JP6707031B2 - Cell separation material and cell separation method - Google Patents

Description

The present invention relates to a cell separating material and a cell separating method using the same. More specifically, the present invention provides a cell separation material suitable for separating rare cells (tumor cells, stem cells, endothelial cells, fetal cells, etc.) present in a liquid from contaminating cells, and collecting and concentrating the rare cells. And a cell separation method using the same.

The main components of cell components in blood are red blood cells, white blood cells, and platelets, but it is known that cells other than these exist in blood. For example, circulating blood tumor cells, circulating stem cells, circulating endothelial cells, etc. (collectively referred to below as “rare cells”) are cells that are extremely rare in whole blood. The detection of these rare cells is considered to be clinically useful.

For example, circulating tumor cells in the blood are found at low concentrations in the blood of cancer patients and are also considered to be the cause of metastatic cancer. If these tumor cells can be detected early, the presence of a primary tumor, tumor metastasis, or recurrence of tumor after treatment can be detected early, and effective treatment can be introduced from the initial stage.
However, there are about 5×10 ⁹ (cells/mL) of red blood cells, about 5×10 ⁶ (cells/mL) of white blood cells, and about 3×10 ⁸ (cells/mL) of platelets in the blood, and the white blood cells of cancer patients At present, it is extremely difficult to detect tumor cells that are present at a ratio of only one in several hundreds of thousands to several tens of millions, and there is a limit in using tumor cells for diagnosis. Therefore, before carrying out the quantitative analysis of tumor cells, a method of removing contaminating cells and concentrating the tumor cells to improve the detection sensitivity and the measurement accuracy is currently developed and used.

For example, as a method for collecting and concentrating circulating tumor cells in blood currently used, negative selection method using magnetic beads immobilized with an antibody against an antigen of a contaminating cell whose surface antigen is known, or tumor cell A positive selection method using magnetic beads to which an antibody against a specific antigen is immobilized can be used. There are also cell sorting methods such as cell sorting using fluorescent antibodies, but both methods require a large amount of antibody and treatment time to sort out a large amount of contaminating cells and a small amount of tumor cells. Therefore, the processing becomes very expensive and the work efficiency is poor. On the other hand, as a method for collecting and concentrating tumor cells without using antibodies, there are density gradient centrifugation methods such as Ficoll separation method and Percoll separation method. Although this method is a method of separating cells by utilizing the difference in specific gravity of cells, it is impossible to separate tumor cells and foreign cells having the same specific gravity because they are separated by specific gravity. In addition, since the operation of washing the Ficoll solution or Percoll solution using a centrifuge after the separation is repeated several times, cell loss occurs, and in the worst case, even a very small number of circulating tumor cells in blood are lost. As a result, an incorrect diagnosis may be made. Red blood cells are the most abundant in blood, and this large amount of red blood cells is a major impediment to the detection of tumor cells. In addition to the above methods, there is a method of hemolyzing erythrocytes as a method of removing erythrocytes in advance (for example, ammonium chloride method or hyperosmotic destruction method). Although this method efficiently lyses and removes erythrocytes, it causes great damage to other cells, making it difficult to analyze and culture tumor cells alive. In addition, some techniques for collecting rare cells such as tumor cells contained in body fluids such as blood have been reported (Patent Documents 1 and 2), but a desired rare cell is selected from a whole blood sample. It is hard to say that this technology is sufficient as a technology to separate and recover it. Further, all of the above methods cannot be processed in a closed system and have a safety problem such as contamination with foreign matter and infection.

The applicant of the present invention also has a method for separating a body fluid containing tumor cells into blood cell components. (a) Capturing tumor cells, white blood cells and platelets in the blood cell separating material by contacting the body fluid containing tumor cells with the blood cell separating material. A step of removing the erythrocyte-rich fraction, (b) a step of separating the tumor cell-enriched fraction from the blood cell separating material using a separation solution, and a step of containing the recovery liquid obtained in the steps (a) and (b) above The proposed method is a cell separation method characterized in that the ratio of erythrocyte removal rate/tumor cell recovery rate is 1.25 or less and 1.00 or more (Patent Document 3). This method simply and quickly removes erythrocyte-rich fractions from body fluids such as blood, bone marrow, cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph fluid or tissue extracts, and collects tumor cell-enriched fractions. However, since the fraction containing tumor cells is collected in the state of containing white blood cells and platelets, it is not a method of selectively collecting tumor cells. Therefore, there has been a demand for a technique capable of more selectively collecting rare cells such as circulating tumor cells in blood.

Japanese Patent No. 5348357 JP, 2014-25918, A JP, 2013-36818, A

In view of the above circumstances, an object of the present invention is to selectively increase rare cells from a liquid in which rare cells such as circulating tumor cells in blood and contaminated cells such as red blood cells, platelets and white blood cells are mixed. It is to provide a cell separation material that can be recovered with a recovery rate.
Another object of the present invention is to provide a cell separation method that can selectively collect rare cells at a high recovery rate using the cell separation material.

The present inventors have earnestly studied to solve such a problem. As a result, it was found that the selective recovery of rare cells is affected by the air permeability and the compression energy in addition to the average fiber diameter of the cell separation material. By using a cell separating material in which these are in a specific range, it was found that the recovery rate of rare cells can be significantly improved, and the removal rate of contaminating cells such as red blood cells, platelets and white blood cells can be improved, and the present invention Has been completed.

