JP6722023B2 - Cell separation filter and method for producing cell concentrate using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell separation filter and a method for producing a cell concentrate using the same.

With the rapid progress of hematology and scientific technology in recent years, the therapeutic effect can be obtained by separating only necessary blood fractions from body fluids such as whole blood, bone marrow, cord blood, and tissue extracts and administering them to patients. A treatment style in which a side effect is suppressed by increasing the dose and not administering a fraction unnecessary for the treatment is widely used.

For example, blood transfusion is one of them. A red blood cell product is a blood product used when bleeding and deficiency of red blood cells or when oxygen is deficient due to the functional decline of red blood cells. Red blood cell preparations do not require white blood cells that induce side effects such as abnormal immune reactions and graft-versus-host disease (GVHD), and require the removal of white blood cells with a filter. In some cases, platelets may be removed in addition to white blood cells.

On the other hand, the platelet product is a blood product used for patients who have bleeding or tend to bleed 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 necessary platelet components are collected.

In addition, in recent years, hematopoietic stem cell transplantation for treating 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 this hematopoietic stem cell, umbilical cord blood is attracting attention in addition to bone marrow and peripheral blood because of advantages such as low burden on the donor and excellent proliferative ability. In recent years, it has been suggested that stem cells are also abundantly present in menstrual blood, and menstrual blood that has been discarded until now may be used as a valuable source of stem cells.

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

The cell separation filter used for the production of such a cell concentrated solution includes a container provided with a first liquid passage port (for example, an inflow port) and a second liquid passage port (for example, an outflow port), and The filter material (for example, non-woven fabric) filled between the liquid port and the second liquid passage port is provided (see, for example, Patent Document 1).
As a flow system of the cell-containing liquid in the container, a cross flow system in which the flow direction of the supply liquid is orthogonal to the filtration direction of the filter medium (see, for example, FIG. 1 and FIG. 2 of Patent Document 1), the supply liquid There is a dead end flow method (for example, see FIGS. 3 and 4 of Patent Document 1) in which the flow direction is the same as the filtering direction of the filter medium.

JP-A-60-194959

Here, in the cross-flow method, since the flow direction of the supply liquid is orthogonal to the filtration direction, it is difficult to deposit particles on the surface of the filter medium and clogging, and is suitable for concentrating a large amount of sample using the entire surface of the filter medium. However, the recovery rate is low.
Further, in the dead end flow method, since the flow direction of the supply liquid is the same as the filtration direction by the filter medium, particles are likely to be accumulated or clogged on the filter medium surface, which is not suitable for concentrating a large amount of sample. Since the recovery pressure applied to the filter medium can be increased, the recovery rate becomes high.

As described above, the cross flow method and the dead end flow method have advantages and disadvantages, and the flow method in the conventional cell separation filter is either the cross flow method or the dead end flow method.
Therefore, in the cell separation filter in the used state, the flow system is fixed to either the cross flow system or the dead end flow system, so that the flow system suitable for each step in the production of the cell concentrate cannot be selected.

In view of the above situation, the present invention is to solve the problem by providing a cell separation filter that can be used by switching to a flow method suitable for each step in the production of a cell concentrate.

The inventor of the present application has conducted extensive studies to solve the above-mentioned problems based on the actual conditions of use of a cell separation filter in the production of a cell concentrate, and by changing the arrangement of the liquid passage port in the container of the cell separation filter, The idea was to switch between the cross flow method and the dead end flow method. Then, the present invention has been completed by proceeding with examination and concept design based on such an idea.

That is, the gist of the present invention is as follows.
[1] A cell separation filter comprising a container in which a first liquid passage port and a second liquid passage port are arranged, and a filter medium filled between the first liquid passage port and the second liquid passage port. ,
The container is provided with a switching mechanism, the switching mechanism is configured to be able to move the arrangement of the first liquid passage port and/or the second liquid passage port , and the switching mechanism is rotationally moved. Cell separation filter selected from at least one selected from the group consisting of a mobile type, a slide type, and a removable type .
[2] The cell separation filter according to [1], wherein the switching mechanism is provided on a first surface of the container and/or a second surface located in a facing direction of the first surface.
[3] The switching mechanism arranges the first liquid passage port and/or the second liquid passage port on a first surface of the container and/or a second surface positioned in a facing direction of the first surface. The cell separation filter according to the above [1] or [2], which is configured to be movable between the central portion and the outermost portion .
[ 4 ] The first liquid passage and the second liquid passage are provided with a pipe connecting portion having a kink prevention mechanism for the pipe connected to the first liquid passage and the second liquid passage. 1] to the cell separation filter according to any one of [ 3 ].
[ 5 ] The connecting part is a hollow columnar base fixed to the container of the cell separation filter,
A pipe connecting portion that fits with the inner surface or the outer peripheral surface of the base,
The cell separation filter according to the above [ 4 ], which is fixed to the base portion by rotating the pipe connection portion at a predetermined angle of 180° or less in the circumferential direction.
[ 6 ] In the connecting portion, the inner cavity surface of the base portion and the outer peripheral surface of the pipe connecting portion are fitted to each other,
The cell separation filter according to the above [ 5 ], wherein a protrusion is provided on the outer peripheral surface of the pipe connecting portion, and a guide for the protrusion is provided on the inner surface of the base portion.
[ 7 ] In the connecting portion, the outer peripheral surface of the base portion and the inner surface of the pipe connecting portion are fitted to each other,
The cell separation filter according to [ 5 ], wherein a protrusion is provided on the outer peripheral surface of the base portion, and a guide for the protrusion is provided on a side surface of the pipe connecting portion.
[ 8 ] The cell separation filter according to any one of [ 4 ] to [ 7 ], wherein the first liquid passage and the second liquid passage further include a liquid leakage prevention mechanism.
[ 9 ] The cell separation filter according to any one of [1] to [ 8 ], wherein the filter medium is a non-woven fabric.
[ 10 ] The cell separation filter according to any one of [1] to [ 8 ], wherein the filter medium is porous cellulose particles.
[ 11 ] The cell separation filter according to the above [ 10 ], wherein the filter medium is obtained by immobilizing a compound having a log P (P is a partition coefficient in an octanol-water system) value of 2.50 or more on the porous cellulose particles.
[ 12 ] The cell separation filter according to the above [ 10 ], wherein the filter medium comprises a tryptophan derivative and a polyanionic compound immobilized on the porous cellulose particles.
[ 13 ] A contact step of introducing a cell-containing liquid from the first liquid passage port or the second liquid passage port of the cell separation filter according to any one of [1] to [ 12 ] and bringing the liquid into contact with a filter medium, and A method for producing a cell concentrate, comprising a collecting step of collecting the cell concentrate from the first liquid passage port or the second liquid passage port of the cell separation filter .
In [14] the recovery step, the to dead-end flow systems by switching mechanism, according to the above [13] to recover a cell concentrate from said first copies liquid inlet or the second through liquid inlet of the cell separation filter Production method.

According to the cell separation filter of the present invention, since the arrangement of the liquid passage port can be moved by the switching mechanism, it is possible to select a flow method suitable for each step in the production of the cell concentrate.
Further, the method for producing a cell concentrate according to the present invention, by introducing the cell-containing solution into the cell separation filter in a cross-flow method by the switching mechanism of the cell separation filter, the accumulation of particles on the filter medium in the cell separation filter and It is possible to suppress an increase in flow resistance due to clogging. Next, by introducing the cell recovery solution into the cell separation filter by the dead-end flow method by the switching mechanism of the cell separation filter, the pressure applied to the filter medium in the cell separation filter can be increased, and the cells trapped in the filter medium can be increased. Can be efficiently collected.

