WO2016158793A1 - Cell separation device and cell separation system - Google Patents

Abstract

A cell separation device comprising a container, in which a liquid supplied from above can be filled, and a partition mechanism, which partitions the inside of the container into upper and lower parts, said cell separation device being characterized in that: the partition mechanism is provided with a partition plate which has through holes and a barrier wall which is in contact with the partition plate and provided with holes communicating with the inside of the lower surface of the upper inside part of the container; and, when the partition plate rotates around the center in the long axial direction of the container as a rotational axis, the upper inside part and the lower inside part of the container are switchable to a communicated state and a blocked state. In the cell separation device according to the present invention, a cell-containing liquid can be easily filled without causing disturbance on the boundary surface between the cell-containing liquid and a separation liquid and, after centrifuging, layered cells can be easily separated and, moreover, target cells can be harvested at a high purity.

Description

Cell separation device and cell separation system

The present invention relates to a cell separation device capable of separating a cell type such as a mononuclear cell using centrifugation and a technique for obtaining the cells using a cell separation system including the cell separation device.

Conventionally, as a general technique for separating cells from blood or the like, a separation solution such as Ficoll-Paque (registered trademark, Pharmacia Fine Chemicals) is used at the bottom of a test tube. A method of installing and centrifuging is known (for example, see Patent Documents 1 and 2). In this method, a step of installing a predetermined amount of Ficoll pack on the bottom of a test tube, a step of pipetting a blood sample onto the Ficoll pack, and a blood component having a specific gravity greater than the specific gravity of Ficoll pack Centrifuging to pass through and a step of collecting the mononuclear cell layer separated above the Ficoll pack with a pipette. However, in the methods described in Patent Documents 1 and 2, the desired cells can be obtained as desired by disrupting the boundary surface when the sample is placed on the separation liquid or mixing with other layers during separation and recovery. It was difficult to obtain with purity.

Therefore, as a technique for coping with such problems, a first container having a first liquid container housing portion and a second container having a second liquid housing portion and provided below the first container, A communication state in which the first container and the second container are attached so as to be relatively rotatable, and the first container and the second container are communicated by relatively rotating the first container and the second container. There has been proposed a cell separation device including a switching mechanism capable of switching between a blocking state in which communication between the first liquid storage unit and the second liquid storage unit is blocked (see, for example, Patent Document 3). However, in the structure in which a plurality of containers and members are connected as described above, the operation becomes complicated, and when the container itself is rotated to switch the communication state / blocking state between the containers, the liquid filled in the container is changed. Since vibration is applied, it is necessary to pay attention to the rotation operation. If the care is neglected, there is a problem that the boundary surface of the stratified cells moves due to the vibration.

Japanese translation of PCT publication 2010-504083 Special table 2007-509601 gazette JP 2013-132240 A

The present invention has been made in view of the above-described problems, and the purpose thereof is to allow easy filling without disturbing the boundary surface with the separation liquid when filling the cell-containing liquid, and the centrifugation. It is an object of the present invention to provide a cell separation device that can easily separate cells stratified after separation and can collect desired cells with high purity.
Another object of the present invention is to provide a cell separation system capable of efficiently collecting desired cells using the above-described cell separation device.

As a result of intensive studies to solve the above problems, the present inventors have provided a partition mechanism that can partition the inside of the container up and down inside the container that can be filled with liquid, and provided with a through hole as the partition mechanism. The present inventors have found that the above-mentioned problems can be solved by adopting a partition plate and configuring the partition plate to rotate about the center in the major axis direction of the container as a rotation axis, thereby completing the present invention.

That is, the present invention provides the following.
(1) A cell separation device for separating a target cell from a cell-containing solution,
A container that can be filled with liquid;
A partition mechanism for partitioning the inside of the container up and down,
The partition mechanism has a partition plate provided with a through hole, and a partition wall provided with a hole communicating with the interior of the container on the upper inner lower surface of the container in contact with the partition plate, and the partition plate is in the major axis direction of the container The cell separation device is configured to be able to switch the upper interior and the lower interior of the container to a communication state or a blocking state by rotating about the center of the container.
(2) The cell separation device according to (1), wherein the surface roughness Ra inside the container is 0.1 to 20 nm.
(3) The cell separation device according to (1) or (2), wherein a cross-sectional area of the through hole is 3 to 25% with respect to a maximum cross-sectional area inside the container.
(4) The cell separation device according to any one of (1) to (3), wherein the volume inside the container is 130% or more of the volume inside the container.
(5) It has at least two holes provided in the through hole and / or the partition wall, and the at least two holes are arranged at equal positions from the center in the long axis direction of the container ( The cell separation device according to any one of 1) to (4).
(6) The cell separation device according to any one of (1) to (5), wherein an inner diameter inside the upper part of the container is configured to gradually reduce the diameter toward a hole provided in the partition wall.
(7) The cell separation device according to any one of (1) to (6), wherein a sealing material is provided around the through hole or around the hole provided in the partition wall.
(8) The cell separation device according to any one of (1) to (7), wherein the cell separation device has a configuration capable of self-supporting with the bottom of the container grounded.
(9) The cell separation device according to (8), wherein the bottom surface of the container is a flat surface.
(10) The outer diameter of the upper and / or lower part of the container is such that the upper and / or lower side surfaces of the container can be fixed to the surface of the container installation adapter provided in the centrifuge. The cell separation device according to any one of (1) to (9), which is configured to be larger than the outer diameter of the other side surface.
(11) The cell separation device according to any one of (1) to (10), wherein a liquid supply and / or discharge port is provided in an upper portion of the container.
(12) The cell separation device according to any one of (1) to (11), wherein a filter having micropores is provided inside the container.
(13) The cell separation device according to (12), wherein the filter has an average pore size of 0.1 to 8 μm.
(14) There are two or more liquid supply or discharge ports, and at least one port is provided below the filter inside the upper part of the container. A cell separation device according to claim 1.
(15) the cell separation device according to any one of (1) to (14);
A cleaning solution bag and / or a filter having micropores;
Consists of
A cell separation system, wherein the cleaning solution bag and / or the filter having the micropores are connected to the cell separation device by a tube.
(16) The cell separation system according to (15), wherein the cell-containing solution bag and the waste solution bag are further connected.

The cell separation device of the present invention includes a container that can be filled with liquid from above and a partition mechanism that partitions the interior of the container up and down, and the partition mechanism is in contact with the partition plate having a through-hole and the partition plate. The upper and lower inner surfaces of the container have a partition wall with a hole communicating with the interior of the container, and the partition plate rotates about the major axis of the container as a rotation axis so that the upper interior and the lower interior of the container Is configured to be able to be switched to a communication state or a shut-off state, and after filling the separation liquid in the lower interior of the container in advance, the partition plate is rotated to keep the upper interior and the lower interior in a shut-off state, After filling the cell-containing liquid into the upper interior of the container from above, the partition plate can be rotated to bring the upper interior and the lower interior into communication with each other. With Easily without disturbing the interface may be filled with a cell separation liquid onto the separated liquid. In addition, after the cell separation device is centrifuged, the partition plate is rotated so that the upper interior and the lower interior of the container are blocked, so that desired cells such as mononuclear cells stratified in the upper interior of the container Can be easily separated from other cells such as erythrocytes settled in the lower interior, and desired cells stratified in the upper interior can be recovered with high purity.