That is, the gist of the present invention is as follows.
[1] A cell separation filter for rare cells, which is composed of fibers having an average fiber diameter of 3 μm or more and 15 μm or less and an air permeability of 25 cc/cm ² /sec or more and 350 cc/cm ² /sec or less and compression energy A plurality of cell separation materials having a WC of 2.7 J/m ² or less are stacked and filled in a container having an inlet and an outlet ,
The filling rate of the cell separation material (total thickness of cell separation material before filling/total thickness of cell separation material after filling) is 1.5 or more and 10 or less,
A cell separation filter, wherein the rare cells are prostate cancer cells, breast cancer cells, lung cancer cells or stem cells .
[2] The cell separation filter according to the above [1], wherein the density of the cell separation material is 6.5×10 ⁴ g/m ³ or more and 1.5×10 ⁵ g/m ³ or less.
[3] The air permeability coefficient M, which is a product of the air permeability (cc/cm ² /sec) of the cell separation material and the thickness (mm) of the cell separation material, is 36 or more and 300 or less, [1] or The cell separation filter according to [2].
[4] The cell separation filter according to any one of [1] to [3], wherein the thickness of the cell separation material is 0.1 mm or more and 3.5 mm or less.
[5] The cell separation filter according to the above [1] to [4], wherein the compression energy WC of the cell separation material is 0.05 J/m ² or more and 1.2 J/m ² or less.
[6] The cell separation filter according to the above [1] to [5], wherein the cell separation material is a nonwoven fabric.
[7] The cell separation filter according to the above [6], wherein the nonwoven fabric is a spunlace nonwoven fabric, a spunbond nonwoven fabric, or a meltblown nonwoven fabric.
[8] The above-mentioned [1] to [7] in which the fibers constituting the cell separation material are made of at least one synthetic polymer selected from the group consisting of polyester, rayon, polyolefin, vinylon, polystyrene, acryl, nylon and polyurethane. ] The cell separation filter described in.
[9] The cell separation filter according to the above [8], wherein the fibers constituting the cell separation material are polyester and polyolefin; rayon and polyolefin; polyester and rayon; or a combination of synthetic polymers of polyester, rayon and vinylon .
[ 10 ] The cell separation filter according to any of [1] to [9], wherein the cell separation material is packed in a compressed state.
[ 11 ] The packing density of the cell separating material (weight of the whole packed cell separating material/volume of the whole packed cell separating material) is 1.0×10 ⁵ g/m ³ or more, 1.0× The cell separation filter according to any one of the above [1] to [10] , which is 10 ⁶ g/m ³ or less.
[ 12 ] The cell separation filter according to any one of the above [1] to [11] , wherein the average pore diameter of the entire cell separation material filled is 1 μm or more and 70 μm or less.
[ 13 ] The cell separation filter according to any one of [1] to [12] , wherein the total thickness of the cell separation material filled is 1 mm or more and 30 mm or less with respect to the flow direction of the liquid.
[ 14 ] A cell separation filter for capturing rare cells and removing red blood cells, white blood cells and platelets by passing a liquid containing rare cells , wherein the rare cells are prostate cancer cells and breast cancer cells The cell separation filter according to any one of [1] to [13] above , which is a lung cancer cell or a stem cell .
[15] the above [1] to the cell separation filter according to any one of to [14] by passed through a liquid containing rare cells abundantly seen contains the rare cells to said cell separation member, and said rare cells A method for cell separation, comprising a step of capturing a fraction of prostate cancer cells, breast cancer cells, lung cancer cells or stem cells .
[ 16 ] The method for cell separation according to the above [ 15 ], wherein the liquid is blood, bone marrow, cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph, urine, saliva or tissue extract.
[ 17 ] The cell separation method according to the above [ 15 ] or [ 16 ], wherein the liquid is blood, cord blood or menstrual blood, and includes the following steps (a) to (c).
(A) a step of passing a liquid containing rare cells from the inlet of the cell separation filter (b) a washing step of passing a washing solution after the step (a) from the inlet of the cell separation filter (c) the (b) After the step), a recovery solution is passed through the outlet of the cell separation filter to recover a rare cell-rich fraction from the inlet of the cell separation filter [ 18 ] The steps (a) and (b) The cell fraction passed through the outlet of the cell separation filter (passage cell fraction) is passed through the inlet of the cell separation filter a plurality of times, and the step (c) is carried out to obtain the passage cell fraction. The cell separation method according to the above [ 17 ], comprising a step of recollecting a fraction containing rare cells mixed in the fraction.
[ 19 ] The method for cell separation according to the above [ 17 ] or [ 18 ], wherein the rare cells recovered in the step (c) can be cultured.
[20] a prior SL (1) to the rare cells enriched fraction was recovered by the method of cell separation according to any one of the cell separation filter, or the [15] - [19] according to any one of [14] The rare cells are prostate cancer cells, breast cancer cells, lung cancer cells or stem cells, red blood cells and platelets are removed by 60% or more, white blood cells are removed by 50% or more, and rare cells can be cultured. Is a rare cell-rich fraction.

By using the cell separating material of the present invention, for example, body fluids such as blood, bone marrow, cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph fluid, urine, saliva or tissue extracts, and liquids such as culture fluid. The rare cells contained in can be separated and efficiently collected from cells mixed with other cells such as red blood cells, platelets and white blood cells.

Further, the rare cell-rich fraction obtained in the present invention has a low erythrocyte, platelet, and white blood cell contamination rate, does not undergo hemolysis even when stored frozen until use, and has very little effect on rare cells. Further, the cells can be collected aseptically, and if the culture solution is used as the recovery solution at the time of cell recovery, the cells recovered as they are can be cultured. Further, even in a liquid in which the rare cell contamination rate is extremely low and rare cells have not been detected so far, the rare cells are recovered by subjecting them to the cell separation method of the present invention after amplifying the rare cells in culture. Therefore, the detection sensitivity can be improved. As described above, the cell separation material, cell separation filter, and cell separation method of the present invention are very useful as a technique for selectively collecting rare cells from various liquids, and a rare cell-rich fraction useful for tests and the like. Can be obtained.

It is a schematic diagram showing an example of a cell separation filter 1 using a cell separation material of the present invention. It is a schematic sectional drawing of the cell separation filter 1 shown in FIG. It is the schematic which shows an example of the cell separation system 13 provided with the cell separation filter 1 shown in FIG.

Hereinafter, the present invention will be described in detail.

(Cell separation material)
The cell separation material of the present invention is composed of fibers having an average fiber diameter of 1.0 μm or more and 50 μm or less, and an air permeability of 10 cc/cm ² /sec or more and 400 cc/cm ² /sec or less and a compression energy WC of 3. It is characterized by being 5 J/m ² or less.

The fibers constituting the cell separation material preferably have an average fiber diameter of 1.0 μm or more and 50 μm or less. If the average fiber diameter is smaller than 1.0 μm, clogging is likely to occur, and if it is larger than 50 μm, rare cells pass without being captured by the cell separation material, and the recovery rate of rare cells is significantly reduced.
The fiber diameter is the width of the fiber in the direction perpendicular to the fiber axis. The average fiber diameter can be measured, for example, by taking a photograph of the cell separation material with a scanning electron microscope and averaging the calculated fiber diameter values obtained from the scale described in the photograph. That is, the average fiber diameter in the present invention means the average value of the fiber diameters measured as described above, and is the average value of the measured fiber diameters of 50 or more, preferably 100 or more. However, when there are many fibers that overlap, when the width cannot be measured because other fibers interfere, and when fibers with significantly different diameters are mixed, that data is excluded to calculate the fiber diameter. ..

In the present invention, by limiting the average fiber diameter of the cell separation material to the above range, rare cells can be efficiently captured and contaminant cells (red blood cells, platelets, white blood cells, etc.) can be removed efficiently. Further, the average fiber diameter is preferably 3 μm or more, more preferably 7 μm or more, and further preferably 10 μm or more from the viewpoint of the separation efficiency of rare cells and foreign cells. The average fiber diameter is preferably 40 μm or less, more preferably 30 μm or less, more preferably 25 μm or less, further preferably 20 μm or less, and particularly preferably 15 μm or less.