1A and 1B are schematic views showing a configuration example of a container of a cell separation filter in which a switching mechanism is a rotary movement type, according to Embodiment 1 of the present invention, in which FIG. , (C) is a vertical sectional perspective view in which a dead end flow system is adopted by a switching mechanism, and (d) is a longitudinal sectional perspective view in which a cross flow system is adopted by a switching mechanism. It is the schematic which shows the structural example of the container of the cell separation filter whose switching mechanism based on Embodiment 2 of this invention is a rotation movement type|formula, (a) is a perspective view, (b) is a disassembled perspective view, (c). 6A is a vertical cross-sectional perspective view in which a dead end flow method is used by a switching mechanism, and FIG. 8D is a vertical cross-sectional perspective view in which a cross flow method is used by a switching mechanism. It is the schematic which shows the structural example of the container of the cell separation filter whose switching mechanism based on Embodiment 3 of this invention is a sliding movement type|system|group, (a) is a perspective view, (b) is a disassembled perspective view, (c). 8) is a partial vertical cross-sectional exploded perspective view of the switching mechanism. It is the same schematic diagram, (a) is the vertical section front view which made the dead end flow system by the change mechanism, (b) is the vertical section front view which made the cross flow system by the change mechanism. It is the schematic which shows the structural example of the container of the cell separation filter whose switching mechanism is a sliding movement type|mold which concerns on Embodiment 4 of this invention, (a) is a vertical cross-sectional front view which made the dead end flow system by the switching mechanism. , (B) are vertical cross-sectional front views of a cross-flow system using a switching mechanism. It is the schematic which shows the structural example of the container of the cell separation filter which the switching mechanism based on Embodiment 5 of this invention is a sliding movement type|formula, (a) is a perspective view, (b) is a partial longitudinal cross-section exploded perspective view. , (C) is a vertical sectional perspective view in which a dead end flow system is adopted by a switching mechanism, and (d) is a longitudinal sectional perspective view in which a cross flow system is adopted by a switching mechanism. It is the same schematic diagram, (a) is the vertical section front view which made the dead end flow system by the change mechanism, (b) is the vertical section front view which made the cross flow system by the change mechanism. It is the schematic which shows the structural example of the container of the cell separation filter by which the switching mechanism based on Embodiment 6 of this invention is a removable attachment type|mold, (a) is a perspective view, (b) is a partial longitudinal cross-section exploded perspective view. , (C) is a vertical cross-sectional front view in which a dead end flow method is adopted by a switching mechanism, and (d) is a vertical cross-sectional front view in which a cross flow method is adopted by a switching mechanism. It is the schematic which shows the structural example of the container of the cell separation filter by which the switching mechanism based on Embodiment 7 of this invention is an attachment/detachment movable type, (a) is a perspective view, (b) is a partial longitudinal cross-section exploded perspective view. , (C) is a vertical cross-sectional front view in which a dead end flow method is adopted by a switching mechanism, and (d) is a vertical cross-sectional front view in which a cross flow method is adopted by a switching mechanism. It is the schematic which shows the structural example of the container of the cell separation filter by which the switching mechanism based on Embodiment 8 of this invention is a detachable movement type|mold, (a) is a perspective view, (b) is a partial longitudinal cross-section exploded perspective view. , (C) is a vertical sectional front view in which a dead end flow method is adopted by a switching mechanism, and (b) is a vertical sectional front view in which a cross flow method is adopted by a switching mechanism. It is a schematic block diagram which shows the example of the circuit which isolate|separates a cell closedly using the cell separation filter of this invention. It is the schematic which shows the structural example of the container of the cell separation filter based on Embodiment 9 of this invention, (a) is a perspective view which made the dead end flow system by the switching mechanism, (b) is sectional drawing. It is sectional drawing of the connection part 35 vicinity of the cell separation filter shown in FIG. It is the schematic which shows the structural example of the connection part 35 of the cell separation filter shown in FIG. 12, (a) is an exploded view of the piping connection part 37, (b) is the connection method of the piping connection part 37 and the base part 36. Is a perspective view, and (c) is a perspective view of a cap 41 that is a closing attachment/detachment body. It is the schematic which shows another structural example of the connection part 35 shown in FIG.14(b), (a) is an exploded view of the pipe connection part 37, (b) is the pipe connection part 37 and the guide 39 of multistage shape. FIG. 6 is a perspective view showing how to connect to a base portion 36 provided with. It is the schematic which shows the other example of the connection part 35 shown in FIG.14(b), (a) shows the state in which the taper shape 39a was provided in the guide 39, (b) shows in the edge part of the guide 39. The state where the fixing hole 39b is provided is shown. It is a schematic diagram of the connection part 35 of the cell separation filter shown in FIG. 12, (a) is a perspective view of the connection part 35 provided with the piping 34, (b) shows a state in which the cap 41 is attached to the base part 36. It is a perspective view shown. It is the schematic which shows the other structural example of the connection part 35 based on Embodiment 9 of this invention, (a) is sectional drawing of the connection part 35, (b) is the pipe connection part 37 and the base part 36. It is a perspective view showing a way of connection, and (c) is a sectional view from the axial center direction of base 16 in the state where piping connection part 37 was made to fit in the cavity of base 16. It is the schematic which shows the structural example of the connection part 35 based on Embodiment 10 of this invention, (a) is sectional drawing of the connection part 35, (b) is connection of the pipe connection part 37a and the base part 36a. It is a perspective view showing a way. It is the schematic which shows the other structural example of the connection part 35 based on Embodiment 10 of this invention, (a) is sectional drawing of the connection part 35, (b) shows the pipe connection part 37a and the base part 36a. It is a perspective view showing how to connect. It is a schematic diagram showing an example of composition near the connecting part 35 which provided the liquid leak prevention mechanism 42, (a) is sectional drawing which shows the aspect in which the liquid leak preventive mechanism 42 is provided in the internal cavity of the pipe connection part 37a. Yes, (b) is a sectional view showing a mode in which the liquid leakage prevention mechanism 42 is provided inside the base portion 36a.

Hereinafter, the present invention will be described in detail.
In the attached drawings, a front view is a view of the container of the cell separation filter, viewed from the side with the first surface facing upward and the second surface facing downward.
In addition, the configuration examples of the container 1 of the cell separation filter shown in FIGS. 1 to 10 are all schematic diagrams for explaining the concept of the invention, and the operability and sealing property of the switching mechanism in them are required. The design should be made with appropriate consideration according to the specifications.

<Cell separation filter>
The cell separation filter according to the present invention includes a container in which a first liquid passage opening and a second liquid passage opening are arranged, and a filter medium filled between the first liquid passage opening and the second liquid passage opening. , A switching mechanism for moving the arrangement of the first liquid passage and/or the second liquid passage.

The container used for the cell separation filter can be made using any structural material. The structural material of the container is not particularly limited, and examples thereof include non-reactive polymers, biocompatible metals, alloys, glass and the like.

Non-reactive polymers include acrylonitrile butadiene styrene terpolymers and other acrylonitrile polymers; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides, polyimides. , Polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene, and the like.

Examples of biocompatible metals and alloys include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, gold-plated alloy iron, platinum-plated alloy iron, cobalt chromium alloy, titanium nitride-coated stainless steel, and the like.

From the viewpoint of sterilization resistance, polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene and the like are preferable as the structural material of the container.

In the present invention, the filter material used for the cell separation filter may be a non-woven fabric or a porous carrier.

