JP2020195330A - Cell separation filter unit and production method of cell concentrated solution - Google Patents

Abstract

To provide a cell separation filter unit capable of achieving a preferable cell collection ratio and storing a separation material therein, and a production method of a cell concentrated solution including a step using the cell separation filter unit.SOLUTION: A cell separation filter unit comprises a peripheral wall part, a first wall part, a second wall part, and a separation material, a first pressing part is provided on the first wall part and a second pressing part is provided on a second wall part. By the first and second pressing parts, an annular region which is provided along an inner face of the peripheral wall part of the separation material filled in the cell separation filter unit is held and compressed, and when a shortest distance between an inner face of the peripheral wall part and an outer periphery of the annular region is L1, and an equivalent circle diameter of a cross section of an internal space of the peripheral wall part calculated from a cross sectional area of an inner space of the peripheral wall part in a radial direction is L2, L1 is 1.00% or greater and 7.75% or smaller of L2.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell separation filter device and a method for producing a cell concentrate, which comprises a step of using the cell separation filter device.

In recent years, with the rapid progress of hematology and scientific technology, the therapeutic effect is achieved by separating only the necessary blood fraction from body fluids such as whole blood, bone marrow, umbilical cord blood, and tissue extracts and administering it to patients. A treatment style that enhances and suppresses side effects by not administering fractions that are not necessary for treatment is widespread.

For example, blood transfusion is one of them. A red blood cell preparation is a blood product used when bleeding or red blood cell deficiency, or when oxygen is deficient due to a decrease in red blood cell function. Erythrocyte preparations do not require leukocytes that induce side effects such as an abnormal immune response or graft-versus-host disease (GVHD), and need to be filtered to remove leukocytes. In some cases, platelets may be removed in addition to white blood cells.

On the other hand, platelet products are blood products used for patients who have bleeding or bleeding tendency due to deficiency of blood coagulation factors. For the production of platelet preparations, unnecessary cells and components other than platelets are removed by centrifugation, and only the required platelet components are collected.

In addition, in recent years, hematopoietic stem cell transplantation for the treatment of leukemia and solid cancer has become popular, and a method of separating and administering a leukocyte group containing hematopoietic stem cells, which is necessary for treatment, has been adopted. As a source of these hematopoietic stem cells, umbilical cord blood is attracting attention in addition to bone marrow and peripheral blood because of its advantages such as less burden on donors and excellent proliferative ability. In recent years, it has been suggested that stem cells are also abundant in menstrual blood, and menstrual blood that has been discarded so far may be used as a valuable stem cell source.

For bone marrow and peripheral blood, it is desired to separate and purify leukocytes except for unnecessary cells and administer them, while for cord blood, banking for relatives has become popular, and cryopreservation is performed until use. Due to necessity, leukocytes are separated and purified for the purpose of preventing erythrocyte hemolysis due to cryopreservation.

Recently, as a cell separation method, a method of collecting leukocytes using a filter material that captures only leukocytes without capturing erythrocytes and platelets has been reported (see Patent Document 1, Patent Document 2, and Patent Document 3). ).

Special Table 2001-518792 International Publication No. 98/32840 Japanese Unexamined Patent Publication No. 10-313855

In a method of recovering cells from a cell-containing solution such as blood using a filter material as described in Patent Document 1, Patent Document 2, and Patent Document 3 as a separating material, a large amount of blood (for example, more than 100 mL) is once collected. The effect of clogging of the separating material by blood cell cells, proteins, impurities, etc. in the blood is large. When the separating material is clogged, the pressure inside the cell separation filter device rises sharply when the cells are backwashed and collected with the recovery solution. At that time, the filter container is greatly deformed, so that the sealing property between the filter container and the separating material is lowered. Then, there is a problem that a gap is formed between the filter container and the separating material, and the recovered liquid is drifted or short-passed, so that the cell recovery rate is lowered.
As described above, there is still room for improvement in the cell recovery rate, and there is a demand for a cell separation filter device capable of achieving a better cell recovery rate.

In view of the above circumstances, an object of the present invention is to use a cell separation filter device that contains a separating material inside and can achieve a good cell recovery rate, and the cell separation filter device. It is an object of the present invention to provide a method for producing a cell concentrate containing a step.

The present inventor has made diligent studies to solve this problem. As a result, in the cell separation filter device including the peripheral wall portion, the first wall portion, the second wall portion, and the separating material, the first pressing portion and the second pressing portion are provided on the first wall portion and the second wall portion, respectively. A portion is provided, and the first retainer portion and the second retainer portion sandwich and compress an annular region of the separating material filled in the cell separation filter device, which is located along the inner surface of the peripheral wall portion, to compress the peripheral wall portion. L1 is the shortest distance between the inner surface of the wall and the outer circumference of the annular region, and L2 is the equivalent circle diameter of the cross section of the internal space of the peripheral wall, which is calculated from the radial cross-sectional area of the internal space of the peripheral wall. It has been found that the above-mentioned problems can be solved by setting L2 to 1.00% or more and 7.75% or less, and the present invention has been completed.

That is, the gist of the present invention is as follows.

[1] A cell separation filter device including a filter container and a separating material filled in the filter container.
The filter container includes a tubular peripheral wall portion, a first wall portion that closes an opening on one end side of the peripheral wall portion, and a second wall portion that closes an opening on the other end side of the peripheral wall portion.
The separating material is filled in the filter container so that a gap is not formed between the separating material and the inner surface of the peripheral wall portion.
A first liquid passage port is formed on the first wall portion, and a first liquid passage port is formed.
A second liquid passage port is formed on the second wall portion, and a second liquid passage port is formed.
A first protrusion and a first holding portion are formed on the inner surface of the first wall portion.
A second retainer is formed on the inner surface of the second wall.
When the filter container is filled with the separating material, the first protrusion cooperates with the second wall portion to sandwich and compress the separating material.
The first pressing portion and the second pressing portion sandwich and compress an annular region of the separating material filled in the filter container, which is located along the inner surface of the peripheral wall portion.
When the shortest distance between the inner surface of the peripheral wall and the outer circumference of the annular region is L1, and the equivalent circle diameter of the cross section of the internal space of the peripheral wall calculated from the radial cross-sectional area of the internal space of the peripheral wall is L2. , L1 is 1.00% or more and 7.75% or less of L2, a cell separation filter device.

[2] The cell separation filter device according to [1], wherein L1 is 1.00% or more and 3.50% or less of L2.

[3] The width W1 of the surface that presses the separating material of the first pressing portion and the width W2 of the surface that presses the separating material of the second pressing portion are 0.75% or more and 6.00% or less of L2, respectively. The cell separation filter apparatus according to [1] or [2].

[4] The width W1 of the surface that presses the separating material of the first pressing portion and the width W2 of the surface that presses the separating material of the second pressing portion are 3.00% or more and 6.00% or less of L2, respectively. The cell separation filter apparatus according to [1] or [2].

[5] The method according to any one of [1] to [4], wherein the width of the surface pressing the separating material of the first pressing portion is narrower than the width of the surface pressing the separating material of the second pressing portion. Cell separation filter device.

[6] The area of the cross section of the internal space of the peripheral wall portion along the main surface of the first wall portion and the main surface of the second wall portion is 16.05 cm ² or more.
The first protrusion is composed of a plurality of first protrusions.
The distance between the first protrusion and the center of the first wall is 10.00 mm or less.
The cell separation filter device according to any one of claims [1] to [5], wherein the maximum distance between adjacent first protrusions among the plurality of first protrusions is 36.74 mm or less.

[7] In any one of [1] to [6], a second protrusion is formed on the inner surface of the second wall in cooperation with the first protrusion to sandwich and compress the separating material. The cell separation filter device according to description.

