JP6788370B2 - A connector for a cell separation device, a cell separation filter with the connector, and a method for producing a cell concentrate using the cell separation filter. - Google Patents

Description

The present invention relates to a connector for a cell separation device, a cell separation filter having the connector, and a method for producing a cell concentrate using the cell separation filter.

In recent years, with the rapid progress of hematology and scientific technology, the therapeutic effect is achieved by separating only the necessary blood fraction from body fluids such as whole blood, bone marrow, umbilical cord blood, and tissue extracts and administering it to patients. 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. Erythrocyte preparations are blood products used when bleeding and erythrocyte deficiency, or when erythrocyte dysfunction causes oxygen deficiency. Erythrocyte preparations do not require leukocytes that induce side effects such as an abnormal immune response or graft-versus-host disease (GVHD), and need to be filtered to remove leukocytes. In some cases, platelets may be removed in addition to white blood cells.

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

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

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

The cell separation filter used for producing a cell concentrate such as these includes a container provided with a first inlet (for example, an inlet) and a second inlet (for example, an outlet), and the first inlet. A filter medium (for example, a non-woven fabric) filled between the liquid port and the second liquid passing port is provided (see, for example, Patent Document 1).
As the flow method of the cell-containing liquid in the container, a cross flow method in which the flow direction of the supply liquid is orthogonal to the filtration direction by the filter medium (see, for example, FIGS. 1 and 2 of Patent Document 1) and the supply liquid There is a dead-end flow method (see, for example, FIGS. 3 and 4 of Patent Document 1) in which the flow direction of the filter medium is the same as the filtration direction by the filter medium.

Japanese Unexamined Patent Publication No. 60-194959

Here, the cross-flow method is suitable for concentrating a large amount of sample using the entire surface of the filter medium because the flow direction of the supply liquid is orthogonal to the filtration direction, so that particles are less likely to be deposited or clogged on the surface of the filter medium. However, the recovery rate is low.
Further, the dead end flow method is not suitable for concentrating a large amount of sample because the flow direction of the supply liquid is the same as the filtration direction by the filter medium, so that particles are likely to be deposited or clogged on the surface of the filter medium. 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 method is fixed to either the cross flow method or the dead end flow method, so that the flow method suitable for each step in the production of the cell concentrate cannot be selected.

Therefore, the inventors of the present application have conducted extensive research to solve the above-mentioned problems based on the actual conditions of use of the cell separation filter in the production of the cell concentrate, and as a result, the arrangement of the liquid passage port in the container of the cell separation filter can be changed. By doing so, I got the idea of switching between the cross flow method and the dead end flow method. Then, they have succeeded in completing a cell separation filter having a plurality of liquid passage ports and capable of facilitating switching of the liquid passage ports according to a desired flow method. However, when connecting the pipe to the liquid passage port of the cell separation device such as the cell separation filter or the container for storing blood, infusion, etc., the pipe was connected to the cell separation filter or the container while rotating, so the pipe was kinked. There was a further problem of occurrence.

Therefore, in view of the above-mentioned situation, the present invention tries to solve the problem without kinking the piping even for the liquid passage portion of the cell separation device such as the cell separation filter or the container in the fixed position. The point is to provide a connector that can be easily connected to.
Another object of the present invention is to provide a cell separation filter equipped with the connector and a method for producing a cell concentrate using the same.

That is, the gist of the present invention is as follows.
[1] A connector that is arranged on the outer surface of a cell separation device and is detachably configured at a liquid passage port where the arrangement can be moved .
A base having a liquid passage connecting portion to be connected to the liquid passing port, and
A connector characterized by having a pipe connection portion to which a pipe is connected and a pipe connection body having a rotating portion that is rotatable with respect to the pipe connection portion.
[2] The connector according to the above [1], wherein the base portion and one end side of the pipe connection body are fitted, and the pipe connection portion is provided on the other end side of the pipe connection body.
[3] The connector according to the above [1] or [2], wherein a fitting mechanism for fitting with the liquid passage portion is provided at the liquid passage port connection portion of the base portion.
[4] The connector according to the above [3], wherein a screw thread screwed with a screw groove provided on an inner peripheral surface of the liquid passage port portion is provided on an outer peripheral surface of the liquid passage port connection portion of the base portion.
[5] The connector according to any one of the above [1] to [4], further provided with a leak prevention mechanism for preventing liquid from leaking between the base and the pipe connecting body.
[6] The connector according to the above [5], wherein the leak prevention mechanism is a check valve.
[7] The connector according to any one of [1] to [6] above, wherein the cell separation device is a cell separation filter or a container.
[8] A cell separation filter in which the connector according to any one of [1] to [6] is attached to a liquid passage port.
[9] A cell separation filter provided with 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 cell according to the above [8], wherein the container is provided with a switching mechanism, and 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. Separation filter.
[10] The cell separation filter according to the above [ 9 ], wherein the switching mechanism is provided on the first surface of the container and / or the second surface located in the facing direction of the first surface.
[11] The switching mechanism arranges the first liquid passage port and / or the second liquid passage port on the first surface and / or the second surface of the container located in the facing direction of the first surface. The cell separation filter according to the above [9] or [10], which is configured to be movable from the central portion to the outermost portion.
[12] The cell separation filter according to any one of [9] to [11], wherein the switching mechanism is selected from at least one group consisting of a rotary movement type, a slide movement type, and a detachable movement type.
[13] The cell separation filter according to any one of [9] to [12] above, wherein the filter medium is a non-woven fabric.
[14] The cell separation filter according to any one of [9] to [12] above, wherein the filter medium is porous cellulose particles.
[15] The cell separation filter according to the above [14], wherein the filter medium is formed by immobilizing a compound having a logP (P is an octanol-partition coefficient in an aqueous system) of 2.50 or more on the porous cellulose particles.
[16] The cell separation filter according to the above [14], wherein the filter medium is formed by immobilizing a tryptophan derivative and a polyanionic compound on the porous cellulose particles.
[17] A contact step of introducing a cell-containing liquid from the first passage port or the second passage port of the cell separation filter according to any one of the above [9] to [16] and bringing the cell-containing liquid into contact with the filter medium, and A method for producing a cell concentrate, which comprises a recovery step of recovering the cell concentrate from the first passage port or the second passage port of the cell separation filter.
[18] The production method according to the above [17], wherein in the contact step, a cross-flow method is adopted by the switching mechanism, and the cell-containing liquid is introduced and brought into contact with the filter medium.
[19] In the recovery step, the dead end flow method is adopted by the switching mechanism, and the cell concentrate is recovered from the first liquid passage port or the second liquid passage port of the cell separation filter [17] or [18]. ] The manufacturing method described in.

According to the connector according to the present invention, a cell separation filter having a fixed cell separation filter or a cell separation device such as a container for storing blood, infusion, etc., particularly a cell separation filter having a plurality of liquid passage ports. When switching the flow method, the pipe can be easily connected to the liquid passage portion at a desired position without generating a kink.
Further, according to the cell separation filter according to the present invention provided with the connector, the arrangement of the liquid passage port can be moved by the switching mechanism, so that a flow method suitable for each step in the production of the cell concentrate can be selected. ..
Further, in the method for producing a cell concentrate according to the invention, particles are deposited on the filter medium in the cell separation filter by introducing the cell-containing solution into the cell separation filter by a cross-flow method by a cell separation filter switching mechanism. 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 captured by the filter medium can be increased. Can be efficiently recovered.

