WO2017179705A1

WO2017179705A1 - Container for cell suspension preparation, and preparation method for cell suspension

Info

Publication number: WO2017179705A1
Application number: PCT/JP2017/015292
Authority: WO
Inventors: 林　真司
Original assignee: 株式会社カネカ
Priority date: 2016-04-15
Filing date: 2017-04-14
Publication date: 2017-10-19
Also published as: JP2019092393A

Abstract

The purpose of the present invention is to provide a method for simply preparing a cell suspension from a living tissue with a high cell recovery rate, and a container used in the method. Provided is a container for cell suspension preparation that is equipped with: a container body 10 that encloses an interior space 13 capable of holding a liquid; a mesh sheet 40 disposed so as to divide the interior space 13 into a first space 11 and a second space 12; an input unit 21 provided to the container body 10; and a discharge unit 31 provided to the container body 10. An input port 20 for pouring liquid into the first space 11 is formed in the input unit 21. A discharge port 30 for discharging liquid from the second space 12 is formed in the discharge unit 31. If the value of the narrow angle formed by the flow direction F of the liquid discharged from the discharge port 30 and a line L normal to the plane conforming to the mesh sheet 40 is taken as θ(º), 90-θ is less than 50. Also provided is a method for preparing a cell suspension from a cell-containing specimen using the container.

Description

Cell suspension preparation container and cell suspension preparation method

The present invention relates to a method for preparing a cell suspension by removing impurities from an enzyme-treated solution of a cell-containing sample such as a collected biological tissue, and a cell suspension preparation container for use in the method.

In the field of cell medicine, when cells used for treatment are administered to a patient, the tissue containing the cells is collected from the living body, then once separated and washed in vitro, and a cell suspension at a concentration suitable for administration. It is a common practice to prepare and administer a solution. As a kind of separation operation, for example, filter separation using a non-woven fabric or centrifugation is known.

For example, Patent Document 1 discloses a blood processing filter including a flexible container having an inlet and an outlet for blood and a sheet-like blood processing filter material arranged so that the inside of the container is separated from the inlet side and the outlet side. It is disclosed. Patent Document 2 discloses a spiral-shaped primary flow path connecting the primary side inlet and the primary side outlet and extending from the membrane periphery to the center of the membrane, and 2 filtrates permeated through the filtration membrane. A plate-type membrane module for collecting a low-molecular-weight substance concentration measurement sample in blood in which a secondary-side flow path leading to a secondary-side outlet is formed is disclosed.

On the other hand, in Patent Document 3, when specific tissue stem cells are obtained from a tissue piece collected from a living tissue using a puncture needle, (1) the pore size is passed through a filter of 200 to 300 μm in a closed system. A step of removing blood cell components, (2) a step of washing with a phosphate buffer containing an antibiotic, (3) a step of subjecting the obtained tissue piece to enzyme treatment to form a cell suspension, and (4) a specific gravity A step of separating light cells from the target cells by floating; (5) a step of passing the suspension of the target cells again through a filter having a pore size of 250 to 300 μm; and (6) a filter having a pore size of 20 to 40 μm. It is disclosed to perform a step of passing, (7) centrifugation and / or a step of obtaining a target cell from a cell suspension by passing through a 400-500 nm filter.

JP 2011-72816 A JP-A-10-99659 JP 2005-287479 A

The separation operation using a filter as disclosed in Patent Documents 1 and 2 is performed to remove undesirable components such as aggregates and leukocytes from a liquid tissue such as blood, and a part of the separation operation is performed inside the filter. Intended to leave. However, for example, when a separation operation using a filter is performed to remove impurities such as undigested material from an enzyme-treated product of adipose tissue, the filter is clogged at an early stage. Therefore, in order to obtain a sufficient amount of cells, a filter having a large area must be used. On the other hand, in the separation operation by centrifugation, the apparatus tends to be large, leading to high costs and further limiting the facilities that can be used. In addition, when washing the cells, the supernatant separated from the cells by centrifugation is removed, but at that time, the cells come into contact with the air and there is a risk of contamination. In the first place, cells that have been pelleted by centrifugation are often damaged and die.

