WO2016002505A1

WO2016002505A1 - Filter having optimized external shape of ridges inside filter

Info

Publication number: WO2016002505A1
Application number: PCT/JP2015/067335
Authority: WO
Inventors: 敬太山下; 尚武前久保; 伸好梅田; 伸彦佐藤
Original assignee: 株式会社カネカ
Priority date: 2014-06-30
Filing date: 2015-06-16
Publication date: 2016-01-07
Also published as: JPWO2016002505A1; JP6646575B2

Abstract

A filter having a container filled with a cell separation material. The filter comprises a fluid introduction port in either an upper section or lower section of the container and a fluid discharge port on the opposite side thereto. The filter has ridges on the surface of the filled cell separation material, that protrude towards either the fluid introduction port side or the fluid discharge port side. The height (y) and the length (x) of the ridges fulfil formula (1) y = -ax² + bx and/or formula (2) y = ax² - bx. The ratio (a/b) between a and b is adjusted to within the range 0.03-0.25. The purpose of the present invention is to provide a cell separation filter having improved cell recovery efficiency and a cell separation method that uses the cell separation filter and whereby target cells can be efficiently recovered.

Description

A filter that optimizes the external shape of the raised part in the filter

The present invention relates to a cell separation filter or a cell separation method using the same.

In recent years, with rapid advances in hematology and scientific technology, only the necessary blood fractions are separated from body fluids such as whole blood, bone marrow, umbilical cord blood, and tissue extracts and administered to patients. In addition, a treatment style that suppresses side effects by not administering fractions that are not necessary for treatment is widespread.

For example, blood transfusion is one of them. An erythrocyte product is a blood product that is used when bleeding and erythrocytes are deficient, or when oxygen is deficient due to reduced function of erythrocytes. The erythrocyte preparation does not require leukocytes that induce side effects such as abnormal immune reactions or graft-versus-host disease (GVHD), and it is necessary to remove leukocytes with a filter. In some cases, platelets may be removed in addition to leukocytes.

On the other hand, the platelet preparation is a blood preparation used for patients who are bleeding or tend to bleed due to lack of blood coagulation factors. For the production of platelet preparations, unnecessary cells and components other than platelets are removed by centrifugation, and only the necessary platelet components are collected.

In addition, in recent years, hematopoietic stem cell transplantation for the treatment of leukemia and solid cancer has been actively performed, and a method of separating and administering leukocyte groups containing hematopoietic stem cells necessary for treatment has been taken. As a source of this hematopoietic stem cell, umbilical cord blood is attracting attention in addition to bone marrow and peripheral blood because of its advantages such as low burden on donors and excellent proliferation ability. In recent years, it has been suggested that there are abundant stem cells in menstrual blood, and menstrual blood that has been discarded may be used as a valuable source of stem cells.

For bone marrow and peripheral blood, it is desirable to separate and purify leukocytes after removing unnecessary cells, but umbilical cord blood is also becoming popular for banking for relatives and is stored frozen until use. From the necessity, leukocytes are separated and purified for the purpose of preventing red blood cell hemolysis due to cryopreservation.

Recently, a method of collecting leukocytes using a filter material that captures only white blood cells without capturing red blood cells and platelets (Patent Document 1, Patent Document 2, and Patent Document 3) has been reported as a cell separation method.
However, when actually separating cells, the cause is unknown, but there are cases where blood does not pass well through the cell separation material packed in the filter material or cells cannot be collected while being captured by the cell separation material. These problems have been problems that cannot be overlooked particularly when cells such as hematopoietic stem cells are collected from blood with a small amount of blood such as umbilical cord blood.

JP 2001-518792 A International Publication No. 98/32840 Japanese Patent Laid-Open No. 10-313855

In view of the above circumstances, an object of the present invention is to provide a cell separation filter with improved cell recovery efficiency.
Another object of the present invention is to provide a cell separation method capable of efficiently collecting desired cells using the cell separation filter.

The present inventors have intensively studied to solve such problems. As a result, the cell recovery rate is significantly improved by forming a raised portion having a predetermined outer shape on the surface of the liquid inlet side or the liquid outlet side in the cell separation material filled in the cell separation filter, In addition, the present inventors have found that blood components that are not recovered but captured by the cell separation material can be significantly reduced, and the present invention has been completed.

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

[1] A filter having a container filled with a cell separation material, having a liquid inlet at either the upper part or the lower part of the container, and a liquid outlet at the opposite side,
The surface of the filled cell separation material has a raised portion that is raised in at least one direction on the liquid inlet side or the liquid outlet side,
The height y and the length x of the raised portion are expressed by the formula (1) and / or the formula (2):
y = −ax ² + bx Formula (1)
y = ax ² −bx Formula (2)
And a filter having one or more ridges in which the ratio of a and b (a / b) is in the range of 0.03 to 0.25.

[2] The filter according to [1], wherein the cell separation material has at least two raised portions and at least one concave portion between the raised portions on the surface of the cell separation material.

[3] When the number of raised portions is n and the number of recessed portions between raised portions is m, the following (c) or (d):
(C) n = m + 1;
(D) n = m + 2;
The filter according to [2], which satisfies the relationship:

[4] The filter according to any one of [1] to [3], wherein a is 0.030 to 0.120 and / or b is 0.3 to 1.5.

[5] The minimum value of the thickness of the cell separation material is 1.20 × 10 to 1.26 × 10 mm, and the maximum thickness of the cell separation material is 1.26 × 10 to 2.10 × 10 mm [1] The filter according to any one of to [4].

[6] The sum of the cross-sectional areas of the raised portions is 2.08 × 10 to 1.09 × 10 ² mm ² , and the cross-sectional areas other than the raised portions are 5.40 × 10 ² to 5.61 × 10 ² mm ² . The filter according to any one of [1] to [5].

[7] protruding to the inside of the container, provided on the upper side or the lower side inside the container with a protrusion capable of pressing the upper or lower surface of the cell separation material incorporated in the container, The filter according to any one of [1] to [6], wherein an area of a tip surface of the protrusion is 30% or less of a surface area of the cell separation material.

[8] The filter according to [7], wherein the shape of the tip surface of the protrusion is linear, rod-shaped, polygonal, elliptical, circular, or a combination of two or more of these shapes.
[9] The filter according to [7] or [8], wherein the filter has at least two protrusions.

