filter was pipetted.
Step 6: The suspension (stirring solution of cells and magnetic particles) on the upper part of the filter was sent again (250 μL / min) from the direction of arrow A in FIG.
Step 7: A cleaning liquid containing no magnetic particles was sent from the direction of arrow A in FIG. As a result, 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, the collected liquid collected on the upper part of the filter is sucked with a pipette, and the cells (white blood cells) and rare cells labeled with magnetic particles together with the collected liquid are used. Was recovered.

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

(Comparative Example 1)
The procedure was the same as in Example 1 except that steps 4 to 6 were not performed. 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 due to magnetic separation could be significantly reduced. Therefore, according to the method of Example 1, it can be said that leukocytes could be magnetically labeled on the filter with high labeling efficiency.

(Example 2)
[Sample preparation]
Fluorescent and Biotin-labeled leukocytes were prepared by the following procedure.
First, blood was collected from humans using a vacuum blood collection tube (EDTA 2K).
Then, leukocytes were separated from the collected whole blood according to the package insert of HetaSep (STEMCELL).
The 1 × 10 ⁶ leukocytes isolated by HetaSep were reacted with a blocking solution (10% goat serum, 0.000001% Avidin and 0.2% BSA dissolved in Dulbecco PBS (−)) at 23 ° C. for 10 minutes. After centrifuging and removing the supernatant, it was resuspended using Dulbecco PBS (-). The supernatant was removed by centrifuging again.
Next, anti-CD45 antibody and anti-CD50 antibody were added to Dalveco PBS (−) in which 10% goat serum, 0.01% Biotin, and 0.2% BSA were dissolved according to the package insert to prepare a primary antibody reaction solution. The cells (leukocytes) 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 PBS (-). Then, the process of centrifuging to remove the supernatant was performed a total of 3 times, and the cells were thoroughly washed.
Alexa594-labeled anti-mouse IgG (Fab) and Biotin-labeled anti-mouse IgG (Fc) were added to Dalveco PBS (-) in which 2 μg / ml Hoechst33342, 10% goat serum and 0.2% BSA were dissolved, respectively, according to the package insert. An antibody reaction solution was prepared. The 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 PBS (-). The steps of centrifuging to remove the supernatant were performed a total of 3 times to thoroughly wash the cells (white blood cells).
This prepared a sample containing fluorescent and Biotin-labeled leukocytes.

[Cell capture]
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). Filtered in seconds).
As the filter 10, a filter having a plurality of through holes and having the following characteristics was used. <Filter characteristics>
-Hole minor axis diameter: 6.5 μm, major axis diameter: 88 μm, area: 572 μm ² , shape: pitch between slit and hole center: 14 μm × 100 μm (minor axis diameter side × major axis diameter side)
・ Pore density: 714 pieces / mm ²
・ Aperture ratio: 41%
・ Film thickness: 5 μm
-Material: Nickel-Filtration area: 28 mm ² .

[Magnetic particle label]
A solution in which 0.2% BSA was dissolved in Dulbecco PBS (−) was sent to the apparatus shown in FIG. 1 and filled in the flow path 8. 200 μL of magnetic particle solution (Neutravidin-labeled magnetic particle suspension, solid content concentration: 0.0125%) was added to the upper surface of the filter 10, and the lower surface of the magnetic particle solution was added so as to be located below the filter 10. A part (100 μL) of the magnetic particle solution was sent and filtered. 200 μL of a solution of 0.2% BSA dissolved in Dulbecco PBS (−) was backfed at a flow rate of 10000 μL / min using a backflow mechanism (in the direction of arrow B in FIG. 1). The solution on the filter 10 (solid content concentration: 0.0062%) was pipette-stirred and then reacted at 23 ° C. for 15 minutes. Then, 300 μL of the magnetic particle solution containing leukocytes on the upper surface of the filter 10 was sent at a flow rate of 250 μL / min (direction of arrow A in FIG. 1), and the reaction was carried out at 23 ° C. for another 15 minutes. As a result, leukocytes were magnetically labeled with magnetic particles.

[Cleaning of magnetic particles]
Then, 1000 μL of Dulbecco PBS (−) in which 1 mg / mL EDTA was dissolved was added to the upper surface of the filter 10, and then the solution was sent at a flow rate of 1000 μL / min (in the direction of arrow A in FIG. 1). This step was performed a total of 4 times to wash the excess magnetic particles on the filter 10.

