WO2016047444A1

WO2016047444A1 - Cell separation material and cell separation method

Info

Publication number: WO2016047444A1
Application number: PCT/JP2015/075602
Authority: WO
Inventors: 愛弓山本; 康弘溝上
Original assignee: 株式会社カネカ
Priority date: 2014-09-24
Filing date: 2015-09-09
Publication date: 2016-03-31
Also published as: JP6707031B2; JPWO2016047444A1

Abstract

The present invention pertains to: a cell separation material comprising fibers having an average fiber diameter of 1.0-50 µm, air permeability of 10-400 cc/cm²/sec, and a compression energy WC of no more than 3.5 J/m²; a cell separation filter characterized by having several layers of the cell separation material laminated and filled into a container having an entry and an exit; and a cell separation method including a step in which fluid including rare cells is caused to pass through the cell separation filter and fractions rich in rare cells are captured in the cell separation material. As a result of the present invention, rare cells can be selectively recovered, at a high recovery rate, from a fluid having mixed therein rare cells such as circulating tumor cells and contaminating cells such as red blood cells, platelets, platelets, and leukocytes.

Description

Cell separation material and cell separation method

The present invention relates to a cell separation material and a cell separation method using the same. More specifically, the present invention relates to a cell separation material suitable for separating rare cells (tumor cells, stem cells, endothelial cells, fetal cells, etc.) present in a liquid from contaminated 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 tumor cells, circulating stem cells, circulating endothelial cells, etc. (hereinafter collectively referred to as “rare cells”) are cells that are extremely rare in whole blood. And 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 considered to be the cause of metastatic cancer. If these tumor cells can be detected early, the presence of the primary tumor, tumor metastasis, or recurrence of the 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) red blood cells, about 5 × 10 ⁶ (cells / mL) white blood cells, and about 3 × 10 ⁸ (cells / mL) platelets in the blood. Currently, it is extremely difficult to detect tumor cells that exist only in a ratio of hundreds of thousands to tens of millions, and there is a limit to using tumor cells for diagnosis. Therefore, a method for improving detection sensitivity and measurement accuracy by removing contaminated cells and concentrating tumor cells before quantitative analysis of tumor cells is currently being developed and used.

For example, currently used methods for collecting and concentrating circulating tumor cells in the blood include negative selection using magnetic beads with immobilized antibodies against antigens of contaminated cells whose surface antigens have been clarified, tumor cells Examples thereof include a positive selection method using magnetic beads on which an antibody against a specific antigen is immobilized. There are cell sorting methods such as cell sorting using fluorescent antibodies, but both methods require a large amount of antibody and processing time to sort out large amounts of contaminating cells and trace amounts of tumor cells. Therefore, the process 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 separation utilizing the specific gravity difference of cells, it is impossible to separate tumor cells and contaminated 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, circulating tumor cells in the blood that are very few are also lost. As a result, an incorrect diagnosis may be made. Red blood cells are the largest number in blood, and this large amount of red blood cells is a major obstacle to detecting tumor cells. In addition to the above-described methods for removing red blood cells in advance, there are methods for hemolyzing red blood cells (for example, the ammonium chloride method and the high osmotic pressure destruction method). Although this method efficiently lyses and removes red blood cells, the damage to other cells is great, making it difficult to analyze and culture tumor cells alive. In addition, several 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 target rare cells are selected from whole blood samples. It is difficult to say that it is sufficient as a separate separation and recovery technology. Furthermore, any of the above methods cannot be processed in a closed system, and has safety problems such as contamination and infection.

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

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

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

The present inventors have intensively studied to solve such problems. As a result, the inventors have 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 separation material in which these are in a specific range, it has been 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. It came to complete.