[10] The cell-containing liquid is introduced from the liquid inlet of the filter according to any one of the above [1] to [9], and is contacted with the cell separation material filled in the filter to obtain leukocytes and / or simple substances. A first step of capturing the nuclei in the cell separator, and
A cell separation method comprising a second step of introducing a collection liquid into the filter and collecting white blood cells and / or mononuclear cells from the cell separation material.

[11] The cell separation method according to [10], wherein the second step is a step of introducing a recovery liquid from a liquid outlet of the filter and recovering leukocytes and / or mononuclear cells from the liquid inlet. .

[12] Further, before the first step, including a step of introducing a physiological saline or a buffer solution from a liquid inlet of the filter and bringing the cell separation material into contact with the physiological saline or the buffer solution, [10] or [11].

[13] Further, by introducing physiological saline or a buffer solution from the liquid inlet of the filter after the first step and before the second step, and leading out from the liquid outlet of the filter. The method for separating cells according to any one of [10] to [12], further comprising a step of removing contaminant components in the filter.

The cells in the blood can be efficiently recovered by using the filter of the present invention.

It is the schematic which shows an example of the filter of this invention. FIG. 2 is a top view of the filter shown in FIG. 1 and a vertical cross-sectional view taken along the line AA of the filter. FIG. 3 is an enlarged vertical sectional view of a main part of the filter shown in FIG. 2. It is the schematic which shows the measurement location at the time of measuring the external shape of a protruding part. It is the schematic which shows an example of the pressing member with a nozzle of a filter. It is a longitudinal cross-sectional view of the cell separation material with which the filter shown in FIG. 2 was filled. 1 is a schematic diagram illustrating a structure of a filter used in Example 1. FIG. 3 is a longitudinal sectional view showing an example of an outer shape of a cell separation material 11 filled in a filter used in Example 1. FIG. 1 is a schematic diagram of a circuit of a cell separation device used in Example 1. FIG. It is the figure at the time of putting the cell separation material taken out from the filter after the cell collection | recovery in Example 1, 3 and Comparative Example 1, and comparing the blood component which remains there, respectively.

Hereinafter, the present invention will be described in detail.

The filter of the present invention is a filter having a container filled with a cell separation material, having a liquid inlet at either the upper part or the lower part of the container and a liquid outlet at the opposite side.
The surface of the filled cell separation material has a raised portion that is raised in at least one direction on the liquid inlet side or the liquid outlet side,
The height y and the length x of the raised portion are expressed by the formula (1) and / or the formula (2):
y = −ax ² + bx Formula (1)
y = ax ² −bx Formula (2)
And one or more raised portions having a ratio of a to b (a / b) in the range of 0.03 to 0.25.

(Cell separation material)
The form of the cell separation material used in the present invention is not particularly limited, and examples thereof include a porous body having a communicating pore structure, a fiber assembly, and a fabric. Preferably it is comprised with a fiber, More preferably, it is a nonwoven fabric.

Examples of the material for the cell separation material include polyolefins such as polypropylene, polyethylene, high density polyethylene and low density polyethylene, polyester, vinyl chloride, polyvinyl alcohol, vinylidene chloride, rayon, vinylon, polystyrene, acrylic (polymethyl methacrylate, poly Hydroxyethyl methacrylate, polyacrylonitrile, polyacrylic acid, polyacrylate, etc.), nylon, polyurethane, polyimide, aramid, polyamide, cupra, polyparaphenylene terephthalamide, carbon, phenol, tetron, pulp, hemp, cellulose, kenaf, chitin, Examples include chitosan, glass, and cotton. Among these, polymers such as polyester, polypropylene, acrylic, rayon, nylon, polybutylene terephthalate, and polyethylene terephthalate can be suitably used. The cell separation material may be composed of a single material among these materials, or may be composed of a composite material obtained by combining a plurality of materials.

The average fiber diameter of the cell separation material used in the present invention is not particularly limited as long as it is appropriately selected according to the type of target cells.

In order to further improve the performance of the cell separation material, the cell separation material may be subjected to a hydrophilic treatment. By hydrophilizing treatment, non-specific capture of cells other than the desired necessary cells can be suppressed, and cell-containing liquid can be passed through the cell separation material without bias to improve performance and improve efficiency of collecting necessary cells. Can be granted. Hydrophilic treatment methods include water-soluble polyhydric alcohols, polymers having hydroxyl groups, cationic groups or anionic groups, or copolymers thereof (for example, hydroxyethyl methacrylate, dimethylaminoethyl methacrylate, or copolymers thereof). Adsorption method, water-soluble polymer (polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, etc.) adsorption method, hydrophilic polymer fixed to hydrophobic membrane (for example, hydrophilic monomer is chemically bonded to the surface) Method), electron beam irradiation method, method of cross-linking insolubilization of hydrophilic polymer by irradiating radiation to cell separation filter in water-containing state, method of insolubilization and immobilization of hydrophilic polymer by heat treatment in dry state , The method of sulfonating the surface of hydrophobic membrane, hydrophilic polymer and sparse A method of forming a membrane from a mixture with a conductive polymer dope, a method of imparting hydrophilic groups to the membrane surface by treatment with an aqueous alkaline solution (NaOH, KOH, etc.), a soaked hydrophobic porous membrane in alcohol, and then treating with a water-soluble polymer aqueous solution A method of insolubilizing with heat treatment or radiation after drying, a method of adsorbing a substance having a surface active action, or the like can be mentioned.

Examples of the hydrophilic polymer include polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, ethylene-vinyl alcohol copolymer, polyhydroxyethyl methacrylate, polysaccharide (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.

Furthermore, in order to improve the adherence of the target cells to the cell separation material, cell adhesion proteins and antibodies against specific antigens expressed on the target stem cells are immobilized on the cell separation material. May be used. Examples of cell adhesion proteins include fibronectin, laminin, vitronectin, collagen and the like. Examples of antibodies include, but are not limited to, CD73, CD90, CD105, CD166, CD140a, CD271, and the like. Examples of immobilization methods include general protein immobilization methods such as cyanogen bromide activation method, acid azide derivative method, condensation reagent method, diazo method, alkylation method, and crosslinking method. It can be used arbitrarily.