When the surface roughness Ra inside the container is 0.1 to 20 nm, it is possible to suppress the adhesion of cells to the surface inside the container and improve the cell recovery rate and purity.

By adjusting the cross-sectional area of the through-hole to 3 to 25% with respect to the maximum cross-sectional area inside the container, cells in the cell-containing liquid filled in the upper part of the container at the time of centrifugation can be Since it can move smoothly to the inside through the lower part, the boundary surface of the stratification after the separation operation becomes more horizontal, and the thickness of the stratification tends to be uniform.

By adjusting the volume inside the upper part of the container to 130% or more of the capacity inside the lower part of the container, a sufficient amount of separation liquid can be filled from the lower part of the container to the vicinity of the partition mechanism, and the density gradient Cell separation using can be performed efficiently.

Centrifugation is achieved by having at least two holes provided in the through hole and / or the partition wall, and the at least two holes are arranged at equal positions from the center in the major axis direction of the container. The movement of the cells can be performed smoothly.

The inner diameter of the upper interior of the container is configured so that the diameter gradually decreases toward the hole provided in the partition wall, so that when the centrifugation process is performed, the cells in the upper interior of the container, In particular, cells having a high density can easily settle quickly inside the container, and target cells such as mononuclear cells can easily form a layer having a uniform thickness inside the container.

By providing a sealing material around the through hole or around the hole provided in the partition wall, the water tightness between the container and the partition plate which is a movable member of the partition mechanism can be improved, and the centrifugal separation is performed. In addition, even if the partition plate is rotated, liquid leakage can be prevented.

The cell separation device is configured to be capable of self-supporting with the bottom of the container grounded, so that the partition plate of the partition mechanism can be rotated without giving large vibration while the cell separation device is self-supporting. As a result, it becomes easy to perform the operation of filling the cell-containing solution, and it is possible to prevent the boundary surfaces of the layers formed after centrifugation from moving. The bottom surface of the container is preferably flat.

The outer diameter of the upper and / or lower part of the container is the other side of the container so that the upper and / or lower side of the container can be fixed to the surface of the container installation adapter provided in the centrifuge. By being configured to be larger than the outer diameter, the cell separation device is firmly fixed in the adapter for container installation and can be centrifuged without vibration during centrifugation by the centrifuge. Becomes uniform, and the cells can be separated with high accuracy.

Since the liquid supply and / or discharge port is provided in the upper part of the container, the operation of supplying the cell-containing liquid and / or the operation of discharging the stratified cells can be performed efficiently.

A filter having fine holes may be provided in the upper interior of the container. In this case, by flipping the cell separation device upside down, the cells layered inside the container and the other liquid medium are separated using the filter, and only the target cells are selectively placed in the cell separation device. Can leave. In addition, after introducing the washing liquid into the upper part of the container, the cell separation device can be similarly turned upside down to discharge the washing liquid from the cell separation device.
The average pore diameter of the filter is preferably 0.1 to 8 μm.

In the state where the cell separation device is erected, there are two or more liquid supply or discharge ports, and at least one port is provided below the filter inside the container. The cell-containing liquid can be supplied from the mouth provided below the filter, and the target cells left in the cell separation device using the filter after centrifugation are provided on the lower side. Can be discharged from the mouth.

The cell separation system of the present invention comprises the cell separation device and a filter having a washing liquid bag and / or fine pores, and the cell separation device has the washing liquid bag and / or the fine pore filter. Are connected by a tube, the washing liquid can be introduced into the cell separation device from the washing liquid bag to wash the cells. Moreover, a cell can be isolate | separated from a liquid medium by using the filter which has a micropore.

In the cell separation system, the cell-containing solution bag and the waste solution bag are further connected by a tube, for example, to facilitate the operation of filling the cell-separating device with the cell-containing solution from the cell-containing solution bag, The operation of discharging the liquid medium other than the cells filled inside the container of the cell separation device after centrifugation into the waste solution bag can be facilitated.

It is a schematic explanatory drawing which shows one embodiment of the cell separation device 1 of this invention. It is a schematic explanatory drawing which shows one embodiment of the partition plate 5 which comprises a partition mechanism. A schematic exploded view of the cell separation device 1 shown in FIG. 1 excluding the lid 19 is shown. It is a schematic explanatory drawing which shows the embodiment of another cell separation device 1a of this invention. It is a schematic explanatory drawing which shows the embodiment of another cell separation device 1b of this invention. It is a schematic explanatory drawing which shows the embodiment of another cell separation device 1c of this invention. It is a schematic explanatory drawing which shows the process of performing a cell separation using the cell separation device 1a of this invention. It is a schematic explanatory drawing which shows one embodiment of the cell separation system 32a of this invention. It is a schematic explanatory drawing which shows one embodiment of another cell separation system 32b of this invention. It is a schematic explanatory drawing which shows one embodiment of another cell separation system 32c of this invention.

The cell separation device of the present invention is a cell separation device for separating a target cell from a cell-containing solution,
A container that can be filled with liquid;
A partition mechanism for partitioning the inside of the container up and down,
The partition mechanism has a partition plate provided with a through hole, and a partition wall provided with a hole communicating with the interior of the container on an upper inner lower surface of the container in contact with the partition plate, and the partition plate is a long axis of the container By rotating about the center of the direction as a rotation axis, the upper inside and the lower inside of the container can be switched to a communication state or a blocking state.

In the present invention, the cell separation device refers to a device that can be inserted into an adapter for container installation of a centrifuge rotor and subjected to a centrifugal separation treatment. The centrifuge is not particularly limited as long as it is a device capable of rotating the cell-containing solution at a high speed and stratifying and separating the cells in the cell-containing solution by the density difference. Especially, the cell separation device of this invention can be used conveniently for the centrifugation method which uses a separation liquid.

In the present invention, the cell-containing liquid refers to a liquid containing cells to be collected. Examples of the cell-containing fluid include blood, lymph fluid, cell culture fluid, umbilical cord blood, bone marrow fluid, and the like.
In the present invention, the cells to be collected include mononuclear cells, hematopoietic stem cells, lymphocytes, monocytes, stromal cells, circulating tumor cells and the like.
Examples of the liquid medium contained in the cell-containing liquid include water, buffer solution, physiological saline, plasma, interstitial fluid, and culture medium.