The material of the fiber is not particularly limited, but from the viewpoint of sterilization resistance and cell safety, polyester (polyethylene terephthalate, polybutylene terephthalate, etc.), rayon, polyolefin (polyethylene, high density polyethylene, low density) Polyethylene, polypropylene, etc.), vinylon, polystyrene, acrylic (polymethylmethacrylate, polyhydroxyethylmethacrylate, polyacrylonitrile, polyacrylic acid, polyacrylate, etc.), nylon, polyurethane, polyvinyl alcohol, polyvinylidene chloride, polyimide, aramid (aromatic) Polyamide), polyamide, cupra, carbon, phenol, pulp, hemp, synthetic polymers such as polycarbonate, natural polymers such as agarose, cellulose, cellulose acetate, chitosan, chitin, and inorganic materials such as glass and metals.
Among them, polyester, rayon, polyolefin, vinylon, polystyrene, acrylic, nylon and polyurethane are preferable from the viewpoint of cell separation efficiency. Further, these materials are not limited to one type alone, and may be used by combining, mixing, and fusing the materials as necessary. When two or more kinds of materials are combined, the combination is not particularly limited, but preferable examples include polyester and polyolefin; rayon and polyolefin; polyester and rayon; or a combination of synthetic polymers including polyester, rayon and vinylon. Further, if necessary, molecules having an affinity for specific cells such as proteins, peptides, amino acids and saccharides may be immobilized on the material.

Examples of the shape of the cell separation material composed of the fibers include a non-woven fabric, a woven fabric, a sponge, a porous body and a mesh, but the non-woven fabric is preferable because it can be easily produced and obtained.

The method for producing the nonwoven fabric is roughly classified into a wet method and a dry method, and further, a resin bond method, a thermal bond method, a spunlace method, a needle punch method, a stitch bond method, a spun bond method, a melt blown method and the like.
Among them, a spunlace nonwoven fabric, a spunpond nonwoven fabric, or a meltblown nonwoven fabric, which is a nonwoven fabric manufactured by a spunlace method, a spunbond method, or a meltblown method, is preferable from the viewpoint of easily capturing rare cells.
Further, the non-woven fabric may be processed by calendar processing, plasma processing, or the like.
Since the fibers are complicatedly entangled and the blood cell separation efficiency is good, so-called split fibers obtained by splitting the composite single yarn into a plurality of fibers are also suitable as the fibers of the nonwoven fabric.

The cell separation material further has an air permeability of 10 cc/cm ² /sec or more and 400 cc/cm ² /sec or less. By limiting the air permeability to the above range, rare cells can be selectively captured by the cell separation material, and the efficiency of separating rare cells and contaminating cells can be increased. The air permeability is preferably 25 cc/cm ² /sec or more, more preferably 40 cc/cm ² /sec or more, and still more preferably 45 cc/cm ² /sec or more, from the viewpoint of separation efficiency between rare cells and contaminant cells. Particularly preferably, it is 47 cc/cm ² /sec or more. Further, the air permeability, preferably 350cc ^/ cm 2 ^/ sec or less, more preferably 330cc ^/ cm 2 ^/ sec or less, more preferably 300cc ^/ cm 2 ^/ sec or less, especially preferably below 275cc ^/ cm 2 ^/ sec is there.
The air permeability can be measured by various methods, and can be easily measured according to, for example, the Frazier type method described in JIS L1096-2010.

The cell separation material has a compression energy WC of 3.5 J/m ² or less. Only by limiting the average fiber diameter and air permeability to the above range and further limiting the compression energy WC to the above range, the trapping efficiency of rare cells can be remarkably improved. The compression energy WC is preferably 2.0 J/m ² or less, more preferably 1.5 J/m ² or less, still more preferably 1.2 J/m ² or less, from the viewpoint of further improving the capture efficiency of rare cells. It is particularly preferably 0.6 J/m ² or less. Also, the compression energy WC is preferably at 0.05 J / ^{m 2} or more, more preferably 0.1 J / ^{m 2} or more, further preferably 0.2 J / ^{m 2} or more.
Among them, in the present invention, the compression energy WC is 0.05 J/m ² or more, from the viewpoint that it has a compression property that makes it easy to fill the cell separation filter and is excellent in the separation ability of the target rare cells. A cell separation material adjusted to 0.2 J/m ² or less is preferable.
The compression energy WC is a parameter representing the compression characteristics of the cell separation material. When a load is applied to the cell separation material, the cell separation material is compressed and deforms in the load direction, and the ease of compression is expressed as the amount of deformation due to the load received by the cell separation material, that is, load x displacement = energy. Therefore, it can be said that the compression energy WC is a parameter indicating the ease of compression of the cell separation material. The larger the numerical value of the compression energy WC, the more easily the cell separating material is compressed, and the smaller the numerical value, the more difficult the cell separating material is compressed.
In addition, the compression energy WC is measured, for example, using a KES compression tester (KES-G5) manufactured by Kato Tech Co., Ltd., at three points under the conditions of a compression speed of 20 μm/sec and a maximum compression load of 50 gf/cm ² . It can be calculated as the average value.

The density of the cell separation material, that is, the basis weight (g/m ² )/thickness (m) may be 2.0×10 ⁴ g/m ³ or more and 1.5×10 ⁵ g/m ³ or less. Above all, it is preferably 6.5×10 ⁴ g/m ³ or more and 1.5×10 ⁵ g/m ³ or less in view of the separation efficiency between rare cells and foreign cells. Further, in order to recover rare cells with a high recovery rate, the density is more preferably 7.0×10 ⁴ g/m ³ or more, further preferably 8.0×10 ⁴ g/m ³ or more. The density of the cell separation material is preferably 1.3 × ¹⁰ 5 g / ^{m 3} or less, more preferably 1.2 × ¹⁰ 5 g / ^{m 3} or less, 1.1 × 10 ⁵ more preferably g / ^{m 3} or less, particularly preferably 1.0 × ¹⁰ 5 g / ^{m 3} or less.

The density indicates a basis weight (g/m ² )/thickness (m), which can also be expressed as weight (g)/unit volume (m ³ ). That is, the density can be obtained by measuring the weight (g) per unit volume (m ³ ) regardless of the form of the cell separating material. Of course, when the basis weight or thickness is described in the catalog of the material used, the density may be calculated from the basis weight (g/m ² )/thickness (m) based on the data.
The thickness of the cell separation material when calculating the density means the thickness in the uncompressed state. As a method of measuring the thickness in the uncompressed state, for example, a pressure is applied to the cell separation material using a KES compression tester (Kato Tech, KES-G5), and the thickness at a low load in the obtained compression curve is measured. It can be the thickness in the non-compressed state.
Further, the basis weight can be calculated, for example, by measuring the weight of the 1 m square cell separating material with a balance.