It is the schematic which shows the example of the connector of this invention, (a) is the vertical sectional front view which shows the structural example of the connector 40. Further, (b) is a vertical sectional front view showing a configuration example of the cap 52. It is an exploded perspective view of the connector 40 shown in FIG. 1 (a). It is the schematic which shows the other example of the connector of this invention, (a) is the vertical sectional front view which shows the structural example of the connector 40a. Further, (b) is a vertical sectional front view showing a configuration example of the cap 52. It is the schematic which shows the example of the cell separation filter provided with the connector 40 shown in FIG. 1 (a). It is the schematic which shows the example of the cell separation filter provided with the connector 40a shown in FIG. 3A. It is a schematic block diagram which shows the example of the circuit which separates cells closedly using a cell separation filter, and is the schematic block diagram which shows the example which used the connector 40 of this invention in the said circuit. It is the schematic which shows the structural example of the container of the cell separation filter whose switching mechanism is a rotary movement type which concerns on Embodiment 1 of this invention, (a) is a perspective view, (b) is a partial longitudinal sectional decomposition perspective view. , (C) is a vertical cross-sectional perspective view in which the dead end flow method is adopted by the switching mechanism, and (d) is a vertical cross-sectional perspective view in which the cross flow method is adopted by the switching mechanism. FIG. 2 is a schematic view showing a configuration example of a container of a cell separation filter whose switching mechanism is a rotary movement type according to a second embodiment of the present invention, (a) is a perspective view, (b) is an exploded perspective view, and (c) ) Is a vertical cross-sectional perspective view in which the dead end flow method is adopted by the switching mechanism, and (d) is a vertical cross-sectional perspective view in which the cross flow method is adopted by the switching mechanism. It is the schematic which shows the structural example of the container of the cell separation filter whose switching mechanism is a slide movement type which concerns on Embodiment 3 of this invention, (a) is a perspective view, (b) is an exploded perspective view, (c). ) Is a partial vertical cross-sectional exploded perspective view of the switching mechanism. In the schematic view, (a) is a vertical cross-sectional front view in which a dead end flow method is adopted by a switching mechanism, and (b) is a vertical cross-sectional front view in which a cross flow method is adopted by a switching mechanism. FIG. 6A is a schematic view showing a configuration example of a container of a cell separation filter in which the switching mechanism is a slide-moving type according to the fourth embodiment of the present invention, and FIG. , (B) is a front view of a vertical cross section in which a cross-flow method is adopted by a switching mechanism. FIG. 5 is a schematic view showing a configuration example of a container of a cell separation filter in which the switching mechanism is a slide moving type according to the fifth embodiment of the present invention, (a) is a perspective view, and (b) is a partial longitudinal sectional exploded perspective view. , (C) is a vertical cross-sectional perspective view in which the dead end flow method is adopted by the switching mechanism, and (d) is a vertical cross-sectional perspective view in which the cross flow method is adopted by the switching mechanism. In the schematic view, (a) is a vertical cross-sectional front view in which a dead end flow method is adopted by a switching mechanism, and (b) is a vertical cross-sectional front view in which a cross flow method is adopted by a switching mechanism. FIG. 6 is a schematic view showing a configuration example of a container of a cell separation filter in which the switching mechanism is removable and movable according to the sixth embodiment of the present invention, (a) is a perspective view, and (b) is a partial vertical cross-sectional exploded perspective view. , (C) is a vertical cross-sectional front view in which the dead end flow method is adopted by the switching mechanism, and (d) is a vertical cross-sectional front view in which the cross flow method is adopted by the switching mechanism. FIG. 7 is a schematic view showing a configuration example of a container of a cell separation filter in which the switching mechanism is removable and movable according to the seventh embodiment of the present invention, (a) is a perspective view, and (b) is a partial vertical cross-sectional exploded perspective view. , (C) is a vertical cross-sectional front view in which the dead end flow method is adopted by the switching mechanism, and (d) is a vertical cross-sectional front view in which the cross flow method is adopted by the switching mechanism. It is the schematic which shows the structural example of the container of the cell separation filter whose switching mechanism is detachable movement type which concerns on Embodiment 8 of this invention, (a) is a perspective view, (b) is a partial longitudinal cross-sectional exploded perspective view. , (C) is a vertical cross-sectional front view in which the dead end flow method is adopted by the switching mechanism, and (b) is a vertical cross-sectional front view in which the cross flow method is adopted by the switching mechanism.

Hereinafter, the present invention will be described in detail with reference to the drawings.

<Connector>
As shown in FIGS. 1 (a) and 3 (a), the connectors 40 and 40a according to the present invention are configured to be detachably attached to the liquid passage port LO arranged on the outer surface of the cell separation device SE. A base 42 having a liquid passage connecting portion 41 connected to the liquid passing port LO, and
It is characterized by having a pipe connecting portion 44 to which the pipe 43 is connected and a pipe connecting body 46 having a rotating portion 45 that is rotatable with respect to the pipe connecting portion 44.

In the present invention, the cell separation device refers to a device used for separating cells, and is not particularly limited, but is a cell used in a cell separation system such as a cell separation filter, a container, a branch plug, and a connecting tube. It may be a separation device.
For example, the cell separation device also includes a cell separation filter having a plurality of liquid passage ports and a container capable of accommodating blood or infusion so that the flow method can be switched. Further, as shown in FIG. 6, the connector 40 shown in FIG. 1A may be used for the connection portion of the piping between the parts such as the bag and the cell separation filter, or the connector 40a shown in FIG. 3A may be used. You may use it.

In the present invention, the term “detachable” means, for example, the liquid passage port LO arranged on the outer surface of the cell separation device SE so that the attachment / detachment of the pipe 43 can be switched to the cell separation device SE such as a cell separation filter or a container. It means that the connectors 40 and 40a can be attached and detached. By providing the connectors 40 and 40a according to the present invention with the above-mentioned configuration, it is possible to connect the liquid passage pipe 43 to the cell separation device SE. In particular, as shown in FIGS. 4 (a), 4 (b), 5 (a), and 5 (b), the liquid passage port used according to the flow type such as the dead end flow method and the cross flow method. In the cell separation filter SF capable of switching the position of the part LO, by using the connectors 40 and 40a according to the present invention, it is possible to smoothly switch the flow method without generating a kink in the pipe 43. Therefore, the time required for switching the flow method can be shortened, and the cell recovery rate can be improved.

In the connectors 40 and 40a according to the present invention, the base portion 42 and one end side of the pipe connection body 46 are fitted, and the pipe connection portion 44 is provided on the other end side of the pipe connection body 46. Be done.

The liquid passage port connection portion 41 of the base portion 42 may have a structure that can be connected to the liquid passage port portion LO of the cell separation device SE and has a detachable structure.
Examples of the shape of the liquid passing port connecting portion 41 include a tubular shape.
Further, as the detachable configuration, from the viewpoint of easily and firmly connecting to the liquid passing port portion LO, the liquid passing port connecting portion 41 has a fitting mechanism that can be fitted with the liquid passing port portion LO. It is preferable to prepare.
The fitting mechanism has a shape in which the liquid passing port connecting portion 41 can be fitted to the liquid passing port LO.