The separation liquid used in the present invention is a liquid adjusted to a specific gravity capable of separating the cells to be collected, and the composition thereof is not particularly limited as long as it does not adversely affect the cells and the stimulation is small. In a buffer solution such as an acid buffered saline, a high molecular compound that is less irritating to cells, such as Ficoll (Sigma), a copolymer of sucrose and epichlorohydrin, polyethylene glycol, dextran, etc. A solution having a specific gravity adjusted can be used. Further, a commercially available blood cell separation liquid, for example, a human lymphocyte specific gravity separation liquid (specific gravity 1.119; Sigma-Aldrich) may be used to mix the separation liquid and the polymer compound so as to have a desired specific gravity. .
The polymer compound used for the preparation of the separation liquid is not particularly limited as long as the stimulation to the cells is small, and examples thereof include Ficoll and dextran, and Ficoll is particularly preferable. In addition, various salts, physiologically active substances, antioxidants and the like can be appropriately blended in the separation liquid. For example, Ficoll-Paque PREMIUM (specific gravity 1.073, 1.077, 1.083) from GE Healthcare Japan may be purchased and used as a separation solution suitable for separating mononuclear cells. Further, it may be prepared by mixing Ficoll solution, Ficoll Conley solution, distilled water, or the like.
Moreover, as a separation liquid for recovering cells other than mononuclear cells, commercially available separation liquids manufactured by GE Healthcare Japan, such as Percoll and Percall plus, can also be used.

The cell separation device includes a container that can be filled with a liquid and a partition mechanism that partitions the inside of the container up and down.
The container that can be filled with the liquid may be designed so that the liquid can be introduced from above. In the present invention, the upper direction refers to the upward direction when the cell separation device 1 is erected, for example, as shown in FIG. The downward direction when the separation device is erected.

The shape and size of the container are not particularly limited as long as the shape and size can be inserted into the adapter for container installation of the centrifuge rotor. For example, a cylindrical container is preferable from the viewpoint of easily filling a liquid inside the container and allowing cells to be easily stratified during centrifugation.

Specific examples of the structural material of the container include a non-reactive polymer, a biocompatible metal, an alloy, and glass. Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene copolymers, polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride, polyamides, polyimides, Examples include polysulfone, polyester, copolyester, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, and polymethylpentene. Among these, polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene, copolyester, and the like are particularly preferable in that they have sterilization resistance.
On the other hand, when using a metal material (biocompatible metal, alloy) as the structural material of the container, for example, stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, Examples thereof include cobalt chromium alloy and titanium nitride-coated stainless steel.

The inner surface of the container is preferably adjusted to a surface roughness Ra of 0.1 to 20 nm. By adjusting the surface roughness Ra in this way, the amount of cells attached to the inner surface of the container can be reduced.
The surface roughness Ra means arithmetic average roughness, and only the reference length is extracted from the roughness curve in the direction of the average line, the x-axis is in the direction of the average line of the extracted portion, and the direction of the vertical magnification is When the y-axis is taken and the roughness curve is expressed by y = f (χ), the value obtained by the following formula is expressed in micrometers (μm). In the formula, “l” indicates a reference length.

The surface roughness Ra can be measured using, for example, a roughness measurement function of an atomic force microscope (for example, “VN-8010” manufactured by Keyence Corporation).

The interior of the container is separated into an upper interior and a lower interior by a partition mechanism. The upper interior of the container is a portion where a cell-containing solution is filled before centrifugation, and the target cells are stratified after centrifugation.
Further, the lower interior of the container is a portion where the separation liquid is filled before centrifugation, and high density cells such as red blood cells settle after centrifugation.

The volume inside the upper part of the container is adjusted to 130% or more of the capacity inside the lower part of the container, and in particular, a separation liquid such as Ficoll pack is filled from the lower part of the container to the vicinity of the partition mechanism. Since the cell-containing liquid can be overlaid on the separation liquid, centrifugation can be performed efficiently. The upper internal capacity with respect to the lower internal capacity is not particularly limited as long as it is 130% or more, and may be 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more. You may design, 500% or less, 490% or less, 480% or less, 470% or less, 460% or less, 450% or less, 440% or less, 430% or less, 420% or less, 410% or less, 400% Hereinafter, it may be designed to be 350% or less, 300% or less, 250% or less, or 200% or less.

In the cell separation device of the present invention, the partition mechanism includes a partition plate provided with the through-hole and a partition wall provided with a hole communicating with the interior of the container on a lower surface inside the container in contact with the partition plate. Yes. The partition plate is a movable member that can rotate about the center in the major axis direction of the container. By rotating the partition plate, the through hole of the partition plate is at least partially overlapped with the hole of the partition wall. The upper interior and the lower interior of the container are in communication with each other, and the partition plate is further rotated to remove the through hole from the hole of the partition wall, whereby the upper interior and the lower interior of the container are blocked. .
In addition, when switching between the communication state and the blocking state by the partition mechanism, the partition wall forming the lower surface inside the upper part of the container does not rotate, so it is necessary to move the liquid such as the cell-containing liquid filled in the upper part. Absent. As a result, the liquid level where the separation liquid and the cell-containing liquid come into contact with each other before centrifugation can be reduced to the cross-sectional integral of the hole, and the disturbance of the liquid level in contact can be minimized. After the separation, the separation operation can be performed without affecting the surface of each layer formed according to the density difference, such as turbulence.

The shape, size, and number of holes provided in the partition wall are not particularly limited, but are matched with the shape, size, and number of through holes of the partition plate from the viewpoint of smoothly moving cells during centrifugation. It is preferable.

It is preferable that the hole provided in the partition wall is arranged so as to at least partially overlap with the through hole arranged on the partition plate.
In addition, the number of holes provided in the partition wall may be one or two or more, but when there are at least two holes, by arranging each hole at an equal position from the center in the major axis direction of the container. In addition, the cells can be moved smoothly during the centrifugation.

In addition, the inner diameter of the upper inside of the container may be configured to gradually reduce toward the hole provided in the partition wall on the upper inner lower surface. The inner diameter inside the upper part of the container refers to the diameter of the lumen portion of the container constituting the upper inner part. In addition, when the shape of the container is a shape other than a cylinder, the maximum diameter of the container lumen is set as the inner diameter of the upper part. In the present invention, the inner diameter of the upper inner portion is configured as described above, so that when the separation process is performed, the cells inside the upper inner portion easily move according to the density difference. Large cells can easily settle quickly inside the container, and as a result, desired cells can easily form a layer having a uniform thickness.
Examples of the diameter reduction include a shape in which the lumen wall of the container is linearly or stepwise reduced, but is not particularly limited.

A partition plate constituting the partition mechanism is disposed inside the container. The shape of the partition plate is not particularly limited as long as it matches the shape inside the container. For example, when the container has a tube shape such as a cylindrical shape, the partition plate is circular, and the container is rotated about the longitudinal axis of the container as an axis to communicate the upper interior with the lower interior. The switching operation between the state and the blocking state can be suitably performed.
The thickness of the partition plate is not particularly limited as long as it can withstand the pressure applied during centrifugation.

The cross-sectional area of the through-hole is adjusted to 3 to 25% with respect to the maximum cross-sectional area inside the container, so that the cell-containing liquid filled in the upper interior of the container at the time of centrifugation Since the cells inside can pass through the through hole and move smoothly downward, the boundary surface of the stratification after the separation operation becomes more horizontal, and the thickness of the stratification can be made uniform easily.
The maximum cross-sectional area inside the container refers to the maximum area of the container lumen.
The cross-sectional area of the through hole and the cross-sectional area of the container are obtained by calculating the cross-sectional area inside the horizontal direction perpendicular to the vertical direction of the container. Further, when there are a plurality of through holes, the sum of the cross sectional areas of all the through holes is taken as the cross sectional area of the through holes.