Further, the thickness of one sheet of the cell separation material in the uncompressed state is preferably 0.1 mm or more and 3.5 mm or less. The thickness is preferably 0.2 mm or more, more preferably 0.3 mm or more from the viewpoint of the separation efficiency between rare cells and contaminating cells, and in order to efficiently capture rare cells in the cell separating material, It is more preferably 2.5 mm or less, still more preferably 2.0 mm or less, and particularly preferably 1.2 mm or less.

The air permeability coefficient M, which is the product of the air permeability (cc/cm ² /sec) of the cell separation material and the thickness (mm) of the cell separation material, is limited to the range of 36 or more and 300 or less. Is preferred. When the air permeability coefficient M is smaller than 36, the removal efficiency of contaminating cells such as red blood cells and white blood cells tends to decrease, and when the air permeability coefficient M is larger than 300, the efficiency of capturing rare cells by the cell separating material decreases. Tend. Further, from the viewpoint of improving the separation efficiency between rare cells and foreign cells, the air permeability coefficient M is preferably 37 or higher, more preferably 72 or higher, and even more preferably 80 or higher. The air permeability coefficient M of the cell separation material is preferably 250 or less, more preferably 200 or less, and further preferably 150 or less.
The air permeability coefficient M is a value defined by the product of air permeability (cc/cm ² /sec) and thickness (mm) of the cell separation material. The air permeability is a parameter that depends on the pore size of the cell separation material, but even if the air permeability is the same, the smaller the thickness of the cell separation material, the smaller the essential air permeability. The degree becomes smaller. Therefore, by multiplying the air permeability and the thickness, it becomes a parameter that represents the essential pore diameter of the cell separation material.

(Cell separation filter)
The cell separation filter of the present invention is characterized in that a plurality of containers having the inlet and the outlet are stacked and filled with the cell separation material.

The form, size, and material of the container filled with the cell separation material are not particularly limited. The form of the container may be any form such as sphere, container, cassette, bag, tube, column and the like. A preferred specific example is a semitransparent cylindrical container having a capacity of about 0.1 mL to 400 mL and a diameter of about 0.1 cm to 15 cm. In addition, a rectangular or square container having a length of about 0.1 cm to 20 cm and a thickness of about 0.1 cm to 5 cm may be used, but the present invention is not limited thereto.

Examples of the container type include a cross flow type and a column type. Either a cross-flow type or a column type can be used, and the type of the container is not particularly limited, but the column type is more preferable from the viewpoint that the recovered liquid can be uniformly introduced.

The column type is, for example, a container with an inlet and an outlet for liquid near the center of the filter surface, or a container with an inlet and an outlet perpendicular to the filter surface, or the filter surface. To a container characterized by the liquid flowing in a direction perpendicular thereto, or a container characterized in that the liquid flows parallel to the compression direction of the separating material.

On the other hand, the cross flow type is represented by a leukocyte removal filter (“Sepa cell” manufactured by Asahi Kasei Medical Co., Ltd., “Pure cell RC” manufactured by Pall Co., Ltd.), and the positions of the inlet and the outlet of the liquid are flat with respect to the filter surface. Of the container with the inlet and outlet located parallel to the filter surface.

The container can be made using any structural material. Specific examples of the structural material include non-reactive polymers, biocompatible metals, alloys, glass and the like. Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides, polyimides. , Polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene, and the like. Examples of the metal material (biocompatible metal, alloy) useful as the material for the container include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, and gold-plated alloy iron, platinum-plated alloy iron, and cobalt-chromium alloy, Examples thereof include titanium nitride-coated stainless steel.
Among these structural materials, structural materials having sterilization resistance are preferable, and specific examples thereof include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene and the like.

A specific example of the cell separation filter in which the container is filled with the cell separation material is the cell separation filter 1 having the structure shown in FIGS. In the cell separation filter 1, a cylindrical container main body 2 having upper and lower openings, a nozzle holding member 3 which is an inner space of the container main body 2 and is disposed near the upper and lower openings, and with the nozzle It is composed of a cell separating material 4 filled between the pressing members 3 and a lid 5 capable of fixing the pressing member 3 with a nozzle near the opening.
The nozzle port 6 of the nozzle-holding member 3 serves as an inlet or an outlet for introducing a liquid into the cell separation filter 1.
The lid 5 may be screwed to the container body 2.
Further, the place where the lid 5 or the pressing member with nozzle 3 and the container body 2 come into contact is not specified, but by disposing the sealing material 7, the liquid introduced into the cell separation filter 1 leaks out. You may be prevented from doing so.

When filling the cell separation material in the container, if necessary, the cell separation material may be cut into an appropriate size, and a plurality of the cells may be stacked in the direction in which the liquid containing rare cells flows. it can. Among them, as shown in FIG. 2, the thickness T of the entire cell separation material 4 to be filled is preferably 1 mm or more and 30 mm or less with respect to the direction D in which the liquid flows. The direction D is a direction connecting the nozzle openings 6 which are the inlets and outlets of the liquid of the cell separation filter 1. The total thickness T of the filled cell separation material 4 is preferably 25 mm or less, more preferably 20 mm or less, still more preferably 15 mm or less, from the viewpoint of efficiently removing contaminating cells. From the viewpoint of reducing the loss of rare cells, it is preferably 3 mm or more, more preferably 5 mm or more.
The total thickness T of the filled cell separation material 4 can be visually measured from the outside using a caliper if the container is a transparent container. Further, even in the case of an opaque container, it can be calculated by measuring the length of the portion filled with the cell separation material 4 in the structure of each part of the container.

In addition, as a method of filling the cell separation material in the container, not only stacking and filling on a flat plate but also winding and rolling in a roll shape may be performed. When used in a roll form, cells may be separated by treating the liquid from the inside to the outside of the roll, or conversely, the liquid may be treated from the outside to the inside of the roll. Good.

When the cell separation material is filled in the container, it is preferable to compress the cell separation material in the flowing direction of the liquid to fill the container, but it may be filled in the container without being compressed depending on the material of the cell separation material. The degree of compression can be appropriately selected according to the material of the cell separation material and the like.

The cell separation filter in which the container is filled with the cell separation material may be produced by using two or more kinds of cell separation materials having different materials and shapes in combination.