That is, the gist of the present invention is as follows.
[1] Consisting of fibers having an average fiber diameter of 1.0 μm or more and 50 μm or less, the air permeability is 10 cc / cm ² / sec or more, 400 cc / cm ² / sec or less, and the compression energy WC is 3.5 J / m ² or less. Cell separation material.
[2] The cell separation material according to [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 separating material and the thickness (mm) of the cell separating material, is 36 or more and 300 or less [1] or The cell separation material according to [2].
[4] The cell separation material according to any one of [1] to [3], wherein the cell separation material has a thickness of 0.1 mm to 3.5 mm.
[5] The cell separation material according to any one of [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 material according to any one of [1] to [5], wherein the cell separation material is a nonwoven fabric.
[7] The cell separation material according to [6], wherein the nonwoven fabric is a spunlace nonwoven fabric, a spunbond nonwoven fabric, or a meltblown nonwoven fabric.
[8] The above [1] to [7], wherein the fiber constituting the cell separation material is composed of at least one synthetic polymer selected from the group consisting of polyester, rayon, polyolefin, vinylon, polystyrene, acrylic, nylon and polyurethane. ] The cell separation material of any one of.
[9] The cell separation material according to [8], wherein the fibers constituting the cell separation material comprise a combination of polyester and polyolefin; rayon and polyolefin; polyester and rayon; or a synthetic polymer of polyester, rayon and vinylon.
[10] A cell separation filter comprising a plurality of the cell separation materials according to any one of [1] to [9] stacked and filled in a container having an inlet and an outlet.
[11] The cell separation filter according to [10], wherein the cell separation material is filled in a compressed state.
[12] The filling rate of the cell separation material (the thickness of the whole cell separation material before filling / the thickness of the whole cell separation material after filling) is 1 or more and 10 or less. Cell separation filter.
[13] The packing density of the cell separation material (weight of the whole cell separation material / volume of the whole cell separation material) is 1.0 × 10 ⁵ g / m ³ or more, 1.0 × The cell separation filter according to any one of [10] to [12], which is 10 ⁶ g / m ³ or less.
[14] The cell separation filter according to any one of [10] to [13], wherein the entire pore size of the cell separation material packed is 1 μm or more and 70 μm or less.
[15] The cell separation filter according to any one of [10] to [14], wherein a thickness of the whole cell separation material packed in a direction in which the liquid flows is 1 mm or more and 30 mm or less.
[16] The cell separation according to any one of the above [10] to [15] for capturing rare cells by passing a liquid containing rare cells and removing red blood cells, white blood cells, and platelets filter.
[17] A step of passing a liquid containing rare cells through the cell separation filter according to any one of [10] to [16], and capturing a fraction containing rare cells in the cell separation material. A cell separation method comprising.
[18] The cell separation method according to [17], wherein the liquid is blood, bone marrow, umbilical cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph fluid, urine, saliva, or tissue extract.
[19] The cell separation method according to [17] or [18], wherein the liquid is blood, umbilical 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 from the inlet of the cell separation filter after the step (a) (c) The (b ) Step of collecting rare cell-rich fraction from the inlet of the cell separation filter by passing the recovered solution through the outlet of the cell separation filter after the step [20] Steps (a) and (b) The cell fraction passed through from the outlet of the cell separation filter (passed cell fraction) was passed through the inlet of the cell separation filter a plurality of times, and the step (c) was further performed, The cell separation method according to [19] above, which further comprises a step of recovering a fraction containing rare cells mixed in the fraction.
[21] The cell separation method according to [19] or [20], wherein the rare cells recovered in the step (c) can be cultured.
[22] The cell separation material according to any of [1] to [9], the cell separation filter according to any of [10] to [16], or the above [17] to [21] A rare cell-rich fraction collected by any of the cell separation methods, wherein erythrocytes and platelets are removed by 60% or more, leukocytes are removed by 50% or more, and rare cells can be cultured. A rare cell-rich fraction.

By using the cell separation material of the present invention, for example, body fluid such as blood, bone marrow, umbilical cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph fluid, urine, saliva or tissue extract, and fluid such as culture fluid The rare cells contained in can be separated and efficiently recovered from mixed cells other than erythrocytes, platelets, leukocytes and the like.

In addition, the rare cell-rich fraction obtained in the present invention has a low contamination rate of red blood cells, platelets and white blood cells, and does not undergo hemolysis even if stored frozen until use, and has very little effect on rare cells. Moreover, it is possible to collect aseptically, and if the culture solution is used as the recovery solution at the time of cell recovery, the recovered cells can be cultured as they are. Further, even in a liquid in which the rare cell contamination rate is extremely small and rare cells have not been detected so far, the rare cells are recovered by amplifying the rare cells by culturing and then using the cell separation method of the present invention. Therefore, 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 recovering rare cells from various liquids, and are useful for examination and the like. Can be obtained.

It is the schematic which shows an example of the cell separation filter 1 using the cell separation material of this 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 has 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. 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. If the average fiber diameter is larger than 50 μm, rare cells pass through without being captured by the cell separation material, so that the collection rate of rare cells is greatly 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 values of the fiber diameter obtained from the scale described in the photograph. That is, the average fiber diameter in the present invention means an average value of the fiber diameters measured as described above, and is an average value of 50 or more, preferably 100 or more fiber diameters measured. However, when there are many overlapping fibers, when other fibers interfere with each other and the width cannot be measured, or when fibers with significantly different diameters are mixed, the fiber diameter is calculated excluding the data. .