(container)
The container filled with the cell separating material has a liquid inlet at either the upper part or the lower part and a liquid outlet at the opposite side.
The liquid introduction port refers to a port for introducing a liquid containing a target cell (also referred to as a cell-containing liquid) into the container from the outside of the container.
The liquid outlet port is a port provided on the opposite side of the liquid inlet port with respect to the vertical direction, and is mainly used to discharge the liquid that has passed through the cell separation material during cell separation operation to the outside of the container. Say.
There may be one or more liquid inlets and liquid outlets, and the number is not particularly limited.
Moreover, there is no limitation in particular as an upper or lower position in which the said liquid inlet or liquid outlet is provided.
Note that the cells captured by the cell separation material may be discharged from the liquid inlet to the outside of the container depending on the configuration of the cell separation device combined with the filter of the present invention.

The shape of the container may be any shape such as a sphere, a container, a cassette, a bag, and a tube as long as the cell separating material can be incorporated therein. Preferable specific examples include, for example, a cylindrical container having a capacity of about 0.1 to 400 mL and a diameter of about 0.1 to 15 cm, a square or a rectangle having a length of about 0.1 to 20 cm, and a thickness of 0. Examples thereof include a square columnar container of about 1 to 5 cm.

For example, as shown in FIGS. 1, 2 (a), and 2 (b), in the cylindrical filter 1, the presser with nozzle that can cover the cylindrical main body 3 and the openings at the top and bottom thereof. The

members

4 and 5 and the

caps

6 and 7 for fixing the main body 3 and the pressing member with

nozzle

4 and 5 are configured.
The holding members with

nozzles

4 and 5 are respectively provided with a liquid inlet 9 for introducing liquid into the container 2 and a liquid outlet 10 for discharging liquid from the container 2. The liquid inlet 9 and the liquid outlet 10 are constituted by nozzles in order to easily connect a tube for feeding a liquid. The shape and size of the nozzle are not particularly limited.
The holding members with

nozzles

4 and 5 have a stopper shape, and are fixed in contact with the inner surface of the main body 3 by being pushed into the lumen of the cylindrical main body 3.
A seal 8 is provided on the contact surface between the

presser members

4 and 5 with the nozzle and the main body 3. The seal 8 ensures the airtightness between the

presser members

4 and 5 with the nozzle and the main body 3 and prevents the entry of microorganisms and the like from the outside. As the seal 8, for example, as shown in FIG. 2 (b), a resin packing may be provided around a groove provided on the surface of the

presser members

4 and 5 with nozzles. There is no particular limitation on the configuration.

The

presser members

4 and 5 with nozzles may be directly fixed to the main body 3 (not shown). The pressing member with the nozzle and the main body can be fixed by, for example, providing a screw on the surface where the pressing member with the nozzle and the main body come into contact. In this case, the

caps

6 and 7 shown in FIG.

In the container 2, the cell separation material 11 is stacked and filled. For example, by using a filter in which two or more layers of cell separation materials 11 having different fiber diameters are stacked, the locations for capturing cells are dispersed, clogging is suppressed, and separation and collection of cells from the filter are also possible. Can be done efficiently. Note that a portion in which cell separation materials having the same fiber diameter are successively laminated is treated as one layer regardless of the number of laminated cell separation materials.

The container 2 may be provided with a cleaning liquid inlet (not shown) for cleaning non-adherent cells remaining in the cell separation material 11 independently on the liquid inlet 9 side, or the liquid outlet 10. A cell recovery solution inlet for recovering cells captured by the cell separation material independently (for flowing the cell recovery solution in the direction opposite to the flow of the cell-containing solution and the washing solution) may be provided. (Not shown).

The container 2 can be manufactured 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. Metal materials (biocompatible metals, alloys) useful as container materials include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt chromium alloy, and nitride. Examples thereof include titanium-coated stainless steel. Particularly preferred is a material having sterilization resistance, and specific examples include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, and polymethylpentene.

(Uplift)
The filter of the present invention has a raised portion that is raised on at least one direction on the liquid inlet side or the liquid outlet side on the surface of the cell separation material filled in the container.
As shown in FIGS. 2 (b), 3 (a), and 3 (b), the raised portion is a raised portion (12 a, 12 a ′, 12 a ″, 12 b, 12 b ′ on the surface of the cell separation material 11. , 12b ″). More specifically, as shown in FIGS. 3 (a) and 3 (b), in the longitudinal section of the cell separation material 11 parallel to the liquid inlet 9 and the liquid outlet 10 of the filter, the liquid inlet 9 side Or it is the part which rose from the lowest part (part 13 if it is the liquid inlet 9 side) of the surface of the cell separation material 11 on the liquid outlet 10 side, and if it is the liquid inlet 9 side, the apex (14a, 14a) ', 14a''). For example, in FIGS. 2 (b), 3 (a), and 3 (b), there are three ridges: a ridge 12 a having a

vertex

14 a, 12 a ′ having a vertex 14 a ′, and 12 a ″ having a vertex 14 a ″. , And the

recesses

15a and 15a ′ are connected to each other as a boundary.

The recessed part between the said protruding parts means the recessed part between protruding parts. For example, the filter 1 shown in FIG. 2B has three raised

portions

12a, 12a ′, 12a ″ on the surface of the cell separation material 11 on the liquid inlet 9 side, and two recessed

portions

15a, 15a ′ therebetween. In addition, the surface of the cell separation material 11 on the liquid outlet 10 side has three raised

portions

12b, 12b ′, 12b ″, and two

concave portions

15b, 15b ′ therebetween.
The

concave portions

15a and 15b between the raised portions are both valley shapes that are recessed in a curved shape between two substantially mountain-shaped raised portions. In the present invention, the concave portion refers to a concave portion in a state where the concave portion can directly contact the cell-containing liquid introduced into the filter. Therefore, as will be described later, by providing the protrusions 16 on the holding

members

4 and 5 with the nozzles, the protrusions and the surface of the cell separation material are formed in the recessed portions generated by pressing the surface of the cell separation material 11. Does not correspond to the recess between the raised portions.

As a method of forming the above-mentioned bulging part and the concave part between the bulging parts on the surface of the cell separation material, a process in which the bulging part or the concave part between the bulging parts is previously formed on the surface of the cell separation material, for example, embossing, etc. And the like.