The material of the nonwoven fabric in the present invention is not particularly limited, from the viewpoint of sterilization resistance and safety to cells, polyethylene terephthalate, polybutylene terephthalate, polyethylene, high density polyethylene, low density polyethylene, polyvinyl alcohol, vinylidene chloride, rayon, Vinylon, polypropylene, acrylic (polymethylmethacrylate, polyhydroxyethylmethacrylate, polyacrylonitrile, polyacrylic acid, polyacrylate), nylon, polyimide, aramid (aromatic polyamide), polyamide, cupra, carbon, phenol, polyester, pulp, Examples include synthetic polymers such as hemp, polyurethane, polystyrene and polycarbonate, natural polymers such as agarose, cellulose, cellulose acetate, chitosan and chitin, and inorganic materials such as glass and metals. Preferred are polyethylene terephthalate, polybutylene terephthalate, polypropylene, acrylic, nylon and polyurethane, which have high cell-capturing ability. More preferred are polyethylene terephthalate, polybutylene terephthalate, and nylon, which have a high ability to capture nucleated cells. In the case of combining two or more kinds of materials to form a fiber, one fiber may be a fiber composed of materials of different components, or a split fiber in which different components are separated and separated. Further, it may be in a form in which fibers made of materials having different components are combined. The term “composite” as used herein is not particularly limited, and examples thereof include a configuration in which two or more kinds of fibers are mixed, a configuration in which individual materials are bonded together, and the like. Furthermore, molecules having an affinity for specific cells such as proteins, peptides, amino acids, and sugars may be immobilized.

In addition, the non-woven fabric may be subjected to a hydrophilic treatment, and the non-specific capture of cells other than nucleated cells is suppressed by the hydrophilic treatment, and the treatment liquid of body fluid or biological tissue passes through the cell separation filter without bias. Therefore, improvement in performance, improvement in recovery rate of necessary cells, and the like can be imparted. As the hydrophilic treatment, a water-soluble polyhydric alcohol, a polymer having a hydroxyl group, a cation group, or an anion group, or a copolymer thereof (eg, hydroxyethyl methacrylate, dimethylaminoethyl methacrylate, or a copolymer thereof) is adsorbed. Method: Adsorption of water-soluble polymer (polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, etc.); Method of immobilizing hydrophilic polymer on hydrophobic membrane; Method of electron irradiation to cell separation filter; Cell in water-containing state Method for cross-linking and insolubilizing a hydrophilic polymer by irradiating a separation filter with radiation; method for sulfonation of the surface of a hydrophobic membrane; method for forming a membrane from a mixture of a hydrophilic polymer and a hydrophobic polymer dope; alkali Method of imparting hydrophilic groups to the membrane surface by aqueous solution treatment (NaOH, KOH, etc.); Method of immersing hydrophobic porous membrane in alcohol, treatment with water-soluble polymer aqueous solution, drying, and insolubilization treatment by heat treatment or radiation. A method of adsorbing a substance having a surface active action, and the like. Examples of the hydrophilic polymer include polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, ethylene-vinyl alcohol copolymer, water-soluble polyhydric alcohol and the like.

The protein fixed to the non-woven fabric is not particularly limited as long as it is a protein having an affinity for specific cells, and specific examples thereof include fibronectin, laminin, vitronectin, collagen and the like. Further, the saccharides to be fixed to the non-woven fabric is not particularly limited as long as it is a saccharide having an affinity for cells, and examples thereof include polysaccharides such as cellulose, chitin and chitosan, and oligosaccharides such as mannose, glucose, galactose and fucose. ..

As the porous carrier in the present invention, cellulose, acetyl cellulose, polysaccharides such as dextrin, and polystyrene, styrene-divinylbenzene copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polyacrylic acid ester, polymethacrylic acid. Included are organic carriers made of synthetic polymers such as esters, polyvinyl alcohol and the like. These have a coating layer consisting of a graft copolymer, such as a hydroxyl group containing polymeric material such as hydroxyethylmethacrylate, a copolymer of polyethylene oxide chain containing monomers and other polymerizing monomers. I don't mind. Of these, synthetic polymers such as cellulose and polyvinyl alcohol are practically preferable because active groups are easily introduced to the surface of the carrier. Porous cellulose particles are particularly preferred.

The porous carrier (particularly porous cellulose particles) is preferably formed by immobilizing a compound having a log P (P is a partition coefficient in an octanol-water system) value of 2.50 or more.
Examples of compounds having a logP value of 2.50 or more include tryptophan derivatives and polyanionic compounds, and the tryptophan derivatives and polyanionic compounds are preferably immobilized on a porous carrier.

The log P value is a parameter of the hydrophobicity of the compound, and the partition coefficient P in a typical octanol-water system is calculated as follows. First, the compound is dissolved in octanol (or water), an equal amount of water (or octanol) is added thereto, and Griffin flask shaker (Griffin & George Ltd. )) for 30 minutes. After that, the mixture is centrifuged at 2000 rpm for 1 to 2 hours, and each concentration of the compound in the octanol layer and the water layer is measured by various methods such as spectroscopic analysis or GLC. P is obtained by substituting these values into the following equation.

P=Coct/Cw (Coct: compound concentration in octanol layer, Cw: compound concentration in water layer)

The logP values of various compounds have been measured by many researchers so far, and the measured values are summarized by C. Hansch et al. (see “Partition Coordinants and There”). Euges; Chemical Reviews (PARTITION COEFFICIENTS AND THEIR USES; Chemical Reviews, 71, 525, 1971).

For compounds for which actual measured values are not known, R. F. Rekker's book "The Hydrophobic Fragmental Constant (THE HYDROPHOBIC FRAGMENTAL CONSTANT)", Elsevier Scientific The value (Σf) calculated using the hydrophobic fragment constant f shown in Publishing Company Amsterdam (Elsevier Sci. Pub. Com., Amsterdam) (1977) is helpful. The hydrophobic fragment constant is a value showing the hydrophobicity of various fragments determined by performing statistical processing based on many measured values of logP, and the sum of the f values of each fragment constituting the compound is logP. It is reported that the values almost match. In the present invention, the logP value also includes Σf.

(Embodiment 1)
As shown in the perspective view of FIG. 1(a), the cell separation filter according to Embodiment 1 of the present invention is located in a circular first surface 1A and in a facing direction of the first surface (parallel to the first surface). It has a circular second surface 1B, and a side surface 1C which is a cylindrical surface connecting the outermost ends of the first surface 1A and the second surface 1B.
The filter medium is housed in the housing space S in the container 1 shown in the longitudinal sectional perspective views of FIGS. 1(c) and 1(d). The filter medium is not shown in order to make the drawings easier to see.

As shown in the partial perspective exploded view of the longitudinal section of FIG. 1(b) and the longitudinal sectional perspective views of FIGS. 1(c) and 1(d), the first surface 1A of the container 1 is located at a position eccentric from its center. A circular guide hole 4A is formed, and a circular guide hole 4B is formed on the second surface 1B facing the first surface 1A at a position eccentric from the center of the second surface 1B in a direction opposite to the eccentric direction of the guide hole 4A.
Then, the circular guide hole 4A formed in the first surface 1A is engaged with the circular guide groove 2A formed in the disk-shaped rotating body 2, so that the rotating body 2 is attached to the main body of the container 1. It is rotatably supported.
Similarly, the circular guide groove 4A formed in the disk-shaped rotating body 3 engages with the circular guide hole 4B formed in the second surface 1B, so that the rotating body 3 is the main body of the container 1. Rotatably supported with respect to.
Further, since the first liquid passage port 11 is attached to a position near the outer periphery of the rotating body 2 and the second liquid passage port 12 is attached to a position close to the outer periphery of the rotating body 3, the second liquid passage port 12 is rotated with respect to the body of the container 1. The positions of the first liquid passage port 11 and the second liquid passage port 12 move according to the positions where the bodies 2 and 3 are rotated.

In this way, the mechanism that rotatably supports the rotating bodies 2 and 3 and moves the liquid passage ports 11 and 12 constitutes the rotational movement type switching mechanisms A1 and B1.
The cell separation filter provided with such switching mechanisms A1 and B1 moves the liquid passage ports 11 and 12 to the central portions of the first surface 1A and the second surface 1B, respectively, as shown in FIG. 1(c). Therefore, the dead end flow method can be realized. Further, as shown in FIG. 1(d), the cross flow method can be realized by moving the liquid passage ports 11 and 12 to the extreme ends of the first surface 1A and the second surface 1B, respectively. Therefore, the flow system can be easily switched by the switching mechanisms A1 and B1.