In the cell separation filter, when the container is filled with the cell separation material, the filling rate of the cell separation material (total thickness of cell separation material before filling/thickness of whole cell separation material after filling) is 1 or more, 10 or more. The following is preferable. When the filling rate is less than 1, rare cells may be lost from the voids formed in the filter, and when the filling rate is more than 10, it is difficult to fill the filter with the cell separation material. Becomes Further, in order to increase the efficiency of capturing rare cells and the efficiency of removing contaminating cells, the filling rate is more preferably 1.2 or more, further preferably 1.5 or more. Further, the filling rate is more preferably 9 or less, further preferably 8 or less, and particularly preferably 7.7 or less.
The total thickness of the cell separation material before being filled in the container is calculated from the sum of the thickness of one cell separation material in the non-compressed state, and the thickness of the whole cell separation material after filling is the caliper. It can be measured by the method described above.

In the cell separation filter of the present invention, the packing density of the cell separation material packed in the container (weight of the whole packed cell separation material/volume of the whole packed cell separation material) is 1.0×10 ⁵ g. /M ³ or more and 1.0×10 ⁶ g/m ³ or less is preferable. When the packing density is less than 1.0×10 ⁵ g/m ³ , rare cells tend to pass through the filter, and the recovery rate of rare cells tends to be significantly reduced. On the other hand, when the packing density exceeds 1.0×10 ⁶ g/m ³ , rare cells are firmly trapped in the packed cell separation material, which makes collection difficult. Furthermore, from the viewpoint of improving the recovery rate of rare cells, the packing density is preferably 1.2×10 ⁵ g/m ³ or more, more preferably 1.4×10 ⁵ g/m ³ or more. The packing density is preferably 9.0×10 ⁵ g/m ³ or less, more preferably 8.0×10 ⁵ g/m ³ or less, and 7.6×10 ⁵ g/m 3. It is more preferably ³ or less.
The total volume of the filled cell separation material may be calculated, for example, by the product of the thickness of the whole filled cell separation material and the area of the cell separation material.

The average pore diameter of the entire cell separation material filled in the cell separation filter is preferably 1 μm or more and 70 μm or less. When the average pore diameter is less than 1 μm, not only rare cells but also foreign cells such as red blood cells and white blood cells cannot pass through the filter, and it becomes difficult to remove the foreign cells. On the other hand, when it exceeds 70 μm, not only contaminating cells but also rare cells are likely to pass through the filter, and it is considered that the recovery rate of rare cells is significantly reduced. Further, from the viewpoint of improving the recovery rate of rare cells, it is preferably 66 μm or less, more preferably 57 μm or less, still more preferably 43 μm or less, and particularly preferably 33 μm or less. The average pore size of the cell separation filter is preferably 3 μm or more, more preferably 6 μm or more, and further preferably 9 μm or more.
The average pore diameter can be measured using, for example, a palm porometer (manufactured by Porus Materials).

By passing a liquid containing rare cells, the cell separation filter of the present invention can capture rare cells and remove contaminating cells such as red blood cells, platelets, and white blood cells.
Here, capturing rare cells means that 30% or more, preferably 40% or more, more preferably 50% or more of the rare cells contained in the liquid in contact with the cell separation material or the liquid passed through the cell separation filter. More preferably, 60% or more, particularly preferably 70% or more, particularly preferably 80% or more, most preferably 90% or more are captured by the cell separating material or the cell separating filter.

Further, the cell separation filter of the present invention removes contaminating cells such as red blood cells, platelets and white blood cells, for example, 60% or more, preferably 70% or more of the red blood cells and platelets contained in the liquid in contact with the cell separation material and the cell separation filter. % Or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, particularly preferably 97% or more, most preferably 99% or more, and 50% or more of leukocytes, preferably This means that 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more, particularly preferably 95% or more, most preferably 97% or more can be removed.

In addition, the cell separation filter of the present invention can freely control the performance of the filter by changing the type of the cell separation material to be filled, the filling method, and the like. That is, it can be designed as a filter having a particularly high recovery rate of rare cells or a filter having a particularly high removal rate of foreign cells.

(Cell separation method)
The present invention relates to a cell separation method including a step of passing a liquid containing rare cells through the cell separation filter to capture a fraction rich in rare cells (rare cell-rich fraction) in the cell separation material. ..

Specifically, a liquid containing rare cells is injected into the container filled with the cell separation material from the inlet side, and a fraction rich in rare cells is captured by the cell separation material.

When flowing the liquid into the container having the inlet and the outlet, the liquid inlet may be set to be higher than the liquid outlet, and the liquid may flow in the same direction as gravity, but You may set it so that it is lower than the outlet, and let the liquid flow in the direction opposite to gravity. By causing the liquid to flow in the direction opposite to the gravity, the liquid uniformly flows into the container and bubbles easily escape, so that the separation efficiency can be further improved. Besides, the direction of flowing the liquid may be a direction perpendicular to gravity, and the direction of flowing the liquid is not limited to the above.

The liquid containing rare cells used in the cell separation method of the present invention is not particularly limited as long as it is a suspension containing rare cells, but is not limited to blood, bone marrow, cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph, Examples include urine, saliva or tissue extract.
Further, it may be a culture solution in which rare cells are cultured in vitro, or a suspension obtained by suspending in a liquid such as physiological saline.

Further, after passing the liquid containing the rare cells, a component other than the rare cells may be removed from the cell separation filter, if necessary, by passing a washing solution from the inlet side.
The wash solution that can be used is not particularly limited as long as it does not damage rare cells, but a common buffer solution such as physiological saline, Ringer's solution, a medium used for cell culture, or a phosphate buffer is preferable.

Next, from the outlet direction of the container, that is, from the direction opposite to the direction of passage of the liquid containing rare cells or the washing solution, the recovered solution is caused to flow, whereby the content of rare cells trapped in the cell separation material is abundant. Images can be collected.

The recovery solution is not particularly limited as long as it is an isotonic solution, but it has a history of use as an injectable agent such as physiological saline, Ringer's solution, dextran sugar injection, and hydroxyethyl starch, a buffer solution, a cell culture medium. Etc.

Further, the viscosity of the recovery solution may be increased in order to increase the recovery rate of the captured rare cells. Therefore, albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, hydroxyethyl cellulose, collagen, hyaluronic acid, gelatin and the like can be added to the above-mentioned recovered solution, but the additives are not limited to these. The viscosity of the recovery solution is not particularly limited, but if the viscosity is too high, the recovery operation tends to be difficult to perform, so 20 mPa·s or less is more preferable. A column type container can be used even at 10 mPa·s or less since the recovery performance does not deteriorate even if a low viscosity recovery solution is used. Furthermore, it is possible to use a recovery solution of 5 mPa·s or less.