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 contaminated cells (erythrocytes, platelets, leukocytes, etc.) can be efficiently removed. 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 separation efficiency between rare cells and contaminating cells. The average fiber diameter is preferably 40 μm or less, more preferably 30 μm or less, more preferably 25 μm or less, still more 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 (polymethyl methacrylate, polyhydroxyethyl methacrylate, polyacrylonitrile, polyacrylic acid, polyacrylate, etc.), nylon, polyurethane, polyvinyl alcohol, polyvinylidene chloride, polyimide, aramid (aromatic) Polyamide), polyamide, cupra, carbon, phenol, pulp, hemp, polycarbonate, synthetic polymers, agarose, cellulose, cellulose acetate Chitosan, natural polymers such as chitin, an inorganic material, metal or the like such as glass.
Among these, polyester, rayon, polyolefin, vinylon, polystyrene, acrylic, nylon, and polyurethane are preferable from the viewpoint of cell separation efficiency. In addition, these materials are not limited to a single type, and may be combined, mixed, and fused as necessary. When two or more kinds of materials are combined, the combination is not particularly limited, but a combination of polyester and polyolefin; rayon and polyolefin; polyester and rayon; or a synthetic polymer composed of polyester, rayon and vinylon is preferable. Further, if necessary, molecules having affinity for specific cells such as proteins, peptides, amino acids, and sugars may be fixed to the material.

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

Nonwoven fabric production methods can be broadly classified into wet methods and dry methods, and further include resin bond methods, thermal bond methods, spun lace methods, needle punch methods, stitch bond methods, spun bond methods, and melt blown methods.
Among these, from the viewpoint of easily capturing rare cells, 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.
The non-woven fabric may be subjected to processing such as calendar processing or plasma processing.
Since the fibers are intertwined in a complicated manner and blood cell separation efficiency is good, so-called divided fibers obtained by dividing a composite single yarn into a plurality of fibers are also suitable as the nonwoven fabric fibers.

The cell separation material further has an air permeability of 10 cc / cm ² / sec to 400 cc / cm ² / sec. By limiting the air permeability to the above range, the rare cells are selectively captured by the cell separation material, and the separation efficiency between the rare cells and the contaminated 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, further preferably 45 cc / cm ² / sec or more, from the viewpoint of separation efficiency between rare cells and contaminating 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. For example, it can be easily measured in accordance with or in accordance with the Frazier method described in JIS L1096-2010.

The cell separation material further has a compression energy WC of 3.5 J / m ² or less. Only when the average fiber diameter and the air permeability are limited to the above ranges and the compression energy WC is limited to the above ranges, 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, further preferably 1.2 J / m ² or less, from the viewpoint of further improving the capture efficiency of rare cells. Particularly preferred is 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 characteristic that can be easily filled into a cell separation filter, and is excellent in the ability to separate target rare cells. A cell separation material adjusted to 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 deformed in the load direction. The ease of compression is expressed as the amount of deformation caused by the load received by the cell separation material, that is, load × displacement = energy. Therefore, the compression energy WC can be said to be a parameter representing the ease of compression of the cell separation material. The larger the numerical value of the compression energy WC, the easier the cell separating material is compressed, and the smaller the numerical value, the more difficult the cell separating material is compressed.
The compression energy WC was measured at three points using, for example, a KES compression tester (KES-G5) manufactured by Kato Tech Co. under conditions of a compression rate of 20 μm / sec and a maximum compression load of 50 gf / cm ² . The average value can be calculated.

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. Among these, from the separation efficiency of rare cells and contaminated cells, it is preferably 6.5 × 10 ⁴ g / m ³ or more and 1.5 × 10 ⁵ g / m ³ or less. Furthermore, in order to collect rare cells at a high recovery rate, the density is more preferably 7.0 × 10 ⁴ g / m ³ or more, and 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 basis weight (g / m ² ) / thickness (m), which can also be expressed as weight (g) / unit volume (m ³ ). That is, the density can also be obtained by measuring the weight (g) per unit volume (m ³ ) regardless of the form of the cell separation material. Of course, when the basis weight or thickness is described in the catalog of the material to be used, the density may be obtained from the basis weight (g / m ² ) / thickness (m) based on the data.
In addition, the thickness of the cell separation material at the time of calculating the density refers to the thickness in an uncompressed state. As a method for measuring the thickness in the uncompressed state, for example, 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 an uncompressed state.
The basis weight can be calculated, for example, by measuring the weight of a 1 m square cell separator with a balance.