(Embodiment 2)
The cell separation filter according to Embodiment 2 of the present invention in the perspective view of FIG. 2A, the exploded perspective view of FIG. 2B, and the longitudinal sectional perspective views of FIGS. Since the same reference numerals as those in the first embodiment indicate the same or corresponding portions, the description of the common portions will be omitted.

As shown in the exploded perspective view of FIG. 2(b) and the longitudinal sectional perspective views of FIGS. 2(c) and (d), the first surface 1A of the container 1 has a pipe line 6A at its center position. A conduit 7A is formed at a position near the outermost end in the radial direction away from, and a support hole 5A is formed at a midpoint between the conduits 6A and 7A. Further, on the second surface 1B facing the first surface 1A, the conduit 6B is located at the center of the second surface 1B and is separated from the center of the conduit 7A in the radial direction, which is the direction opposite to the radial separation direction. The conduit 7B is formed at a position in the vicinity of the extreme end, and the support hole 5B is formed at a midpoint between the conduits 6B and 7B.
Here, the conduit 6A is electrically connected from the first surface 1A to the accommodation space S vertically below the first surface 1A. The conduit 6B is electrically connected from the second surface 1B to the accommodation space S vertically above the second surface 1B.
Here, the conduit 7A is bent from the first surface 1A so as to extend vertically downward with respect to the first surface 1A, and then is bent so as to horizontally extend inward in the radial direction with respect to the first surface 1A. It is conducting up to. The conduit 7B is bent so as to extend vertically upward from the second surface 1B with respect to the second surface 1B and then horizontally inward in the radial direction with respect to the second surface 1B, and is electrically connected to the accommodation space S. There is.

Then, since the spindle 2B at the center of the disk-shaped rotating body 2 is fitted into the support hole 5A formed in the first surface 1A, the rotating body 2 is rotatably supported with respect to the main body of the container 1. ..
Similarly, since the support shaft 5B formed in the second surface 1B is fitted with the support shaft 3B at the center of the disk-shaped rotating body 3, the rotating body 3 is rotatable with respect to the main body of the container 1. Supported.
Therefore, the mechanism for moving the first liquid passage 11 and the second liquid passage 12 in the second embodiment is the rotary movement type switching mechanism A1, B1 similar to that in the first embodiment.

As shown in FIG. 2(c), the cell separation filter including the switching mechanisms A1 and B1 has the first liquid passage port 11 communicating with the conduit 6A at the center position of the first surface 1A, and The dead end flow system is realized by connecting the second liquid passage port 12 to the conduit 6B at the center position of the two surfaces 1B. Further, as shown in FIG. 2(d), the first liquid passage 11 communicates with the conduit 7A at the position near the end of the first surface 1A, and the pipe at the position near the end of the second surface 1B. By connecting the second liquid passage port 12 to the passage 7B, the cross flow method can be realized. Therefore, the flow system can be easily switched by the switching mechanisms A1 and B1.

(Embodiment 3)
Books in the perspective view of FIG. 3A, the exploded perspective view of FIG. 3B, the exploded perspective view of the partial vertical cross section of FIG. 3C, and the front view of the vertical cross section of FIGS. 4A and 4B. In the cell separation filter according to the third embodiment of the invention, the same reference numerals as those in the first embodiment indicate the same or corresponding portions, and thus the description of the common portions will be omitted.

As shown in the exploded perspective view of FIG. 3( b) and the perspective views of FIGS. 4( a) and 4 (b ), the first surface 1</b>A of the container 1 extends radially from its central portion to the outermost end portion. A rectangular accommodation opening 1D that extends to the vicinity is formed, and the second surface 1B facing the first surface 1A extends from the center portion thereof in a radial direction opposite to the radial direction in which the accommodation opening 1D extends and extends to the extreme end. A rectangular accommodation opening 1E that extends to the vicinity of the portion is formed.
The slide guide body 8 having the through holes 8A and 8B formed at the front and rear positions in the longitudinal direction is housed in the housing opening 1D formed in the first surface 1A.
Similarly, in the accommodation opening 1E formed in the second surface 1B, the slide guide body 9 having the through holes 9A and 9B formed at the front and rear positions in the longitudinal direction is accommodated.
Further, the closing slide body 13A, the liquid passage slide body 10A having the first liquid passage port 11, and the closing slide body 13B are slidably supported by the slide guide body 8. Further, the closing slide body 14A, the liquid passage slide body 10B provided with the second liquid passage port 12, and the closing slide body 14B are slidably supported by the slide guide body 9.

Therefore, the first liquid passage port 11 can be moved by sliding the liquid passage slide body 10A having the first liquid passage port 11 along the slide guide body 8 and the second liquid passage port 12 can be moved. The second liquid passage port 12 can be moved by sliding the provided liquid passage slide body 10B along the slide guide body 9.

In this way, the mechanism for slidably supporting the liquid passage slide bodies 10A, 10B and moving the first liquid passage port 11 and the second liquid passage port 12 constitutes a slide movement type switching mechanism A2, B2.
As shown in FIG. 4A, while closing the through hole 8B with the closing slide body 13B, the liquid passage slide body 10A is moved to communicate with the first liquid passage port 11 through the through hole 8A. By closing the hole 9B with the closing slide body 14B and moving the slide body 10B to communicate with the through hole 9A, the dead end flow system is realized. Further, as shown in FIG. 4B, while closing the through hole 8A with the closing slide body 13A, the slide body 10A is moved to communicate with the first liquid passage port 11 through the through hole 8B. By closing the slide body 14A for closing and moving the slide body 10B to communicate with the through hole 9B, the cross flow method is realized. Therefore, the flow system can be easily switched by the slide movement type switching mechanisms A2, B2.

(Embodiment 4)
In the cell separation filter according to the fourth embodiment of the present invention in the vertical cross-sectional front views of FIGS. 5A and 5B, the same reference numerals as those in the third embodiment indicate the same or corresponding portions, and therefore are common. The description of the part is omitted.

As shown in the vertical sectional front views of FIGS. 5A and 5B, the through holes 8A and 8B of the slide guide body 8 communicate with the conduit 6A and the conduit 7A, respectively, and the through holes of the slide guide body 9 are formed. 9A and 9B communicate with the conduit 6B and the conduit 7B, respectively.
A mechanism for moving the first liquid passage port 11 and the second liquid passage port 12 in the fourth embodiment is a slide movement type switching mechanism A2, B2 similar to that in the third embodiment, as shown in FIG. Easy to use the dead-end flow method in which the flow direction of the supply liquid is the same as the filtration direction by the filter medium, and the cross flow system in which the flow direction of the supply liquid is orthogonal to the filtration direction by the filter medium as shown in FIG. 5B. Can be switched to.

(Embodiment 5)
The perspective view of FIG. 6A, the partial exploded perspective view of the vertical section of FIG. 6B, the perspective view of the vertical section of FIGS. 6C and 6D, and the vertical section of FIGS. 7A and 7B. In the cell separation filter according to the fifth embodiment of the present invention in the front view, the same reference numerals as those in the third embodiment indicate the same or corresponding portions, and the description of the common portions will be omitted.

The liquid passage slide body 10A including the first liquid passage port 11 according to the present embodiment also serves as a closing slide body that closes the through holes 8A and 8B shown in the fourth embodiment. 3 has a rectangular shape longer than the liquid passage slide body 10A shown in FIG. 3, is supported slidably in the radial direction in a state of being engaged with the guide groove 1F, and is radial from the central portion of the first surface 1A. The first liquid passage port 11 projects outward through the elongated hole 1H extending to.
Similarly, the liquid passage slide body 10B including the second liquid passage port 12 according to the present embodiment also serves as a closing slide body that closes the through holes 9A and 9B shown in the fourth embodiment. In addition, it has a rectangular shape longer than the liquid passage slide body 10B shown in the third embodiment, and is supported slidably in the radial direction while being engaged with the guide groove 1G. The second liquid passage port 12 projects outward through the elongated hole 1I extending in the radial direction opposite to the radial direction in which the elongated hole 1H extends from the central portion.