[8] Regarding the internal space of the peripheral wall portion, the area of the cross section along the main surface of the first wall portion and the main surface of the second wall portion is 16.05 cm ² or more.
The second protrusion is composed of a plurality of second protrusions.
The distance between the second protrusion and the center of the second wall is 10.00 mm or less.
The cell separation filter device according to [7], wherein the maximum distance of the adjacent second protrusions among the plurality of second protrusions is 36.74 mm or less.

[9] The first protrusion is composed of a plurality of first protrusions.
The second protrusion is composed of a plurality of second protrusions.
Each of the plurality of first protrusions extends radially with respect to the central portion of the first wall portion.
The plurality of first protrusions are arranged along the first holding portion, and the plurality of first protrusions are arranged along the first holding portion.
Each of the plurality of second protrusions extends radially with respect to the central portion of the second wall portion.
The cell separation filter device according to [7] or [8], wherein the plurality of second protrusions are arranged along the second retainer.

[10] The cell separation filter device according to [9], wherein the number of the first protrusions and the number of the second protrusions are independently 3 or more and 10 or less.

[11] The cell separation filter device according to any one of [1] to [10], wherein the peripheral wall portion and the first wall portion are integrally formed.

[12] The cell separation filter device according to any one of [1] to [11], wherein the separating material is a non-woven fabric or a porous body containing porous cellulose particles.

[13] A cell-containing liquid containing cells is passed through one of a first liquid passage port and a second liquid passage port in the cell separation filter device according to any one of [1] to [12]. By introducing from the liquid port and passing through the separating material, cells can be captured by the separating material, and
The recovery solution is introduced into the cell separation filter device, and the cells trapped in the separation material are released into the recovery solution to generate a cell concentrate.
A method for producing a cell concentrate, which comprises collecting the cell concentrate from the cell separation filter device.

[14] The cell concentrate according to [13], wherein the recovered liquid is introduced from the other liquid passage of the first liquid passage and the second liquid passage, and the cell concentrate is collected from one of the liquid passages. Production method.

According to the present invention, a cell separation filter device that contains a separating material inside and can achieve a good cell recovery rate, and a cell concentrate containing a step of using the cell separation filter device are produced. Methods and can be provided.

It is sectional drawing of the cell separation filter apparatus which shows typically the state which the annular region of the separating material was compressed by the 1st presser part and the 2nd presser part in the vicinity of the peripheral wall part. It is a perspective view which shows the cell separation filter apparatus which concerns on embodiment of this invention. It is an exploded perspective view of the cell separation filter apparatus of FIG. FIG. 3 is a sectional view taken along line III-III of FIG. It is an exploded sectional view of FIG. 4A. FIG. 3 is a plan view of the separating material accommodating portion (particularly, the bottom portion (first wall portion)) of FIG. 3 as viewed from the inner surface side. FIG. 3 is a plan view of the lid portion (second wall portion) of FIG. 3 as viewed from the inner surface side. It is a figure which shows the outline of the circuit of the cell separation device used in an Example and a comparative example.

Hereinafter, embodiments of the present invention will be described with reference to the drawings as necessary.

≪Cell separation filter device≫
The cell separation filter device 1 includes a filter container 2 and a separating material 5 filled in the filter container 2. Hereinafter, the "cell separation filter device" will also be referred to as a "filter device". Hereinafter, the "filter container" is also referred to as a "container".
In the filter device 1, the cells to be separated are captured by the separating material 5 by passing a cell-containing solution containing the cells to be separated into the filter device 1. The cells captured in the separating material 5 are released from the separating material 5 and collected by introducing the cell recovery solution into the filter device 1.
In the filter device 1, the filter container 2 is provided with a configuration described later, so that the target cells can be recovered from the cell-containing liquid with a high recovery rate.

<Filter container>
The filter container 2 includes a tubular peripheral wall portion 30, a first wall portion 10, and a second wall portion 20. The first wall portion 10 closes the opening on one end side of the tubular peripheral wall portion 30. The second wall portion 20 closes the opening on the other end side of the tubular peripheral wall portion 30 with respect to the opening closed by the first wall portion 10.

The first wall portion 10 and the second wall portion 20 may be integrally formed with the peripheral wall portion 30, or may be separate from the peripheral wall portion 30. However, in order to fill the filter container 2 with the separating material 5, at least one of the first wall portion 10 and the second wall portion 20 is usually attached to the peripheral wall portion 30 so as to be detachable from the peripheral wall portion. It is provided as a separate body.

In the container 2, a separating material 5 for separating cells, which will be described later, is filled in the container 2 so that a gap is not formed between the container 2 and the inner surface of the peripheral wall portion 30. This is to prevent a decrease in the cell recovery rate due to the cell-containing liquid not passing through the separating material 5 and the cell-containing liquid short-passing through the gap between the inner surface of the peripheral wall portion 30 and the separating material 5.

The first liquid passage port 11 is formed in the first wall portion 10. A second liquid passage port 21 is formed in the second wall portion 20. The number of the first liquid passage port 11 and the second liquid passage port 21 is not particularly limited. One first liquid passage port 11 may be formed on the first wall portion 10, or two or more first liquid passage ports 11 may be formed. Since the container 2 can be easily manufactured and the operation of connecting the tube for supplying the cell-containing liquid to the filter device 1 (not shown in FIG. 1) is easy, the first liquid passage port 11 is provided. , It is preferable that one is formed on the first wall portion 10. The number of the second liquid passage ports 21 in the second wall portion 20 is the same as the number of the first liquid passage ports 11 in the first wall portion 10.

Since it is easy to manufacture the container 2 and it is easy to evenly supply the cell-containing liquid to the separating material 5, the first liquid passage port 11 and the second liquid passage port 21 are the first, respectively. It is preferable that one wall portion 10 and the second wall portion 20 are formed so as to have an opening at the center or substantially the center of the main surface.
Here, the main surface of the first wall portion 10 and the main surface of the second wall portion 20 are the main surface of the first wall portion 10 and the second wall portion 20 when the space in the container 2 is filled with the separating material 5. Represents a surface facing or in contact with the separating material 5.
Further, the center of the main surface is the center of the circle when the shape of the main surface is a circle. When the shape of the main surface is a convex polygon, the center of the circle inscribed in the convex polygon is set as the center of the main surface.

The shapes of the first liquid passage port 11 and the second liquid passage port 21 are particularly limited as long as the liquid can be introduced into the filter device 1 and the liquid in the filter device 1 can be discharged to the outside of the filter device 1. Not done. Since it is easy to connect a tube for circulating the liquid inside and outside the filter device 1, it is preferable that the first liquid passage port 11 and the second liquid passage port 21 are each composed of nozzles. There is no particular limitation on the shape and size of the nozzle.

A first protrusion 12 and a first pressing portion 13 are formed on the inner surface of the first wall portion 10. A second pressing portion 23 is formed on the inner surface of the second wall portion 20.
The first protrusion 12 cooperates with the second wall 20 to sandwich and compress the separating material 5. As a result, the separating material 5 is compressed as a whole.
Further, in the separating material 5, the separating material 5 is densely compressed in the region in contact with the first protrusion 12 and the region in the vicinity thereof. On the other hand, in the region where the first protrusion 12 does not come into contact with the first protrusion 12, the separating material 5 loosens.

In this way, the entire separating material 5, that is, the inner layer is compressed, and a dense portion and a loose portion are formed in the separating material 5, so that cells can be easily captured in the loose portion of the separating material 5. Therefore, when the recovery liquid is introduced into the filter device 1 after the cells are captured, the cells can be easily released from the separating material 5 into the recovery liquid.

The first projection portion 12 is preferably supported by the first pressing portion 13 formed in an annular shape along the inner surface of the peripheral wall portion 30.
The number of the first protrusions 12 is not particularly limited as long as the separating material 5 can be compressed to a desired degree. When a plurality of first protrusions 12 are formed, the first protrusions 12 may be arranged at equal intervals or may be arranged irregularly. The first protrusions 12 are preferably arranged at equal intervals from the viewpoint that the liquid containing cells can be easily permeated into the separating material 5.