In addition, as a pretreatment of the step of capturing the fraction rich in rare cells in the cell separation material as described above, a step of immersing the cell separation material in a pretreatment solution such as physiological saline or a buffer solution is performed. Good. This operation is not always necessary, but by immersing the cell separation material in the pretreatment solution, a liquid containing rare cells can be uniformly passed through the entire cell separation material, and improvement in separation efficiency is expected. It The pretreatment solution does not have to be the same as the washing solution, but if it is the same, a solution bag or the like connected to the cell separation filter during the treatment can be shared, which simplifies the circuit system and improves operability. From the viewpoint, it is preferable that they are the same. The amount of liquid for the pretreatment is practically preferably 1 to 100 times the filter capacity. The buffer solution that can be used is not particularly limited, but a common buffer solution such as physiological saline, Ringer's solution, a medium used for cell culture, or a phosphate buffer solution is preferable.

Further, in addition to rare cells, when a liquid containing contaminating cells such as red blood cells, platelets, white blood cells, for example, blood, cord blood or menstrual blood is used, by performing the following (a) to (c) steps, Rare cells can be recovered with higher efficiency, and contaminating cells can be removed with higher efficiency.
(A) a step of passing a liquid containing rare cells from the inlet of the cell separation filter (b) a washing step of passing a washing solution after the step (a) from the inlet of the cell separation filter (c) the (b) Rare cell recovery step of collecting a rare cell-rich fraction from the inlet of the cell separation filter by passing a recovery solution through the outlet of the cell separation filter after the step

In the step (a), a liquid containing rare cells may be passed through the cell separation filter, and a red blood cell-rich fraction that has passed through without being captured by the cell separation material may be removed from the cell separation filter. it can. In this step, when the liquid is passed from the liquid inlet side of the container filled with the cell separation material, the liquid may be fed from the container containing the liquid by a natural drop through the liquid feeding circuit, or by using a pump You may send liquid. Alternatively, a syringe containing a liquid containing rare cells may be directly connected to the container and the plunger of the syringe may be manually pushed. When the liquid is passed through the pump, the separation efficiency tends to decrease when the liquid feeding speed is too fast, and the treatment time tends to take when the liquid feeding speed is too slow. Examples of the liquid transfer rate include, but are not limited to, a rate of 0.1 mL/min to 100 mL/min.

In the step (b), by passing the washing solution through the filter, the fraction rich in white blood cells and platelets can be removed by passing through the filter. The same cleaning solution as described above may be used.
When the cleaning solution is passed, it may be fed by gravity or by a pump. The flow rate in the case of sending a liquid by a pump is similar to that in the case of sending a liquid containing rare cells, and examples thereof include a speed of 0.1 to 100 mL/min, but are not limited thereto. The amount of washing solution depends on the volume of the container used.If the amount of washing solution is too small, the amount of white blood cells and platelets remaining in the filter will be large, and if the amount of washing solution is too large, rare cells will pass through the filter. Therefore, it is preferable to wash with a liquid amount of about 0.5 to 100 times the filter capacity, since the process will take a long time.

In the present invention, the cell fraction (passage cell fraction) passed from the outlet of the cell separation filter in the steps (a) and (b) is passed through the inlet of the cell separation filter a plurality of times. The method further includes the step of re-collecting the fraction containing rare cells mixed in the passage cell fraction by carrying out the step (c).

In the step (c), rare cells trapped in the cell separation material are separated from the cell separation material and collected. Specifically, the recovery solution is injected into the filter filled with the cell separation material from the outlet side in the steps (a) and (b), and the rare cells are introduced from the inlet side in the steps (a) and (b). Collect. That is, the liquid passing direction in the step (c) is opposite to that in the steps (a) and (b). When injecting the recovery solution, the recovery solution may be put in a syringe or the like in advance, and the plunger of the syringe may be pushed out using a hand or a device. The amount and flow rate of the recovered solution vary depending on the volume of the container used and the amount to be processed, but it is preferable to adjust the flow rate to 0.5 to 20 mL/sec with a liquid volume of about 1 to 100 times the filter volume. It is not limited to this.

In the cell separation method of the present invention, it is possible to select a method having a particularly high recovery rate of rare cells or a method having a particularly high removal rate of contaminating cells. For example, the cell separation efficiency is controlled by controlling the liquid passing rate in the step (a), the washing liquid amount and the liquid passing rate in the step (b), and the recovered liquid amount and the liquid passing rate in the step (c). It is possible to control

Further, as the cell separation method of the present invention, a cell separation system using the cell separation filter may be configured. The cell separation system contains, in addition to the cell separation filter, an inlet and an outlet of a liquid containing rare cells, a washing solution and a recovery solution, a bag containing the liquid containing rare cells, and a liquid containing foreign cells that have passed through the filter. It is practical and preferable to simultaneously have a waste liquid bag, a bag for storing rare cells collected from the filter, and the like. In this case, the cell separation filter has an inlet for the liquid containing rare cells and an outlet for the liquid, and further has an inlet and an outlet for the washing solution used to remove contaminating cells such as red blood cells. Moreover, it is preferable that an inlet for introducing the recovery solution is also provided independently of the outflow portion. The inlet of the washing solution and the collection solution connected to the filter may function as the outlet of the liquid after washing the contaminating cells and collecting the rare cells, and further, each bag, syringe, etc. are connected via a three-way stopcock or the like. May be.

For example, the cell separation system 13 as shown in FIG. 3 can be used. In the cell separation system 13, the cell separation filter 1 is set such that the liquid inlet 8 is vertically downward and the liquid outlet 9 is vertically upward. Is discharged from the outlet 9 on the upper side in the vertical direction inside the cell filter 1.
At the inlet 8 of the cell separation filter 1, via a three-way stopcock 10a, a bag 11 containing a liquid containing rare cells and a bag 12 containing a washing solution (right side in the figure), and cells A bag (collection bag) 20 for containing the liquid collected from the separation filter 1 and a means 21 (lower side in the figure) for collecting the liquid collected in the collection bag or the like are connected by a tube.
Below the three-way stopcock 10a, a collection bag 20 and a means 21 for collecting the liquid collected in the collection bag and the like are connected by a tube via the three-way stopcock 10c.
Further, the outlet 9 of the cell separation filter 1 is connected via a three-way stopcock 10d to a bag (waste liquid bag) 23 containing the liquid that has passed through the cell separation filter 1 and a bag 24 containing the collected solution. ing.

In the cell separation system 13, the bags 11 and 12 are arranged vertically above the cell separation filter 1 and the outlet 9, so that the liquid contained in the bag 11 or the bag 12 is either Can also be introduced into the cell separation filter 1 by utilizing gravity, and any of the introduced liquid is discharged from the outlet 9 and stored in the waste liquid bag 23. In addition, the switching of the liquid passage from the bag 11 or the bag 12 can be performed by the three-way stopcock 10b.
As described above, rare cells can be captured by the cell separation material in the cell separation filter 1.