As for the shape of the through-hole, the shape seen from the major axis direction of the container includes a circle, an ellipse, a triangle, a quadrangle, a polygon, and the like. From the viewpoint of easy sedimentation of high density cells such as red blood cells, a circular shape is preferable.

The number of the through holes may be one, but is preferably 2 or more, more preferably 3 or more, and still more preferably 4 or more, and there is no particular limitation.
The through hole only needs to be formed in the partition plate, and the position thereof is preferably arranged so that at least a part thereof overlaps with the hole arranged in the partition wall on the upper inner lower surface of the container. It is not limited. Further, the number of the through holes may be one or two or more, but when there are at least two, the through holes are arranged at equal positions from the center in the long axis direction of the container. Cells can be moved smoothly during separation.

Among them, it has at least two holes provided in the through hole and / or the partition wall, and the at least two holes are arranged at equal positions from the center in the major axis direction of the container. By aligning the positions of the through hole and the hole at the time of centrifugation, it is possible to smoothly move the cells in the upper part of the container to the lower part through the holes.

By having the sealing material around the through hole or around the hole provided in the partition wall, the water tightness between the container and the partition plate which is a movable member of the partition mechanism can be further improved.
Seal materials include nitrile rubber, fluorine rubber, urethane rubber, silicone rubber, ethylene propylene rubber, hydrogenated nitrile rubber, chloroprene rubber, acrylic rubber, butyl rubber, chlorosulfonated polyethylene, epichlorohydrin rubber, natural rubber, fluorine resin, etc. There is no particular limitation.

Also, by providing a handle on the side surface of the partition plate, the partition plate can be easily rotated using fingers of the hand. The configuration of the handle is not particularly limited as long as the shape allows the partition plate to be rotated with a finger.

Examples of a method for attaching the partition plate to the partition wall on the upper inner lower surface of the container so as to be rotatable about the center in the major axis direction of the container as a rotation axis include, for example, the partition surface on the upper inner lower surface, the surface of the partition plate, It is possible to include a configuration in which a convex portion provided on the surface of the partition plate is fitted to the side wall of the upper inner lower surface while contacting each other.

Further, by providing a convex portion below the partition plate and fitting the side surface of the convex portion and the side surface inside the lower portion of the container so as to contact each other, the partition plate can be arranged more stably inside the container.

In addition to the partition on the upper inner lower surface of the container as described above, a partition may be provided on the lower inner side of the container.

In the present invention, the upper part and the lower part of the container partitioned by the partition plate may be composed of separate container members or may be composed of an integral container member. In the case of another container member, for example, the lower end of the upper container member is fitted to the partition plate, and the side surface of the partition plate is fitted to the side surface of the upper end of the lower container member, Can be further fitted to the side surface of the upper container member. Moreover, when it is an integral container member, a container can be produced with the said partition plate using 3D printer etc.

It is preferable that the bottom of the container is configured to be able to stand by itself while the cell separation device of the present invention is grounded. By providing this self-supporting configuration, the rotation operation of the partition plate of the partition mechanism can be performed without giving a large vibration while the cell separation device is self-supporting, and as a result, the operation of filling the cell-containing liquid can be performed. It becomes easy to perform, and it can prevent that the boundary surface of each layer formed after centrifugation moves.
As the self-supporting configuration, the bottom surface of the container is preferably flat.

The upper part of the container may be open, but a lid is preferably provided from the viewpoint of preventing contamination of bacteria and the like. The shape of the lid is not particularly limited as long as the lid can be firmly fixed to the upper portion of the container.

Further, the outer diameter of the upper and / or lower part of the container is the side of another container so that the upper and / or lower side of the container can be fixed to the surface of the container installation adapter provided in the centrifuge. It is preferable that the outer diameter is larger. By being configured in this manner, the cell separation device can rotate without vibration in the container installation adapter during the centrifugation operation by the centrifuge, so that the thickness of the stratification becomes uniform and the accuracy of Good centrifugation can be performed.

A liquid supply and / or discharge port may be provided in the upper part of the container. By providing the liquid supply and / or discharge port at the top of the container, the operation of supplying the cell-containing liquid and / or the operation of discharging the stratified cells can be performed efficiently. Specifically, the mouth may be provided on the lid or the side surface of the container.
There is no particular limitation on the shape and size of the mouth provided in the upper part of the container.

In addition, by providing a filter having fine holes inside the container, for example, when the container is turned upside down, the liquid medium passes through the filter and is discharged from the cell separation device by gravity, Only the stratified cells can be selectively left in the cell separation device, and the cells can be easily washed.

The “micropore” refers to a pore smaller than the target cell. Among these, the average pore diameter of the filter is preferably 0.1 to 8 μm from the viewpoint of easily leaving desired cells in the cell separation device.
The average pore diameter may be measured with a palm porometer (for example, manufactured by PMI).

The filter includes a layered filter, but is not particularly limited. Filter materials include stainless steel, nickel, polyester, polyolefin, polyacrylonitrile, polyamide, polystyrene, polyalkyl (meth) acrylate, polyvinyl chloride, polychloroprene, polyurethane, polyvinyl alcohol, polyvinyl acetate, polysulfone, polyethersulfone, polybutadiene. Butadiene-acrylonitrile copolymer, styrene-butadiene copolymer, ethylene-vinyl alcohol copolymer, cellulose diacetate, ethyl cellulose and the like, but there is no particular limitation.

Among them, in the cell separation device of the present invention, there are two or more liquid supply or discharge ports, and at least one port is provided below the filter inside the container. In a state where the cell separation device is erected, the cell-containing liquid can be supplied to the upper inside from the mouth provided below the filter, and after centrifugation, the filter is used to store the cell-containing liquid in the cell separation device. The cells left in can be discharged from the mouth provided on the lower side.

The cell separation device of the present invention having the above-described configuration can form a cell separation system by connecting to another cell separation instrument. For example, the cell separation device of the present invention comprises a cleaning solution bag and / or a filter having fine pores, and the washing solution bag and / or the fine pore filter is connected to the cell separation device by a tube. The system that is. By adopting such a configuration, the cells are washed by introducing the washing solution from the washing solution bag into the cell separation device after centrifugation, or further, the cells are separated from the washing solution by using a filter having micropores. be able to.

Further, the tube is used for introducing or deriving a liquid such as a cell-containing liquid, a washing liquid, and a collecting liquid to each cell separation instrument such as a cell separation device constituting the cell separation system. The tube only needs to be flexible enough to be attached to each cell separation instrument placed at a desired position, and the material, size, shape, etc. are not particularly limited.