Moreover, it is preferable that the thickness of one sheet of the cell separation material in the uncompressed state is 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 separation efficiency between rare cells and contaminating cells. Further, in order to efficiently capture rare cells in the cell separation material, More preferably, it is 2.5 mm or less, More preferably, it is 2.0 mm or less, Especially preferably, it is 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 separator and the thickness (mm) of the cell separator, is limited to a range of 36 to 300. 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 be reduced. When the air permeability coefficient M is larger than 300, the efficiency of capturing rare cells in the cell separation material is reduced. Tend. Furthermore, from the viewpoint of improving the separation efficiency between rare cells and contaminating cells, the air permeability coefficient M is preferably 37 or more, more preferably 72 or more, and further preferably 80 or more. Further, the air permeability coefficient M of the cell separation material is preferably 250 or less, more preferably 200 or less, and even more preferably 150 or less.
The air permeability coefficient M is a value defined by the product of the air permeability (cc / cm ² / sec) and the thickness (mm) of the cell separation material. The air permeability is a parameter that depends on the size of the pore size of the cell separation material, but even if the air permeability is the same, the smaller 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 representing 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 the cell separation materials are stacked and filled in a container having an inlet and an outlet.

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 a sphere, a container, a cassette, a bag, a tube, or a column. Preferable specific examples include, for example, a translucent 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 column container having a length of about 0.1 cm to 20 cm and a thickness of about 0.1 cm to 5 cm can be used, but the present invention is not limited thereto.

The container type includes a cross flow type and a column type. Either the cross flow type or the 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 introduced uniformly.

The column type means, for example, a container having a liquid inlet and outlet near the center of the surface with respect to the filter surface, or a container in which the inlet and outlet portions are positioned perpendicular to the filter surface, or the filter surface. The container is characterized in that the liquid flows in a direction perpendicular to the above, or the container characterized in that the liquid flows in parallel to the compression direction of the separating material.

On the other hand, as represented by the leukocyte removal filter (“Sepacel” manufactured by Asahi Kasei Medical Co., Ltd., “Pure Cell RC” manufactured by Paul Co., Ltd.) This refers to a container in which an inlet and an outlet are 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, and glass. Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymers; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides and polyimides , Polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene and the like. As for the metal material (biocompatible metal, alloy) useful as the material of the container, stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt-chromium alloy, Examples include titanium nitride-coated stainless steel.
Among these structural materials, structural materials having sterilization resistance are preferable, and specific examples include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, and polymethylpentene.

Specific examples of the cell separation filter in which the container is filled with the cell separation material include the cell separation filter 1 having the structure shown in FIGS. In the cell separation filter 1, a cylindrical container body 2 provided with openings at the top and bottom, a presser member 3 with a nozzle disposed in the vicinity of the top and bottom openings in the inner space of the container body 2, with the nozzle It is comprised from the lid | cover 5 which can fix the cell separation material 4 filled between the pressing members 3 and the said pressing member 3 with a nozzle near the said opening part.
The nozzle port 6 of the pressing member with nozzle 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 presser member 3 with the nozzle contacts the container body 2 is not specified, but the liquid introduced into the cell separation filter 1 leaks out by disposing the sealing material 7. You may make it prevent doing.

When filling the container with the cell separation material, the cell separation material may be cut into an appropriate size as necessary, and used in a state where a plurality of layers are stacked in the direction in which a liquid containing rare cells flows. it can. In particular, as shown in FIG. 2, the thickness T of the whole 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 nozzle ports 6 that are liquid inlets and outlets of the cell separation filter 1. The total thickness T of the filled cell separating material 4 is preferably 25 mm or less, more preferably 20 mm or less, and even more preferably 15 mm or less, from the viewpoint of efficiently removing contaminating cells. Further, from the viewpoint of reducing the loss of rare cells, it is preferably 3 mm or more, and more preferably 5 mm or more.
In addition, if the said container is a transparent container, the thickness T of the said filled cell separation material 4 whole can be visually measured using a caliper from the outside, for example. Moreover, even if it is an opaque container, it can be calculated by measuring the length of the part filled with the cell separating material 4 in the structure of each part of the container.

In addition, as a method of filling the container with the cell separation material, the container may be packed in a roll shape as well as stacked on a flat plate. When used in the form of a roll, 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 filling the cell separating material into the container, it is preferable to compress the cell separating material in the flowing direction of the liquid and fill the container. However, depending on the material of the cell separating material, the container may be filled without being compressed. The degree of compression can be appropriately selected according to the material of the cell separation material.

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

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 the cell separation material before filling / total thickness of the cell separation material after filling) is 1 or more. The following is preferable. When the packing ratio is less than 1, rare cells may be lost from the voids generated in the filter. When the packing ratio is greater than 10, it is difficult to fill the filter with a cell separation material. It becomes. Furthermore, in order to increase the capture efficiency of rare cells and at the same time increase the removal efficiency of contaminating cells, the filling rate is more preferably 1.2 or more, and further preferably 1.5 or more. The filling rate is more preferably 9 or less, further preferably 8 or less, and particularly preferably 7.7 or less.
The thickness of the whole cell separation material before filling the container is calculated from the total thickness of one cell separation material in the uncompressed state, and the thickness of the whole cell separation material after filling is calculated using the calipers. Can be measured by the conventional method.