Next, when collecting the rare cells captured by the cell separation material in the cell separation filter 1, the collection solution is introduced from the bag 24 into the cell separation filter 1 through the outlet 9 to remove the rare cells. It is separated from the cell separation material and collected in the collection bag 20 as a suspension.
When the volume of the suspension is large, for example, the collection bag 20 is detached from the cell separation system 13 and centrifuged to precipitate rare cells, and then the cell separation system 13 is again provided. The liquid in the supernatant in the attached/collection bag 20 may be discharged from the means 21 for collecting the liquid collected in the collection bag or the like.

In the cell separation system, the cell collection bag is connected to the inlet and outlet of each of the solutions described above, whereby cells can be separated aseptically and in a closed system. Further, it is preferable that each bag can be separated after use, and may have a shape like a commonly used blood bag, but a flat plate type cartridge system or the like may also be used. As the form of each bag, a cell culturable bag, a bag having freeze storage resistance, or the like may be selected as necessary.

In the above-described cell separation method, the stress applied to the rare cells is extremely low as compared with the conventional centrifugation method, and therefore the rare cells contained in the recovery solution can be cultured as they are.

In addition, the rare cells may be cryopreserved in a cryopreservation tube or the like at a cryogenic temperature such as in liquid nitrogen or a deep freezer.
When cryopreserving rare cells, a cryoprotective agent is added to the recovery solution in order to protect the rare cells from damage due to freezing. The type of cryoprotective agent added is not particularly limited, but dimethyl sulfoxide, dextran, albumin, hydroxyethyl starch, etc. can be used. The cryoprotectants may be used alone or in combination of several kinds.

In the above cryopreservation method, the temperature during storage is not particularly limited, but is preferably −196° C. to −30° C., more preferably −196° C. to −50° C., in order to prevent a decrease in activity of rare cells. It is preferably -196°C to -80°C.

The survival rate of rare cells after cryopreservation is preferably 80% or more, more preferably 85% or more.

The rare cells recovered in the present invention include tumor cells, stem cells, endothelial cells, fetal cells and the like.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

(Examples 1-27, Comparative Examples 1-4)
As shown in FIGS. 1 and 2, a cylindrical container body (2) having an inner diameter of 26 mm is filled with a round-shaped non-woven fabric (4) having a diameter of 26 mm, and the upper and lower openings of the container body (2) are filled. A holding member (3) with a nozzle was inserted, and a cap (5) was screwed onto the pressing member (3) to produce a cell separation filter (1).
Regarding the non-woven fabric used in each of the examples and comparative examples, Table 1 shows the type of resin, the non-woven fabric manufacturing method, the average fiber diameter, the compression energy WC, the air permeability, the thickness, the density, and the air permeability coefficient M. Table 1 shows the total thickness, packing density, packing rate, and average pore size of the cell separation material packed in the cell separation filter.

Next, the cell separation filter was used to separate and examine rare cells and cells other than rare cells (red blood cells, platelets and white blood cells) (hereinafter referred to as contaminating cells). As rare cells, human prostate cancer cell line PC3, human breast cancer cell line MCF-7, human lung cancer cell line A549, and human adipose-derived stem cells (ASC) were used as model cells. In order to make a clear distinction between the recovery rate of rare cells and the removal rate of foreign cells, an experimental system that does not include foreign cells in the study of the recovery rate of rare cells should be included in the study system of the removal rate of foreign cells in the study of the removal rate of foreign cells. It was an experimental system.

In examining the recovery rate of rare cells, rare cells were recovered from the liquid containing rare cells by the following procedure.
(1) A liquid in which rare cells were suspended was passed through the inlet of the cell separation filter. Table 2 shows the type and number of rare cells that have passed, the type and amount of solution in which the cells have been suspended, and the rate of passage of the cell suspension.
(2) After the above step, the washing solution was passed through the inlet of the cell separation filter. Table 2 shows the type and amount of the cleaning solution and the liquid passing rate of the cleaning solution.
(3) After the above step, the recovered solution was passed through the outlet of the cell separation filter, and the rare cell-rich fraction was recovered from the inlet of the cell separation filter. Table 2 shows the type and amount of the recovered solution.
(4) The number of rare cells in the rare cell-rich fraction obtained in the above step was measured by an automatic cell counting device (GE Healthcare, CYTORECON).

In the examination of the removal rate of foreign cells, foreign cells were removed from the liquid containing foreign cells by the following procedure.
(1) Human peripheral blood was passed through the inlet of the cell separation filter. Table 2 shows the liquid volume and the liquid flow rate of the human peripheral blood that was passed.
(2) After the above step, the washing solution was passed through the inlet of the cell separation filter. Table 2 shows the type and amount of the cleaning solution and the liquid passing rate of the cleaning solution.
(3) After the above step, the recovered solution was passed through the outlet of the cell separation filter, and the contaminating cells remaining in the filter were recovered from the inlet of the cell separation filter to obtain a contaminating cell fraction. Table 2 shows the type and amount of the recovered solution.
(4) Count the number of blood cells in the peripheral blood before passing through the filter and the number of blood cells in the contaminating cell fraction obtained in the above step with a hemocytometer (Sysmex, K-4500) did. Here, hematopoietic cells refer to red blood cells, white blood cells, and platelets.

Table 3 shows the recovery rate of rare cells and the removal rate of contaminating cells. The recovery rate of rare cells was calculated by dividing the number of cells recovered from the cell separation filter by the number of cells passed through the filter. The removal rate of contaminating cells was calculated by subtracting from 1 the value obtained by dividing the number of cells recovered from the cell separation filter by the number of cells passed through the filter (contamination cell recovery rate).

From the results shown in Table 3, in the cell separation filters of Examples 1 to 27, the recovery rate of rare cells was 35% or more, which was higher than that of Comparative Examples 1 to 4, regardless of the type of rare cells. There is. Further, from the results of Examples 1 to 17, it can be seen that the removal rate of leukocytes is 51% or more, the removal rate of red blood cells is 98% or more, and the removal rate of platelets is 79% or more.

Among the examples, the cell separation filters of Examples 1 to 4, 6 to 23, 26 and 27 in which the packing density of the cell separation material filled in the cell separation filter is 1.0×10 ⁵ g/m ³ or more. In Examples 1 to 4, 6 to 12, 15 to 23, 26 and 27, the rare cell recovery rate is as high as 43% or more and the air permeability coefficient M of the cell separation material is 300 or less. It can be seen that the cell separation filter of No. 1 has an even higher rare cell recovery rate of 51% or more.