For example, as shown in FIG. 1A, when the liquid passage port LO is a hole provided on the outer surface of the cell separation device SE (for example, the attachment / detachment holes 17A and 17B of FIGS. 14, 15, and 16). , 18A, 18B, 19A, 19B), as the fitting mechanism, a screw groove 47 is provided on the inner peripheral surface of the hole, and a screw thread 48 screwed with the screw groove 47 is provided in the liquid passage connection portion 41. Examples thereof include a configuration provided on the outer peripheral surface. With such a configuration, the liquid passage port connection portion 41 and the liquid passage port portion LO can be firmly fixed.
Further, although not shown, a magnet may be provided around the hole, and a magnet of a pole different from the hole or a ferromagnetic material such as iron may be provided on the surface of the liquid passage connecting portion 41 in contact with the periphery of the hole. .. By using the magnet in this way, the connection between the liquid passage port connection portion 41 and the liquid passage port portion LO can be easily and surely performed. Further, by providing an anisotropic magnet or a ferromagnetic material such as iron, for example, when pressure is applied during cell collection in the cell separation filter, it is generated in the direction perpendicular to the end face of the liquid passage port LO. Although it does not come off due to the magnetic force, it can be easily removed due to its anisotropic characteristics when the liquid passage port connecting portion 41 is rotated.
Further, although not shown, a rubber stopper may be provided in which a bottle needle is formed at the end of the liquid passage port connection portion 41 and a notch is provided in the liquid passage port portion LO. In this case, there is a configuration in which the liquid can be passed only when the bottle needle is inserted into the notch of the rubber stopper of the liquid passage port LO.

Further, as shown in FIG. 3A, when the liquid passage port LO is a cylinder 49 protruding toward the outer surface side of the cell separation device SE (for example, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11, the first liquid passage port 11 and the second liquid passage port 12) of FIGS. 12, 12), as the fitting mechanism, on the inner peripheral surface of the cylinder 49, the outer circumference of the end portion of the liquid passage port connection portion 41. An embodiment in which the surfaces are fitted can be mentioned.
In this case, the surfaces on which the cylinder 49 and the liquid passage connecting portion 41 are fitted and come into contact with each other may be smooth. Further, although not shown on the contact surface, the liquid passage port connecting portion 41 and the cylinder 49 can be more firmly fixed by providing a screw thread and a screw groove screwed with the screw thread.
Further, a magnet is provided on the contact surface between the cylinder 49 and the liquid passage port connection portion 41, and the contact surface of the cylinder 49 and the contact surface on the liquid passage port connection portion 41 side have poles different from those on the cylinder 49 side. An example is a configuration in which a ferromagnetic material such as a magnet or iron is provided. By using the magnet in this way, the connection between the liquid passage port connecting portion 41 and the cylinder 49 can be performed more reliably. Further, by providing an anisotropic magnet or a ferromagnetic material such as iron, for example, when pressure is applied during cell collection in the cell separation filter, it is generated in the direction perpendicular to the end face of the liquid passage port LO. Although it does not come off due to the magnetic force, it can be easily removed due to its anisotropic characteristics when the liquid passage port connecting portion 41 is rotated.
Further, although not shown, a rubber stopper may be provided in which a bottle needle is formed at the end of the liquid passage port connection portion 41 and a notch is provided in the liquid passage port portion LO. In this case, there is a configuration in which the liquid can be passed only when the bottle needle is inserted into the notch of the rubber stopper of the liquid passage port LO.

As shown in FIGS. 1A and 3A, the pipe connecting body 46 is connected to a pipe connecting portion 44 and the pipe connecting portion 44 to which a pipe 43 for passing liquid to the cell separation device SE is connected. On the other hand, it has a rotating portion 45 that is rotatable.

The pipe connecting portion 44 may have a shape that can be fixed to the inner surface or the outer surface of the pipe 43 in a state in which liquid can pass therethrough.

The pipe connecting portion 44 is rotatably held by the rotating portion 45.
The shape of the rotating portion 45 may be a tubular shape from the viewpoint of being easily rotated by hand. The axial cross-sectional shape of the rotating portion 45 includes a circle, a quadrangle, a polygon, and the like, but is not particularly limited.

As a mode in which the pipe connecting portion 44 is rotatably held by the rotating portion 45, for example, as shown in FIG. 1A, a recess 50 is provided on the outer peripheral surface of the pipe connecting portion 44, and the recess is provided. A convex portion 51 that fits the 50 may be provided on the inner surface of the rotating portion 45. Further, as shown in FIG. 3A, a convex portion 51a may be provided on the outer peripheral surface of the pipe connecting portion 44, and a concave portion 50a matching the convex portion 51a may be provided on the inner surface of the rotating portion 45.

The rotating portion 45 is used for fixing to the cell separation device SE. As the fixing mode, an appropriate mode can be selected according to the shape of the liquid passage port LO arranged on the outer surface of the cell separation device SE. For example, as shown in FIG. 1A, if the liquid passage port LO is a hole, the rotating portion 45 is fixed to the base 42 previously fixed to the hole-shaped liquid passage LO. Can be mentioned. Further, as shown in FIG. 3A, if the liquid passage port LO is a cylinder 49, the rotating portion 45 may be fixed to the cylinder 49.
In either case, by rotating the rotating portion 45 about the center line thereof, the pipe 43 connected / fixed to the pipe connecting portion 44 is not rotated, and the rotating portion 45 is passed through the base portion 42 or the cylinder 49. The pipe 43 can be connected to the cell separation device SE.

Further, a seal 53 may be provided between the pipe connecting portion 44 and the rotating portion 45 constituting the connectors 40 and 40a according to the present invention in order to prevent liquid from leaking. In particular, a silicone rubber packing or an O-ring can be preferably used as the seal 53.

Further, in the connectors 40 and 40a according to the present invention, a liquid leakage prevention mechanism for preventing liquid from leaking from between the base portion 42 and the pipe connecting portion 44 is further provided at any position. You may. Specifically, the connectors 40 and 40a shown in FIGS. 1 (a) and 3 (a) may be provided at appropriate positions in the lumen of the base portion 42. Further, a liquid leakage prevention mechanism may be provided at an appropriate position in the lumen of the pipe connection portion 44. Further, a liquid leakage prevention mechanism may be used in combination at both an appropriate position in the cavity of the base portion 42 and an appropriate position in the cavity of the pipe connection portion 44.
Examples of such a liquid leakage prevention mechanism include check valves such as a ball type, a disc type, and a swing type.

Further, as shown in FIGS. 4 (a), 4 (b), 5 (a), and 5 (b), in the cell separation filter having a plurality of liquid passage ports LO, the connectors 40 and 40a according to the present invention. A cap 52 can be attached to the liquid passage port LO to which the is not attached as a closing attachment / detachment body. An appropriate method can be selected for the cap 52 according to the form of the liquid passage port LO arranged on the outer surface of the cell separation device SE. For example, as shown in FIG. 1A, if the liquid passage port LO is a hole, the cap 52 is fixed to the base 42 fixed to the hole-shaped liquid passage LO. Further, as shown in FIG. 3A, if the liquid passage port LO is a cylinder 49, the cap 52 is fixed to the cylinder 49.
Further, although not shown, the cap may have a shape that closes the cavity that is the liquid passage portion of the pipe connecting portion 44 in the connectors 40 and 40a.

Each part of the connectors 40 and 40a can be made by using any structural material. The structural material of each part is not particularly limited, and examples thereof include non-reactive polymers, biocompatible metals, alloys, and glass.

Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides and 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, as well as gold-plated ferroalloys, platinum-plated ferroalloys, cobalt chromium alloys, and titanium nitride-coated stainless steels.

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 connector.