In addition, a cell-containing liquid bag and a waste liquid bag may be further connected to the cell separation system. Such a configuration facilitates the operation of filling the cell separation device into the cell separation device from the cell-containing solution bag before centrifugation, and the cell separation device is filled inside the container of the cell separation device after centrifugation. The operation of discharging the liquid medium other than the cells to the waste solution bag can be facilitated.
At the time of centrifugation, the cell separation device may be detached from the cell separation system and attached to a centrifuge, and the cell separation device may be connected to the cell separation system again after centrifugation.

As the washing solution bag, the cell-containing solution bag, and the waste solution bag, commercially available known bags may be used, and there is no particular limitation.

The filter having the fine pores is intended to concentrate and wash cells. The average pore diameter of the filter may be 0.1 to 8 μm.
What is necessary is just to connect the filter which has the said micropore to the desired position of the tube which connects each bag and a cell separation device. For example, what is necessary is just to produce the container which incorporates the filter which has the said micropore, and has an inlet / outlet, and connects the inlet / outlet of this container to the said tube.

In addition, a tube connecting each bag and the cell separation device is provided with a plurality of tubes so that a washing solution, a cell-containing solution, a waste solution, and the like can be efficiently passed when a desired cell is collected and washed multiple times. It is preferable to provide this branch. Specific examples of the branching include a method of using a branching pipe such as a bifurcated, trifurcated or quadrant, to which a three-way cock, a four-way cock, a clamp, and a syringe can be connected, but there is no particular limitation.

When recovering the target cells from the cell separation device, a recovery liquid may be introduced into the cell separation device, and the target cells may be recovered together with the recovery liquid in a cell recovery bag connected to the cell separation system. The cell collection bag may be connected to the cell separation system in advance, or a tube connectable to the cell collection bag, that is, a spiked tube, a luer adapter (male, female) tube, or an SCD connection. You may go.
Moreover, as a cell collection bag, a bag having resistance to cryopreservation, a bag capable of cell culture, and the like can be used.

In addition, another cell separation instrument may be connected to the cell separation system via a tube depending on the purpose.
Examples of the cell separation instrument include a cell recovery liquid introduction port used for introducing a cell recovery liquid into a container, a hollow fiber filter module used for concentrating cells, and concentration using the hollow fiber filter module. Examples thereof include a circulation bag used for retaining the cell-containing liquid when performing the above, an air filter for adjusting the pressure in the cell separation system, and the like.
In addition, there is no limitation in particular about the installation position of the cell separation instrument in the said cell separation system.

Hereinafter, embodiments of the cell separation device will be described with reference to the drawings.

1 (a) and 1 (b) show an example of a cell separation device 1 used in the present invention. 1A is a top view of the cell separation device 1, and FIG. 1B is a cross-sectional view of the cell separation device 1 from the side surface direction. FIG. 2 shows a top view of the partition plate 5 provided inside the cell separation device 1 shown in FIG.

The cell separation device 1 includes a container 2 that can be filled with liquid from above and a partition mechanism 3 that partitions the inside of the container 2 up and down.

Examples of the shape of the container 2 include a cylindrical shape as shown in FIGS. 1 (a) and 1 (b). However, there is no particular limitation.

Further, the surface inside the container 2 is adjusted to have a surface roughness Ra of 0.1 to 20 nm, so that the amount of cells that adhere to the inside surface of the container 2 and cannot be separated and collected can be reduced. it can.

The interior of the container 2 is partitioned into an upper interior 6 and a lower interior 7 by the partition mechanism 3. The upper inside 6 of the cell separation device 1 is a portion where a cell-containing liquid is filled before centrifugation, and a target cell is stratified after centrifugation.
The lower interior 7 is a portion where the separation liquid is filled before centrifugation, and high density cells such as red blood cells settle after centrifugation.

It is preferable to adjust the capacity of the upper interior 6 of the container 2 to 130% or more of the capacity of the lower interior 7 of the container 2.

The partition mechanism 3 is a mechanism capable of partitioning the inside of the container 2 up and down. The partition plate 5 provided with a through hole 4 and the container 2 on the lower surface of the upper interior 6 of the container 2 in contact with the partition plate 5. A partition wall 8 having a hole 9 communicating with the inside of the container 2 is provided, and the partition plate 5 rotates about the long axis direction center (the center axis of the container 2) C of the container 2 as a rotation axis. By aligning or removing the positions of the hole 4 and the hole 9, the upper inner part 6 and the lower inner part 7 of the container 2 can be switched to a communication state or a blocking state.

The partition mechanism 3 includes a partition plate 5 provided with a partition wall 8 and a through-hole 4 on the lower surface of the upper inside 6 of the container 2 in contact with the partition plate 5.
For example, as shown in FIG. 1B, the upper interior 6 and the lower interior 7 of the container 2 are in communication with each other by overlapping the through hole 4 of the rotated partition plate 5 with the hole 9 provided in the partition wall 8. It becomes. Although not shown, the partition plate 5 is rotated about the major axis C of the container 2 as a rotation axis, and the position of the through hole 4 is moved to a position that does not overlap with the hole 9. 6 and the lower interior 7 can be cut off.

The holes 9 provided in the partition wall 8 on the lower surface of the through hole 4 and the upper inner 6 are arranged at equal positions from the center C in the major axis direction of the container 2, so that the upper inner 6, the lower inner 7, Can be smoothly performed to set the communication state or the shut-off state.

As for the shape of the through-hole 4, the shape seen from the major axis direction of the container 2 includes a circle, an ellipse, a triangle, a quadrangle, a polygon, and the like. From the viewpoint that cells with a high density, for example, erythrocytes, tend to settle toward the lower inside 7, a circular shape is preferable.

The number of the through holes 4 may be one, but is preferably 2 or more, more preferably 3 or more, and still more preferably 4 or more, and is not particularly limited.

The cross-sectional area of the through hole 4 is preferably adjusted to 3 to 25% with respect to the maximum cross-sectional area inside the container 2. By adjusting the cross-sectional area of the through-hole 4 to the above range, cells in the cell-containing liquid filled in the upper inside 6 of the container 2 pass through the through-hole 4 and move to the lower inside 7 during centrifugation. This can be performed smoothly, the boundary surface of the stratification after the separation operation becomes more horizontal, and the thickness of the stratification can be made uniform easily.
The maximum cross-sectional area inside the container 2 refers to the maximum area of the container lumen.
In addition, when a plurality of through holes are provided, it is preferable to design the sum of the cross-sectional areas of the plurality of through holes to be 3 to 25% with respect to the maximum cross-sectional area inside the container.

The partition plate 5 having the through hole 4 is disposed inside the container 2 so as to contact the lower surface of the partition wall 8. The shape of the partition plate 5 is not particularly limited as long as it matches the shape inside the container 2.
The thickness of the partition plate 5 is not particularly limited as long as it can withstand the pressure applied during centrifugation.