[Cell recovery]
1000 μL of a solution of 0.2% BSA dissolved in Dulbecco PBS (−) was backfed at a flow rate of 10000 μL / min (14 mm / sec) using a backflow mechanism (in the direction of arrow B in FIG. 1). Then, 1100 μL of the solution on the filter 10 was completely recovered by pipette suction.

[Evaluation of labeling efficiency]
The recovered suspension was magnetically separated, and leukocytes that were not magnetically separated (not captured by a magnet) were judged to have not been magnetically labeled (or were not sufficiently labeled), and the labeling efficiency was evaluated. went.
That is, the collected cells were magnetically separated using a neodymium magnet. The number of white blood cells 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 (white blood cell count after magnetic separation (LEUK-a)).
On the other hand, among the suspensions collected after labeling, the leukocyte concentration in 100 μL of the suspension not used for magnetic separation was counted using a blood cell counter (Moxi-Z), and the number of leukocytes 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 unlabeled 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 carried out except that a sample newly prepared by the same preparation method as in Example 2 was used and the flow rate at the time of backflow feeding of the suspension of magnetic particles was set to the flow rate shown in Table 1 below. .. The results are shown in Table 1 below.

(Comparative example 3)
The same as in Example 2 was carried out except that a sample newly prepared by the same preparation method as in Example 2 was used and the backflow liquid was not sent in the magnetic particle labeling. That is, after a part (100 μL) of the magnetic particle solution (200 μL) added to the upper surface of the filter is sent and filtered, the solution on the filter is stirred with a pipette and then reacted at 23 ° C. for 30 minutes to perform magnetic particle labeling. It was. The results are shown in Table 1 below.

As shown in Table 1, labeling by the methods of Examples 2-5 reduces the unlabeled rate, i.e. the proportion of unlabeled (or poorly labeled) leukocytes to less than 1%. I was able to. That is, it can be said that according to the methods of Examples 2 to 5, leukocytes could be magnetically labeled on the filter with high labeling efficiency.

(Example 6)
Using a sample newly prepared by the same method as in Example 2, a part of the magnetic particle liquid was fed and the backflow feed after filtration was divided into two times of 100 μL each (total feed amount: 200 μL). ), The same as in Example 2. The results are shown in Table 2 below.

(Example 7)
The procedure was the same as in Example 2 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 any of the methods of Examples 6 and 7, leukocytes could be magnetically labeled on the filter with high labeling efficiency. Further, it was found that the desired effect can be obtained by performing the backflow feeding liquid at least once after the feeding liquid feeding and filtering of the magnetic particle liquid.
The reason why the non-labeling rate differs between Example 7 and Example 2 under the same treatment conditions is considered to be the difference in the sample used, specifically, the difference in the leukocytes contained in the sample. In the above example, the magnetic particles are labeled using the CD antigen expressed on the surface of leukocytes, but the expression of the CD antigen has inter-individual variation (individual difference) and inter-specimen difference resulting from intra-individual variation. It is known. It is considered that the non-labeling rate was different due to this difference between the samples. In addition, in the method of the present disclosure, it has been confirmed that sufficient reproducibility of the labeling rate can be obtained for the same sample.

(Example 8)
The procedure was the same as in Example 2 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)
A sample newly prepared by the same method as in Example 2 was used, and the same procedure as in Example 8 was carried out except that pipette stirring was not performed. The results are shown in Table 3 below.

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

(Comparative Example 4)
Same as 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 was not fed (FIG. 2B) before the backflow feed. I 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 liquid added to the upper surface of the filter prior to the backflow feeding. That is, it can be seen that the labeling efficiency is improved by immersing both sides of the filter in the suspension and then backflowing.

2. Cell capture and recovery test with a filter (Experimental Example 1)
Rare cells in a blood sample were collected using the rare cell capture device shown in FIG.

As the filter, a filter having the following characteristics was used. Both SNU-1 and SW620 can capture the filter having the following characteristics.
<Filter characteristics>
Hole minor axis diameter: 6.5 μm, major axis diameter: 88 μm, area: 572 μm ² , shape: pitch between the centers of slit holes (minor axis diameter side x major axis diameter side): 14 μm × 100 μm
Pore density: 714 pieces / mm ²
Aperture ratio: 41%
Film thickness: 5 μm
Material: Nickel Filtration area: 79mm ²

The rare cell capture / recovery device shown in FIG. 4 including the above filter and a liquid feeding unit including a backflow liquid feeding mechanism was configured. An aluminum member was used for the member support portion 46 for fixing the filter. Using the rare cell capture / recovery device, the rare cells were captured by filter filtration and the captured cells were recovered from the filter under the conditions shown in Table 5 below. Specifically, it was as follows.