The connectors 40 and 40a can be manufactured by combining each part based on a conventional method. For example, in the case of the connector 40 shown in FIG. 1A, as shown in FIG. 2, the rotating portion 45 is sandwiched between the upper 44a and the lower 44b of the pipe connecting portion 44, and the upper 44a and the lower 44b are sandwiched. The contact surface is fixed with an adhesive or the like, and then the pipe 43 is connected to the end of the upper portion 44a of the pipe connection portion 44. On the other hand, the liquid passage port connecting portion 41 of the base 42 is inserted into the liquid passage port LO provided on the outer surface of the cell separation device SE to stop the rotation. Then, while inserting the end portion of the pipe connecting portion 44b into the cavity of the base portion 42, the rotating portion 45 is rotated by hand so that the end surface of the rotating portion 45 is pressed against the outer surface of the base portion 42. Can be fixed.

<Cell separation filter>
In the cell separation filter SF according to the present invention, as shown in FIGS. 4 (a), 4 (b), 5 (a), and 5 (b), the connectors 40 and 40a are connected to the liquid passage port LO. The attached cell separation filter SF.
4 (a) and 4 (b) show the cell separation filter SF to which the connector 40 shown in FIG. 1 (a) and the cap 52 shown in FIG. 1 (b) are attached, and FIG. 5 (a) shows. 5 (b) shows the cell separation filter SF to which the connector 40a shown in FIG. 3 (a) and the cap 52 shown in FIG. 3 (b) are attached.

Further, the cell separation filter SF may be a general cell separation filter, but in particular, even if the cell separation filter can switch the flow method, the connectors 40 and 40a can be suitably connected.
Hereinafter, a cell separation filter capable of switching the flow method will be described with reference to the drawings.
In the drawing, a front view is a view of the cell separation filter container with the first surface facing up and the second surface facing down.
Further, the configuration examples of the cell separation filter container 1 shown in FIGS. 7 to 16 are all schematic views for explaining the concept of the invention, and the operability and sealing property of the switching mechanism in them are required. The design may be made with appropriate consideration according to the specifications.

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

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, and glass.

Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides and 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, as well as gold-plated ferroalloys, platinum-plated ferroalloys, cobalt chromium alloys, and titanium nitride-coated stainless steels.

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, examples of the filter medium used for the cell separation filter include a non-woven fabric and a porous carrier.

In the present invention, the material of the non-woven fabric is not particularly limited, but from the viewpoint of sterilization resistance and cell safety, polyethylene terephthalate, polybutylene terephthalate, polyethylene, high-density polyethylene, low-density polyethylene, polyvinyl alcohol, vinylidene chloride, rayon, etc. Viniron, polypropylene, acrylic (polymethylmethacrylate, polyhydroxyethylmethacrylate, polyacrylic nitrile, polyacrylic acid, polyacrylate), nylon, polyimide, aramid (aromatic polyamide), polyamide, cupra, carbon, phenol, polyester, pulp, Examples thereof include synthetic polymers such as hemp, polyurethane, polystyrene and polycarbonate, natural polymers such as agarose, cellulose, cellulose acetate, chitosan and chitin, inorganic materials such as glass and metals. Preferably, polyethylene terephthalate, polybutylene terephthalate, polypropylene, acrylic, nylon, or polyurethane having high cell capture ability. More preferably, polyethylene terephthalate, polybutylene terephthalate, and nylon having high nucleated cell capture ability. When two or more kinds of materials are combined into a fiber, the form of the fiber may be a fiber in which one fiber is made of a material of different components, or a split fiber in which different components are peeled and divided. Further, the fiber may be a composite of fibers made of different materials. The compounding referred to here is not particularly limited, and examples thereof include a form composed of a mixture of two or more types of fibers, a form composed of a single material, and the like. Furthermore, molecules having an affinity for a specific cell, such as proteins, peptides, amino acids, sugars, etc., may be immobilized.

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

The protein to be fixed to the non-woven fabric is not particularly limited as long as it is a protein having an affinity for a specific cell, and specific examples thereof include fibronectin, laminin, vitronectin, and collagen. The saccharide 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. ..

The porous carrier in the present invention includes polysaccharides such as cellulose, acetyl cellulose and dextrin, polystyrene, styrene-divinylbenzene copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polyacrylic acid ester, and polymethacrylic acid. Includes organic carriers made of synthetic polymers such as esters and polyvinyl alcohol. They have a coating layer made of a graft copolymer, such as a hydroxyl group-containing polymer material such as hydroxyethyl methacrylate, a polyethylene oxide chain-containing monomer, or a copolymer of another polymerized monomer. It doesn't matter. Of these, synthetic polymers such as cellulose and polyvinyl alcohol are practically preferred because the active groups are easily introduced onto the surface of the carrier. In particular, porous cellulose particles are most preferable.

It is preferable that the porous carrier (particularly the porous cellulose particles) is formed by immobilizing a compound having a logP (P is an octanol-partition coefficient in an aqueous system) of 2.50 or more.
Examples of the compound having a logP value of 2.50 or more include a tryptophan derivative and a polyanionic compound, and it is preferable that the tryptophan derivative and the polyanionic compound are immobilized on the porous carrier.

The logP value is a parameter of the hydrophobicity of the compound, and the partition coefficient P in a typical octanol-aqueous system can be obtained as follows. First, the compound is dissolved in octanol (or water), and an equal amount of water (or octanol) is added thereto, and Griffin flask shaker (Griffin & George Ltd.) (Griffin & George Ltd.). ), Shake for 30 minutes. After that, centrifugation is performed at 2000 rpm for 1 to 2 hours, and the concentrations of the compounds in the octanol layer and the aqueous layer are measured spectroscopically or by various methods such as GLC. By substituting these values into the following equation, P can be obtained.

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

Many researchers have measured the logP values of various compounds so far, but these measured values have been organized by C. Hansch et al. ("Partition Cofisients and There"). -Eugees; Chemical Reviews (PARTITION COEFFICIENTS AND THEIR USES; Chemical Reviews, Vol. 71, p. 525, 1971).

For compounds for which the measured values are unknown, RF Rekker wrote his book "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) can be used as a reference. The hydrophobic fragment constant is a value indicating the hydrophobicity of various fragments determined by statistical processing based on a large number of logP measured values, and the sum of the f-numbers of each fragment constituting the compound is logP. It is reported that it almost matches the value. In the present invention, the logP value also includes Σf.

(Embodiment 1)
As shown in the perspective view of FIG. 7A, the cell separation filter according to the first embodiment of the present invention is located on the circular first surface 1A in the 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 end portions of the first surface 1A and the second surface 1B.
Further, the filter medium is accommodated in the accommodation space S in the container 1 shown in the vertical sectional perspective views of FIGS. 7 (c) and 7 (d). The illustration of the filter medium is omitted in order to make the drawings easier to see.