The shape of the first protrusion 12 may be a straight line or a curved line. For example, the shape of the first protrusion 12 may be an arc shape, an S shape, or a zigzag shape. Further, the shape of the first protrusion may be not only radial but also lattice-shaped or dot-shaped having a gap for permeating the cell-containing liquid into the separating material 5.

It is preferable that the second protrusion 22 is also formed on the inner surface of the second wall 20 as well as the first wall 10. The second protrusion 22 cooperates with the first protrusion 12 to sandwich and compress the separating material 5. In this case, the above-mentioned roughness is also formed on the surface of the separating material 5 that comes into contact with the second protrusion 22.
The cells captured in the separating material 5 are recovered by being released from the separating material 5 into the cell collecting liquid by supplying the cell collecting liquid into the container 2.
When the surface of the separating material 5 in contact with the second protrusion 22 is also made coarse and dense, the cell recovery liquid easily flows from the rough portion of the separating material 5 toward the central portion of the separating material 5, and the separating material 5 is easily separated. It is easy to release the cells captured in 5.

The number and shape of the second protrusions 22 are the same as those of the first protrusions 12.

The first pressing portion 13 and the second pressing portion 23 sandwich and compress an annular region of the separating material 5 filled in the container, which is located along the inner surface of the peripheral wall portion 30.
In the annular region 50, the shortest distance between the inner surface of the peripheral wall portion 30 and the outer circumference of the annular region 50 is L1, and the cross section of the internal space of the peripheral wall portion 30 is calculated from the radial cross-sectional area of the internal space of the peripheral wall portion 30. When the equivalent circle diameter is L2, L1 is compressed so as to be 1.00% or more and 7.75% or less of L2. L1 is preferably 1.00% or more and 3.50% or less of L2 from the viewpoint of easily increasing the recovery rate of desired cells.
As the specific length of L1, for example, 0.50 mm or more and 6.00 mm or less is preferable, 0.80 mm or more and 5.00 mm or less is more preferable, and 1.00 mm or more and 4.00 mm or less is further preferable.
It is particularly preferable that the ratio of L1 to L2 is within the above range and the specific length of L1 is within the above range.
As the specific length of L2, for example, it is preferably 40.00 mm or more and 80.00 mm or less, and more preferably 45.00 mm or more and 70.00 mm or less.

Since the separating material 5 has pores, the size of the separating material 5 changes to some extent by compression.
Here, if L1 is too small with respect to L2, the end portion of the separating material 5 is compressed, and the end portion of the separating material 5 is pulled toward the center side of the main surface of the separating material 5. Then, a gap is formed between the peripheral wall portion 30 and the end portion of the separating material 5, and the sealing property of the separating material 5 is lowered. In this case, the cell-containing solution or the recovery solution short-passes between the peripheral wall portion 30 and the separating material 5, so that the desired cell recovery rate tends to decrease.
On the other hand, when the annular region 50 near the outer peripheral portion of the separating material 5 is compressed by being sandwiched between the first pressing portion 13 and the second pressing portion 23 so that L1 and L2 satisfy the above requirements, FIG. As shown, when observing a cross section perpendicular to the surface direction of the separating material 5, the end portion of the separating material 5 on the peripheral wall portion 30 side of the annular region 50 spreads in a fan shape.
For this reason, when the annular region 50 of the separating material 5 is compressed, the separating material 5 is pressed against the peripheral wall portion 30 in the vicinity of the central portion in the thickness direction of the separating material 5. 5 is deformed.

In this way, when the cells in the cell-containing liquid are captured by the separating material 5 using the filter device 1, the annular region 50 is compressed with the separating material 5 appropriately protruding to the outside of the annular region 50. Then, when the separating material 5 is pressed against the peripheral wall portion 30, the cell-containing liquid and the recovered liquid flowing from the central side of the main surface of the separating material 5 toward the vicinity of the peripheral wall portion 30 are transferred to the peripheral wall portion 30 and the separating material 5. It is possible to suppress a decrease in cell recovery rate due to short pass or knitting by passing through a very narrow gap between the cells.
As a result, by using the filter device 1 having the above-mentioned predetermined configuration, the target cells can be recovered from the cell-containing liquid with a high recovery rate.

Here, if the ratio of L1 to L2 is too small, L1 is too short, so that the amount of deformation of the separating material 5 is small, and the cells are contained by passing through a very narrow gap between the peripheral wall portion 30 and the separating material 5. It is difficult to suppress the short path of the liquid.
On the other hand, if the ratio of L1 to L2 is too large, the area of the main surface of the separating material 5 inside the first pressing portion 13 and the second pressing portion 23 decreases, so that the utilization efficiency of the separating material 5 decreases. ..

The width W1 of the surface that presses the separating material 5 of the first pressing portion 13 and the width W2 of the surface that presses the separating material 5 of the second pressing portion 23 are 0.75% or more and 6.00 of the above-mentioned L2, respectively. % Or less, more preferably 3.00% or more and 6.00% or less.
The width W1 of the surface that presses the separating material 5 of the first pressing portion 13 and the width W2 of the surface that presses the separating material 5 of the second pressing portion 23 are 0.50 mm or more and 3.50 mm or less, respectively. Is preferable, and more preferably 2.00 mm or more and 3.50 mm or less.
It is particularly preferable that the ratio of W1 and W2 to L2 is within the above range, and the width values of W1 and W2 are within the above range.
The wider the width is, the easier it is to suppress the short path of the cell-containing liquid to the vicinity of the peripheral wall portion 30, but if the width is too wide, the utilization efficiency of the separating material 5 is lowered. In this respect, when the width is within the above range, it is easy to obtain a sufficient effect on suppressing the short path of the cell-containing solution without excessively lowering the utilization efficiency of the separating material 5.

The width of the surface of the first pressing portion 13 that presses the separating material 5 is preferably narrower than the width of the surface of the second pressing portion 23 that presses the separating material 5.
Here, in the container 2, stress is concentrated in the groove between the inner surface of the peripheral wall portion 30 and the first holding portion 13 due to the pressure of the cell recovery liquid introduced from the second liquid passage port 21 when the cell recovery liquid is passed. As a result, the vicinity of the groove of the container 2 may be deformed.
However, if the width of the surface of the first pressing portion 13 that presses the separating material 5 is narrower than the width of the surface that presses the separating material 5 of the second pressing portion 23, such concentration of stress can be easily relaxed.

Each member constituting the container 2 can be manufactured by using an arbitrary structural material. Specific examples of the structural material include non-reactive polymers, biocompatible metals, alloys, and glass. Non-reactive polymers include acrylic nitrile polymers such as acrylonitrile butadiene styrene terpolymer (ABS); polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride. ; Polyethylene, polyimide, polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene and the like can be mentioned. Metallic materials (biocompatible metals, alloys) useful as materials for the container 2 include stainless steel, titanium, platinum, tantalum, gold, and their alloys, as well as gold-plated alloy iron, platinum-plated alloy iron, and cobalt chromium alloy. Examples include stainless steel coated with titanium nitride. Particularly preferably, it is a material having sterilization resistance. Specific examples thereof include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene and the like.

<Separation material>
The form of the separating material 5 is not particularly limited, and examples thereof include a porous body having a communicating hole structure, an aggregate of fibers, and a woven fabric. It is preferably a porous body containing porous cellulose particles. Further, it is preferably a woven fabric or a non-woven fabric composed of fibers, and more preferably a non-woven fabric.