On the other hand, Patent Document 3 describes the use of a syringe-like device for enzyme treatment of living tissue in a closed system, but specific means for supplying enzymes to living tissue while maintaining the closed system. Is not disclosed. In order to actually supply an enzyme to a living tissue while maintaining a closed system inside the syringe-like device, a complicated device is required and it is considered that the realization is not easy.

Therefore, an object of the present invention is to provide a method for preparing a cell suspension easily from a cell-containing sample such as a living tissue with a high cell recovery rate, and a container used therefor.

In view of the above problems, the present inventors have provided a mesh sheet for removing impurities from an enzyme-treated solution of a cell-containing sample such as a living tissue, and suspended the cell without causing clogging or the like. The present inventors have found a container having a constitution capable of preparing a liquid and a method for preparing a cell suspension using the container. The enzyme treatment solution may be obtained by performing an enzyme treatment of the cell-containing sample inside the container, or may be prepared outside the container. The gist of the present invention is as follows.

(1) A cell suspension preparation container,
A container body containing an internal space capable of holding liquid;
A mesh sheet arranged to divide the internal space into a first space and a second space;
A charging unit provided in the container body, and formed with a liquid charging port into the first space;
A discharge part provided in the container body, and formed with a discharge port for liquid from the second space;
With
The container in which 90-θ is less than 50, where θ (°) is a narrow angle formed between the flow direction of the liquid discharged from the discharge port and the normal line of the surface along the mesh sheet .
(2) The container according to (1), wherein the mesh sheet has a pore diameter of 50 to 300 μm.
(3) The container according to (1) or (2), wherein the container body has flexibility.
(4) The container according to any one of (1) to (3), wherein at least a part of the container body is made of a transparent or translucent material.
(5) The container according to any one of (1) to (4), wherein the mesh sheet has a porosity of 40% or more.
(6) Of the portion exposed to the internal space of the mesh sheet, a region having an area of 50% or more with respect to the area when the portion is viewed in plan is a region where 90-θ is less than 50. (1) A container according to any one of (5).
(7) A cell suspension preparation container,
A container body containing an internal space capable of holding liquid;
A mesh sheet arranged to divide the internal space into a first space and a second space;
A charging unit provided in the container body, and formed with a liquid charging port into the first space;
A discharge part provided in the container body, and formed with a discharge port for liquid from the second space;
With
The container body includes a first side wall and a second side wall facing each other with the mesh sheet interposed therebetween,
The first side wall and the mesh sheet surround the first space;
The second side wall and the mesh sheet surround the second space;
The insertion portion is disposed between the peripheral portion of the first side wall and the peripheral portion of the mesh sheet so as to communicate the first space and the outside via the input port,
The discharge part is arranged between the peripheral part of the second side wall and the peripheral part of the mesh sheet so as to communicate the second space and the outside through the discharge port,
The said container by which said 1st side wall, said 2nd side wall, and said mesh sheet are integrated in the peripheral part.
(8) The charging unit and the discharging unit are disposed at positions facing the container body, and the first side wall and the second of the end of the charging unit on the first space side The width along the direction facing the side wall is W1,
The width of the end of the discharge portion on the second space side along the direction in which the first side wall and the second side wall face each other is W2,
The average value of W1 and W2 is W,
The distance between the end of the input portion on the first space side and the end of the discharge portion on the second space side is D
When
The container according to (7), wherein D / W is 12 or more.
(9) The container according to (7) or (8), wherein the first side wall and the second side wall have flexibility.
(10) A method for preparing a cell suspension from a cell-containing sample,
(A) a container body containing an internal space capable of holding a liquid;
A mesh sheet arranged to divide the internal space into a first space and a second space;
A charging unit provided in the container body, and formed with a liquid charging port into the first space;
A discharge part provided in the container body, and formed with a discharge port for liquid from the second space;
A cell-containing sample and an enzyme are introduced through the inlet, and an enzyme treatment solution is generated by performing an enzyme reaction in the first space, and / or an enzyme treatment of the cell-containing sample is conducted through the inlet. The process of adding liquid,
(B) The enzyme treatment liquid is passed through the mesh sheet to remove impurities, a cell suspension is generated on the second space side, and the generated cell suspension is discharged through the discharge port. A process comprising the steps of:
(11) When the narrow angle formed by the flow direction of the cell suspension discharged through the discharge port and the normal of the surface along the mesh sheet is θ (°) in step (b) The method according to (10), wherein the cell suspension is discharged so that 90-θ is less than 50.
(12) The method according to (10) or (11), wherein the container is the container according to any one of (1) to (9).
(13) The cell-containing sample is one or more selected from the group consisting of fat, skin, blood vessel, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle, prostate, intestine, amniotic membrane, placenta and umbilical cord The method according to any one of (10) to (12), which is derived.
(14) The method according to any one of (10) to (13), wherein the input port and / or the discharge port are sealed with silicon rubber and / or a needleless port.
(15) the discharge port is connected to a cell suspension treatment apparatus capable of washing and concentrating the cell suspension in a closed system;
(C) The method according to any one of (10) to (14), further comprising a step of washing and concentrating the cell suspension discharged from the discharge port with the device in a closed system to obtain a cell concentrate. the method of.