The mechanism for moving the first liquid passage port 11 and the second liquid passage port 12 in the fifth embodiment is the sliding movement type switching mechanism A2, B2 similar to those in the third and fourth embodiments. Then, the arrangement of the first liquid passage port 11 can be continuously changed within a range in which the first liquid passage port 11 can move in the long hole 1H, and the second liquid passage port 12 moves in the long hole 1I. The arrangement of the second liquid passage port 12 can be continuously changed within the range that can be achieved.

(Embodiment 6)
8A is a perspective view, FIG. 8B is an exploded perspective view, and FIGS. 8C and 8D are vertical cross-sectional perspective views according to the sixth embodiment of the present invention. Since the same reference numerals as those in the first embodiment indicate the same or corresponding portions, the description of the common portions will be omitted.

As shown in the exploded perspective view of FIG. 8B and the vertical cross-sectional perspective views of FIGS. 8C and 8D, the first surface 1A of the container 1 has the attachment/detachment hole 17A at the center position. An attachment/detachment hole 18A is formed at a position in the vicinity of the outermost end that is radially separated from the container 1. The attachment/detachment hole 17B is formed at the center of the second surface 1B of the container 1 in the radial direction from the center of the attachment/detachment hole 18A. The attachment/detachment hole 18B is formed at a position in the vicinity of the outermost end that is radially away from the center, which is the opposite direction.
Then, one of the liquid passage attachment/detachment body 15A having the first liquid passage port 11 and the closing attachment/detachment body 16A is attached to the attachment/detachment holes 17A and 18A formed in the first surface 1A, respectively. One of the liquid passage attachment/detachment body 15B having the second liquid passage port 12 and the closing attachment/detachment body 16B is attached to the attachment/detachment holes 17B and 18B formed in 1B, respectively.

As described above, the mechanism for replacing the fluid passing removable body 15A and the closing removable body 16A with respect to the removable holes 17A and 18A, which includes the removable holes 17A and 18A and the liquid passing removable body 15A and the closing removable body 16A, is provided. , A detachable and movable switching mechanism A3.
In addition, a mechanism for replacing the liquid passing removable body 15B and the closing removable body 16B with respect to the removable holes 17B and 18B, which includes the removable holes 17B and 18B and the liquid passing removable body 15B and the closing removable body 16B, is attached and detached. A movable switching mechanism B3 is configured.
According to such a switching mechanism A3, B3, as shown in FIG. 8(c), the first liquid passage 11 and the second liquid passage 12 are provided at the central portions of the first surface 1A and the second surface 1B, respectively. The dead-end flow method can be realized by moving it to. Further, as shown in FIG. 8(d), the cross flow method can be realized by moving the first liquid passage port 11 and the second liquid passage port 12 to the extreme ends of the first surface 1A and the second surface 1B, respectively. .. Therefore, the flow system can be easily switched by the detachable moving switching mechanism.

(Embodiment 7)
The cell separation filter according to Embodiment 7 of the present invention in the perspective view of FIG. 9A, the exploded perspective view of FIG. 9B, and the longitudinal sectional perspective views of FIGS. The same reference numerals as in Embodiment 6 indicate the same or corresponding portions, and the description of the common portions will be omitted. The pipelines 6A, 6B, 7A and 7B are similar to those of the second embodiment shown in FIG. 2 and the fourth embodiment shown in FIG.

The mechanism for moving the first liquid passage 11 and the second liquid passage 12 in the seventh embodiment is the detachable movement type switching mechanism A3, B3 similar to the sixth embodiment.
According to such switching mechanisms A3 and B3, as shown in FIG. 9(c), the first liquid passage 11 and the second liquid passage 12 are provided at the central portions of the first surface 1A and the second surface 1B, respectively. The dead-end flow method can be realized by moving it to. Further, as shown in FIG. 9(d), the cross flow system can be realized by moving the first liquid passage port 11 and the second liquid passage port 12 to the extreme ends of the first surface 1A and the second surface 1B, respectively. .. Therefore, the flow system can be easily switched by the detachable moving switching mechanism.
Here, in the state of FIG. 9C, the first liquid passage 11 communicates with the conduit 6A at the center of the first surface 1A, and the second passage 6B communicates with the conduit 6B at the center of the second surface 1B. The liquid port 12 communicates.
Further, in the state of FIG. 9D, the first liquid passage 11 communicates with the conduit 7A at the position near the end of the first surface 1A, and the conduit at the position near the end of the second surface 1B. The second liquid passage port 12 communicates with 7B.

(Embodiment 8)
In the cell separation filter according to the eighth embodiment of the present invention in the perspective view of FIG. 10A, the exploded perspective view of FIG. 10B, and the vertical cross-sectional perspective views of FIGS. 10C and 10D. The same reference numerals as in Embodiment 6 indicate the same or corresponding portions, and the description of the common portions will be omitted.
This embodiment eliminates the attachment/detachment hole 18A formed in the first surface 1A and the attachment/detachment hole 18B formed in the second surface in the sixth embodiment, and forms the attachment/detachment hole 19A in the upper portion of the side surface 1C, Positions symmetrical to the attachment/detachment hole 19A on the side surface 1C with reference to a point equidistant from the first surface 1A and the second surface 1B through the centers of the attachment/detachment holes 17A and 17B at the center of the first surface 1A and the second surface 1B. That is, the attachment/detachment hole 19B is formed in the lower portion of the side surface 1C. One of the liquid passage attachment/detachment body 15A having the first liquid passage port 11 and the closing attachment/detachment body 16A is attached to the attachment/detachment hole 17A formed in the first surface 1A and the attachment/detachment hole 19A in the upper portion of the side surface 1C, respectively. Then, one of the liquid passage attachment/detachment body 15B having the second liquid passage port 12 and the closing attachment/detachment body 16B are attached to the attachment/detachment holes 17B and 18B formed in the second surface 1B.

In the present embodiment, a mechanism that replaces the liquid passage attachment/detachment body 15A having the first liquid passage port 11 and the closing attachment/detachment body 16A with respect to the attachment/detachment holes 17A and 19A constitutes the detachable movement type switching mechanism A3. ..
In addition, a mechanism for replacing the fluid passing attachment/detachment body 15B and the closing attachment/detachment body 16B with respect to the attachment/detachment holes 17B, 19B, which includes the attachment/detachment holes 17B, 19B, and the fluid passage attachment/detachment body 15B and the closing attachment/detachment body 16B, A movable switching mechanism B3 is configured.
According to such switching mechanisms A3 and B3, as shown in FIG. 9(c), the first liquid passage 11 and the second liquid passage 12 are provided at the central portions of the first surface 1A and the second surface 1B, respectively. The dead-end flow method can be realized by moving it to. Further, as shown in FIG. 9(d), the cross flow system can be realized by moving the first liquid passage port 11 and the second liquid passage port 12 to the extreme ends of the first surface 1A and the second surface 1B, respectively. .. Therefore, the flow system can be easily switched by the detachable moving switching mechanism.

In the cell separation filters according to the first to eighth embodiments, the first liquid passage port 11 and the second liquid passage port 12 are connected to the first liquid passage port 11 and the second liquid passage port 12 by a pipe 34. The connecting portion 35 of the pipe 34 having the kink prevention mechanism may be provided.

(Embodiment 9)
For example, as shown in the perspective view of FIG. 12( a ), the sectional view of FIG. 12( b ), and the sectional view of FIG. 13, the connecting portion 35 is fixed to the container 1 of the cell separation filter and is hollow. A columnar base portion 36 and a pipe connecting portion 37 that fits with the inner cavity surface of the base portion 36 are provided.