In addition, from the viewpoint of easily forming a raised portion or the like without depending on the material of the cell separating material, the distal end surface of the holding member with

nozzles

4 and 5 inserted into the lumen of the main body 3 of the container 2 is shown in FIG. In addition, by providing the

protrusions

16a and 16b, the surface of the cell separation material 11 filled in the container 2 can be pressed. And the said

protrusion parts

16a and 16b protrude to the lumen | bore side of the said container 2, and it protrudes by pressing the surface of the upper side or lower part side of the said cell separation material 11 with which the said container 2 is filled. The part can be formed.

The shape of the tip surfaces of the

protrusions

16a and 16b is not particularly limited, and examples thereof include a linear shape, a rod shape, a polygonal shape, an elliptical shape, a circular shape, or a shape obtained by combining two or more of these shapes.
In particular, when at least two protrusions are combined, when the surface of the cell separation material is pressed with the protrusions, distortion occurs between the protrusions, and the recesses between the protrusions are easily formed.
For example, as shown in FIG. 2 (a), the periphery of the cell separation material 11 is pressed into a round shape with a substantially circular protrusion 16a, and further, a rod-shaped protrusion 16b from the periphery to the center at intervals of approximately 120 °. When the pressure is pressed, distortion occurs on the surface of the cell separation material 11 that is not pressed, and a plurality of ridges and the ridges of these ridges are formed as shown in FIGS. 2 (b), 3 (a), and 3 (b). A plurality of substantially valley-shaped recesses are formed therebetween. Specifically, the raised

portions

12a and 12a '' are large raised portions formed on the surface of the cell separation material 11 surrounded by the substantially circular protruding

portions

16a and 16b in three directions, and the protruding portions 12a ′. Is a small raised portion near the center in the downward direction of the liquid inlet 9. Therefore, four raised portions are formed on the surface of the cell separating material 11 on the liquid inlet 9 side. Moreover, since the recessed part 15a arises between the said large protruding part 12a and the small protruding part 12a '(it is the same also between the said protruding parts 12a''and12a'), the cell separation material 11 by the side of the liquid inlet 9 is produced. There are a total of three recesses on the surface of

The area of the front end surface of the protrusion is preferably 30% or less of the surface area of the cell separation material from the viewpoint of facilitating formation of a raised portion, particularly a recessed portion between raised portions.
The area of the tip surface of the protrusion may be calculated by measuring using a ruler or the like, and the vertical length (y) and horizontal length (x) in the cross-sectional area of the tip surface are measured. The origin (0, 0) for determining the approximate area may be calculated by measuring a plurality of points other than the origin and performing integral calculation using Excel.

As shown in FIG. 5, the tip surface of the protrusion may be adjusted so that the heights of all the protrusions coincide with each other, or the tip surface may be adjusted to change the height of the protrusion. You may adjust so that there may be a three-dimensional level | step difference.

In the present invention, the height y and the length x of at least one raised portion among the raised portions are represented by the formula (1) and / or the formula (2):
y = −ax ² + bx Formula (1)
y = ax ² −bx Formula (2)
And the ratio of a and b (a / b) is in the range of 0.03 to 0.25.
Note that a and b are both constants and are numerical values exceeding 0.

The expression (1) is an expression showing the outer shape of a substantially parabolic ridge on the upper surface in a longitudinal section parallel to the vertical direction of the cell separation material.
Moreover, said Formula (2) is a type | formula which shows the external shape of the substantially parabolic bulge part in a lower surface in the longitudinal cross section parallel to the up-down direction of a cell separation material.
Therefore, the filter of the present invention is a filter in which the outer shape of at least one raised portion of the raised portions formed on either of the upper and lower surfaces of the cell separation material satisfies the formula (1) and / or the formula (2). Say.

The position of the longitudinal section for confirming the outer shape of the raised portion is not particularly limited as long as a substantially parabolic section can be confirmed.
The height and length of the cross section of the raised portion shown in the longitudinal section are the height y of the raised portion and the length x of the raised portion, respectively. For both y and x, the lowest position of the raised portion is set as the origin (0, 0). For y, the upward height is indicated by “+”, and the downward height is indicated by “−”.

The a is an index indicating the size of the bulge in the bulge that satisfies the formula (1) or the formula (2), and the higher the value of the a, the higher the bulge is in the upward or downward direction. Become. On the other hand, the smaller the value of a, the lower the bulge is in the upward or downward direction.
From the viewpoint of maintaining the structure and function of the filter itself and performing ideal fluid flow during cell recovery, the a is preferably 0.030 to 0.120, and 0.036 to 0.115. It is more preferable. The b is preferably from 0.3 to 1.5, and more preferably from 0.4 to 1.4.

By defining the origins (0, 0) of y and x in the longitudinal section of the raised portion, measuring a plurality of points other than the origin, and inputting those values into calculation software to calculate approximate curves to calculate a and b The formula (1) or the formula (2) including the above can be calculated.
As the points to be measured, at least 5 points including the origin are preferable.
In addition, what is necessary is just to set the said origin in the lowest position where the protruding part in a longitudinal cross section arises. The origin is determined for each raised portion.

For example, when measuring the outer shape of the upward protruding portion 12a shown in FIG. 4A, the origin O1 is first determined, and then the positions of four points (R1, R2, R3, R4) from the origin O1 are set to R1. It is measured as (y1, x1), R2 (y2, x2), R3 (y3, x3), R4 (y4, x4). Then, the numerical formula (1) can be calculated by inputting these numerical values into spreadsheet software and calculating an approximate curve.

On the other hand, when measuring the outer shape of the downward raised portion 12b shown in FIG. 4B, an origin O2 different from the raised portion 12a is determined, and then four points (R5, R6, R7, R8) from the origin O2. ) Is measured as R5 (-y5, x5), R6 (-y6, x6), R7 (-y7, x7), R8 (-y8, x8).
Then, the numerical formula (2) can be calculated by inputting these numerical values into spreadsheet software and calculating an approximate curve.

In the filter of the present invention, in the above formula (1) or formula (2), the ratio of a and b (a / b) is adjusted to a range of 0.03 to 0.25.
By adjusting the a / b within the above range, there is an effect that the cell collection efficiency is increased.
Regarding the technical relationship between the a / b and the cell recovery rate, the cell separation material has an ideal shape suitable for cell recovery, and has a shape with a plurality of ridges on the surface. It is conceivable that, when the cell is collected, ideal liquid flow is performed, and as a result, the cell collection efficiency is improved.
The a / b is more preferably 0.05 to 0.10, and further preferably 0.06 to 0.093.