Further, the inner diameter of the upper inner portion 6 of the container 2 may be configured to gradually reduce toward the hole 9 provided in the partition wall 8 on the lower surface of the upper inner portion 6. The inner diameter D of the upper inner part 6 of the container 2 refers to the diameter of the lumen portion of the container 2 constituting the upper inner part 6, and specifically, as shown in FIG. 8 is the length between the lumen walls 10 forming the upper interior 6 of the container 2. Further, the configuration in which the outer diameter D is gradually reduced toward the hole 9 provided in the partition wall 8 on the lower surface of the upper inner 6 is, for example, as shown in FIG. A configuration in which the horizontal thickness of the lumen wall 10 increases toward the hole 9, and the vertical thickness of the partition wall 8 gradually decreases from the vicinity of the center C of the partition wall 8 to the edge of the hole 9. Such a structure is mentioned. In FIG. 1B, the inner diameter (D1 + D2) of the container 2 in the vicinity of the hole 9 is configured to be smaller than the inner diameter D above it, and becomes smaller as it goes downward, and the hole 9 at the lowermost position. It becomes equal to the diameter.
Although not illustrated in the drawings, the inner wall 10 of the container 2 may have a shape in which the diameter is gradually reduced.

Further, as shown in FIGS. 1B and 2, a handle 11 may be provided on the side surface of the partition plate 5. About the structure of the said handle 11, what is necessary is just a shape which can rotate the partition plate 5 with a finger, and there is no limitation in particular.

Moreover, as a method of attaching the partition plate 5 to the partition wall 8 on the lower surface of the upper inside 6 of the container 2 so as to be rotatable about the center C in the major axis direction of the container 2 as a rotation axis, for example, as shown in FIG. As described above, the outer surface of the partition wall 5 on the lower surface of the upper inner 6 and the surface of the partition plate 5 are brought into contact with the surface of the partition plate 5 on the outer surface of the side wall 12 on the lower surface of the upper inner 6. For example, the inner surface of the convex portion 13 may be fitted. In this case, the shape of the side wall 12 on the lower surface of the upper inner 6 as viewed from the major axis direction of the container 2 and the shape of the convex portion 13 provided on the surface of the partition plate 5 are both circular. The partition plate 5 can rotate with the center C in the long axis direction of the container 2 as a rotation axis.

Further, as shown in FIG. 1B, a convex portion 14 is provided on the lower surface of the partition plate 5 separately from the upper surface, and the outer surface of the convex portion 14 and the lower inner portion 7 of the container 2 are configured. When the partition plate 5 is rotated in the cell separation device 1, it can be moved more stably by fitting the upper side surface 15 to be brought into contact with each other.
In this case, the shape of the lower convex portion 14 of the partition plate 5 and the shape of the side wall 15 of the lower inner portion 7 as seen from the major axis direction of the container 2 are both circular.

Further, as shown in FIGS. 1B and 2, by having a sealing material 16 around the through-hole 4, watertightness between the container 2 and the partition plate 5 that is a movable member of the partition mechanism 3. Can be further enhanced.
Further, the position where the sealing material 16 is installed may be provided around the hole 9 provided in the partition wall on the lower surface of the upper inside 6 of the container 2 although not shown.
Further, as shown in FIG. 1B, the sealing material 16 is provided on the contact surface between the container 2 and the partition plate 5, for example, the side wall 12 on the lower surface of the upper inner 6 or the outer surface of the lower convex portion 14. Thus, when the partition plate 5 is rotated, the liquid can be prevented from leaking from the contact surface.

Also, the upper inner portion 6 and the lower inner portion 7 of the container 2 partitioned by the partition plate 5 may be configured by different container members as shown in FIGS. For example, the inner surface of the upper convex portion 13 of the partition plate 5 is fitted into the outer surface 12 at the lower end of the upper container member 2a, and then the upper surface of the lower container member 2b is inserted into the outer surface of the upper convex portion 13 of the partition plate 5. There is a method in which the side surface 17 is fitted and the upper inner surface 17 of the lower container member 2b is fixed to the outer surface 12 of the upper container member 2a. As shown in FIG. 3, the lower container member 2b is fitted with the handle 11 of the partition plate 5, and the groove 40 or the upper container member that enables rotational movement about the center C in the major axis direction of the container 2 is used. A hole 42 is provided for fitting and fixing the fixture 41 provided on the outer surface of the lower end of 2a.

Although not shown, the upper inner part 6 and the lower inner part 7 of the container 2 partitioned by the partition plate 5 may be constituted by an integral container member. The integral container member can be manufactured together with the partition plate so as to have a predesigned shape using a 3D printer or the like.

The cell separation device 1 of the present invention may have a configuration that can stand by itself with the bottom of the container 2 grounded. Examples of the self-supporting configuration include making the bottom surface 18 of the container 2 flat as shown in FIG.

The upper portion of the container 2 may be open, but a lid 19 is preferably provided from the viewpoint of preventing loss of target cells and contamination of other cells. The shape of the lid 19 is not particularly limited. Moreover, it is preferable that the lid 19 and the upper part of the container 2 are firmly fixed. For the fixing, for example, a spiral groove that becomes a male screw on the outside of the container 2 and a female screw on the inside of the lid 19 may be formed, but there is no particular limitation.

The outer diameter of the upper part and / or the lower part of the container 2 is such that the outer surface of the upper part and / or the lower part of the container 2 can be fixed to the surface of the adapter for container installation provided in the centrifuge. It may be configured to be larger than the outer diameter of the side surface. For example, as shown in FIG. 1 (b), the outer diameter of the outer surface 20 of the lid 19 and the outer diameter of the outer surface 21 of the lower part of the lower container member 2b are outside the outer surface 22 of the upper container member 2a. What is necessary is just to be comprised larger than a diameter. In this case, by adjusting the outer diameter of the outer surface 20 of the lid 19 and the outer diameter of the outer surface 21 of the lower container member 2b to be the same size, a container installation adapter for a commercially available centrifuge The cell separation device 1 can be stably installed.
The outer diameter including the handle 11 of the partition plate 5 is adjusted to be substantially the same as or smaller than the outer diameter of the outer surface 20 of the lid 19 and the outer diameter of the outer surface 21 of the lower container member 2b. To do.

A liquid supply and / or discharge port may be provided in the upper part of the container 2. For example, like the cell separation device 1a shown in FIGS. 4 (a) and 4 (b), the liquid or gas supply and / or discharge ports 23a, 23b, and 23c are provided in the lid 19 provided on the upper portion of the container 2. May be provided. In this case, for example, the use may be divided so that the port 23a is a supply port for liquids such as a cell-containing liquid and a washing solution, the port 23b is a gas supply port, and the port 23c is a cell recovery port and / or a waste liquid discharge port. .
There is no particular limitation on the shape and size of the mouths 23a, 23b, 23c provided in the upper part of the container.

In addition, about the cell separation device 1a shown to FIG. 4 (a), 4 (b), the structure except having demonstrated above should just be the same as the structure of the cell separation device 1 shown in FIG.

Further, as in the cell separation device 1b shown in FIGS. 5 (a) and 5 (b), a filter 24 having fine holes may be provided in the upper inside 6 of the container 2. The average pore size of the filter 24 is preferably 0.1 to 8 μm from the viewpoint of easily leaving desired cells in the cell separation device. The filter 24 may be a layered filter.