As shown in the perspective view of FIG. 14( b ), the base portion 36 is in a state in which the peripheral edges of the attachment/detachment holes 17 A, 17 B, 18 A, 18 B, 19 A, 19 B of the container 1 are bulged in a thick tubular shape. Alternatively, hollow columnar members may be attached to the attachment/detachment holes 17A, 17B, 18A, 18B, 19A and 19B. A guide 39 for inserting the protrusion 38 provided on the pipe connecting portion 37 is provided on the inner surface of the base portion 36.

For example, the pipe connection part 37 includes a first liquid passage port 11 (or a second liquid passage port 12) as shown in the exploded perspective view of FIG. 14A and the perspective view of FIG. 14B. It is a tubular member. A protrusion 38 is provided on the outer peripheral surface 37a of the end opposite to the first liquid passage 11 (or the second liquid passage 12) so as not to come off the pipe connection portion 37. ing.
Further, as shown in FIGS. 15(a) and 15(b), the guide 39 provided on the base portion 36 may have multiple stages. By using the multi-stage guide 39, the fixing strength between the base portion 36 and the protrusion portion 38 can be increased, and it is possible to sufficiently cope with the change in pressure generated when the liquid is introduced into the cell separation filter, and to recover the cells. Can be suitably performed. The multi-stepped shape is not particularly limited as long as two or more steps are provided so that the protrusion 38 inserted to the end of the guide 39 does not easily return to the inlet of the guide 39.
Further, as shown in the perspective view of FIG. 14B, the guide 39 of the base portion 36 may have a shape in which the inner cavity surface and the outer peripheral surface of the base portion 36 penetrate.

As a method of fixing the pipe connecting portion 37 to the base portion 36, for example, the end portion of the pipe connecting portion 37 on the side of the protruding portion 38 is put into the inner cavity of the base portion 36, and the protruding portion 38 is fixed to the guide 39. The pipe connecting part 37 is fixed to the base part 36 by rotating the pipe connecting part 37 so as to follow (FIG. 17A).
In addition, the width of the guide 39 may be designed in a tapered shape 39a that gradually narrows by the time the pipe connecting portion 37 reaches the position where the pipe connecting portion 37 is fixed to the base portion 36 (FIG. 16A). It is preferable that the protrusion 38 is fixed so as to be removably tightened and fixed so that the protrusion 38 does not easily fall off and can withstand pressure. Further, at a position where the pipe connecting portion 37 is fixed to the base portion 36, a fixing hole 39b having a width wider than the width of the guide is provided at an end portion of the guide 39 so that the fixing position can be confirmed. The protrusion 38 may be designed to be fixed in a possible state (FIG. 16B).
The predetermined angle for rotating the pipe connecting portion 37 may be 180° or less, and is preferably 90° or less, more preferably 45° or less from the viewpoint of the balance between the kink prevention effect and the strength of the base 36. ..
The predetermined angle can be adjusted by the length of a guide 39 provided in the circumferential direction of the base portion 36.

Further, a seal 40 may be provided at an arbitrary position between the pipe connecting portion 37 and the base portion 36 from the viewpoint of preventing liquid leakage. In particular, as the seal 40, a silicone rubber packing or an O-ring can be preferably used.

Further, in the cell separation filter, as shown in FIGS. 12(a), 12(b), 14(c) and 17(b), the closing attachment/detachment body is provided in the base portion 36 of the attachment/detachment hole that does not pass the liquid. The cap 41 can be attached as the. The cap 41 may have the same configuration as the pipe connection part 37, except that the cap 41 closes the first liquid passage port 11 (or the second liquid passage port 12 ).

Further, as shown in the sectional view of FIG. 18A and the perspective view of FIG. 18B, the guide 39 of the base portion 36 may be formed in a groove shape. By using the groove-shaped guide 39, it is possible to increase the strength of the base portion 36 and sufficiently cope with a change in pressure that occurs when a liquid is introduced into the cell separation filter, and it is possible to suitably recover cells. it can.
Further, the width of the groove-shaped guide 39 may be gradually narrowed by the time the pipe connecting portion 37 reaches a position where the pipe connecting portion 37 is fixed to the base portion 36. Alternatively, the height of the groove of the groove-shaped guide 39 may be gradually reduced by the time the pipe connecting portion 37 reaches the position where the pipe connecting portion 37 is fixed to the base portion 36. It is preferable that the protrusion 38 is fixed so as to be removably tightened and fixed so that the protrusion 38 does not easily fall off and can withstand pressure. Further, as shown in FIG. 18C, at the position where the pipe connecting portion 37 is fixed to the base portion 36, the groove is provided at the end of the groove-shaped guide 39 so that the fixing position can be confirmed. It may be designed to have a fixing hole 39b wider than the groove of the groove-shaped guide 39 and wider than the width of the groove-shaped guide 39 so that the protrusion 38 is fixed in a removable state. .. Note that FIG. 18C shows a cross-sectional view from the axial center direction of the base 16 in a state where the pipe connecting portion 37 is fitted in the inner cavity of the base 16.

(Embodiment 10)
As another aspect of the connecting portion 35 of the pipe 34 having the kink prevention mechanism, for example, as shown in the cross-sectional view of FIG. 19(a) and the perspective view of FIG. 19(b), the outer peripheral surface of the base portion 36a, A configuration may be adopted in which the inner surface of the pipe connection portion 37a is fitted. In this structure, the protrusion 38 is provided on the outer peripheral surface of the base 36a. A guide 39 for the protrusion 38 is provided on the side surface of the pipe connecting portion 37a.

Further, the guide 39 provided on the pipe connecting portion 37a may be multistage. By using the multi-stage guide 39, the fixing strength between the pipe connecting portion 37a and the guide 39 can be increased, and it is possible to sufficiently cope with the change in the pressure generated when the liquid is introduced into the cell separation filter, and to recover the cells. Can be suitably performed. The multi-stepped shape is not particularly limited as long as two or more steps are provided so that the protrusion 38 inserted to the end of the guide 39 does not easily return to the inlet of the guide 39.
Further, as shown in the perspective view of FIG. 19B, the guide 39 of the pipe connecting portion 37 may have a shape in which the inner cavity surface and the outer peripheral surface of the pipe connecting portion penetrate.
Further, the width of the guide 39 may be tapered so as to gradually narrow until the pipe connecting portion 37 reaches a position where the pipe connecting portion 37 is fixed to the base portion 36a. It is preferable that the protrusion 38a is fixed so as to be removably tightened and fixed so that it does not easily fall off and can withstand pressure. Further, at the position where the pipe connecting portion 37a is fixed to the base portion 36a so that the fixed position can be confirmed, the guide 39 has a fixing hole having a width larger than the width of the guide, and is in a removable state. The protrusion 38a may be designed to be fixed.

The predetermined angle for rotating the pipe connecting portion 37a may be 180° or less, and is preferably 90° or less, more preferably 45° or less from the viewpoint of the balance between the kink prevention effect and the strength of the base 36.
The predetermined angle can be adjusted by the length of the guide 39 provided on the pipe connecting portion 37a.
Further, by closing the first liquid passage port 11 (or the second liquid passage port 12) of the pipe connection portion 37a, it is possible to provide a closing attachment/detachment body.

In the cell separation filter according to the tenth embodiment, as shown in the cross-sectional view of FIG. 20(a) and the perspective view of FIG. 20(b), the protrusion 38a and the base 36a are formed on the inner surface of the pipe connecting portion 37a. A groove-shaped guide 39 may be provided on the outer peripheral surface.
Further, the width of the groove-shaped guide 39 may be tapered so as to be gradually narrowed by the time the pipe connecting portion 37 reaches a position where the pipe connecting portion 37 is fixed to the base portion 36. Alternatively, the groove height of the groove-shaped guide 39 may be gradually reduced by the time the pipe connecting portion 37 reaches a position where the pipe connecting portion 37 is fixed to the base portion 36. It is preferable that the protrusion 38 is fixed so as to be removably tightened and fixed so that the protrusion 38 does not easily fall off and can withstand pressure. In addition, at the position where the pipe connecting portion 37 is fixed to the base portion 36, the end portion of the groove-shaped guide 39 is wider than the width of the groove-shaped guide 39 so that the fixing position can be confirmed. Then, it may be designed such that it has a fixing hole higher than the groove of the groove-shaped guide 39 and the protrusion 38 is fixed in a removable state.