In the filter of the present invention, from the viewpoint of increasing cell recovery efficiency, at least one ridge is provided on at least one surface on the liquid inlet side or the liquid outlet side, and at least one concave portion is provided between the ridges. It is preferable.

In the present invention, when the number of the raised portions on the surface of the cell separating material is n and the number of the recessed portions between the raised portions is m, the following (c) or (d):
(C) n = m + 1;
(D) n = m + 2;
By satisfying this relationship, cell recovery efficiency can be increased.
Note that n and m are integers, and n ≧ 2 and m ≧ 1.

In addition, the raised part and the recessed part between raised parts said by said Formula (c), (d) are the raised part and recessed part between raised parts seen in the longitudinal cross-section of the cell separation material with which the filter was filled.

Examples of the cell separation material satisfying the relationship of the formula (c) include a cell separation material having a raised portion on one of the liquid inlet side and the liquid outlet side. For example, when the pressing member with nozzle 4 shown in FIG. 5 is used on the liquid inlet side or the liquid outlet side of the filter 1, four raised portions and three recessed portions between the raised portions are generated on the surface of the cell separation material 11. Therefore, the relationship of the formula (c) is satisfied.
As a cell separation material of another form satisfying the relationship of the formula (c), for example, a case where a nozzle having a different number of protrusions 16b on the holding member 4 with nozzle from the number shown in FIG. 5 is used. Can be mentioned. For example, when there are two protrusions 16b, there are two oval ridges between the protrusions and one circular ridge at the center, and there is a recess between the oval ridge and the circular ridge. Since there is one, three raised portions and two recessed portions between the raised portions are generated on the surface of the cell separation material.
Further, when there are four protrusions 16b, there are four elliptical bulges between the protrusions and a circular bulge at the center, and one recess between the elliptical bulge and the circular bulge. For this reason, five raised portions and four recessed portions between the raised portions are formed on the surface of the cell separation material.

Examples of the cell separation material that satisfies the relationship of the formula (d) include cell separation materials that have raised portions on both the liquid inlet side and the liquid outlet side. For example, when the pressing member with nozzle 4 shown in FIG. 5 is used on the liquid inlet side and the liquid outlet side of the filter 1, the raised portions and the recessed portions between the raised portions are formed on the upper and lower surfaces. The cell separation material has eight ridges and six ridges between the raised portions, and satisfies the relationship of the above formula (d).
Examples of the cell separation material in another form that satisfies the relationship of the formula (d) include, for example, a case where a nozzle different from the number of the protrusions 16b illustrated in FIG. For example, when the pressing member with nozzle 4 having two protrusions 16b is used, six ridges and four recesses between the ridges are formed on the surfaces of the upper and lower cell separation materials, and there are four protrusions 16b. In the case where the pressing member with nozzle 4 is used, 10 raised portions and 8 recessed portions between raised portions are generated on the surface of the cell separating material.

In the conventional cell separation filter, the surface of the cell separation material to be filled is generally suppressed in a substantially flat shape with respect to the vertical direction by a lid, a partition wall or the like. Therefore, the raised portions, especially the recessed portions between the raised portions, have a shape that cannot be seen on the surface of the cell separation material filled in the conventional filter.

In the filter of the present invention, the minimum thickness of the cell separation material is preferably 1.20 × 10 to 1.26 × 10 mm. Further, the maximum value of the thickness of the cell separation material is preferably 1.26 × 10 to 2.10 × 10 mm, and more preferably 1.55 × 10 to 2.05 × 10 mm.

In the filter of the present invention, with respect to the maximum value of the thickness of the cell separating material, for example, in FIG. 2B, the thickness of the cell separating material including the raised

portions

12a and 12b is the thickness of the cell separating material including the other raised portions. When larger, this thickness X becomes the maximum value of the cell separation material 11.
Further, the minimum value of the thickness of the cell separating material 11 is, for example, the thickness Y between the surfaces of the cell separating material 11 in the portion pressed by the protrusion 16a in FIG.
Since the raised portion on the surface of the cell separation material 11 exists in a three-dimensional shape, the longitudinal section of the cell separation material 11 is observed at various angles with respect to the center of the cell separation material, What is necessary is just to measure the maximum thickness and the minimum thickness.
The longitudinal section of the filter is observed by imaging with X-ray CT, and the length of each part of the longitudinal section of the cell separation material 11 is determined by CT data viewer (product name “myVGL2” manufactured by Volume Graphics). .2 ").

In the filter of the present invention, the sum of the cross-sectional areas of the raised portions is 2.08 × 10 to 1.09 × 10 ² mm ² , and the cross-sectional area other than the raised portions is 5.40 × 10 ² to 5.61 ×. By setting it to 10 ² mm ² , there is an advantage that it is possible to adjust the outer shape of the raised portion more suitable for cell recovery.

The cross-sectional area of the cell separation material refers to a vertical cross-sectional area when the cell separation material is cut in parallel to the vertical direction of the container.
The cross-sectional area of the raised portion refers to a cross-sectional area when the cell separation material is cut in the vertical direction at a portion including a position where the height of the raised portion is maximum with respect to the upward or downward direction. In addition, the cross-sectional area other than the raised portion refers to an area obtained by subtracting the cross-sectional area of the raised portion from the total cross-sectional area measured by cutting the cell separating material in the vertical direction as described above.
For example, FIG. 6 shows a longitudinal section of the cell separation material 11 of the filter 1 shown in FIG.
The cross-sectional area of the cell separation material is all the cross-sectional areas (α + β + α ′) shown in the figure.
On the other hand, regarding the cross-sectional area of the raised portion, there are a cross-sectional area α on the liquid inlet 9 side and a cross-sectional area α ′ on the liquid outlet 10 side, and the sum (α + α ′) thereof becomes the cross-sectional area of the raised portion.
And the remaining cross-sectional area ((beta)) which pulled the cross-sectional area of the protruding part from the cross-sectional area of the said cell separation material becomes cross-sectional area other than a protruding part.
The ratio (α + α ′) / β is more preferably adjusted to a range of 0.23 to 0.42.