In the cell separation filter of the present invention, the packing density of the cell separation material filled in the container (weight of the whole cell separation material / volume of the whole 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 , the rare cells are likely to pass through the filter, so the recovery rate of the rare cells tends to be remarkably 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, making it difficult to recover. 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, and 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. More preferably, it is ³ or less.
In addition, what is necessary is just to calculate the volume of the said filled cell separation material by the product of the thickness of the said whole filled cell separation material, and the area of a cell separation material, for example.

Moreover, it is preferable that the average pore diameter of the whole cell separation material filled in the cell separation filter is 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 contaminated cells such as erythrocytes and leukocytes cannot pass through the filter, making it difficult to remove the contaminated cells. On the other hand, when it exceeds 70 μm, it is highly possible that not only contaminated cells but also rare cells pass through the filter, and the recovery rate of rare cells is significantly reduced. Furthermore, 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 diameter 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).

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 by passing a liquid containing rare cells.
Here, capturing the 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, it means that 60% or more, particularly preferably 70% or more, particularly preferably 80% or more, and most preferably 90% or more is captured by the cell separation material or the cell separation filter.

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 of red blood cells and platelets contained in the liquid in contact with the cell separation material and the cell separation filter, preferably 70. % Or more, more preferably 80% or more, more 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 It 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, and most preferably 97% or more can be removed.

Further, the cell separation filter of the present invention can freely control the performance of the filter by changing the type of 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 rare cell recovery rate, or can be designed as a filter having a particularly high contamination cell removal rate.

(Cell separation method)
The present invention relates to a cell separation method including a step of allowing a liquid containing rare cells to flow through the cell separation filter and capturing a fraction (rare cell rich fraction) rich in rare cells 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 a 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. It may be set so that it is lower than the outlet, and the liquid may flow in the direction opposite to gravity. By flowing the liquid in the direction opposite to the gravity, the liquid flows uniformly into the container and the bubbles are easily removed, so that the separation efficiency can be further improved. In addition, the direction in which the liquid flows may be perpendicular to gravity, and the direction in which the liquid flows 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 blood, bone marrow, umbilical cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph fluid, Examples include urine, saliva or tissue extract.
Alternatively, it may be a culture solution obtained by culturing rare cells in vitro or a suspension obtained by suspending in a liquid such as physiological saline.

In addition, after passing the liquid containing the rare cells, components other than the rare cells may be removed from the cell separation filter by passing a washing solution from the inlet side as necessary.
The washing solution that can be used is not particularly limited as long as it does not damage rare cells, but general buffer solutions such as physiological saline, Ringer's solution, medium used for cell culture, and phosphate buffer are preferable.

Next, by flowing the recovery solution from the outlet direction of the container, that is, from the direction opposite to the direction in which the liquid containing the rare cells or the washing solution is passed, the fraction containing the rare cells captured in the cell separation material is abundant. The image can be collected.

The recovered solution is not particularly limited as long as it is an isotonic solution, but has been used as an injectable agent such as physiological saline, Ringer's solution, dextran sugar injection, hydroxyethyl starch, buffer solution, cell culture medium Etc.

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

In addition, as a pretreatment for 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. Also good. This operation is not necessarily required, 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 an improvement in separation efficiency is expected. The This pretreatment solution need not be the same as the washing solution, but if it is the same, the solution bag connected to the cell separation filter can be shared during the treatment, which simplifies the circuit system and improves operability. It is preferable that it is the same from a viewpoint. The amount of the pretreatment liquid is practically and preferably about 1 to 100 times the filter capacity. The buffer that can be used is not particularly limited, but general buffers such as physiological saline, Ringer's solution, a medium used for cell culture, and phosphate buffer are preferable.

In addition to rare cells, when using a liquid containing contaminating cells such as red blood cells, platelets, white blood cells, for example, blood, umbilical cord blood or menstrual blood, the following steps (a) to (c) are carried out, Rare cells can be recovered with higher efficiency and contaminated 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 from the inlet of the cell separation filter after the step (a) (c) The (b ) After the step, a rare cell-recovering step in which a recovery solution is passed through the outlet of the cell separation filter and a rare cell-rich fraction is recovered from the inlet of the cell separation filter.

In the step (a), a liquid containing rare cells is passed through the cell separation filter, and a fraction rich in erythrocytes that have passed without being captured by the cell separation material is 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 naturally dropped from the container in which the liquid is placed 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 pushed by hand. When the liquid is passed by the pump, the separation efficiency tends to decrease if the liquid feeding speed is too fast, and the processing time tends to be longer if it is too slow. Examples of the liquid feeding speed include a speed of 0.1 mL / min to 100 mL / min, but are not limited thereto.