The cell separation device 1b has a liquid supply port 23a, a gas supply port 23b, and a discharge port 23c in the lid 19 at the top of the container 2. Further, in the liquid supply port 23a, as shown in FIG. 5B, another tube 25 is inserted from the port provided in the lid 19, and the end of the tube 25 is passed through the filter 24 in the upper interior 6. Thus, the position of the liquid supply port 23 a can be provided below the filter 24. Thus, by providing the position of the liquid supply port 23a below the filter 24, the cell-containing liquid can be put into the container 2 without passing through the filter 24 while the lid 19 is fixed to the cell separation device 1b. Can be supplied. In addition, after the cell separation device 1b is centrifuged, the liquid supply port 23a is closed, the discharge port 23c is opened, and the vertical position of the cell separation device 1b is inverted, so that the cell separation is performed using the filter 24. The liquid medium of the cell-containing liquid can be discharged from the discharge port 23c while leaving the target cell in the device 1b.

Further, as in the cell separation device 1c shown in FIGS. 6 (a) and 6 (b), the gas supply port 23b and the liquid discharge port 23c are provided on the upper surface of the lid 19 on the upper side of the container 2 and provided inside the container 2. Alternatively, the liquid supply port 23 a may be provided on the outer surface of the container 2 below the filter 24.

The configurations of the cell separation devices 1b and 1c shown in FIGS. 5 and 6 other than those described above may be the same as those of the cell separation device 1 shown in FIG.

Next, a cell separation method using the cell separation device of the present invention will be described. The cell separation can be performed, for example, by the procedure shown in FIG.

First, the partition plate 5 of the cell separation device 1a of the type having the three ports 23a, 23b, 23c in the lid 19 is rotated to rotate the through hole 4 and the hole 9 provided in the partition wall 8 on the lower surface of the upper inside 6 of the container 2. Are put into communication between the upper interior 6 and the lower interior 7 of the container 2.
Next, the separation liquid 26 is injected from the upper opening 23a of the cell separation device 1a to fill the lower inside 7 ((a) in the figure).
Next, by rotating the partition plate 5 and shifting the position of the through hole 4 of the partition plate 5 from the hole 9 of the partition wall 8, the upper interior 6 and the lower interior 7 of the container 2 are blocked, and then the cell The cell-containing liquid 27 is filled from the upper port 23a of the separation device 1a ((b) in the figure).
Next, the partition plate 5 of the cell separation device 1a is rotated so that the position of the through hole 4 of the partition plate 5 overlaps the hole 9 of the partition wall 8 so that the upper interior 6 and the lower interior 7 of the container 2 communicate with each other. In this state, the cell separation device 1a is attached to the centrifuge, and centrifugation is performed to stratify the cell components ((c) in the figure). In addition, at the time of centrifugation, in order to prevent contamination of germs inside the cell separation device 1a, the mouths 23a, 23b and 23c may be respectively covered.
Next, the cell separation device 1a is taken out of the centrifuge and the mononuclear spheres 28 stratified inside the container 2 are collected. In the case where the cell-containing liquid 27 is blood, in the cell separation device 1a after centrifugation, the red blood cell layer 29 (lower internal 7) and the separation liquid layer 30 (lower internal 7 and The upper interior 6), the mononuclear sphere 28 layer (upper interior 6), and the plasma layer 31 (upper interior 6) are stratified. When collecting the mononuclear sphere 28, the partition plate 5 is rotated so that the position of the through hole 4 of the partition plate 5 is shifted from the hole 9 of the partition wall 8, so that the upper interior 6 and the lower interior 7 of the container 2 are shifted. And the mononuclear cells 28 can be efficiently recovered by easily preventing contamination of other cells such as red blood cells in the lower interior 7.

Next, a cell separation system using the cell separation device will be described.
What is necessary is just to select suitably as a cell separation instrument connected to a cell separation device in the said cell separation system according to the process objective.

For example, the cell separation device of the present invention comprises a cleaning solution bag and / or a filter having fine pores, and the washing solution bag and / or the fine pore filter is connected to the cell separation device by a tube. The system that is.
In this cell separation system, cells can be washed by introducing the washing solution from the washing solution bag into the cell separation device after centrifugation, or the cells can be separated from the washing solution by using a filter having micropores. .

Specifically, there are a cell separation system 32a shown in FIG. 8, a cell separation system 32b shown in FIG. 9, and a cell separation system 32c shown in FIG.

The cell separation system 32a shown in FIG. 8 uses a cell separation device 1a having three openings in the lid 19, and the cell separation device 1a includes a cell-containing liquid bag 35 containing a cell-containing liquid, a cell An air filter 34 for passing gas through the separation device 1a and a washing bag 37 used for washing desired cells in the cell separation device 1a after centrifugation are connected via a tube to separate the cells. A clip 46 is provided. The washing bag 37 includes a washing solution bag 33 containing washing solution, a waste solution bag 36 for containing waste solution generated in the cell separation system 32a, and a cell recovery bag 38 for containing desired cells via tubes. It is connected.

In the cell separation system 32b shown in FIG. 9, the cell separation device 1a has a tube containing a cell-containing solution bag 35, an air filter 34, a washing solution bag 33, a cell collection bag 38, a waste solution bag 36, and a cell collection solution introduction port 43. Connected through. The cell collection liquid introduction port 43 is used for introducing the cell collection liquid into the cell separation system 32b.

In the cell separation system 32c shown in FIG. 10, the cell-containing liquid bag 35, the air filter 34, and the circulation bag 44 are connected to the cell separation device 1a through a tube. Connected to the circulation bag 44 are a washing liquid bag 33, a cell collection bag 38, and a hollow fiber filter module 45 for concentrating the cell-containing liquid. A circuit capable of circulating the cell-containing liquid is formed between the circulation bag 44 and the hollow fiber filter module 45, and a waste liquid bag 36 is further connected to the hollow fiber filter module 45.

The tubes constituting the cell separation systems 32a, 32b, and 32c are not shown in the portion where the three tubes are connected, but a three-way cock or a stopper can be provided to arbitrarily stop the liquid flow. It has become.

In addition, when the cell separation device 1a is subjected to centrifugation, the cell separation device 1a is detached from the cell separation systems 32a, 32b, and 32c and attached to a centrifuge. Further, after the centrifugation, the cell separation device 1a is connected to the cell separation systems 32a, 32b, and 32c again.

Next, a method for performing cell separation using the cell separation systems 32a, 32b, and 32c will be described.

In the cell separation system 32a shown in FIG. 8, after the cell-containing liquid is supplied from the cell-containing liquid bag 35 to the cell separation device 1a before the centrifugation, the cell separation device 1a is removed and the centrifugation process shown in FIG. 7 is performed. . After centrifugation, the cell separation device 1a is attached to the cell separation system 32a.
Next, the cell separation device 1 a is turned upside down to introduce a liquid containing mononuclear cells into the washing bag 37.
Further, a cleaning solution such as physiological saline is introduced into the cleaning bag 37 from the cleaning solution bag 33 to dilute and wash the mononuclear cells.
Thereafter, the washing bag 37 is removed from the cell separation system 32a and centrifuged to settle the mononuclear cells, and then the lower part of the washing bag 37 is stopped with a clip 46 at a position where no mononuclear cells are present.
Next, the cleaning bag 37 is turned upside down to introduce waste liquid containing physiological saline into the waste liquid bag 36. In addition, you may perform this washing | cleaning operation in multiple times.
Next, a small amount of cleaning solution is supplied from the cleaning solution bag 33 to the cleaning bag 37, the clip 46 is removed, the mononuclear cells are suspended, and then the mononuclear cells are recovered in the cell recovery bag 38.