(cell)
The cells targeted in the present invention may be white blood cells or mononuclear cells, and are 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 and other multipotent biological stem cells, T cells, B cells, killer T cells (cytotoxic T cells) ), Lymphocyte cells such as NK cells, NKT cells, regulatory T cells, macrophages, monocytes, dendritic cells, granulocytes, erythrocytes, platelets, etc., nerve cells, muscle cells, fibroblasts, hepatocytes, Examples include somatic cells such as cardiomyocytes or cells that have undergone treatment such as gene introduction or differentiation.

(Cell separation method)
As described above, the filter of the present invention is suitably used for collecting various cells, particularly leukocytes, hematopoietic stem cells and / or mononuclear cells.
Examples of leukocytes include granulocytes such as neutrophils, eosinophils and basophils in peripheral blood, and mononuclear cells such as monocytes and lymphocytes.

As a cell separation method using the filter, a cell-containing liquid is introduced from the liquid inlet of the filter and brought into contact with a cell separation material filled in the filter, whereby leukocytes and / or mononuclear cells are separated from the cell separation material. A first step to be captured by, and
Examples include a method including a second step of introducing a collection solution into the filter and collecting white blood cells and / or mononuclear cells from the cell separation material.

The cell-containing liquid can be used without particular limitation as long as it is a suspension containing cells containing the leukocytes, hematopoietic stem cells and / or mononuclear cells. For example, biological tissue such as the umbilical cord can be treated with an enzyme. Suspension, blood and bone marrow fluid, umbilical cord blood and other body fluids, blood and bone marrow fluid after crushing treatment, extraction treatment, decomposition treatment, ultrasonic treatment, etc., density gradient centrifugation treatment, filtration treatment, enzyme treatment and degradation treatment And suspensions prepared by pretreatment such as sonication and so on.

Further, it may be a suspension obtained by culturing or proliferating cells such as leukocytes exemplified above using a culture solution or a stimulating factor in vitro.

In the first step, when the cell-containing liquid is introduced from the liquid inlet of the filter, the cell-containing liquid is introduced into the filter by pressurizing the cell-containing liquid, By contacting with the cell separation material filled in the filter, leukocytes and / or mononuclear cells are captured by the cell separation material. The degree of pressurization is not particularly limited.

As the cell separation material, a material capable of capturing leukocytes and / or mononuclear cells may be used.
In addition, blood components other than leukocytes may be removed as unnecessary components or may be collected as necessary, and there is no particular limitation.

In the second step, the collection liquid is introduced into the filter that has captured white blood cells, etc., so that the white blood cells and the like are separated from the filter into the collection liquid, and the collection liquid containing the white blood cells and the like is collected from the liquid inlet. By introducing it into a bag or the like, white blood cells and the like can be collected.
The recovered liquid is introduced into the filter from the liquid outlet of the filter.

The recovered solution is not particularly limited as long as it is a solution that is isotonic with cells, and examples include those that have been used as injection solvents such as physiological saline and Ringer's solution, buffers, and culture media for cell culture. . In particular, a medium that can be cultured as it is after passing through the culture step is preferable, and when it is used as it is for the treatment without passing through the culture step, safety such as an isotonic solution that has been used for infusions such as physiological saline is guaranteed. It is preferable to use the recovered liquid.

In the present invention, the first step and the second step may be performed at the room temperature or may be performed at a refrigeration temperature. When performing under refrigeration temperature, processing the refrigerated cell containing liquid is mentioned. Examples of storage of the cell-containing liquid include storage by a refrigerator set at a refrigeration temperature, storage by a water bath, storage by dry ice, and the like. It is preferable to store in a refrigerator because of its versatility. The refrigeration 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 die, and if the storage temperature exceeds 6 ° C, the bacteria may propagate and cause contamination.

In addition, from the viewpoint of adjusting the state of the cell separation material and facilitating the capture of cells, before the first step, a physiological saline or buffer solution is introduced from the liquid inlet of the filter, and the cell separation material and the physiological Saline or buffer may be contacted.

In addition, after the first step and before the second step, physiological saline or a buffer solution is introduced from the liquid inlet of the filter and led out from the liquid outlet of the filter, thereby By removing the contaminating components, unnecessary components can be reduced in the collected cells.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

(Example 1)
As shown in FIG. 7, 112 cylindrical non-woven fabrics (cell separators 11) made of polybutylene terephthalate are filled in a cylindrical container body 3 made of polycarbonate having a diameter of 52 mm in a stacked state. The structure shown in FIGS. 1, 2 (a) and 2 (b) is obtained by inserting the

presser members

4 and 5 shown in FIG. 5 into the upper and lower openings and screwing them with

caps

6 and 7 from above. The filter 1 which has was produced.

In the filter 1, the protrusions 16b of the two upper and lower

nozzle pressing members

4, 5 are aligned, and the thickness of the nonwoven fabric pressed by the tip surface of the protrusion 16b is the minimum value of the cell separation material. I did it. In addition, the distance between the tip surfaces of the upper and lower protrusions 16b was adjusted to 12 mm (the minimum value of the thickness of the cell separation material).

When the vertical cross section of the cell separation material 11 was observed at a position including the cross section of the protrusion 16b of the holding members with

nozzles

4 and 5, as shown in FIG. 8, the raised

portions

12a, 12a ′, The raised

portions

12b and 12b ′ and the recessed portion 15b were confirmed on the surface of the recessed portion 15a and the liquid outlet port side. The raised portions 12 a ′ and 12 b ′ are both between the protruding portions 16 b and are large raised portions having a substantially elliptical shape when viewed from the upper surface. The raised

portions

12 a and 12 b are both of the cell separating material 11. It was near the center and was a small bulge with a substantially circular shape when viewed from above. Therefore, there were a total of 8 raised parts and 6 recessed parts between raised parts.
As a result of observing the longitudinal section of the cell separating material 11 from various other angles, the thickness of the cell separating material was maximum in this section, and the height of the raised portion was higher.
Next, the origins O1, O2, O3, and O4 of the respective heights and lengths of the four raised portions (12a, 12a ′, 12b, and 12b ′) are determined, and the raised portions at a plurality of points are determined from the respective origins. Height and length were measured. The results are shown in Table 1.

Using the spreadsheet function of Microsoft's spreadsheet software “Excel”, the approximate curve was calculated by selecting “secondary” in “polynomial approximation” for the above ridges. .
Upper raised portion 12a: y = −0.0865x ² + 1.051x
Upper raised portion 12a ′: y = −0.1153x ² + 1.231x
Lower raised portion 12b: y = 0.0729x ² −0.9183x
Lower ridge 12b ′: y = 0.0473x ² −0.8018x

Further, the position of the longitudinal section to be observed is rotated by about 3 ° from the center of the cell separation material, and the longitudinal section including the raised portion on the upper surface next to the protruding portion 16b is observed. The origin was determined, and the height and length of the raised portions at a plurality of points from the origin were measured. The results are shown in Table 2.