First, cells were captured by filtration. 7.5 ml of a blood sample containing SNU-1 stained with Celltracker green was passed through at a constant pressure from the direction of arrow A in FIG. 4 at a constant pressure (ΔP1 = 0.75 kPa: filtration flow rate 0.25 ml / min) ( The liquid flow at this low pressure may utilize the head pressure, or may use the same or separate pump as the backflow liquid feeding mechanism.) As a result, SNU-1 was captured on the filter. Then, after washing the filter by passing 4 mL of PBS (−) twice, 900 μL of ammonium chloride solution (ACS) was passed three times and hemolyzed at room temperature for 10 minutes. 4 mL of PBS (−) was passed through and washed, and the liquid in the device was replaced. After removing the flow path connecting the supply port 41 and the separation portion 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 removed, 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.
Then, the cells trapped in the filter were collected. PBS (-) (recovered liquid, viscosity: 0.95 mPa · s) using a backflow liquid feeding mechanism from the direction opposite to the liquid flow direction in which the cells were captured in the filter (arrow B direction 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, and the entire amount was collected. 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-1s in the recovered liquid by the number of SNU-1s captured on the filter. The results are shown in Table 5 below.

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

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

(Experimental Example 4)
The cells were captured and recovered in the same manner as in Experimental Example 1 except that the flow rate of the recovered solution was set to 6,000 ml / min. The results are shown in Table 5 below.

(Experimental Example 5)
The cells were captured and recovered in the same manner as in Experimental Example 1 except that the volume of the recovered solution was 0.25 ml and the flow rate was 600 ml / min. The results are shown in Table 5 below.

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

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

(Experimental example 8)
The 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 (the liquid was sent 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 (delivery over about 30 minutes), and the blood sample volume 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 (delivery over about 10 minutes), and the blood sample volume was 4 ml. The results are shown in Table 5 below.

(Experimental example 11)
First, cells were captured by filter filtration. 2 ml of a blood sample containing SW620 stained with Celltracker green was passed through 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). / Minute). As a result, SW620 was captured on the filter below. Hemolysis treatment was performed in the same manner as in Experimental Example 1, and after washing with PBS (-), immobilization treatment and cell membrane permeation treatment were performed on a filter. The immobilization treatment was carried out by passing a 4% paraformamide: PFA (PBS (−) solution) to fill the vicinity of the filter, and then reacting at room temperature for 15 minutes. The cell membrane permeation treatment was carried out 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 replaced with the liquid inside the device. The flow path connecting the supply port 41 and the separation portion was removed, sealed with a cover glass, and then the number of SW620s captured on the filter was counted by microscopic observation.
After counting, the cover glass was removed, 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.
Then, the cells trapped in the filter were collected. 1 ml of PBS (-) (recovery solution) was passed through the filter from the direction opposite to the direction in which the cells were captured (arrow B in FIG. 4) using a reverse flow feeding mechanism (flow rate 6). 000 ml / min). The collected liquid collected on the upper part of the filter was sucked with a pipette, and the entire amount was collected. The number of SW620s in the recovered liquid was counted by microscopic observation. The recovery rate was determined by dividing the number of SW620s in the recovered liquid by the number of SW620s captured on the filter. The results are shown in Table 5 below.
<Filter characteristics>
Hole minor axis diameter: 6.5 μm, major axis diameter: 9.8 μm, area: 50 μm ² , shape: pitch between centers of elliptical holes (minor axis diameter side x major axis diameter side): 19 μm × 14 μm
Pore density: 3,759 pieces / mm ²
Aperture ratio: 19%
Film thickness: 5 μm
Material: Nickel Filtration area: 20 mm ²

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

(Experimental example 13)
Immobilization treatment with 5 mg / ml dimethylsverimidate: DMS (PBS (-) solution) for 10 minutes at room temperature, cell membrane permeation treatment with 0.2% Triton-X100 (PBS (-) solution) only. Cells were captured and recovered in the same manner as in Experimental Example 11 except for the change. The results are shown in Table 5 below.