This specification includes the disclosure of Japanese Patent Application No. 2016-082522, which is the basis of the priority of the present application.

According to the method of the present invention, a cell suspension can be prepared in a closed system without an open system operation such as centrifugation from a cell-containing sample such as a collected biological tissue or an enzyme-treated solution of a cell-containing sample. it can. Further, by using the cell suspension preparation container of the present invention in the method, it is possible to prepare a cell suspension with an excellent throughput and cell recovery rate without causing clogging of the mesh. .

It is a perspective view which shows one Embodiment of the container 1 for cell suspension preparation of this invention. FIG. 1 is a cross-sectional view taken along line AA of the cell suspension preparation container 1 of FIG. 1-1. FIG. 2 is a cross-sectional view taken along the line BB of the cell suspension preparation container 1 of FIG. 1-1. FIG. 1 is a perspective view showing a state in which a liquid is placed in the internal space 13 of the cell suspension preparation container 1 of the present invention shown in FIG. 1-1. FIG. 2 is a cross-sectional view taken along the line CC of the cell suspension preparation container 1 of FIG. 2-1. FIG. 2 is a schematic end view of the vicinity of a discharge part 31 of a CC line cutting part of the cell suspension preparation container 1 of FIG. 2-1. It is the schematic of one Embodiment of the cell suspension processing apparatus connected to the container for cell suspension preparation of this invention. A graph showing the relationship between 90-θ and the amount of cell suspension treated, where θ (°) is the narrow angle formed by the flow direction of the cell suspension and the normal of the surface along the mesh sheet It is. 10 is a graph showing the relationship between 90-θ and the cell recovery rate when the narrow angle formed by the flow direction of the cell suspension and the normal of the surface along the mesh sheet is θ (°). It is the graph which showed the relationship between the pore diameter of a mesh sheet | seat, and a cell recovery rate. It is a graph which shows the result in Example 6 and Comparative Example 3. FIG. 8 shows an enzyme treatment container 810 for preparing an enzyme treatment solution by containing a cell-containing sample and an enzyme, and a cell suspension containing the prepared enzyme treatment solution. 1 is a schematic diagram showing a configuration of a cell suspension preparation system 800 including a cell suspension preparation container 1 according to an embodiment of the present invention and a collection container 820 that collects the generated cell suspension. is there.