In addition, in the cell separation filters according to the first to tenth embodiments, the first liquid passage port and the second liquid passage port may further include a liquid leakage prevention mechanism. The liquid leakage prevention mechanism is a mechanism for preventing the liquid from flowing out from the inside of the cell separation filter or the pipe 34 when the pipe connection portion 37a is removed.
For example, as shown in the cross-sectional view of FIG. 21A, a liquid leakage prevention mechanism 42 is provided in the inner cavity of the pipe connecting portion 37a having the first liquid passage port 11.
Further, the liquid leakage prevention mechanism 42 may be provided in the inner cavity of the base portion 36a as shown in the sectional view of FIG.
Further, the liquid leakage prevention mechanism 42 may be used in combination at two positions shown in FIGS. 21(a) and 21(b).
Further, the positions of the inner cavity of the pipe connecting portion 37a where the liquid leakage prevention mechanism 42 is provided and the inner cavity of the base portion 36a may be positions other than those shown in FIGS. 21(a) and 21(b). ..
Examples of the liquid leakage prevention mechanism include ball-type, disc-type, swing-type, and other check valves.

<Method for producing cell concentrate>
The method for producing a cell concentrate of the present invention comprises a contact step of introducing a cell-containing solution from the first liquid passage port 11 or the second liquid passage port 12 of the cell separation filter and bringing it into contact with a filter medium, and a cell separation filter SF. A collecting step of collecting the cell concentrate from the first liquid passage port 11 or the second liquid passage port 12.

The cell-containing fluid in the present invention means a body fluid containing nucleated cells such as peripheral blood, bone marrow, umbilical cord blood, menstrual blood and tissue extract, or these body fluids such as physiological saline, calcium ion and magnesium ion. It may be a Ringer's solution containing a valent cation, a medium used for cell culture such as RPMI, MEM, IMEM, DMEM, a solution diluted with a phosphate buffer solution such as PBS, or a cell roughly separated from a body fluid. Also, the animal species is not particularly limited as long as it is a mammal, and examples thereof include humans, cows, mice, rats, pigs, monkeys, dogs, cats and the like. Even when the cell-containing liquid contains an anticoagulant, the type of anticoagulant is not limited, and examples thereof include heparin, low-molecular-weight heparin, fusan (nafomostat methylate), EDTA, ACD (acid-citrate-dextrose) liquid, Examples include citric acid anticoagulant such as CPD (citrate-phosphate-dextrose) solution. Storage conditions are not limited as long as the purpose of use is not affected.

The nucleated cell in the present invention is not particularly limited as long as it is a cell containing a nucleus, for example, mononuclear cell, leukocyte, granulocyte, neutrophil, eosinophil, basophil, erythroblast, bone marrow. Blast cells, promyelocytic cells, myelocytic cells, postmyelocytic cells, lymphocytes, monocytes, macrophages, T lymphocytes, B lymphocytes, NK cells, NK/T cells, dendritic cells, multinucleated giant cells, epithelial cells, endothelium Examples thereof include cells, mesenchymal cells, mesenchymal stem cells, hematopoietic stem cells, ES cells, iPS cells and stem cells.
The cell concentrate in the present invention is a liquid containing nucleated cells, which is a liquid having a larger number of nucleated cells in the liquid or a higher cell density than the liquid containing cells. The stem cells in the present invention refer to cells that are separated from the body fluid and have pluripotency and self-renewal ability.

The mesenchymal stem cells in the present invention include osteoblasts, chondrocytes, vascular endothelial cells, cardiomyocytes, or fat, cementoblasts that are periodontal tissue-constituting cells, and periodontal ligament fibroblasts by the addition of differentiation-inducing factors. Cells that differentiate into etc. Hematopoietic stem cells are cells that differentiate hematopoietic cells such as leukocytes, lymphocytes, neutrophils, eosinophils, basophils, granulocytes, monocytes, macrophages, erythrocytes, platelets, megakaryocytes, and dendritic cells. Is.

The step of recovering the cell concentrate from the cell separation filter in the present invention is a step of collecting a liquid containing a large amount of nucleated cells obtained by passing through the filter, and the cell concentrate may be collected depending on the properties of the cell separation filter. Should be collected.

If the filter medium filled in the filter has an affinity for the cells and contaminants to be removed, the cell-containing liquid is introduced from the inlet of the filter, and after the contact step of contacting the filter medium and the cell-containing liquid, The cell concentrate can be collected from the outlet. When recovering the cell concentrate from the outlet of the filter, it is preferable to introduce the recovery liquid from the inlet of the filter to recover the cell concentrate from the outlet. Examples of the cells to be removed include granulocytes, monocytes, platelets, B lymphocytes, lymphocytes having a specific subclass, and the like. Specifically, a method in which granulocytes are captured and removed by a filter and a mononuclear cell-containing fraction is collected can be mentioned.

If the filter medium filled in the filter has an affinity for the nucleated cells to be collected, introduce the cell-containing liquid from the inlet of the filter and let out the cells and contaminants to be removed from the outlet. After that, the cell concentrate can be collected from the inlet. When recovering the cell concentrate from the inflow port of the filter, it is preferable to introduce the recovery solution from the outflow port of the filter and recover the cell concentrate from the inflow port. Examples of cells to be removed include erythrocytes, T lymphocytes, lymphocytes having a specific subclass, and the like. Specifically, a method of passing unnecessary cells and collecting the stem cells captured by the filter can be mentioned.

Further, when the cell concentrate is recovered by introducing the recovery liquid from the outlet of the filter, the washing liquid is introduced from the inlet of the filter before the collection, so that the cells to be removed can be more efficiently removed. It is possible to let out foreign substances and contaminants from the outlet. The washing solution is not particularly limited as long as it can wash away only the cells and contaminants to be removed, and may be, for example, physiological saline, Ringer's solution, medium used for cell culture, general buffer such as phosphate buffer, or these solutions. Examples include a solution to which serum or protein is added.

The recovery liquid is preferably used for the purpose of recovering the nucleated cells trapped in the filter or the mononuclear cells remaining in the filter. The liquid to be recovered is not particularly limited, and examples thereof include physiological saline, divalent cations such as magnesium ion and calcium ion, saccharides, serum, liquids containing proteins, buffers, media, plasma and liquids containing them. To be In order to increase the recovery rate of the nucleated cells captured by the filter, the recovery solution may have a higher viscosity. The substance added to the recovery solution to increase the viscosity is not particularly limited, and examples thereof include albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, hydroxyl cellulose, collagen, hyaluronic acid, gelatin and the like.

The rate and method of passing the cell-containing liquid and the recovery liquid are not particularly limited, and for example, a method of passing by using gravity, a method of passing through while maintaining a constant flow rate using a roller clamp and a syringe pump, high Examples include a method in which pressure is applied and the solution is passed all at once. For the purpose of recovering the nucleated cells trapped in the filter, a method of applying a high pressure to the recovered solution and passing it at once in view of the efficiency of recovering the nucleated cells is preferable. The method of passing a liquid manually at a flow rate of 90 mL/min or more using a syringe is preferable in that more nucleated cells can be collected.