As shown in the partial vertical cross-sectional exploded perspective view of FIG. 7 (b) and the vertical cross-sectional perspective view of FIGS. 7 (c) and 7 (d), the first surface 1A of the container 1 is located at a position eccentric from the center thereof. 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 in the direction opposite to the eccentric direction of the guide hole 4A.
Then, since the circular guide groove 2A formed in the disk-shaped rotating body 2 engages with the circular guide hole 4A formed in the first surface 1A, the rotating body 2 with respect to the main body of the container 1. It is rotatably supported.
Similarly, since the circular guide groove 3A formed in the disk-shaped rotating body 3 engages with the circular guide hole 4B formed in the second surface 1B, the rotating body 3 is the main body of the container 1. It is rotatably supported against.
Further, since the first liquid passage port 11 is attached to the position near the outer circumference of the rotating body 2 and the second liquid passage port 12 is attached to the position closer to the outer circumference of the rotating body 3, it rotates with respect to the main 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 the cell separation filter SF shown in FIGS. 7 (a), 7 (b), (c) and (d), the cylindrical first liquid passage port 11 and the second liquid passage port 12 are all shown in FIG. Although the connector 40a shown in 3 (a) is attached, the first liquid passage port 11 and the second liquid passage port 12 may be hole-shaped liquid passage ports, and in this case, FIG. 1 (a). The connector 40 shown in is attached.

The mechanism for rotatably supporting the rotating bodies 2 and 3 and moving the liquid passage ports 11 and 12 constitutes the rotary movement type switching mechanisms A1 and B1.
As shown in FIG. 7C, 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. By doing so, the dead end flow method can be realized. Further, as shown in FIG. 7D, the cross-flow method can be realized by moving the liquid passage ports 11 and 12 to the end ends of the first surface 1A and the second surface 1B, respectively. Therefore, the flow method can be easily switched by the switching mechanisms A1 and B1.

(Embodiment 2)
It is carried out in the cell separation filter according to the second embodiment of the present invention in the perspective view of FIG. 8A, the exploded perspective view of FIG. 8B, and the vertical sectional perspective view of FIGS. 8C and 8D. Since the same reference numerals as those in Form 1 indicate the same or corresponding parts, the description of the common parts will be omitted.

As shown in the exploded perspective view of FIG. 8B and the vertical sectional perspective views of FIGS. 8C and 8D, the pipe line 6A is centered at the center position of the first surface 1A of the container 1. A pipeline 7A is formed at a position near the end end radially separated from the conduit 7A, and a support hole 5A is formed at a midpoint between the pipelines 6A and 7A. Further, on the second surface 1B facing the first surface 1A, the pipeline 6B is separated from the center in the radial direction at the center position, which is the direction opposite to the radial separation direction from the center of the pipeline 7A. A pipeline 7B is formed at a position near the end end, and a support hole 5B is formed at a midpoint between the pipelines 6B and 7B.
Here, the pipeline 6A conducts from the first surface 1A to the accommodation space S vertically downward with respect to the first surface 1A. The pipeline 6B conducts from the second surface 1B to the accommodation space S vertically upward with respect to the second surface 1B.
Here, the pipeline 7A extends vertically downward from the first surface 1A with respect to the first surface 1A, and then bends so as to extend horizontally inward in the radial direction with respect to the first surface 1A. Is conducting. The pipeline 7B extends vertically upward from the second surface 1B to the second surface 1B, and then bends so as to extend horizontally inward in the radial direction with respect to the second surface 1B, and conducts to the accommodation space S. There is.

Then, since the support shaft 2B at the center of the disk-shaped rotating body 2 fits into the support hole 5A formed on 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 3B at the center of the disk-shaped rotating body 3 fits into the support hole 5B formed on the second surface 1B, the rotating body 3 can rotate with respect to the main body of the container 1. Be supported.
Therefore, the mechanism for moving the first liquid passage port 11 and the second liquid passage port 12 in the second embodiment is the rotary movement type switching mechanisms A1 and B1 similar to the first embodiment.

In the cell separation filter provided with such switching mechanisms A1 and B1, as shown in FIG. 8C, the first liquid passage port 11 communicates with the pipe line 6A at the center position of the first surface 1A, and the first liquid passage port 11 is connected. The dead end flow method is realized by communicating the second liquid passage port 12 with the pipeline 6B at the center position of the two surfaces 1B. Further, as shown in FIG. 8D, the first liquid passage port 11 communicates with the pipeline 7A located near the end of the first surface 1A, and the pipe at the position near the end of the second surface 1B. The cross-flow method can be realized by communicating the second liquid passage port 12 with the road 7B. Therefore, the flow method can be easily switched by the switching mechanisms A1 and B1.
In the cell separation filter SF shown in the perspective view of FIG. 8 (a), the exploded perspective view of FIG. 8 (b), and the vertical sectional perspective views of FIGS. 8 (c) and 8 (d), the cylindrical first The connector 40a shown in FIG. 3A is attached to both the first liquid passage port 11 and the second liquid passage port 12, but the first liquid passage port 11 and the second liquid passage port 12 have a hole shape. It may be a liquid passage port, and in this case, the connector 40 shown in FIG. 1A is attached.

(Embodiment 3)
The book in the perspective view of FIG. 9A, the exploded perspective view of FIG. 9B, the partial vertical sectional sectional perspective view of FIG. 9C, and the vertical sectional front view of FIGS. 10A and 10B. In the cell separation filter according to the third embodiment of the present invention, since the same reference numerals as those of the first embodiment indicate the same or corresponding parts, the description of the common parts will be omitted.

As shown in the exploded perspective view of FIG. 9B and the perspective views of FIGS. 10A and 10B, the first surface 1A of the container 1 extends from the central portion in the radial direction to the outermost portion. A rectangular accommodating opening 1D extending to the vicinity is formed, and the second surface 1B facing the first surface 1A extends from the central portion in the radial direction opposite to the radial direction in which the accommodating opening 1D extends. A rectangular accommodating opening 1E extending to the vicinity of the portion is formed.
The accommodation opening 1D formed on the first surface 1A accommodates the slide guide body 8 having through holes 8A and 8B formed at front and rear positions in the longitudinal direction.
Similarly, the accommodation opening 1E formed on the second surface 1B accommodates the slide guide body 9 having through holes 9A and 9B formed at front and rear positions in the longitudinal direction.
Further, the closing slide body 13A, the liquid passing slide body 10A provided with the first liquid passing 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 passing slide body 10B provided with the second liquid passing 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 provided with 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.

The mechanism for slidably supporting the slide bodies 10A and 10B for liquid passage and moving the first liquid passage port 11 and the second liquid passage port 12 constitutes the slide movement type switching mechanisms A2 and B2.
As shown in FIG. 10A, while closing the through hole 8B with the closing slide body 13B, the liquid passing slide body 10A is moved to communicate with the first liquid passing port 11 through the through hole 8A. The dead end flow method is realized by moving the slide body 10B and communicating it with the second liquid passage port 12 through the hole 9A while closing the hole 9B with the closing slide body 14B. Further, as shown in FIG. 10B, 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, and the through hole 9A is The cross-flow method is realized by moving the slide body 10B and communicating it with the second liquid passage port 12 through the hole 9B while closing it with the closing slide body 14A. Therefore, the flow method can be easily switched by the slide movement type switching mechanisms A2 and B2.
The perspective view of FIG. 9A, the exploded perspective view of FIG. 9B, the partial vertical sectional sectional perspective view of FIG. 9C, and the vertical sectional front view of FIGS. 10A and 10B. In the cell separation filter SF in the above, the above-mentioned connectors 40 and 40a may be provided in the cylindrical first liquid passage port 11 and the second liquid passage port 12.

(Embodiment 4)
In the cell separation filter according to the fourth embodiment of the present invention in the vertical sectional front view of FIGS. 11A and 11B, the same reference numerals as those of the third embodiment indicate the same or corresponding parts, and thus are common. The description of the part is omitted.