As the material of the separating material 5, for example, polyolefin (for example, polypropylene, polyethylene, high density polyethylene, low density polyethylene, etc.), polyester, vinyl chloride, polyvinyl alcohol, vinylidene chloride, rayon, vinylon, polystyrene, acrylic resin (for example, For example, polymethylmethacrylate, polyhydroxyethylmethacrylate, polyacronitrile, polyacrylic acid, polyacrylate, etc.), nylon (eg, aliphatic polyamide, aromatic polyamide (aramid)), polyurethane, polyimide, cupra, Kevlar, carbon, Examples thereof include phenol resin, tetron, pulp, hemp, cellulose, kenaf, chitin, chitosan, glass, cotton and the like. Among them, polymers such as polyester, polypropylene, acrylic, rayon, nylon, polybutylene terephthalate, and polyethylene terephthalate can be preferably used. The separating material 5 may be made of a single material among these materials, or may be made of a composite material in which a plurality of materials are combined.

The average fiber diameter of the separating material 5 may be appropriately selected according to the type of target cells and is not particularly limited.

In order to further improve the performance of the separating material 5, the separating material 5 may be hydrophilized.
The hydrophilization treatment has the effects of suppressing non-specific capture in cells other than the target required cells, improving the ability to allow the cell-containing liquid to pass through the separating material 5 evenly, and improving the recovery efficiency of the required cells. Can be done.

As a hydrophilic treatment method,
A method for adsorbing a water-soluble polyhydric alcohol, a polymer having a hydroxyl group, a cation group, an anionic group, or a copolymer thereof (for example, hydroxyethyl methacrylate, dimethylaminoethyl methacrylate, or a copolymer thereof);
Method of adsorbing water-soluble polymers (polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, etc.);
A method of fixing a hydrophilic polymer to a hydrophobic membrane (for example, a method of chemically bonding a hydrophilic monomer to the surface);
Method of irradiating electron beam;
A method of cross-linking and insolubilizing a hydrophilic polymer by irradiating a cell separation filter with water in a water-containing state;
A method of insolubilizing and fixing a hydrophilic polymer by heat treatment in a dry state;
How to sulfonate the surface of a hydrophobic membrane;
A method of forming a film from a mixture of a hydrophilic polymer and a hydrophobic polymer dope;
A method of imparting a hydrophilic group to the film surface by treatment with an aqueous solution of alkali (NaOH, KOH, etc.);
A method in which a hydrophobic porous membrane is immersed in alcohol, treated with a water-soluble polymer aqueous solution, dried, and then insolubilized by heat treatment, radiation, or the like;
Examples thereof include a method of adsorbing a substance having a surface-active action.

Examples of the hydrophilic polymer include polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, ethylene-vinyl alcohol copolymer, polyhydroxyethyl methacrylate, polysaccharides (cellulose, chitin, chitosan, etc.), water-soluble polyhydric alcohol and the like.

Examples of the hydrophobic polymer include polystyrene, polyvinyl chloride, polyolefin (polyethylene, polypropylene, etc.), acrylic, urethane, vinylon, nylon, polyester and the like.

Further, in order to improve the adhesion of the cells to be recovered to the separating material 5, an antibody against the cell adhesion protein and the specific antigen expressed on the target stem cell is fixed on the separating material 5. It may be changed. Examples of cell-adherent proteins include fibronectin, laminin, vitronectin, collagen and the like. Examples of the antibody include, but are not limited to, CD73, CD90, CD105, CD166, CD140a, CD271 and the like. Further, as the immobilization method, for example, a general protein immobilization method such as cyanogen bromide activation method, acid azide derivative method, condensation reagent method, diazo method, alkylation method, cross-linking method and the like can be used. It can be used arbitrarily.

The thickness of the separating material 5 is not particularly limited. The thickness of the separating material 5 is appropriately determined in consideration of the distance between the first protrusion 12 and the second wall 20 or the distance between the first protrusion 12 and the second protrusion 22. .. The thickness of the separating material 5 when the distance between the first protrusion 12 and the second wall 20 or the distance between the first protrusion 12 and the second protrusion 22 is 4.00 mm to 12.00 mm. Is preferably 7.20 mm to 21.60 mm.

The cell separation filter device 1 is configured by filling the container 2 with the separating material 5 described above.

Hereinafter, a preferred embodiment of the cell separation filter device 1 will be described with reference to FIGS. 2 to 5. It should be noted that the various constituent requirements described below for the cell separation filter device 1 shown in FIGS. 2 to 5 are for cell separation having the above-mentioned essential constituent requirements, unless they are not applicable due to shape restrictions or the like. It can be applied to the filter device alone or in combination of two or more requirements.

As shown in FIG. 2, the container 2 includes a separating material accommodating portion 3, a lid portion (second wall portion) 20 for covering the separating material accommodating portion 3, and a separating material accommodating portion 3 and a lid portion 20. A cap 40 for fixing is provided.
As shown in FIG. 3, the filter device 1 is configured by accommodating the separating material 5 made of a non-woven fabric or the like in the container 2.

In the separating material accommodating portion 3, the tubular peripheral wall portion 30 forming a space for filling the separating material 5 and the bottom portion (first wall portion) 10 that closes the space on one end side of the peripheral wall portion 30 are integrated. It is formed in. Hereinafter, the separating material accommodating portion is also simply referred to as “accommodating portion”.

The shape of the cross section along the main surface of the bottom portion 10 of the accommodating portion 3 may be circular or may be a shape other than circular such as a polygon.
Preferred specific examples of the shape of the accommodating portion 3 include, for example, a cylindrical shape having a capacity of about 1 to 400 mL, an inner diameter of about 45 to 60 mm, and a thickness of about 1 to 50 mm, and a piece having a length of about 1 to 100 mm inside the container. Examples thereof include a square cylinder having a thickness of about 1 to 50 mm and a square or a rectangle.
A lid 20 is provided on the other end side of the peripheral wall portion 30 of the accommodating portion 3 in order to close the space.

As shown in FIG. 3A, a liquid introduction port (first liquid passage port) 11 for introducing a liquid into the container 2 is formed in the central portion of the bottom portion 10 of the storage portion 3. Further, a liquid outlet (second liquid passage port) 21 for discharging the liquid from the container 2 is formed in the central portion of the lid portion 20.
The liquid inlet 11 and the liquid outlet 21 are composed of nozzles in order to facilitate connection of a tube for feeding the liquid. The shape and size of the nozzle are not particularly limited.
For convenience, the liquid inlet port 11 and the liquid outlet port 21 are referred to, but when the filter device 1 is used, the liquid may be discharged from the liquid inlet port 11 or the liquid may be introduced from the liquid outlet port 21.

The lid portion 20 has a plug shape, and is fixed in contact with the inner surface of the peripheral wall portion 30 of the accommodating portion 3 by pushing it into the lumen of the accommodating portion 3.
It is preferable that a seal 8 is provided on the contact surface between the lid portion 20 and the peripheral wall portion 30 of the accommodating portion 3. With this seal 8, the airtightness between the lid portion 20 and the accommodating portion 3 can be ensured, and leakage of the cell-containing liquid and cell recovery liquid from the inside of the container 2 and invasion of microorganisms and the like from the outside can be prevented. Examples of the seal 8 include providing a resin packing (O-ring) around a groove provided on the side surface of the lid 20, but the arrangement and configuration of the seal 8 are not particularly limited.

The lid portion 20 may be directly fixed to the accommodating portion 3 (not shown). The lid and the accommodating portion can be fixed by providing, for example, a screw on the surface where the lid and the accommodating portion come into contact with each other. In this case, the cap 40 shown in FIGS. 2 to 4B becomes unnecessary.

In the container 2, the separating material 5 is laminated and filled. For example, by using a filter in which two or more layers of separating materials 5 having different fiber diameters are laminated, the places where cells are captured are dispersed, the occurrence of clogging is suppressed, and the separation and recovery of cells from the filter are also efficient. Can be done The portion in which the separating materials 5 having the same fiber diameter are continuously laminated is treated as one layer regardless of the number of the separated separating materials 5 laminated.