FIG. 11 shows an example of a circuit for closing cells using the cell separation filter of the present invention in a closed manner.
In the circuit shown in FIG. 11, the container 20 containing the cell-containing liquid has a liquid passage port and is connected to the three-way stopcock 25 through a pipe 28 through which the liquid can pass. Similarly, the container 21 containing the priming liquid also has a liquid passage port, and is connected to the three-way stopcock 25 through a pipe 29 through which the liquid can pass. The three-way stopcock 25 and the three-way stopcock 26 are connected by a pipe 30 through which liquid can pass. The container 22 that stores the cell concentrate has a liquid passage port, and is connected to the three-way stopcock 26 through a pipe 31 through which the liquid can pass. In the cell separation filter of the present invention, the inflow port is connected to the three-way stopcock 27, and the outflow port is connected to the three-way stopcock 26. The container 23 that stores the cell recovery liquid has a liquid passage port, and is connected to the three-way stopcock 27 through a pipe 32 through which the liquid can pass. Similarly, the container 24 for containing the priming liquid and the cell-containing liquid that has passed through the cell separation filter SF has a liquid passage port, and is connected to the three-way stopcock 27 through a liquid passage pipe 33.

A specific procedure for producing a cell concentrate using the circuit shown in FIG. 11 is as follows.
First, in order to remove air in the cell separation filter, improve the cell trapping efficiency, and perform priming for the purpose of ensuring a blood flow path, the priming liquid contained in the container 21 is removed. , Pass through the cell separation filter and flow into the container 24. At this time, the three-way stopcock 25 connects the pipe 29 and the pipe 30 and shuts off the flow to the pipe 28. The three-way stopcock 26 connects the pipe 30 to the cell separation filter and shuts off the flow to the pipe 31. Furthermore, the three-way stopcock 27 connects the cell separation filter and the pipe 33 and shuts off the flow to the pipe 32.

Next, in order to bring the cell-containing liquid into contact with the filter medium in the cell separation filter, the cell-containing liquid contained in the container 20 is passed through the cell separation filter and flown into the container 24. At this time, the three-way stopcock 25 connects the pipe 28 and the pipe 30 and shuts off the flow to the pipe 29. The three-way stopcock 26 connects the pipe 30 to the cell separation filter and shuts off the flow to the pipe 31. Furthermore, the three-way stopcock 27 connects the cell separation filter and the pipe 33 and shuts off the flow to the pipe 32.

Finally, in order to collect the cell concentrated liquid, the cell recovery liquid contained in the container 23 is passed through the cell separation filter and flown into the container 22. At this time, the three-way stopcock 27 electrically connects the cell separation filter and the pipe 32 and blocks the flow to the pipe 33. Further, the three-way stopcock 26 connects the cell separation filter to the pipe 31 and shuts off the flow to the pipe 30.
The circuit preferably comprises a three-way stopcock, roller clamps, clamps, etc. to control the flow of liquid.

By introducing the cell-containing solution into the cell separation filter and bringing it into contact with the filter medium in the cross-flow system by the switching mechanism of the cell separation filter, the flow of the cell-containing solution is The increase in flow resistance due to accumulation and clogging can be moderated, and a large amount of cell-containing liquid can be filtered using the entire surface of the filter medium.
Further, by performing the collecting step for collecting the cell concentrate after the contacting step in a state where the dead end flow system is set by the switching mechanism of the cell separation filter, the flow direction of the collected solution is the same as the filtering direction by the filter medium. Therefore, the cell recovery rate can be improved.

A1, B1 Rotational movement type switching mechanism A2, B2 Sliding movement type switching mechanism A3, B3 Detachable movement type switching mechanism S Storage space SF Cell separation filter 1 Container 1A First surface 1B Second surface 1C Side surface 1D, 1E Storage Openings 1F, 1G Guide grooves 1H, 1I Oblong holes 2, 3 Rotating bodies 2A, 3A Guide grooves 2B, 3B Support shafts 4A, 4B Guide holes 5A, 5B Support holes 6A, 6B, 7A, 7B Pipe lines 8, 9 Slide guides Body 8A, 8B, 9A, 9B Through hole 10A, 10B Liquid passing slide body 11 First liquid passing port 12 Second liquid passing port 13A, 13B, 14A, 14B Closing slide body 15A, 15B Liquid passing removable body 16A , 16B Closure attachable/detachable body 17A, 17B, 18A, 18B, 19A, 19B Attachment/detachment hole 20 Container containing cell-containing liquid 21 Container containing priming liquid 22 Container containing cell concentrated liquid 23 Cell recovery liquid contained A container 24 for storing a priming liquid and a cell-containing liquid that have passed through a cell separation filter 25, 26, 27 Three-way stopcocks 28 20 and 25 connected to liquid-permeable pipes 29 21 and 25 Liquid-permeable pipes 30 25 and 26 connected to liquid-permeable pipes 31 23 and 27 connected to liquid-permeable pipes 32 24 and 27 connected to liquid-permeable pipes 34 Piping 35 connected to the first liquid passage 11 and the second liquid passage 12 Connection portions 36, 36a of the pipe 34 having a kink prevention mechanism Base portions 37, 37a Pipe connection portions 38, 38a Projection portion 39 Guide 39a Tapered guide 39b Guide fixing hole 40 Seal 41 Cap 42 Liquid leakage prevention mechanism

Claims (14)

A container in which a first liquid passage and a second liquid passage are arranged, and a cell separation filter comprising a filter medium filled between the first liquid passage and the second liquid passage,
The container is provided with a switching mechanism, the switching mechanism is configured to be able to move the arrangement of the first liquid passage port and/or the second liquid passage port , and the switching mechanism is rotationally moved. Cell separation filter selected from at least one selected from the group consisting of a mobile type, a slide type, and a removable type . The cell separation filter according to claim 1, wherein the switching mechanism is provided on a first surface of the container and/or a second surface located opposite to the first surface. The switching mechanism arranges the first liquid passage opening and/or the second liquid passage opening from a central portion of a first surface of the container and/or a second surface located in a facing direction of the first surface. The cell separation filter according to claim 1 or 2, which is configured to be movable between the outermost ends . The first copies liquid inlet and a second through fluid port, claim and a connecting portion of the pipe with a kink prevention mechanism of the piping connected to the first copies liquid inlet and a second through fluid port 1-3 The cell separation filter according to any one of 1. The connecting portion is fixed to the container of the cell separation filter, a hollow columnar base,
A pipe connecting portion that fits with the inner surface or the outer peripheral surface of the base,
The cell separation filter according to claim 4 , wherein the pipe connection part is fixed to the base part by rotating the pipe connection part at a predetermined angle of 180° or less in the circumferential direction. In the connecting portion, the inner surface of the base portion and the outer peripheral surface of the pipe connecting portion are fitted together,
The cell separation filter according to claim 5 , wherein a protrusion is provided on an outer peripheral surface of the pipe connecting portion, and a guide for the protrusion is provided on an inner surface of the base portion. In the connecting portion, the outer peripheral surface of the base portion and the inner surface of the pipe connecting portion are fitted,
The cell separation filter according to claim 5 , wherein a protrusion is provided on an outer peripheral surface of the base portion, and a guide for the protrusion is provided on a side surface of the pipe connecting portion. The cell separation filter according to any one of claims 4 to 7 , wherein the first liquid passage port and the second liquid passage port further include a liquid leakage prevention mechanism. Cell separation filter according to any of claims 1-8 wherein the filter media is a nonwoven fabric. Cell separation filter according to any of claims 1-8 wherein the filter media is a porous cellulose particles. The cell separation filter according to claim 10 , wherein the filter medium is obtained by immobilizing a compound having a log P (P is a partition coefficient in an octanol-water system) value of 2.50 or more on the porous cellulose particles. The cell separation filter according to claim 10 , wherein the filter medium comprises a tryptophan derivative and a polyanionic compound immobilized on the porous cellulose particles. A contact step of introducing a cell-containing liquid from the first liquid passage port or the second liquid passage port of the cell separation filter according to any one of claims 1 to 13 and contacting with a filter medium, and the cell separation filter of the above. A method for producing a cell concentrate, comprising a collecting step of collecting the cell concentrate from the first liquid passage or the second liquid passage . The manufacturing method according to claim 13 , wherein in the collecting step, a dead end flow system is used by the switching mechanism to collect the cell concentrate from the first liquid passage port or the second liquid passage port of the cell separation filter.

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