With respect to the outer shape of the raised portion, an approximate curve was calculated using a spreadsheet function of Microsoft software “Excel”, and the result was as follows.
y = −0.0367x ² + 0.4315x

The state of the cell separation material filled in the filter 1 was as follows.

(Thickness of cell separation material)
Maximum thickness of cell separation material in filter (X): 18.46 mm
Minimum thickness (Y) of cell separation material in the filter: 12.09 mm
Thickness of the cell separation material from the concave portion between the raised portions on the liquid inlet side to the concave portion between the raised portions on the liquid outlet side: 13.96 to 16.92 (mm)

(Cross sectional area of cell separation material)
Sum of cross-sectional areas of raised portions: 144.93 to 182.46 mm ²
Cross-sectional area other than the raised portion: 545.86 mm ²

(Example 2)
The filter 1 was produced in the same manner as in Example 1 except that the protrusions 16b of the upper and lower two

presser members

4 and 5 in the filter 1 were not aligned and fixed by rotating by 60 ° from the center. .

Next, the origin of each height and length is determined for arbitrarily selected ridges (12a, 12b) on the upper side and the lower side, and the height and length of the ridges at a plurality of points are measured from the origin. did. The results are shown in Table 3.

Using the spreadsheet function of Microsoft's software “Excel”, the approximate curve was calculated by selecting “second order” in “polynomial approximation” for the above-mentioned outer shape of the raised portion. .
Upper raised portion 12a: y = −0.0387x ² + 0.6829x
Lower raised portion 12b: y = 0.0362x ² −0.4469x

(Example 3)
A filter 1 was produced in the same manner as in Example 1 except that the number of cell separation materials to be laminated was 140. The minimum value of the thickness of the cell separation material was not changed, and the compression was strengthened by changing the number of stacked layers so that the cell separation material was more easily raised.

Then, the origin O of each height and length was determined for the upper raised portion 12a, and the height and length of the raised portions at a plurality of points were measured from the origin. The results are shown in Table 4.

Using the spreadsheet function of Microsoft software “Excel”, the approximate curve of the raised portion was calculated by selecting “secondary” in “polynomial approximation” and found the following.
Upper raised portion 12a: y = −0.1009x ² + 1.4385x

From the results of Examples 1 to 3, regarding the outer shapes of various raised portions on the surface of the cell separation material packed in the filter 1, the outer shape of the upper raised portion is the above formula (1), and the outer shape of the lower raised portion. It can be seen that can be expressed by the above equation (2).

(Comparative Example 1)
Example 1 except that it was pressed in advance so that the height of the laminated state was 12 mm, and was filled with non-woven fabric (112 sheets) in which the surface of the cell separation material was substantially planar and the height of the raised portions was suppressed. Similarly, filter 1 was produced.

Next, the origin of height and length was determined for the lower raised portion 12b, and the height and length of raised portions at a plurality of points were measured from the origin. The results are shown in Table 5.

Using the spreadsheet function of the software “Excel” manufactured by Microsoft Corporation, an approximation curve was calculated by selecting “second order” in “polynomial approximation” for the raised portion, and the results were as follows.
Raised portion 12b: y = 0.163x ² −0.2543x

(Test Example 1)
Next, the circuit shown in FIG. 9 is connected to the liquid inlet 9 and the liquid outlet 10 of the cell separation filter 1 obtained in Examples 1 to 3 and Comparative Example 1 to produce a cell separation device 25. did.

In the circuit shown in FIG. 9, a tube 17a is connected to the liquid inlet 9, and the tube 17a is connected to a means 18 for containing a cell suspension and a means 19 for containing a priming physiological saline. The tube 17b and the tube 17c connected to the means (collection bag) 20 for storing the collected liquid that has passed through the filter and the means 21 for collecting the collected liquid collected in the collection bag or the like are connected via the flow path switching means 22a. Connected.
The tube 17b was connected with a means 18 for containing a cell suspension and a means 19 for containing a priming physiological saline via a flow path switching means 22b.
The tube 17c is connected to a means 20 for storing the recovered liquid that has passed through the filter and a means 21 for recovering the recovered liquid collected in a recovery bag or the like via a flow path switching means 22c.
Further, a tube 17d is connected to the liquid outlet 10, and a means (waste liquid bag) 23 for containing the cell suspension that has passed through the filter and a means 24 for collecting the recovered liquid are connected via the flow path switching means 22d. Connected.

Next, a cell separation operation was performed using the cell separation device 25. The operation of each flow path switching unit was appropriately performed according to the type of liquid passing through the filter 1 and the target unit for liquid transfer.

First, as means 19 for storing the priming physiological saline, a priming operation is performed by manually passing 150 mL of physiological saline through the filter 1 using a syringe, and the physiological saline that has passed through the filter 1 is passed through the waste liquid bag 23. It was collected.
Next, 100 mL of CPD anticoagulated fresh bovine blood (blood preservation solution C solution (CPD): bovine peripheral blood containing 20% CPD mixed with blood = 1: 4) is filtered from means 18 for containing the cell suspension. Then, the liquid was passed through using gravity, and the leukocytes in the blood were captured by the cell separation material. The passing liquid was collected in the waste liquid bag 23.
Thereafter, a HES (hydroxyethyl starch) solution was manually passed through the liquid outlet 10 into the filter 1 as a means 24 for recovering the recovered liquid, and white blood cells were recovered from the liquid inlet 9 into the recovery bag 20. .
A blood cell counter (trade name “K-4500”, manufactured by Sysmex Corporation) was used for blood count of blood before treatment and blood count of the solution after recovery, and the leukocyte recovery rate was calculated from each measurement result.

In the filters obtained in Examples 1 and 2, cell (leukocyte) recovery was 75% to 82%. In the filter obtained in Example 3, the cell recovery rate was 76%, and no decrease in cell recovery rate (performance) was observed.
On the other hand, in Comparative Example 1, the cell recovery rate was 65%, and a decrease in the cell recovery rate (performance) was observed.