Further, the container 2 is provided with a cleaning liquid introduction port for cleaning non-adherent cells remaining in the separating material 5 independently on the liquid introduction port 11 side (not shown), or on the liquid outlet port 21 side. It may be provided with a cell recovery liquid introduction port (for flowing the cell recovery liquid from the direction opposite to the flow of the cell-containing liquid and the washing liquid) for independently collecting the cells captured in the separating material 5 (shown in the figure). Z).

<Cross-sectional area inside the container>
The area of the cross section along the main surface of the bottom portion (first wall portion) 10 of the accommodating portion 3 and the main surface of the lid portion (second wall portion) 20 in the space inside the container 2 is 16.05 cm ² (container). It is preferable that the inner diameter in the radial direction of 2 is Φ45.20 mm) or more.
More preferably, the cross-sectional area of the space inside the container 2 is 24.07 cm ² (inner diameter of the container 2 Φ55.00 mm) or more and 33.18 cm ² (inner diameter of the container 2 Φ65.00 mm) or less.
Further, it is preferable that the value obtained by dividing the cross-sectional area of the space in the container 2 by the minimum distance (for example, 5 mm to 12 mm) between the first protrusion 12 and the second protrusion 22 is 20 or more and 67 or less. ..

By increasing the area inside the container 2, that is, the area inside the filter device 1 to 16.05 cm ² (inner diameter Φ45.20 mm) or more in this way, clogging of the separating material 5 can be reduced, and the separating material 5 can be reduced. It is easy for 5 to efficiently capture cells. As a result, the amount of blood processed can be increased and the cell recovery rate can be improved.
Further, by increasing the area inside the container 2, that is, the area inside the filter device 1 to 24.07 cm ² or more and 33.18 cm ² or less (inner diameter Φ55.00 mm or more and Φ65.00 mm or less), the amount of blood processed is as described above. Can be increased and the cell recovery rate can be improved.

If the area inside the container 2, that is, the area inside the filter device 1 is further increased to 50.27 cm ² (inner diameter of the container 2 Φ80.00 mm), the cell recovery rate may decrease. This is because the amount of cells captured in the separating material 5 increases, but the collected liquid introduced at a predetermined pressure at the time of collection concentrates in the central portion of the separating material 5, and thus is captured in the outer peripheral portion of the separating material 5. This is probably due to the inability to recover the cells. However, even in this case, the cell recovery rate can be increased by adjusting the supply method of the recovery liquid so that the recovery liquid is also supplied to the outer peripheral portion of the separating material 5.

<First protrusion>
As shown in FIGS. 3 and 5, the inner surface of the bottom portion 10 of the accommodating portion 3 is filled with the separating material 5 in cooperation with the lid portion 20 when the space inside the container 2 is filled with the separating material 5. A plurality of first protrusions 12 projecting from the inner surface toward the space inside the container 2 are formed so as to be sandwiched and compressed.
Each of the first protrusions 12 extends radially with respect to the central portion of the bottom 10 of the accommodation portion 3, and the first protrusions 12 are arranged along the peripheral wall portion 30 of the accommodation portion 3.
The first protrusion 12 is supported by a first pressing portion 13 formed in an annular shape along the peripheral wall portion 30.
The first protrusions 12 may be arranged at equal intervals or may be arranged irregularly. The first protrusions 12 are preferably arranged at equal intervals from the viewpoint that the liquid containing cells can be easily permeated into the separating material 5.

Next, the distance between the adjacent first protrusions 12 of the first protrusions 12 will be described. In the adjacent first protrusion 12, the distance between the tips of the bottom 10 of the accommodating portion 3 on the central portion side is the minimum, and the distance between the ends on the peripheral wall portion 30 side is the maximum. From this, in the following, the distance between the tips on the central side of the adjacent first protrusion 12 is referred to as the "minimum distance", and the distance between the ends of the adjacent first protrusion 12 on the peripheral wall portion 30 side is "maximum". Expressed as "distance".

As shown in FIG. 5, the minimum distance A between the adjacent first protrusions 12 is preferably equal to or larger than the inner diameter (for example, about 2.5 mm) of the liquid introduction port (first liquid passage port) 11. ..
More preferably, the minimum distance A of the adjacent first protrusions 12 is 3.79 mm (center radius R8.75 mm described later, several tens of first protrusions 12) or more 13.74 mm (center radius R8.75 mm, The number of the first protrusions 12 is 3) or less.
The ratio of the minimum distance A of the first protrusion 12 to the inner diameter of the liquid introduction port 11 (for example, about 2.5 mm) is 1.5 (center radius R8.75 mm, several tens of first protrusions 12). ) Or more and 5.5 (center radius R8.75 mm, number of first protrusions 12 is 3) or less.

In this way, by setting the minimum distance A between the adjacent first protrusions 12 to be equal to or larger than the inner diameter of the liquid introduction port 11 (for example, about 2.5 mm), the separating material 5 can be appropriately compressed. Cells can be efficiently captured in the separating material 5. In addition, blood and the recovery liquid can be efficiently passed between the first protrusions 12, and cells can be uniformly captured from the central portion to the outer peripheral portion of the separating material 5. As a result, the cell recovery rate can be improved.
Further, by setting the minimum distance A of the adjacent first protrusions 12 to 3.79 mm or more and 13.74 mm or less, the cell recovery rate can be improved as described above.

Further, the center distance between the first protrusion 12 and the center of the bottom 10 of the accommodating portion 3, in other words, the center radius R of the region surrounded by the first protrusion 12 is preferably 10.00 mm or less.
In this case, when the separating material 5 is compressed by the first protrusion 12, the inner layer of the region surrounded by the tip of the first protrusion 12 in the separating material 5 can be appropriately compressed, while the non-woven fabric in this region is loosened. Can be done. Therefore, cells can be efficiently captured in this region, and as a result, the cell recovery rate can be improved.
On the other hand, if the central radius is larger than 10.00 mm, the inner layer of the region of the separating material 5 surrounded by the tip of the first protrusion 12 cannot be appropriately compressed. Therefore, in this region, it is presumed that the cells penetrate to the inner layer and the recovery rate decreases when the recovery liquid is introduced (see Examples described later).

The number of the first protrusions 12 is 3 (inner diameter Φ55.00 mm of the container 2, the circumferential distance C40.00 mm of the first protrusion 12 described later) or more and 10 (inner diameter Φ65.00 mm of the container 2, the first). It is preferable that the circumferential distance of 1 protrusion 12 is C20.00 mm) or less.
By increasing the number of the first protrusions 12 in this way, the separating material 5 can be appropriately compressed, and the cells can be efficiently captured in the separating material 5. Therefore, the cell recovery rate can be improved.

Further, the maximum distance B of the adjacent first protrusions 12 among the first protrusions 12 is 36.74 mm (inner diameter of the container 2 Φ65.00 mm, the number of the first protrusions 12 is 5) or less. preferable.
More preferably, the maximum distance B of the adjacent first protrusions 12 is 19.31 mm (inner diameter Φ55.00 mm of the container 2, several tens of first protrusions 12) or more 36.74 mm (inner diameter Φ65 of the container 2). 00 mm, the number of the first protrusions 12 is 5) or less.
In other words, the circumferential distance C along the intermediate surface between the inner surface and the outer surface of the peripheral wall portion 30 of the adjacent first protrusion 12 is 20.00 mm or more and 40.00 mm or less.
As a result, a large number of first protrusions 12 can be provided, the separating material 5 can be appropriately compressed, and cells can be efficiently captured in the separating material 5. Therefore, the cell recovery rate can be improved.

Based on the above, more preferably, the cross-sectional area of the space inside the container 2 is 28.27 cm ² (inner diameter Φ60.00 mm of the container 2), and the central radius R of the first protrusion 12 is 8.75 mm. The number of one protrusion 12 is nine. That is, it is more preferable that the minimum distance A of the adjacent first protrusions 12 is 4.47 mm and the maximum distance B is 19.50 mm.