In the step (b), by passing the washing solution through the filter, a fraction rich in white blood cells and platelets can be passed through the filter and removed. The same cleaning solution as described above may be used.
When passing the cleaning solution, it may be sent by natural fall or by a pump. The flow rate when the liquid is sent by a pump is similar to that when a liquid containing rare cells is sent, and the speed is 0.1 to 100 mL / min, but is not limited thereto. The amount of the washing solution varies depending on the volume of the container used. However, if the amount of the washing solution is too small, the amount of white blood cells and platelets remaining on the filter will increase. If the amount of the washing solution is too large, rare cells will pass through the filter. It is preferable to perform cleaning with a liquid amount of about 0.5 to 100 times the filter capacity.

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

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

In the cell separation method of the present invention, a method with a particularly high rare cell recovery rate can be selected, or a method with a particularly high contamination cell removal rate can be selected. For example, the cell separation efficiency is controlled by controlling the flow rate in the step (a), the amount and flow rate of the washing solution in the step (b), and the amount and flow rate of the recovered liquid in the step (c). Can be controlled.

Further, as the cell separation method of the present invention, a cell separation system using the cell separation filter may be configured. In addition to the cell separation filter, the cell separation system contains the inlet and outlet of the liquid containing the rare cells, the washing solution and the recovery solution, the bag containing the liquid containing the rare cells, and the liquid containing the contaminated cells that have passed through the filter. It is practical and preferable to simultaneously provide 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 through which a liquid containing rare cells flows in and an outlet through which it flows, and further has an inflow section and an outflow section for a cleaning solution used to remove contaminating cells such as red blood cells. Moreover, it is preferable that an inlet for introducing the recovered solution is also provided independently of the outflow part. The inlet of the cleaning solution and recovery solution connected to the filter may function as an outlet for liquid after washing with contaminated cells or after collecting rare cells, and each bag or syringe is connected via a three-way stopcock. May be.

For example, a cell separation system 13 as shown in FIG. 3 can be used. In the cell separation system 13, since the cell separation filter 1 is set so that the liquid inlet 8 is vertically downward and the liquid outlet 9 is vertically upward, the liquid introduced from the inlet 8 is set. Is discharged from the outlet 9 on the upper side in the vertical direction in the cell filter 1.
The inlet 8 of the cell separation filter 1 is provided with a bag 11 containing a liquid containing rare cells and a bag 12 containing a washing solution (right side in the figure) via a three-way cock 10a, and a cell. A bag (collection bag) 20 for storing 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 cock 10a, a means 21 for collecting the liquid collected in the collection bag 20 and the collection bag is connected by a tube via the three-way cock 10c.
The outlet 9 of the cell separation filter 1 is connected via a three-way cock 10d to a bag (waste solution bag) 23 for storing the liquid that has passed through the cell separation filter 1 and a bag 24 for storing the recovered 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 the liquid contained in the bag 11 or the bag 12 is either Can be introduced into the cell separation filter 1 by using gravity, and all of the introduced liquid is discharged from the outlet 9 and stored in the waste liquid bag 23. In addition, switching of the liquid flow from the bag 11 or the bag 12 can be performed by the three-way cock 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 recovered solution is introduced from the bag 24 into the cell separation filter 1 through the outlet 9, and the rare cells are introduced into the cell separation filter 1. It is separated from the cell separation material and collected in the collection bag 20 as a suspension.
Further, when the amount of the suspension is large, for example, the collection bag 20 is removed from the cell separation system 13 and centrifuged to settle rare cells, and then the cell separation system 13 is returned to the cell separation system 13 again. The supernatant liquid in the collection bag 20 may be attached and 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 solution described above, so that cells can be separated in a sterile and closed system. Each bag is preferably used after being used, and may be shaped like a commonly used blood bag, but may be a flat cartridge type. As the form of each bag, a bag capable of cell culture, a bag having cryopreservation resistance, and the like may be selected as necessary.

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

Further, the rare cells may be cryopreserved at a cryogenic temperature such as liquid nitrogen or a deep freezer while being put in a cryopreservation tube or the like.
When the rare cells are cryopreserved, a cryoprotectant is added to the collected solution for the purpose of protecting the rare cells from damage caused by freezing. The type of cryoprotectant added is not particularly limited, and dimethyl sulfoxide, dextran, albumin, hydroxyethyl starch, and the like can be used. The cryoprotectant may be used alone or in combination of several kinds.

In the cryopreservation method, the temperature at the time of 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 the activity of rare cells. The temperature is preferably -196 ° C to -80 ° C.