In the cell separation system 32b shown in FIG. 9, after the cell-containing liquid is supplied from the cell-containing liquid bag 35 to the cell separation device 1a before centrifugation, the cell separation device 1a is removed and the centrifugation process shown in FIG. 7 is performed. . After centrifugation, the cell separation device 1a is attached to the cell separation system 32b.
Next, the cell separation device 1 a is turned upside down, the liquid containing mononuclear cells is passed through the filter 24, and the waste liquid is introduced into the waste liquid bag 36.
Further, a cleaning liquid such as physiological saline is introduced into the filter 24 from the cleaning liquid bag 33 to clean the mononuclear cells. In addition, you may perform this washing | cleaning operation in multiple times.
Thereafter, a small amount of cell recovery liquid is introduced into the filter 24 from the cell recovery liquid introduction port 43, and the mononuclear cells are recovered in the cell recovery bag 38.

In the cell separation system 32c shown in FIG. 10, after the cell-containing liquid is supplied from the cell-containing liquid bag 35 to the cell separation device 1a before centrifugation, the cell separation device 1a is removed and the centrifugation process shown in FIG. 7 is performed. . After centrifugation, the cell separation device 1a is attached to the cell separation system 32c.
Next, the cell separation device 1 a after centrifugation is turned upside down to introduce a liquid containing mononuclear cells into the circulation bag 44. After introducing a cleaning solution such as physiological saline from the cleaning solution bag 33 into the circulation bag 44, the solution is circulated using the hollow fiber filter module 45 to concentrate and wash the mononuclear cells, and the waste solution is introduced into the waste solution bag 36. . In addition, you may perform this washing | cleaning operation in multiple times.
Thereafter, the mononuclear cells in the circulation bag 44 are collected in the cell collection bag 38.

Any of the cell separation systems 32a, 32b, 32c having the above-described configuration is easy to fill the cell separation device 1a with the cell-containing solution from the cell-containing solution bag 35, and after the centrifugation. Since the liquid medium other than the cells filled in the upper interior 6 of the cell separation device 1a can be easily discharged to the waste liquid bag 36, desired cells can be efficiently recovered.

While many of the advantages of the invention encompassed herein have been described above, it will be understood that this disclosure is merely exemplary in many respects. Without departing from the scope of the present invention, various changes can be made in details, particularly in matters such as the shape, size and arrangement of parts.
Of course, the scope of the invention is limited by the language set forth in the appended claims.

DESCRIPTION OF SYMBOLS 1, 1a, 1b, 1c Cell separation device 2 Container 2a Upper container member 2b Lower container member 3 Partition mechanism 4 Through-hole 5 Partition plate 6 Upper interior 7 Lower interior 8 Partition 9 Hole 10 Lumen wall 11 Handle 12 Upper container member The outer surface of the lower end of 2a 13 The upper convex part of the partition plate 14 The lower convex part of the partition plate 15 The upper side surface of the lower inside 7 of the container 2
16 Sealing material 17 Upper inner surface of the lower container member 2b 18 Bottom surface of the container 19 19 Lid 20 Outer surface of the lid 19 21 Outer surface of the lower container member 2b 22 Outer surface of the upper container member 2a 23 Port 24 Filter 25 Tube 26 Separation liquid 27 Cell-containing liquid 28 Mononuclear cell 29 Red blood cell layer 30 Separation liquid layer 31 Plasma layer 32a, 32b, 32c Cell separation system 33 Washing solution bag 34 Air filter 35 Cell-containing solution bag 36 Waste solution bag 37 Washing bag 38 cells Collection bag 40 Groove 41 Fixing tool of outer surface 12 at the lower end of upper container member 2a 42 Hole for fixing fixing tool 43 Cell collection liquid introduction port 44 Circulation bag 45 Hollow fiber filter module 46 Clip

Claims (16)

1. A cell separation device for separating a target cell from a cell-containing liquid,
   A container that can be filled with liquid;
   A partition mechanism for partitioning the inside of the container up and down,
   The partition mechanism has a partition plate provided with a through hole, and a partition wall provided with a hole communicating with the interior of the container on an upper inner lower surface of the container in contact with the partition plate, and the partition plate is a long axis of the container A cell separation device configured to be capable of switching between an upper inside and a lower inside of a container between a communication state and a blocking state by rotating about the center of the direction as a rotation axis.
2. The cell separation device according to claim 1, wherein the inner surface roughness Ra of the container is 0.1 to 20 nm.
3. The cell separation device according to claim 1 or 2, wherein a cross-sectional area of the through hole is 3 to 25% with respect to a maximum cross-sectional area inside the container.
4. The cell separation device according to any one of claims 1 to 3, wherein a capacity inside the upper part of the container is 130% or more of a capacity inside the lower part of the container.
5. The at least two holes provided in the through hole and / or the partition wall, and the at least two holes are arranged at equal positions from the center in the longitudinal direction of the container. 5. The cell separation device according to any one of 4 above.
6. The cell separation device according to any one of claims 1 to 5, wherein an inner diameter of an upper part of the container is configured to be gradually reduced toward a hole provided in the partition wall.
7. The cell separation device according to any one of claims 1 to 6, further comprising a sealing material around the through hole or around the hole provided in the partition wall.
8. The cell separation device according to any one of claims 1 to 7, wherein the cell separation device has a configuration capable of being self-supported with the bottom of the container grounded.
9. The cell separation device according to claim 8, wherein the bottom surface of the container is flat.
10. The outer diameter of the upper part and / or the lower part of the container is the other side of the container so that the upper and / or lower side surface of the container can be fixed to the surface of the adapter for container installation provided in the centrifuge. The cell separation device according to any one of claims 1 to 9, wherein the cell separation device is configured to be larger than the outer diameter.
11. The cell separation device according to any one of claims 1 to 10, wherein a liquid supply and / or discharge port is provided in an upper part of the container.
12. The cell separation device according to any one of claims 1 to 11, wherein a filter having fine pores is provided inside the container.
13. The cell separation device according to claim 12, wherein the filter has an average pore diameter of 0.1 to 8 µm.
14. The liquid supply or discharge port is provided in two or more, and at least one of the ports is provided below the filter inside the upper part of the container. Cell separation device.
15. A cell separation device according to any one of claims 1 to 14;
    A cleaning solution bag and / or a filter having micropores;
    Consists of
    A cell separation system, wherein the cleaning solution bag and / or the filter having the micropores are connected to the cell separation device by a tube.
16. The cell separation system according to claim 15, wherein the cell-containing liquid bag and the waste liquid bag are further connected.
    
    
    

Images