The shape of the cross section of the first protrusion 12 perpendicular to the direction in which the first protrusion 12 protrudes is not particularly limited. Since the first protrusion 12 and the separating material 5 can be easily brought into contact with each other on a wide surface, it is typically preferable that one side is a rectangle that comes into contact with the separating material 5.

The width W of the first protrusion 12 is preferably 1.50 mm or more and 3.50 mm or less.
If the width of the first protrusion 12 is thinner than 1.50 mm, the first protrusion 12 locally bites into the separating material 5, making it difficult to properly compress the entire portion 12.
On the other hand, when the width of the first protrusion 12 is thicker than 3.50 mm, the cell recovery rate tends to decrease. This is due to the following reasons. When the filter device 1 is thinned, the compressive stress on the separating material 5 by the first protrusion 12 becomes small, and the region of the separating material 5 with which the first protrusion 12 abuts becomes rough. Then, cells also enter this region, and it becomes difficult to collect them when passing the recovery liquid for cell recovery.

Here, when the cross-sectional area inside the container 2 is increased, the pressure of the recovered liquid introduced from the liquid outlet 21 at the time of passing the recovered liquid for cell recovery causes the inner surface of the peripheral wall portion 30 of the accommodating portion 3 and the first pressing portion. Stress is concentrated in the groove between 13 and the groove of the accommodating portion 3 may be deformed. When the vicinity of the groove of the accommodating portion 3 is deformed, the sealing property between the first pressing portion 13 of the accommodating portion 3 and the separating material 5 is lowered to form a gap, and the recovered liquid flows unevenly from this gap. More specifically, the degree of compression of the region compressed by the first pressing portion 13 of the separating material 5 decreases, and the recovered liquid tends to flow out in this region. Therefore, it is difficult to sufficiently release the cells trapped in the separating material 5 by the recovery solution, and it is difficult to achieve the desired cell recovery rate, but it may decrease.
In this regard, according to the present embodiment, an appropriately large number of first protrusions 12 can be formed in the accommodating portion 3, and deformation in the vicinity of the groove of the accommodating portion 3 due to the pressure of the collected liquid at the time of cell collection is reduced. can do. Therefore, it is possible to maintain the column strength while preventing a decrease in the sealing property between the first pressing portion 13 of the accommodating portion 3 and the separating material 5.

<Second protrusion>
As shown in FIGS. 3 and 6, when the space inside the container 2 is filled with the separating material 5, the inner surface of the lid 20 cooperates with the first protrusion 12 of the bottom 10 of the accommodating portion 3. A plurality of second protrusions 22 projecting from the inner surface toward the space inside the container 2 are formed so as to sandwich and compress the separating material 5. The shape of the second protrusion 22 may be the same as that of the first protrusion 12 described above.

That is, each of the second protrusions 22 extends radially with respect to the central portion of the lid 20, and the second protrusions 22 extend to the accommodation portion 3 when the lid portion 20 covers the accommodation portion 3. It is provided so as to be arranged along the peripheral wall portion 30 of the.
The second protrusion 22 is supported by a second pressing portion 23 formed in an annular shape along the peripheral wall portion 30.

Further, as shown in FIG. 6, the minimum distance A between the adjacent second protrusions 22 is equal to or larger than the inner diameter (for example, about 2.5 mm) of the liquid outlet (second liquid passage) 21. preferable.
Preferably, the minimum distance A of the adjacent second protrusions 22 is 3.79 mm (center radius R8.75 mm described later, several tens of second protrusions 22) or more 13.74 mm (center radius R8.75 mm, first. 2 The number of protrusions 22 is 3) or less.
The ratio of the minimum distance A of the second protrusion 22 to the inner diameter (for example, about 2.5 mm) of the liquid outlet 21 is 1.5 (center radius R8.75 mm, several tens of second protrusions 22). ) Or more and 5.5 (center radius R8.75 mm, number of second protrusions 22 is three) or less.

Further, the center distance between the second protrusion 22 and the center of the lid 20, in other words, the center radius R of the region surrounded by the second protrusion 22 is preferably 10.00 mm or less.

The number of the second protrusions 22 is 3 (inner diameter Φ55.00 mm of the container 2, the circumferential distance C40.00 mm of the second protrusion 22 described later) or more and 10 (inner diameter Φ65.00 mm of the container 2, the second). The circumferential distance of the two protrusions 22 is preferably C20.00 mm) or less.

Further, the maximum distance B of the adjacent second protrusions 22 of the second protrusions 22 is 36.74 mm (inner diameter of the container 2 Φ65.00 mm, the number of the second protrusions 22 is five) or less. preferable.
More preferably, the maximum distance B of the adjacent second protrusions 22 is 19.31 mm (inner diameter Φ55.00 mm of the container 2, several tens of second protrusions 22) or more 36.74 mm (inner diameter Φ65 of the container 2). 00 mm, the number of the second protrusions 22 is 5) or less.
Further, the circumferential distance C along the intermediate surface between the inner surface and the outer surface of the peripheral wall portion 30 of the adjacent second protrusion 22 is preferably 20.00 mm or more and 40.00 mm or less.

Here, if the cross-sectional area inside the container 2 is increased, the lid portion 20 may be deformed by the pressure of the recovered liquid introduced from the liquid outlet 21 at the time of passing the recovered liquid for cell recovery. When the lid portion 20 is deformed, a gap is generated due to a decrease in the sealing property between the second protrusion 22 of the lid portion 20 and the separating material 5, and the recovered liquid flows out from this gap (in other words, the second in the separating material 5). 2 The degree of compression of the region compressed by the protrusion 22 decreases, and the recovered liquid flows unevenly in this region). Therefore, the cells trapped in the separating material 5 cannot be sufficiently released by the recovery solution, and the cell recovery rate may decrease.
In this regard, according to the present embodiment, it is possible to form an appropriately large number of second protrusions 22 on the lid portion 20 and reduce the deformation of the lid portion 20 due to the pressure of the recovery liquid at the time of cell recovery. it can. Therefore, it is possible to maintain the column strength while preventing a decrease in the sealing property between the second protrusion 22 of the lid portion 20 and the separating material 5.

Based on the above, more preferably, the area of the cross section of the space inside the container 2 is 28.27 cm ² (inner diameter of the container 2 is Φ60.00 mm), and the central radius R of the second protrusion 22 is 8.75 mm. The number of 2 protrusions 22 is nine. That is, the minimum distance A of the adjacent first protrusions 12 is 4.47 mm, and the maximum distance B is 19.50 mm.

Further, the width W of the second protrusion 22 is preferably 1.50 mm or more and 3.50 mm or less.

In the present embodiment, the second protrusion 22 is formed on the inner surface of the lid 20, but the lid 20 does not have to include the second protrusion 22. In this case, when the space inside the container 2 is filled with the separating material 5 on the inner surface side of the lid portion 20, the separating material 5 is supported in a state where liquid can flow from one side to the other side of the separating material 5. A support member may be provided. Examples of the support member include a mesh and a perforated plate.

As described above, by accommodating the separating material 5 described later in the container 2 of the present embodiment to form the filter device 1, the blood processing amount can be increased and the cell recovery rate can be improved. Can be done. Further, it is possible to reduce the deformation of the lid portion 20 due to the pressure of the recovered liquid at the time of cell recovery, and it is possible to reduce the deformation of the lid portion 20 between the first pressing portion 13 of the accommodating portion 3 and the separating material 5, and the second protrusion portion of the lid portion 20. It is possible to maintain the column strength while preventing a decrease in the sealing property between the 22 and the separating material 5.

In the present embodiment described above, the peripheral wall portion 30 and the bottom portion (first wall portion) 10 are integrally formed of a housing portion 3, a lid portion (second wall portion) 20, and a cap 40. The container 2 was illustrated. However, the configuration of the container 2 is not limited to this. For example, the peripheral wall portion 30 and the bottom portion (first wall portion) 10 in the accommodating portion 3 may be separate bodies. Specifically, the accommodating portion 3 is composed of a tubular peripheral wall portion whose bottom side is also open, and a bottom lid portion (first wall portion) similar to the lid portion (second wall portion) 20 described above. May be done. In this case, the liquid introduction port 11, the first protrusion 12, and the first pressing portion 13 may be formed on the bottom lid portion (first wall portion).