As shown in the vertical cross-sectional front view of FIGS. 11A and 11B, the through holes 8A and 8B of the slide guide body 8 communicate with the pipeline 6A and the conduit 7A, respectively, and the through holes of the slide guide body 9 are communicated with each other. 9A and 9B communicate with the pipeline 6B and the pipeline 7B, respectively.
The mechanism for moving the first liquid passage port 11 and the second liquid passage port 12 in the fourth embodiment is the slide-movable switching mechanisms A2 and B2 similar to those in the third embodiment, as shown in FIG. 11 (a). 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 method in which the flow direction of the supply liquid is orthogonal to the filtration direction by the filter medium as shown in FIG. 11B. Can be switched to.
In the cell separation filter SF in the vertical sectional front view of FIGS. 11 (a) and 11 (b), the above-mentioned connector 40 is connected to the cylindrical first liquid passage port 11 and the second liquid passage port 12. , 40a may be provided.

(Embodiment 5)
A perspective view of FIG. 12 (a), a partial vertical cross-sectional exploded perspective view of FIG. 12 (b), a vertical sectional perspective of FIGS. 12 (c) and 12 (d), and a vertical section of FIGS. 13 (a) and 13 (b). In the cell separation filter according to the fifth embodiment of the present invention in the front view, the same reference numerals as those of the third embodiment indicate the same or corresponding parts, and thus the description of the common parts will be omitted.

The liquid passing slide body 10A provided with the first liquid passing port 11 in the present embodiment also serves as a closing slide body for closing the through holes 8A and 8B shown in the fourth embodiment. It has a rectangular shape longer than the liquid-passing slide body 10A shown in 3, and is supported so as to be slidable in the radial direction while being engaged with the guide groove 1F, and is radially supported from the central portion of the first surface 1A. The first liquid passage port 11 projects outward through the elongated hole 1H extending to the outside.
Similarly, the liquid passing slide body 10B provided with the second liquid passing port 12 in the present embodiment also serves as a closing slide body for closing the through holes 9A and 9B shown in the fourth embodiment. In addition, it has a rectangular shape longer than the liquid-passing slide body 10B shown in the third embodiment, and is supported so as to be slidable in the radial direction while being engaged with the guide groove 1G, and has a second surface 1B. 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 slide movement type switching mechanisms A2 and B2 similar to the third and fourth embodiments, but the fifth embodiment. Then, the arrangement of the first liquid passage port 11 can be continuously changed within the range in which the first liquid passage port 11 can move in the elongated hole 1H, and the second liquid passage port 12 moves in the elongated hole 1I. The arrangement of the second liquid passage port 12 can be continuously changed within the possible range.
A perspective view of FIG. 12 (a), an exploded perspective view of FIG. 12 (b), a partial longitudinal sectional exploded perspective view of FIGS. 12 (c) and 12 (d), and FIGS. 13 (a) and 13 (b). In the cell separation filter SF in the front view of the vertical cross section, the above-mentioned connectors 40 and 40a may be provided in the cylindrical first liquid passage port 11 and the second liquid passage port 12.

(Embodiment 6)
The cell separation filter according to the sixth embodiment of the present invention in the perspective view of FIG. 14 (a), the exploded perspective view of FIG. 14 (b), and the vertical sectional perspective view of FIGS. 14 (c) and 14 (d). Since the same reference numerals as those in Form 1 indicate the same or corresponding parts, the description of the common parts will be omitted.

As shown in the exploded perspective view of FIG. 14 (b) and the vertical sectional perspective views of FIGS. 14 (c) and 14 (d), the attachment / detachment hole 17A is centered at the center position of the first surface 1A of the container 1. An attachment / detachment hole 18A is formed at a position near the end end radially separated from the container 1, and an attachment / detachment hole 17B is formed at the center position of the second surface 1B of the container 1 in the radial separation direction from the center of the attachment / detachment hole 18A. The attachment / detachment hole 18B is formed at a position near the end end, which is in the opposite direction to the above direction and is separated from the center in the radial direction.
The attachment / detachment holes 17A, 17B, 18A, 18B serve as liquid passage ports.
The attachment / detachment holes 17A and 18A formed on the first surface 1A are formed on the second surface 1B by attaching one of the connector 40 which is the liquid passage attachment / detachment body 15A and the cap 52 which is the closing attachment / detachment body 16A, respectively. One of the connector 40, which is the liquid-passing attachment / detachment body 15B, and the cap 52, which is the closing attachment / detachment body 16B, is attached to the attachment / detachment holes 17B and 18B, respectively.

As described above, the mechanism for replacing the connector 40 and the cap 52 with respect to the detachable holes 17A and 18A, which are composed of the detachable holes 17A and 18A and the connector 40 (15A) and the cap 52 (16A), is the detachable movable switching mechanism A3. To configure.
Further, a mechanism for replacing the connector 40 and the cap 52 with respect to the detachable holes 17B and 18B, which are composed of the detachable holes 17B and 18B and the connector 40 (15B) and the cap 52 (16B), constitutes the detachable movable switching mechanism B3. To do.
According to such switching mechanisms A3 and B3, as shown in FIG. 14C, the dead end flow method can be realized by connecting the connector 40 to the attachment / detachment hole 17A and the attachment / detachment hole 17B, respectively. Further, as shown in FIG. 14D, the cross-flow method can be realized by connecting the connectors 40 to the attachment / detachment holes 18A and the attachment / detachment holes 18B, respectively. Therefore, the flow method can be easily switched by the detachable movable switching mechanism. The cap 52 is connected to the attachment / detachment hole to which the connector 40 is not connected.
In the cell separation filter SF shown in FIGS. 14 (a), (b), (c) and (d), the hole-shaped first liquid passage port 11 and the second liquid passage port 12 are all shown in FIG. Although the connector 40 shown in a) is attached, the first liquid passage port 11 and the second liquid passage port 12 may be a cylindrical liquid passage port, and in this case, it is shown in FIG. 3 (a). The connector 40a is attached.

(Embodiment 7)
The cell separation filter according to the seventh embodiment of the present invention in the perspective view of FIG. 15 (a), the exploded perspective view of FIG. 15 (b), and the vertical sectional perspective views of FIGS. 15 (c) and 15 (d). Since the same reference numerals as those of Form 6 indicate the same or corresponding parts, the description of the common parts will be omitted. The pipelines 6A, 6B, 7A, and 7B are the same as those in FIG. 8 of the second embodiment and FIG. 11 of the fourth embodiment.

The flow-type switching mechanism in the seventh embodiment is the same detachable and movable switching mechanism A3 and B3 as in the sixth embodiment.
According to such switching mechanisms A3 and B3, as shown in FIG. 15C, the dead end flow method can be realized by connecting the connector 40 to the attachment / detachment hole 17A and the attachment / detachment hole 17B, respectively. Further, as shown in FIG. 15D, the cross-flow method can be realized by connecting the connectors 40 to the attachment / detachment holes 18A and the attachment / detachment holes 18B, respectively. Therefore, the flow method can be easily switched by the detachable movable switching mechanism. The cap 52 is connected to the attachment / detachment hole to which the connector 40 is not connected.
Here, in the state of FIG. 15C, the first liquid passage port 11 communicates with the pipe line 6A at the center position of the first surface 1A, and the second communication port 6B at the center position of the second surface 1B. The liquid port 12 communicates.
Further, in the state of FIG. 15 (d), the first liquid passage port 11 communicates with the conduit 7A located 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.
In the cell separation filter SF shown in FIGS. 15 (a), 15 (b), (c) and (d), the hole-shaped first liquid passage port 11 and the second liquid passage port 12 are all shown in FIG. 1 ( Although the connector 40 shown in a) is attached, the first liquid passage port 11 and the second liquid passage port 12 may be a cylindrical liquid passage port, and in this case, it is shown in FIG. 3 (a). The connector 40a is attached.