(Test Example 2)
Cell separation materials were taken out from the filters used in Examples 1 and 3 and Comparative Example 1 after white blood cell collection, and placed on white absorbent paper.
As a result, as shown in FIG. 10, the cell separation material obtained in Examples 1 and 3 had almost the original color (white) of the cell separation material, whereas the cell obtained in Comparative Example 1 A large amount of blood-derived components remained from the separating material and stained red.
Therefore, as in the filter 1 used in Examples 1 and 3, a raised portion that protrudes in at least one direction on the liquid inlet side or the liquid outlet side on the surface of the packed cell separation material. It can be seen that the formation and the outer shape of the cell separation material satisfy a predetermined formula can reduce the recovery loss of blood components and can recover the target cells more efficiently.

The cells collected in the present invention can be provided by culturing and proliferating the cells, or can be used without being proliferated, and may be used as therapeutic cells. Specific treatment subjects include ischemic diseases and vascular diseases, but are not limited thereto.

Cells to be transplanted into a cartilage injury patient by differentiation induction with a differentiation inducer or the like, cells to be transplanted into a bone disease patient, cells to be transplanted into a myocardial disease patient or vascular disease patient, neural tissue However, the present invention is not limited to these cells.
As the differentiation inducer, those capable of inducing differentiation of the target cells are preferably used, but as the differentiation inducer for cartilage, dexamethasone, TGFβ, insulin, transferrin, ethanolamine, proline, ascorbic acid, pyruvate Salt, selenium, etc .; bone differentiation inducers include dexamethasone, β-glycerophosphate, vitamin C, ascorbate, etc .; myocardial differentiation inducers include EGF, PDGF, 5-azacytidine Examples of the differentiation inducer to nerve include EGF, bFGF, bHLH and the like; Examples of the differentiation inducer to blood vessel include bFGF, VEGF and the like.

The cells collected or grown according to the present invention may be stored frozen. From the point that damage to cells can be reduced, it is preferably cryopreserved using liquid nitrogen. Alternatively, cryopreserved cells can be thawed and transplanted into humans or animals, used for research, or cultured again.

The pharmaceutical composition can be produced using the cells collected or grown according to the present invention. A pharmaceutical composition can be produced by mixing the cells with a pharmaceutically acceptable additive. Examples of pharmaceutically acceptable additives include coagulants, nutrient sources such as vitamins, and antibiotics.

DESCRIPTION OF SYMBOLS 1 Filter 2 Container 3

Main body

4, 5 Holding member with a

nozzle

6, 7 Cap 8 Seal 9 Liquid inlet 10 Liquid outlet 11

Cell separation material

12a, 12a ', 12b, 12b' Protrusion part 13 Surface of the cell separation material 11

Lowest part

14a, 14a ′, 14a ″ apex of

ridges

15a, 15a ′, 15b, 15b ′ recesses between

ridges

16, 16a, 16b

protrusions

17a, 17b, 17c, 17d tube 18 containing cell suspension Means for carrying out 19 Means for containing physiological saline for priming 20 Means for containing collected liquid that has passed through filter (collection bag)
21 Means for collecting the collected liquid collected in a collection bag, etc. 22a, 22b, 22c, 22d Channel switching means 23 Means for storing the cell suspension that has passed through the filter (waste liquid bag)
24 Means for Collecting Recovery Solution 25 Cell Separation Device

Claims

A filter having a container filled with a cell separation material, having a liquid inlet at either the upper part or the lower part of the container, and a liquid outlet at the opposite side;
The surface of the filled cell separation material has a raised portion that is raised in at least one direction on the liquid inlet side or the liquid outlet side,
The height y and the length x of the raised portion are expressed by the formula (1) and / or the formula (2):
y = −ax 2 + bx Formula (1)
y = ax 2 −bx Formula (2)
And a filter having one or more ridges in which the ratio of a and b (a / b) is in the range of 0.03 to 0.25.
The filter according to claim 1, wherein the surface of the cell separation material has at least two raised portions and at least one concave portion between the raised portions.
When the number of raised portions is n and the number of recessed portions between raised portions is m, the following (c) or (d):
(C) n = m + 1;
(D) n = m + 2;
The filter according to claim 2, satisfying the relationship:
The filter according to any one of claims 1 to 3, wherein a is 0.030 to 0.120 and / or b is 0.3 to 1.5.
The minimum value of the thickness of the cell separation material is 1.20 × 10 to 1.26 × 10 mm, and the maximum value of the thickness of the cell separation material is 1.26 × 10 to 2.10 × 10 mm. The filter according to crab.
The sum of the cross-sectional areas of the raised portions is 2.08 × 10 to 1.09 × 10 2 mm 2 , and the cross-sectional areas other than the raised portions are 5.40 × 10 2 to 5.61 × 10 2 mm 2. The filter according to any one of 5 to 5.
Protruding portions protruding on the inside of the container and capable of pressing the upper or lower surface of the cell separating material incorporated in the container are provided on the upper or lower side inside the container, and the protruding portion The filter according to any one of claims 1 to 6, wherein the tip end surface has an area of 30% or less of the surface area of the cell separation material.
The filter according to claim 7, wherein the shape of the tip surface of the protrusion is linear, rod-shaped, polygonal, elliptical, circular, or a combination of two or more of these shapes.
The filter according to claim 7 or 8, wherein the filter has at least two protrusions.
A cell-containing liquid is introduced from the liquid inlet of the filter according to any one of claims 1 to 9, and is contacted with a cell separation material filled in the filter, whereby leukocytes and / or mononuclear cells are separated from the cell separation material. A first step to be captured by, and
A cell separation method comprising a second step of introducing a collection liquid into the filter and collecting white blood cells and / or mononuclear cells from the cell separation material.
The cell separation method according to claim 10, wherein the second step is a step of introducing a recovery liquid from a liquid outlet of the filter and recovering leukocytes and / or mononuclear cells from the liquid inlet.
Furthermore, before said 1st process, the process which introduce | transduces physiological saline or a buffer solution from the liquid inlet of a filter, and makes a cell separation material and a physiological saline or buffer solution contact is included. The cell separation method according to 1.
Further, after the first step and before the second step, a physiological saline or a buffer solution is introduced from the liquid inlet of the filter and led out from the liquid outlet of the filter. The cell separation method according to any one of claims 10 to 12, further comprising a step of removing a contaminating component.