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

Examples of rare cells collected 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 in Examples, but the present invention is not limited only to the following Examples.

(Examples 1 to 27, Comparative Examples 1 to 4)
As shown in FIGS. 1 and 2, a cylindrical container body (2) having an inner diameter of 26 mm is filled with a nonwoven fabric (4) cut into a round shape with a diameter of 26 mm, and the upper and lower openings of the container body (2) are filled. The holding member with nozzle (3) was inserted and screwed with a cap (5) from above to produce a cell separation filter (1).
In addition, about the nonwoven fabric used in each Example and the comparative example, the kind of resin, the manufacturing method of a nonwoven fabric, an average fiber diameter, compression energy WC, air permeability, thickness, density, and air permeability coefficient M are shown in Table 1. Table 1 shows the thickness, packing density, packing rate, and average pore diameter of the whole cell separating material packed in the cell separation filter.

Next, separation examination of rare cells and cells other than rare cells (red blood cells, platelets and white blood cells) (hereinafter referred to as contaminated cells) was performed using the cell separation filter. As the 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 clearly distinguish the recovery rate of rare cells and the removal rate of contaminated cells, the recovery system for rare cells should be an experimental system that does not include contaminated cells, and the removal rate of contaminated cells must include rare cells. The experimental system was not possible.

In the examination of the rare cell recovery rate, the rare cells were collected from the liquid containing the rare cells by the following procedure.
(1) A liquid in which a rare cell was suspended was passed through the inlet of the cell separation filter. Table 2 shows the type and number of rare cells that passed through, the type and volume of the solution in which the cells were suspended, and the flow rate of the cell suspension.
(2) After the 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 flow rate of the cleaning solution.
(3) After the 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 with an automatic cell counting device (GE Healthcare, CYTORECON).

In the examination of the removal rate of the contaminated cells, the contaminated cells were removed from the liquid containing the contaminated cells by the following procedure.
(1) Human peripheral blood was passed through the inlet of the cell separation filter. Table 2 shows the volume and speed of human peripheral blood that has passed through.
(2) After the 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 flow rate of the cleaning solution.
(3) After the 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 contaminated cell fraction. Table 2 shows the type and amount of the recovered solution.
(4) The number of blood cells in the peripheral blood before passing through the filter and the number of blood cells in the contaminated cell fraction obtained in the above step are measured with a blood cell counter (Sysmex, K-4500). did. Here, the blood cell represents a red blood cell, a white blood cell, and a platelet.

Table 3 shows the collection rate of rare cells and the removal rate of contaminating cells. The rare cell recovery rate 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 (contaminated cell recovery rate).

From the results shown in Table 3, the cell separation filters of Examples 1 to 27 have a high recovery rate of 35% or more compared to Comparative Examples 1 to 4, regardless of the type of rare cells. Yes. From the results of Examples 1 to 17, it can be seen that leukocyte removal rate is 51% or more, red blood cell removal rate is 98% or more, and platelet removal rate is 79% or more, so that contaminated cells can also be removed.

Among the Examples, the cell separation filters according to Examples 1 to 4, 6 to 23, 26, and 27 in which the packing density of the cell separation material packed 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 has a higher rare cell recovery rate of 51% or more.

Furthermore, in the results of the recovery rate of prostate cancer cell line PC3, which is a kind of rare cell, Examples 1 to 4, 6, 9, and 11 have a significantly high recovery rate of rare cells of 70% or more, and leukocytes. Removal rate of 90% or more, red blood cell removal rate of 99% or more, and platelet removal rate of 95% or more, which is a cell separation filter with excellent rare cell concentration efficiency.

Moreover, in the cell separation filters of Examples 26 and 27, the recovery rate is as high as 68% or more regardless of the type of cancer cell, and thus it is used for examination diagnosis of cancer patients of various cancer types. It is thought that it is a cell separation filter that can.

On the other hand, Example 1, 2, Example 3, 4, Example 5, 6, Example 7, 8, Example 9, 10, Example 16, 17, Example 20, 21 using the same nonwoven fabric. Comparing these results, it can be seen that by adjusting the packing density and packing rate of the cell separation filter to be high and reducing the average pore size of the cell separation filter, the recovery rate of rare cells is improved.
Moreover, from the results of Examples 23, 24, and 25, using 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 an air permeability coefficient M of 300 or less. In Example 23 in which the packing density is adjusted to 1.0 × 10 ⁵ g / m ³ or more, it can be seen that the recovery rate of rare cells is significantly improved as compared with 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 is high, the collection of rare cells The rate is remarkably low at 15.1% or less.

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.