As shown in FIGS. 2 to 4B, as described above, the filter device 1 is configured by filling the space inside the container 2 with the separating material 5.

≪Manufacturing method of cell concentrate≫
By the treatment using the cell separation filter device described above, a cell concentrate containing desired cells can be suitably produced.

<Cell>
The target cell is not particularly limited. For example, artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), mesenchymal stem cells, adipose-derived mesenchymal cells, adipose-derived stromal stem cells, pluripotent adult stem cells, bone marrow stromal cells, hematopoietic stem cells. Pluripotent living stem cells, T cells, B cells, killer T cells (cytotoxic T cells), NK cells, NKT cells, control T cells and other lymphoid cells, macrophages, monospheres, etc. Examples thereof include somatic cells such as dendritic cells, granulocytes, erythrocytes, platelets, nerve cells, muscle cells, fibroblasts, hepatocytes, and myocardial cells, or cells that have undergone treatment such as gene introduction or differentiation.

Among such cells, leukocytes, hematopoietic stem cells and / or mononuclear cells are preferable because the effect of increasing the recovery rate is likely to appear remarkably.
Examples of leukocytes include granulocytes such as neutrophils, eosinophils and basophils in peripheral blood, and monocytes such as monocytes and lymphocytes.
Hereinafter, a method for producing a cell concentrate containing at least one of these cells will be described.

<Manufacturing method of cell concentrate>
A suitable method for producing a cell concentrate containing cells is
The cell-containing liquid containing cells is introduced into the above-mentioned filter device 1 from the liquid introduction port 11 and passed through the separating material 5, so that the cells are captured by the separating material 5.
The recovery liquid is introduced into the filter device 1 and the cells trapped in the separating material 5 are released into the recovery liquid to generate a cell concentrate.
Collecting the cell-containing liquid from the filter device 1 and
It is a method including.

The cell-containing solution is not particularly limited as long as it is a suspension containing cells containing leukocytes and / or mononuclear cells. For example, the density gradient of suspensions after subjecting biological tissues such as umbilical cords to enzyme treatment, crushing treatment, extraction treatment, decomposition treatment, ultrasonic treatment, etc., body fluids such as blood and bone marrow fluid, umbilical cord blood, and blood and bone marrow fluid. Examples of cell-containing liquids include cell suspensions prepared by pretreatment such as centrifugation, filtration, enzyme treatment, decomposition treatment, and ultrasonic treatment.
In addition, the cell-containing solution may be a suspension obtained by culturing or proliferating the above-mentioned cells such as leukocytes in vitro using a culture solution, a stimulating factor, or the like.

When the cell-containing liquid is introduced from the liquid introduction port 11 of the filter device 1, a differential pressure may be generated between the liquid introduction port 11 and the liquid outlet 21. The method of generating the differential pressure is not particularly limited, and a method of introducing the cell-containing liquid into the filter device 1 by pressurizing the cell-containing liquid, or a method of depressurizing the liquid outlet 21 side into the filter device 1 Examples thereof include a method of inhaling a cell-containing liquid.
In this way, the cell-containing liquid is supplied into the filter device 1, the cell-containing liquid is brought into contact with the separating material 5, and the cells are captured by the separating material 5.
The degree of the differential pressure is not particularly limited as long as the container 2 and the separating material 5 are not damaged or the cells contained in the cell-containing solution are not excessively crushed.
As the separating material 5, a material capable of capturing cells may be used.

Next, by supplying the recovery liquid to the separating material 5 in which the cells are captured, the cells are released from the separating material 5 into the recovery liquid to generate a cell concentrate containing the cells. Leukocytes and the like can be recovered by introducing the recovery liquid containing the leukocytes and the like into a bag or the like dedicated to recovery from the liquid introduction port.
The recovered liquid is introduced into the filter device 1 from the liquid outlet 21 or the liquid introduction port 11. The method for recovering the cell concentrate containing cells from the inside of the filter device 1 is not particularly limited, but typically, the cell-containing liquid containing monocytes is recovered from the liquid introduction port 11.
Since monocytes trapped in the separating material 5 are easily released into the collecting liquid by the flow of the collecting liquid, the recovered liquid is introduced into the filter device 1 so as to pass through the separating material 5 from the liquid outlet 21. Is preferable. In this case, it is preferable to perform a so-called backwash operation in which the cell concentrate containing cells is collected as it is from the liquid introduction port 11 while the recovery liquid is introduced from the liquid outlet 21.

The recovered solution is not particularly limited as long as it is a solution that is isotonic with cells, and examples thereof include those that have been used as injection solvents such as physiological saline and Ringer's solution, buffer solutions, and media for cell culture. In particular, a medium that can be cultured as it is is preferable when it undergoes the culturing step, and when it is used as it is for treatment without undergoing the culturing step, the safety of an isotonic solution that has been used for infusion of physiological saline or the like is guaranteed. It is preferable to use the recovered solution.

The operation described above may be performed at room temperature or at a refrigerating temperature. When carried out at a refrigerated temperature, treatment of the refrigerated cell-containing liquid can be mentioned. Examples of the storage of the cell-containing liquid include storage in a refrigerator set to a refrigerating temperature, storage in a water bath, and storage in dry ice. It is preferable to store in a refrigerator because of its versatility. The refrigerating temperature is preferably 1 ° C. or higher and 6 ° C. or lower, more preferably 3 ° C. or higher and 5 ° C. or lower. If the refrigeration temperature is less than 1 ° C, the cells will die, and if stored above 6 ° C, bacteria may grow and cause contamination.

Further, from the viewpoint of adjusting the state of the separating material 5 and facilitating the capture of cells, a physiological saline solution or a buffer solution is used from the liquid inlet 11 or the liquid outlet 21 before supplying the cell-containing liquid into the filter device 1. May be introduced into the filter device 1 to bring the separating material 5 into contact with the saline solution or the buffer solution.

Before the recovery solution is used to generate a cell concentrate containing monocytes, a physiological saline solution or a buffer solution is introduced from the liquid inlet 11 and led out from the liquid outlet 21 to remove contaminants in the filter. This makes it possible to reduce unnecessary intracellular components that are recovered.

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

(Examples 1 to 9)
As shown in FIG. 3, a polyester non-woven fabric (with a basis weight of 35 g) cut into a round shape according to the diameter of the accommodating portion 3 in a cylindrical accommodating portion 3 having a height (inner dimension) and a diameter (inner diameter) shown in Table 1. A filter having a structure as shown in FIGS. 2 and 4A by filling 45 sheets of / m ² and separating material 5), inserting the lid 20 into the opening of the accommodating portion 3, and screwing from above with a cap 40. Device 1 was manufactured.

Next, the circuit shown in FIG. 7 was connected to the liquid inlet 11 and the liquid outlet 21 of the filter device 1 to fabricate the cell separation device 100.

In the circuit shown in FIG. 7, a tube 111 was connected to the liquid inlet 11. The tube 111 contains a tube 112 connected to a means 120 for accommodating a cell suspension and a means 125 for accommodating physiological saline for priming, and a means (collection bag) for accommodating a recovery liquid that has passed through the filter device 1. The tube 113 connected to the 130 and the means 135 for collecting the recovered liquid collected in the collection bag or the like was connected via the flow path switching means 116.
A means 120 for accommodating a cell suspension and a means 125 for accommodating physiological saline for priming were connected to the tube 112 via a flow path switching means 117.
A means 130 for accommodating the collected liquid that has passed through the filter device 1 and a means 135 for collecting the collected liquid collected in a collection bag or the like are connected to the tube 113 via the flow path switching means 118.
Further, a tube 114 is connected to the liquid outlet 21, and a means (waste liquid bag) 145 for accommodating the cell suspension that has passed through the filter device 1 and a means 140 for collecting the recovered liquid via the flow path switching means 119. Connected with.