(Embodiment 8)
The cell separation filter according to the eighth embodiment of the present invention in the perspective view of FIG. 16 (a), the exploded perspective view of FIG. 16 (b), and the vertical sectional perspective view of FIGS. 16 (c) and 16 (d). Since the same reference numerals as those of Form 6 indicate the same or corresponding parts, the description of the common parts will be omitted.
In the present embodiment, the attachment / detachment hole 18A formed on the first surface 1A and the attachment / detachment hole 18B formed on the second surface in the sixth embodiment are eliminated, and the attachment / detachment hole 19A is formed on the upper portion of the side surface 1C. Point-symmetrical position with 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 center of the attachment / detachment holes 17A and 17B at the center of the first surface 1A and the second surface 1B. A detachable hole 19B is formed in the lower part of the side surface 1C. One of the connector 40 and the cap 52 is attached to the attachment / detachment hole 17A formed on the first surface 1A and the attachment / detachment hole 19A at the upper part of the side surface 1C, respectively, and the attachment / detachment holes 17B and 18B formed on the second surface 1B are provided. , One of the connector 40 and the cap 52 is attached, respectively.

In the present embodiment, the mechanism for replacing the connector 40 and the cap 52 with respect to the attachment / detachment holes 17A and 19A constitutes the attachment / detachment movable switching mechanism A3.
Further, a mechanism for replacing the connector 40 and the cap 52 with respect to the detachable holes 17B and 19B, which are composed of the detachable holes 17B and 19B and the connector 40 and the cap 52, constitutes the detachable movable switching mechanism B3.
According to such switching mechanisms A3 and B3, as shown in FIG. 16C, the dead end flow method can be realized by connecting the connector 40 to the attachment / detachment hole 17A and the attachment / detachment hole 17B, respectively. Further, as shown in FIG. 16D, the cross-flow method can be realized by connecting the connectors 40 to the attachment / detachment holes 19A and the attachment / detachment holes 19B, respectively. Therefore, the flow method can be easily switched by the detachable movable switching mechanism.
In the cell separation filter SF shown in FIGS. 16 (a), 16 (b), (c) and (d), the hole-shaped first liquid passage port 11 and the second liquid passage port 12 are all shown in FIG. Although the connector 40 shown in a) is attached, the first liquid passage port 11 and the second liquid passage port 12 may be a cylindrical liquid passage port, and in this case, it is shown in FIG. 3 (a). The connector 40a is attached.

<Manufacturing method of cell concentrate>
The method for producing a cell concentrate of the present invention includes a contact step of introducing a cell-containing solution from the first passage port 11 or the second passage port 12 of the cell separation filter and bringing it into contact with a filter medium, and a cell separation filter SF. It has a recovery step of recovering 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 is a body fluid containing nucleated cells such as peripheral blood, bone marrow, umbilical cord blood, menstrual blood, and tissue extract, or these body fluids are 2 such as physiological saline, calcium ion, and magnesium ion. It may be a Ringer solution containing a valent cation, a medium diluted with a medium such as RPMI, MEM, IMEM, DMEM used for cell culture, a phosphate buffer solution such as PBS, or a crudely separated cell from a body fluid. 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, and cats. When the cell-containing solution contains an anticoagulant, the type of anticoagulant is not limited, and for example, heparin, low molecular weight heparin, fusan (nafomostat methylate), EDTA, ACD (acid-citrate-dextrose) solution, etc. Examples thereof include citric acid anticoagulant such as CPD (citrate-phospate-dextrose) solution. The 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, and for example, monocytes, leukocytes, granulocytes, neutrophils, eosinophils, basal spheres, erythroblasts, and bone marrow Sprouts, anterior bone marrow cells, bone marrow cells, posterior bone marrow cells, lymphocytes, monocytes, macrophages, T lymphocytes, B lymphocytes, NK cells, NK / T cells, dendritic cells, polynuclear 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 refers to a liquid containing nucleated cells, which has a larger number of nucleated cells or a higher cell density in the liquid than the cell-containing liquid. The stem cell in the present invention refers to a cell isolated from body fluid and having pluripotency and self-renewal ability.

The mesenchymal stem cells in the present invention include osteoblasts, chondrocytes, vascular endothelial cells, myocardial cells, cementoblasts which are fat and periodontal tissue constituent cells, and periodontal ligament fibroblasts by adding a differentiation-inducing factor. It is a cell that differentiates into. 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 recovering a liquid containing a large amount of nucleated cells obtained through the filter, and the cell concentrate depends on the properties of the cell separation filter. It is only necessary to be able to collect.

If the filter medium filled in the filter has an affinity for cells and impurities to be removed, the cell-containing solution is introduced from the inflow port of the filter, and after the contact step of bringing the filter medium into contact with the cell-containing solution, after the contact step. The cell concentrate can be recovered from the outlet. When recovering the cell concentrate from the outlet of the filter, it is preferable to introduce the recovered solution from the inlet of the filter and collect the cell concentrate from the outlet. Examples of cells to be removed include granulocytes, monocytes, platelets, B lymphocytes, lymphocytes having a specific subclass, and the like. Specific examples thereof include a method in which granulocytes are captured by a filter and removed to recover a mononuclear cell-containing fraction.

If the filter medium filled in the filter has an affinity for nucleated cells for recovery, introduce the cell-containing solution from the inlet of the filter and let out the cells and impurities for removal from the outlet. After that, the cell concentrate can be recovered from the inflow port. When collecting the cell concentrate from the inlet of the filter, it is preferable to introduce the recovered solution from the outlet of the filter and collect the cell concentrate from the inlet. Examples of cells for removal include erythrocytes, T lymphocytes, lymphocytes having a specific subclass, and the like. Specific examples thereof include a method of passing unnecessary cells and collecting stem cells captured by a filter.

In addition, when the recovered solution is introduced from the outlet of the filter to recover the cell concentrate, the cells to be removed are more efficiently introduced by introducing the washing solution from the inlet of the filter before the recovery. And impurities can be discharged from the outlet. The washing solution is not particularly limited as long as it can wash away only the cells and impurities 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 solutions containing serum or protein.

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

The speed and method of passing the cell-containing liquid and the recovered liquid are not particularly limited, and for example, a method of passing liquid using gravity, a method of passing liquid while keeping the flow velocity constant using a roller clamp or a syringe pump, and a high method. Examples include a method of applying pressure to pass the liquid at once. For the purpose of recovering the nucleated cells captured in the filter, a method of passing the recovered solution at once by applying a high pressure is preferable from the viewpoint of the nucleated cell recovery efficiency. A method of manually passing the liquid using a syringe or using a pump at a flow rate of 90 mL / min or more is preferable in that more nucleated cells can be collected.