DESCRIPTION OF SYMBOLS 1 Cell separation filter 2 Container body 3 Holding member with nozzle 4 Cell separation material 5 Lid 6 Nozzle port 7 Sealing material 8 Inlet 9 Outlet 10 Three-way stopcock 11 Bag containing liquid containing rare cells 12 Containing washing solution 13 Cell separation system 20 Bag for collecting liquid recovered from the cell separation filter 1 (Recovery bag)
21 Means for collecting liquid collected in a collection bag (syringe, etc.)
23 Bag for storing liquid that has passed through the cell separation filter 1 (waste liquid bag)
24 Bag containing the recovery solution T Thickness of the whole cell separation material 4 filled in the cell separation filter 1 D Flow direction of the liquid introduced into the cell separation filter 1

Claims

A cell having an average fiber diameter of 1.0 μm or more and 50 μm or less, an air permeability of 10 cc / cm 2 / sec or more, 400 cc / cm 2 / sec or less, and a compression energy WC of 3.5 J / m 2 or less Separation material.
The cell separation material 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 the product of the air permeability (cc / cm 2 / sec) of the cell separator and the thickness (mm) of the cell separator, is 36 or more and 300 or less. Cell separation material.
The cell separation material according to any one of claims 1 to 3, wherein a thickness of the cell separation material is 0.1 mm or more and 3.5 mm or less.
The cell separation material 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 material according to any one of claims 1 to 5, wherein the cell separation material is a nonwoven fabric.
The cell separation material according to claim 6, wherein the nonwoven fabric is a spunlace nonwoven fabric, a spunbond nonwoven fabric or a meltblown nonwoven fabric.
The fiber constituting the cell separation material comprises at least one synthetic polymer selected from the group consisting of polyester, rayon, polyolefin, vinylon, polystyrene, acrylic, nylon, and polyurethane. The cell separation material described in 1.
The cell separation material according to claim 8, wherein the fibers constituting the cell separation material comprise a combination of polyester and polyolefin; rayon and polyolefin; polyester and rayon; or a synthetic polymer of polyester, rayon and vinylon.
A cell separation filter, wherein a plurality of the cell separation materials according to any one of claims 1 to 9 are stacked and filled in a container having an inlet and an outlet.
The cell separation filter according to claim 10, wherein the cell separation material is filled in a compressed state.
The cell separation filter according to claim 10 or 11, wherein a filling rate of the cell separation material (thickness of the whole cell separation material before filling / total thickness of the cell separation material after filling) is 1 or more and 10 or less.
The packing density of the cell separation material (weight of the whole cell separation material / volume of the whole cell separation material) is 1.0 × 10 5 g / m 3 or more, 1.0 × 10 6 g The cell separation filter according to any one of claims 10 to 12, which is / m 3 or less.
The cell separation filter cage according to any one of claims 10 to 13, wherein an average pore diameter of the whole cell separation material filled is 1 µm or more and 70 µm or less.
The cell separation filter according to any one of claims 10 to 14, wherein a thickness of the whole cell separation material filled in a direction in which the liquid flows is 1 mm or more and 30 mm or less.
The cell separation filter according to any one of claims 10 to 15, which captures rare cells and removes red blood cells, white blood cells and platelets by passing a liquid containing rare cells.
A cell separation method comprising a step of passing a liquid containing rare cells through the cell separation filter according to any one of claims 10 to 16, and capturing a fraction rich in rare cells in the cell separation material. .
The cell separation method according to claim 17, wherein the liquid is blood, bone marrow, umbilical cord blood, menstrual blood, peritoneal fluid, pleural fluid, lymph fluid, urine, saliva, or tissue extract.
The cell separation method according to claim 17 or 18, wherein the liquid is blood, umbilical cord blood or menstrual blood, and comprises 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 from the inlet of the cell separation filter after the step (a) (c) The (b ) After the step, a rare cell-recovering step in which a recovery solution is passed through the outlet of the cell separation filter and a rare cell-rich fraction is recovered from the inlet of the cell separation filter.
The cell fraction passed from the outlet of the cell separation filter in the steps (a) and (b) (passed cell fraction) is passed through the inlet of the cell separation filter a plurality of times, and (c) The cell separation method according to claim 19, further comprising a step of re-collecting a fraction containing rare cells mixed in the passing cell fraction by performing the step.
21. The cell separation method according to claim 19 or 20, wherein the rare cells collected in the step (c) can be cultured.
The cell separation material according to any one of claims 1 to 9, the cell separation filter according to any one of claims 10 to 16, or the cell according to any one of claims 17 to 21. A rare cell-rich fraction collected by a separation method, wherein 60% or more of red blood cells and platelets have been removed, and 50% or more of white blood cells have been removed, and rare cells can be cultured. .