A cell separation operation was performed using the cell separation device 100. The operation of each flow path switching means was appropriately performed according to the type of liquid to be passed through the filter device 1 and the means for sending the liquid.
First, the priming operation of the filter device 1 was performed using 50 mL to 150 mL of the physiological saline solution of the means 125 for accommodating the physiological saline solution, and the physiological saline solution that had passed through the cell separation filter device 1 was collected in the waste liquid bag 145.
Next, 130 mL of leukocyte concentrate (bovine peripheral blood anticoagulated by CPD) is passed through the filter device 1 using gravity from the means 120 for accommodating the cell suspension, and the passing liquid is collected in the waste liquid bag 145. did.
The leukocyte concentrate used in the above operation has a white blood cell count of 1.0 × 10 ⁹ cells to 4.0 × 10 from a buffy coat obtained by centrifuging bovine blood anticoagulated by CPD at 3000 rpm for 30 minutes. ^It was prepared to have ⁹ cells.
Then, using the flow path switching means 117, 100 mL of the physiological saline solution of the means 125 for accommodating the physiological saline solution was passed through the filter device 1 by gravity, and the passing liquid was collected in the waste liquid bag 145.
Finally, 50 mL of physiological saline was manually introduced from the liquid outlet 21 of the filter device 1 using a syringe as a means 140 for collecting the recovered liquid at a flow rate of 10 to 50 mL / sec, and connected to the liquid introduction port 11. It was collected in the collection bag 130.
The leukocyte concentration of the recovered solution (cell concentrate) and the cell suspension (cell-containing solution) before treatment was measured by a blood cell counter (K-4500, Sysmex Corporation). Then, the white blood cell count was calculated from the volumes of the cell suspension and the recovery solution before the treatment, and the cell recovery rate (white blood cell recovery rate) was determined.

According to Table 1, a good cell recovery rate can be realized when the above-mentioned L1 which is the shortest distance between the peripheral wall portion 30 and the annular region 50 is 1.00% or more and 7.75% or less of L2. I understand.
According to the comparison between Example 1 and Example 4 in which the widths of the presser portions W1 and W2 are the same, it can be seen that the longer L1 is relative to L2, the lower the cell recovery rate tends to be. ..
According to the comparison between Example 3 and Example 4 in which L1 is the same, it can be seen that the wider the widths of the pressing portions W1 and W2, the better the cell recovery rate.

1 Cell separation filter device 2 Filter container 3 Separation material storage 5 Separation material 8 Seal 10 Bottom (1st wall)
11 Liquid inlet (first liquid inlet)
12 1st protrusion 13 1st presser foot 20 Lid (2nd wall)
21 Liquid outlet (second liquid outlet)
22 Second protrusion 23 Second presser foot 30 Peripheral wall 100 Cell separation device 111 Tube 112 Tube 113 Tube 114 Tube 116 Flow path switching means 117 Flow path switching means 118 Flow path switching means 119 Flow path switching means 120 Cell suspension Means for accommodating liquid 125 Means for accommodating physiological saline for priming 130 Means for accommodating recovered liquid (recovery bag)
135 Means for collecting the recovered liquid 140 Means for collecting the recovered liquid 145 Means for accommodating the cell suspension (waste liquid bag)

Claims (14)

A cell separation filter device including a filter container and a separating material filled in the filter container.
The filter container includes a tubular peripheral wall portion, a first wall portion that closes an opening on one end side of the peripheral wall portion, and a second wall portion that closes an opening on the other end side of the peripheral wall portion.
The separating material is filled in the filter container so that a gap is not formed between the separating material and the inner surface of the peripheral wall portion.
A first liquid passage port is formed on the first wall portion.
A second liquid passage port is formed on the second wall portion.
A first protrusion and a first holding portion are formed on the inner surface of the first wall portion.
A second pressing portion is formed on the inner surface of the second wall portion.
When the separating material is filled in the filter container, the first protruding portion cooperates with the second wall portion to sandwich and compress the separating material.
The first pressing portion and the second pressing portion sandwich and compress an annular region of the separating material filled in the filter container, which is located along the inner surface of the peripheral wall portion.
The shortest distance between the inner surface of the peripheral wall portion and the outer circumference of the annular region is L1, and the circular equivalent diameter of the cross section of the internal space of the peripheral wall portion calculated from the radial cross-sectional area of the internal space of the peripheral wall portion is L2. A cell separation filter device in which the L1 is 1.00% or more and 7.75% or less of the L2. The cell separation filter device according to claim 1, wherein the L1 is 1.00% or more and 3.50% or less of the L2. The width W1 of the surface of the first pressing portion that presses the separating material and the width W2 of the surface of the second pressing portion that presses the separating material are 0.75% or more and 6.00% or less of L2, respectively. The cell separation filter device according to claim 1 or 2. The width W1 of the surface of the first pressing portion that presses the separating material and the width W2 of the surface of the second pressing portion that presses the separating material are 3.00% or more and 6.00% or less of L2, respectively. The cell separation filter device according to claim 1 or 2. The cell according to any one of claims 1 to 4, wherein the width of the surface of the first pressing portion that presses the separating material is narrower than the width of the surface of the second pressing portion that presses the separating material. Separation filter device. The area of the cross section of the internal space of the peripheral wall portion along the main surface of the first wall portion and the main surface of the second wall portion is 16.05 cm ² or more.
The first protrusion is composed of a plurality of first protrusions.
The distance between the first protrusion and the center of the first wall is 10.00 mm or less.
The cell separation filter device according to any one of claims 1 to 5, wherein the maximum distance of the adjacent first protrusions among the plurality of first protrusions is 36.74 mm or less. The invention according to any one of claims 1 to 6, wherein a second protrusion is formed on the inner surface of the second wall in cooperation with the first protrusion to sandwich and compress the separating material. Cell separation filter device. The area of the cross section of the internal space of the peripheral wall portion along the main surface of the first wall portion and the main surface of the second wall portion is 16.05 cm ² or more.
The second protrusion is composed of a plurality of second protrusions.
The distance between the second protrusion and the center of the second wall is 10.00 mm or less.
The cell separation filter device according to claim 7, wherein the maximum distance between the adjacent second protrusions among the plurality of second protrusions is 36.74 mm or less. The first protrusion is composed of a plurality of first protrusions.
The second protrusion is composed of a plurality of second protrusions.
Each of the plurality of first protrusions extends radially with respect to the central portion of the first wall portion.
The plurality of first protrusions are arranged along the first holding portion.
Each of the plurality of second protrusions extends radially with respect to the central portion of the second wall portion.
The cell separation filter device according to claim 7 or 8, wherein the plurality of second protrusions are arranged along the second retainer portion. The cell separation filter device according to claim 9, wherein the number of the first protrusions and the number of the second protrusions are independently 3 or more and 10 or less. The cell separation filter device according to any one of claims 1 to 10, wherein the peripheral wall portion and the first wall portion are integrally formed. The cell separation filter device according to any one of claims 1 to 11, wherein the separating material is a non-woven fabric or a porous body containing porous cellulose particles. A cell-containing liquid containing cells is placed in the cell separation filter device according to any one of claims 1 to 12 from one of the first passage and the second passage. By introducing and passing the separating material, the cells are captured by the separating material, and
A recovery solution is introduced into the cell separation filter device, and the cells trapped in the separation material are released into the recovery solution to generate a cell concentrate.
A method for producing a cell concentrate, which comprises collecting the cell concentrate from the cell separation filter device. 13. The thirteenth aspect of the present invention, wherein the recovered liquid is introduced from the other liquid passing port of the first liquid passing port and the second liquid passing port, and the cell concentrate is collected from the one liquid passing port. Method for producing cell concentrate.

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