(Experimental example 14)
First, cells were captured by filter filtration. 2 ml of a blood sample containing unstained SW620 was passed through from the direction of arrow A in FIG. 4 at a constant pressure in the same manner as in Experimental Example 1 (ΔP1 = 0.4 kPa: filtration flow rate 0.25 ml / min). .. As a result, SW620 was captured on the filter (the same filter as in Experimental Example 1 except that the filtration area was 20 mm ² ). Hemolyzed in the same manner as in Experimental Example 1, washed with PBS-EDTA, immobilized with 1% PFA (PBS (-) solution) at room temperature for 10 minutes on a filter, and 0.2% Triton-X100 (PBS). The (-) solution) was passed through the cell membrane permeation treatment. It was then treated with FcR blocking and stained with a fluorescently labeled anti-cytokeratin antibody. It was washed with PBS-EDTA and the liquid in the device was replaced. The flow path connecting the supply port 41 and the separation portion was removed, sealed with a cover glass, and then the number of SW620s captured on the filter was counted by microscopic observation.
After counting, the cover glass was removed, 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.
Then, the cells trapped in the filter were collected. 1 ml of PBS-EDTA (recovery solution) was passed through the filter from the direction opposite to the direction in which the cells were captured (arrow B in FIG. 4) using a backflow feeding mechanism (flow rate 6,). 000 ml / min). The collected liquid collected on the upper part of the filter was sucked with a pipette, and the entire amount was collected. The number of SW620s in the recovered liquid was counted by microscopic observation. The recovery rate was determined by dividing the number of SW620s in the recovered liquid by the number of SW620s captured on the filter. The results are shown in Table 5 below.

(Experimental Example 15)
First, cells were captured by filter filtration. Using a resin member as a member for fixing the filter, 8 ml of a blood sample containing SUN-1 stained with Celltracker green and SW620 stained with Celltracker flow rate was determined from the direction of arrow A in FIG. 4 over about 32 minutes. The solution was passed at a flow rate (filtration flow rate 0.25 ml / min). As a result, SNU-1 and SW620 were captured on the filter (the same filter as in Experimental Example 1). Then, after washing the filter with 2 mL of PBS-EDTA 4 times, pass 900 μL of ACS 3 times and hemolyze at room temperature in the same manner as in Experimental Example 1 (to improve the accuracy of counting after recovery). The device was washed 4 times with 2 mL of PBS-BSA to replace the liquid in the device.
Then, the cells captured by the filter were collected. 1 ml of PBS-BSA (recovered solution, viscosity: 1.50 mPa · s) is passed from the direction opposite to the direction in which the cells were captured (arrow B in FIG. 4) using a reverse flow feeding mechanism. (Flow rate 600 ml / min). Then, after stirring the recovered liquid on the upper part of the filter twice with a pipette, 500 μl of the recovered liquid collected on the upper part of the filter was sucked and collected with a pipette, and then after further stirring twice, the remaining total amount was similarly recovered. The numbers of SNU-1 and SW620 in the recovered liquid were 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)
Cells were captured and recovered in the same manner as in Experimental Example 15 except that the flow rate of the recovered solution was set to 316 ml / min (liquid passing pressure 58 kPa). The results are shown in Table 5 below.

(Experimental Example 17)
The cells were captured and recovered 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 to be used were NCI-H1650 (human lung cancer) stained with Celltracker green. went. The results are shown in Table 5 below.

(Experimental Example 18)
First, cells were captured by filter filtration. A resin member was used as a member for fixing the filter, and 8 ml of a blood sample containing unstained NCI-H1650 was passed through at a constant flow rate from the direction of arrow A in FIG. 4 at a constant flow rate (filtration flow rate 0.1 ml). / Minute). As a result, NCI-H1650 was captured on a filter (the same filter as in Experimental Example 1 except that the filtration area was 28 mm ² ). Then, cell staining such as hemolysis treatment, immobilization / cell membrane permeation treatment, and antibody staining is performed on the captured NCI-H1650, and finally, the device is washed with a sucrose / glucose-based isotonic solution. Liquid replacement was performed.
Then, the cells captured by the filter were collected. 1 ml of the isotonic solution (recovery solution, viscosity: 1.75 mPa · s) is passed from the direction opposite to the direction in which the cells were captured (the direction of arrow B in FIG. 4) using a backflow flow mechanism. Liquid (flow rate 600 ml / min). Then, the recovered liquid on the upper part of the filter was stirred twice with a pipette, 250 μl of the recovered liquid collected on the filter was recovered, and then the recovered liquid collected on the filter was further stirred twice, and the remaining total amount was similarly recovered. The number of NCI-H1650 in the recovered 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 recovered in the same manner as in Experimental Example 18 except that the volume of the recovered solution was 0.5 ml. The results are shown in Table 5 below.