FIG. 6 shows an example of a circuit for closedly separating cells using the cell separation filter of the present invention.
In the circuit shown in FIG. 6, the container 20 containing the cell-containing liquid is provided with a liquid passage port, and is connected to the three-way stopcock 25 through a pipe 28 capable of passing the liquid. Similarly, the container 21 for accommodating the liquid for priming also has a liquid passage port and is connected to the three-way stopcock 25 through a pipe 29 capable of passing the liquid. The three-way stopcock 25 and the three-way stopcock 26 are connected by a pipe 30 capable of passing liquid. The container 22 for accommodating the cell concentrate is provided with a liquid passage port, and is connected to the three-way stopcock 26 through a pipe 31 capable of passing the liquid. In the cell separation filter of the present invention, the inlet is connected to the three-way stopcock 27 and the outlet is connected to the three-way stopcock 26. The container 23 for storing the cell recovery liquid is provided with a liquid passage port, and is connected to the three-way stopcock 27 through a pipe 32 capable of passing the liquid. Similarly, the container 24 for storing the priming liquid and the cell-containing liquid that has passed through the cell separation filter SF is provided with a liquid passage port, and is connected to the three-way stopcock 27 through a liquid-permeable pipe 33.

The specific procedure for producing a cell concentrate using the circuit shown in FIG. 6 is as follows.
First of all, in order to perform priming for the purpose of removing air in the cell separation filter, improving the cell capture efficiency, and securing the blood flow path, the priming liquid stored in the container 21 is used. , Pass through a cell separation filter and flow into container 24. At this time, the three-way stopcock 25 conducts the pipe 29 and the pipe 30 to block the flow to the pipe 28. The three-way stopcock 26 conducts the pipe 30 and the cell separation filter to block the flow to the pipe 31. Further, the three-way stopcock 27 conducts the cell separation filter and the pipe 33 to block 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 flows into the container 24. At this time, the three-way stopcock 25 conducts the pipe 28 and the pipe 30 to block the flow to the pipe 29. The three-way stopcock 26 conducts the pipe 30 and the cell separation filter to block the flow to the pipe 31. Further, the three-way stopcock 27 conducts the cell separation filter and the pipe 33 to block the flow to the pipe 32.

Finally, in order to collect the cell concentrate, the cell recovery solution contained in the container 23 is passed through a cell separation filter and flowed into the container 22. At this time, the three-way stopcock 27 conducts the cell separation filter and the pipe 32 to block the flow to the pipe 33. Further, the three-way stopcock 26 conducts the cell separation filter and the pipe 31 to block the flow to the pipe 30.
The circuit preferably includes a three-way stopcock, roller clamp, clamp, etc. to control the flow of liquid.

By performing the contact step of introducing the cell-containing liquid into the cell separation filter and bringing it into contact with the filter medium in a cross-flow system by the switching mechanism of the cell separation filter, the flow of the cell-containing liquid causes the deposition of particles on the surface of the filter medium. The increase in flow resistance due to 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 recovery step of recovering the cell concentrate after the contact step in a state of using the dead end flow method by the switching mechanism of the cell separation filter, the flow direction of the recovery liquid is the same as the filtration direction by the filter medium. Therefore, the cell recovery rate can be improved.

A1, B1 Rotational movement type switching mechanism A2, B2 Slide movement type switching mechanism A3, B3 Detachable movement type switching mechanism S Storage space SF Cell separation filter 1 Container 1A First side 1B Second side 1C Side 1D, 1E Opening 1F, 1G Guide groove 1H, 1I Long hole 2,3 Rotating body 2A, 3A Guide groove 2B, 3B Support shaft 4A, 4B Guide hole 5A, 5B Support hole 6A, 6B, 7A, 7B Pipe line 8, 9 Slide guide Body 8A, 8B, 9A, 9B Through hole 10A, 10B Slide body for liquid passage 11 First liquid passage port 12 Second liquid passage port 13A, 13B, 14A, 14B Slide body for closing 15A, 15B Detachable body for liquid passage 16A , 16B Closing / detaching body 17A, 17B, 18A, 18B, 19A, 19B Detachable hole 20 Container containing cell-containing liquid 21 Container containing liquid for priming 22 Container containing cell concentrate 23 Container containing cell recovery solution Container 24 Containers 25, 26, 27 for storing priming liquid and cell-containing liquid that have passed through the cell separation filter Connect to the liquid-permeable pipes 29 21 and 25 connected to the three-way activation plugs 28 20 and 25. Liquid-permeable pipe 30 Connected to liquid-permeable pipes 31 25 and 26 Liquid-permeable pipe 32 connected to 23 and 27 Liquid-permeable pipe 40 connected to 24 and 27 40a Connector 41 Liquid flow port connection 42 Base 43 Piping 44 Piping connection 44a Upper 44b Lower 45 Rotating part 46 Piping connector 47 Thread groove 48 Thread 49 Cylindrical 50, 50a Concave 51, 51a Convex 52 Cap 53 Seal SE cell Separation device LO liquid flow port

Claims (18)

It is a connector that is arranged on the outer surface of the cell separation device and is detachably configured at the liquid passage port where the arrangement can be moved.
A base having a liquid passage connecting portion to be connected to the liquid passing port, and
A connector characterized by having a pipe connection portion to which a pipe is connected and a pipe connection body having a rotating portion that is rotatable with respect to the pipe connection portion. The connector according to claim 1, wherein the base portion and one end side of the pipe connection body are fitted, and the pipe connection portion is provided on the other end side of the pipe connection body. The connector according to claim 1 or 2, wherein a fitting mechanism for fitting with the liquid passage portion is provided at the liquid passage port connection portion of the base portion. The connector according to claim 3, wherein a screw thread screwed with a screw groove provided on the inner peripheral surface of the liquid passage port portion is provided on the outer peripheral surface of the liquid passage port connection portion of the base portion. The connector according to any one of claims 1 to 4, further provided with a leak prevention mechanism for preventing liquid from leaking between the base and the pipe connection. The connector according to claim 5, wherein the leak prevention mechanism is a check valve. The connector according to any one of claims 1 to 6, wherein the cell separation device is a cell separation filter or a container. A cell separation filter in which the connector according to any one of claims 1 to 6 is attached to a liquid passage port. A cell separation filter provided with 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 cell separation according to claim 8, wherein the container includes a switching mechanism, and 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. filter. The cell separation filter according to claim 9, wherein the switching mechanism is provided on the first surface of the container and / or the second surface located in the facing direction of the first surface. The switching mechanism arranges the first liquid passage port and / or the second liquid passage port from the center of the second surface located in the facing direction of the first surface and / or the first surface of the container. The cell separation filter according to claim 9 or 10, which is configured to be movable between the ends. The cell separation filter according to any one of claims 9 to 11, wherein the switching mechanism is selected from at least one group consisting of a rotary movement type, a slide movement type, and a detachable movement type. The cell separation filter according to any one of claims 9 to 12, wherein the filter medium is a non-woven fabric. The cell separation filter according to any one of claims 9 to 12, wherein the filter medium is porous cellulose particles. The cell separation filter according to claim 14, wherein the filter medium is obtained by immobilizing a compound having a logP (P is an octanol-partition coefficient in an aqueous system) value of 2.50 or more on the porous cellulose particles. The cell separation filter according to claim 14, wherein the filter medium is formed by immobilizing a tryptophan derivative and a polyanionic compound on the porous cellulose particles. A contact step of introducing a cell-containing liquid from the first passage port or the second passage port of the cell separation filter according to any one of claims 9 to 16 and bringing the cell-containing liquid into contact with the filter medium, and the cell separation filter. A method for producing a cell concentrate, which comprises a recovery step of recovering the cell concentrate from the first passage or the second passage . The production method according to claim 17, wherein in the recovery step, a dead end flow method is adopted by the switching mechanism, and the cell concentrate is recovered from the first liquid passage port or the second liquid passage port of the cell separation filter.

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