Furthermore, as a result of the recovery rate of the prostate cancer cell line PC3, which is a kind of rare cells, Examples 1 to 4, 6, 9, and 11 show that the recovery rate of rare cells is remarkably high at 70% or more, and leukocytes The removal rate is 90% or more, the removal rate of red blood cells is 99% or more, and the removal rate of platelets is 95% or more, which is a cell separation filter with excellent concentration efficiency of rare cells.

Further, with the cell separation filters of Examples 26 and 27, the recovery rate was as high as 68% or more regardless of the type of cancer cells, so it was used for inspection and diagnosis of cancer patients of various cancer types. It is considered to be a cell separation filter that can be used.

On the other hand, the same non-woven fabric is used in Examples 1 and 2, Examples 3 and 4, Examples 5 and 6, Examples 7 and 8, Examples 9 and 10, Examples 16 and 17, and Examples 20 and 21. Comparing each of the above results, it can be seen that the recovery rate of rare cells is improved by adjusting the packing density and packing rate of the cell separation filter to be high and reducing the average pore diameter of the cell separation filter.
Further, from the results of Examples 23, 24 and 25, a nonwoven fabric having a compression energy of 1.2 J/m ² or less, a density of 6.5×10 ⁴ g/m ³ or more and a permeability coefficient M of 300 or less was used. In Example 23 in which the packing density was adjusted to 1.0×10 ⁵ g/m ³ or more, the recovery rate of rare cells was significantly improved as compared with the other Examples 24 and 25.

On the other hand, in the cell separation filters of Comparative Examples 1 to 4 using the cell separation material having a compression energy WC of 3.6 J/m ² , although the removal rate of red blood cells, platelets and white blood cells was high, recovery of rare cells was performed. The rate is significantly lower than 15.1%.

Therefore, it can be seen that rare cells such as tumor cells and stem cells can be selectively and efficiently recovered by using the cell separation filter filled with the cell separation material of the present invention.

1 Cell Separation Filter 2 Container Body 3 Nozzle Pressing Member 4 Cell Separation Material 5 Lid 6 Nozzle Mouth 7 Sealing Material 8 Inlet 9 Outlet 10 Three-way Stopcock 11 Bag Containing Liquid Containing Rare Cells 12 Retaining Wash Solution Bag 13 Cell separation system 20 Bag containing liquid collected from cell separation filter 1 (collection bag)
21 Means (syringe, etc.) for collecting the liquid collected in a collecting bag, etc.
23 Bag for storing liquid that has passed through the cell separation filter 1 (waste liquid bag)
24 Bag Containing Recovered Solution T Total Thickness of Cell Separation Material 4 Filled in Cell Separation Filter 1 Direction of Flow of Liquid Introduced into Cell Separation Filter 1

Claims (18)

A cell separation filter for rare cells, which is composed of fibers having an average fiber diameter of 3 μm or more and 15 μm or less, an air permeability of 25 cc/cm ² /sec or more, 350 cc/cm ² /sec or less, and a compression energy WC of 0. A plurality of cell separation materials of 0.05 J/m ² or more and 2.7 J/m ² or less are stacked and filled in a container having an inlet and an outlet,
The cell separation material is a non-woven fabric,
The filling rate of the cell separation material (total thickness of cell separation material before filling/thickness of whole cell separation material after filling) is 1.5 or more and 10 or less,
A cell separation filter, wherein the rare cells are prostate cancer cells, breast cancer cells, lung cancer cells or stem cells. The cell separation filter according to claim 1, wherein the density of the cell separation material is 6.5×10 ⁴ g/m ³ or more and 1.5×10 ⁵ g/m ³ or less. The air permeability coefficient M, which is a product of the air permeability (cc/cm ² /sec) of the cell separation material and the thickness (mm) of the cell separation material, is 36 or more and 300 or less. Cell separation filter. The cell separation filter according to any one of claims 1 to 3, wherein the thickness of the cell separation material is 0.1 mm or more and 3.5 mm or less. The cell separation filter according to any one of claims 1 to 4, wherein the compression energy WC of the cell separation material is 0.05 J/m ² or more and 1.2 J/m ² or less . The cell separation filter according to any one of claims 1 to 5, wherein the non-woven fabric is a spun lace non-woven fabric, a spun bond non-woven fabric or a melt blown non-woven fabric. Fibers constituting the cell separation material, polyester, rayon, polyolefin, vinylon, polystyrene, acrylic, any one of claim 1 to 6 comprising at least one synthetic polymer selected from the group consisting of nylon and polyurethane The cell separation filter according to 1. The cell separation filter according to claim 7 , wherein the fibers constituting the cell separation material are composed of polyester and polyolefin; rayon and polyolefin; polyester and rayon; or a combination of synthetic polymers of polyester, rayon and vinylon. Cell separation filter according to any one of the cell separation material, is filled in a compressed state claims 1-8. The packing density of the cell separating material (weight of the whole packed cell separating material/volume of the whole packed cell separating material) is 1.0×10 ⁵ g/m ³ or more, 1.0×10 ⁶ g /M ³ or less, the cell separation filter according to any one of claims 1 to 10 . The cell separation filter according to any one of claims 1 to 11 , wherein the average pore diameter of the entire cell separation material filled is 1 µm or more and 70 µm or less. The cell separation filter according to any one of claims 1 to 12 , wherein the thickness of the entire cell separation material filled is 1 mm or more and 30 mm or less with respect to the direction in which the liquid flows. By passed through a liquid containing rare cells, to capture the rare cells, and red blood cell separation filter according to any one of claims 1 to 12 for the removal of leukocytes and platelets. A step of passing a liquid containing rare cells through the cell separation filter according to any one of claims 1 to 13 to capture a fraction rich in rare cells in the cell separation material, and A method for separating cells, wherein the rare cells are prostate cancer cells, breast cancer cells, lung cancer cells or stem cells. 15. The cell separation method according to claim 14 , wherein the liquid is blood, bone marrow, cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph, urine, saliva or tissue extract. The cell separation method according to claim 14 or 15 , wherein the liquid is blood, cord blood, or menstrual blood, and includes the following steps (a) to (c).
(A) a step of passing a liquid containing rare cells from the inlet of the cell separation filter (b) a washing step of passing a washing solution after the step (a) from the inlet of the cell separation filter (c) the (b) Rare cell recovery step of collecting a rare cell-rich fraction from the inlet of the cell separation filter by passing a recovery solution through the outlet of the cell separation filter after the step The cell fraction (passed cell fraction) passed from the outlet of the cell separation filter in the steps (a) and (b) is passed a plurality of times from the inlet of the cell separation filter, and further, the step (c) is performed. The cell separation method according to claim 16 , further comprising the step of re-collecting a fraction containing rare cells mixed in the passage cell fraction by carrying out the step. The cell separation method according to claim 16 or 17 , wherein the rare cells recovered in the step (c) can be cultured .

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