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

(Experimental Example 21)
Except that the filtration area was 28 mm ² , the filtration flow rate was 1 ml / min, the volume of the recovered solution 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. The cells were captured and recovered in the same manner as in Experimental Example 15. The results are shown in Table 5 below.

(Experimental Example 22)
The cells were captured and recovered in the same manner as in Experimental Example 21 except that the cells to be used were SW620 stained with Celltracker orange. The results are shown in Table 5 below.

(Experimental Example 23)
The cells were captured and recovered in the same manner as in Experimental Example 21 except that the cells to be used were NCI-H1650 stained with Celltracker green. The results are shown in Table 5 below.

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

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

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

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

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

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

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

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

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

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

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

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

(Comparative Experiment Example 5)
The cells were captured and recovered in the same manner as in Comparative Experimental Example 3 except that the cells to be 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 with a recovery rate of more than 70% in all the experimental examples. In addition, a high recovery rate could be achieved with a small amount of recovered liquid.

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

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

Claims (14)

A method of labeling particles trapped in 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 captured,
By sending a part of the suspension from the filter surface toward the other filter surface, the suspension containing the magnetic beads is in contact with both sides of the filter .
The suspension containing the magnetic beads is reverse-fed from the state where the suspension containing the magnetic beads is in contact with both sides of the filter from the other filter surface toward the filter surface in which the particles are captured. as well as
A magnetic labeling method comprising delivering and discharging the suspension from the filter surface to the other filter surface . The labeling method according to claim 1, which comprises stirring the suspension containing the magnetic beads on the filter surface in which the particles are captured after the reverse liquid feeding. This includes passing the collected liquid through the filter in which the cells are captured from the direction opposite to the flow direction of the sample for capturing the cells, and sucking and collecting the collected liquid accumulated 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 in the filter by sending a sample containing the particles to the filter. The labeling method according to claim 4, wherein the flow velocity of the sample is 0.01 mm / sec to 100 mm / sec per the opening area of the filter. The labeling method according to claim 4 or 5, wherein the flow rate of the reverse feed solution is made faster than 1/10 of the flow rate of the sample. The labeling method according to any one of claims 1 to 6, wherein the flow velocity of the reverse feed solution is 0.01 mm / sec or more per the opening area of the filter. A device for labeling particles with magnetic beads,
The filter part that can hold the filter and
An introduction flow path into which a suspension containing magnetic beads can be introduced into the filter portion,
A discharge channel capable of holding or discharging the suspension that has passed through the filter unit,
A liquid feeding means capable of supplying the suspension to the filter unit, and
A control unit for controlling the liquid feeding means is provided.
The control unit sends a part of the suspension from one filter surface toward the other filter surface so that the suspension containing the magnetic beads is in contact with both sides of the filter. A labeling device that controls the liquid feeding means so that the suspension containing the magnetic beads is reversely fed from the other filter surface from the state where the suspension containing the magnetic beads is in contact with both surfaces of the filter. .. The labeling device according to claim 8, further comprising a means for stirring the liquid at or near the surface of the filter. Further provided with a recovery means for recovering the particles captured in the filter section,
The control unit passes the collected liquid through the filter in which the cells are captured from the direction opposite to the liquid passing direction of the sample for capturing the cells, and sucks the collected liquid collected on the filter. The labeling device according to claim 8 or 9, wherein the collection 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 means so as to supply a sample containing particles to be labeled to the filter unit. The labeling device according to claim 11, wherein the control unit controls the liquid feeding means so as to feed the sample at a flow rate of 0.01 mm / sec to 100 mm / sec per opening area of the filter. The labeling device according to claim 11 or 12, wherein the control unit controls the liquid feeding means so that the flow rate of the reverse liquid is faster than 1/10 of the flow rate of the sample. The labeling device according to any one of claims 8 to 13, wherein the control unit controls the liquid feeding means so that the flow velocity of the back feeding liquid is 0.01 mm / sec or more per the opening area of the filter. ..