(Cell suspension preparation container)
FIGS. 1-1 to 1-3 are schematic views showing one embodiment of the cell suspension preparation container of the present invention. The cell suspension preparation container 1 of this embodiment is arranged so that a container body 10 containing an internal space 13 capable of holding a liquid and the internal space 13 are divided into a first space 11 and a second space 12. The mesh sheet 40, the container main body 10 and the charging section 21 provided with the liquid inlet 20 into the first space 11, and the container main body 10 and the liquid from the second space 12 are provided. The discharge part 31 in which the discharge port 30 of this was formed is provided. FIGS. 1-1 to 1-3 schematically show an example of the shape of the cell suspension preparation container 1 of the present embodiment when no contents are contained in the internal space 13. FIGS. 2-1 and 2-2 show an example of the shape when the liquid is filled in the internal space 13 of the cell suspension preparation container 1 of the present embodiment. However, in FIGS. 2-1 and 2-2, the depiction of the liquid is omitted for simplicity of illustration. Further, in order to fill the internal space 13 of the cell suspension preparation container 1 with a liquid, the discharge port 30 needs to be sealed, but depiction of means for sealing is also omitted.

(Container body)
The container body 10 that constitutes the outer shell of the internal space 13 that can hold the liquid can be provided with a mesh sheet 40 in the manner described later, and should not affect the cells that are held inside. For example, any material can be used. The container body 10 and the mesh sheet 40 include, for example, a first side wall 15 and a second side wall 16 that face each other with the mesh sheet 40 therebetween, and the first side wall 15 and the mesh sheet 40 surround the first space 11. The second side wall 16 and the mesh sheet 40 can be configured to surround the second space 12. FIGS. 1-1 to 1-3 represent such embodiments. In this embodiment, the first side wall 15, the second side wall 16, and the mesh sheet 40 are integrated at the first side wall peripheral part 150, the second side wall peripheral part 160 and the mesh sheet peripheral part 41.

It is preferable that at least a part of the container body 10 is made of a transparent or translucent material because the state of the cell suspension inside can be visually observed. In addition, it is preferable that at least a part, preferably the whole, be flexible because the internal cell suspension can be physically separated by clamping as necessary. It is preferable that the container main body 10 does not contain an adhesive at a portion exposed to the internal space 13. When the container main body 10 includes the first side wall 15 and the second side wall 16, the manufacturing process can be simplified if the material of the side wall can be bonded by thermal fusion without using an adhesive. This is preferable because it does not need to consider the influence of the adhesive on the cells. Specific examples of the material of the container body 10 include vinyl chloride, soft vinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polyolefin such as polyethylene and polypropylene, hydrogenated product of styrene-butadiene-styrene copolymer, and Examples thereof include thermoplastic elastomers such as styrene-isoprene-styrene copolymers or hydrogenated products thereof, and mixtures of thermoplastic elastomers with polyolefins and softeners such as ethylene-ethyl acrylate. It is preferable that the inner surface defining the internal space 13 of the container body 10 is pear-finished because residual liquid can be reduced when the cell suspension is discharged.

When the container body 10 has flexibility, it is normal that the shape changes when the liquid is accommodated in the internal space 13. In the embodiment of the cell suspension preparation container 1 shown in FIGS. 1-1 to 1-3, when the container body 10 in which the first wall portion 15 and the second wall portion 16 are combined has flexibility, When the liquid is accommodated in the space 13, as shown in FIGS. 2-1 and 2-2, the central portions of the first wall portion 15 and the second wall portion 16 bulge outwardly, The volume of 13 increases. Each treatment such as an enzyme treatment, which will be described later, and removal of impurities through the mesh sheet 40 can be performed in the internal space 13 whose volume has increased.

(Mesh sheet)
The mesh sheet 40 is disposed so as to divide the internal space 13 into a first space 11 and a second space 12. As the mesh sheet, one that can allow liquid such as water to pass therethrough, and preferably allows necessary cells to pass therethrough is used. The mesh sheet 40 preferably has a pore diameter in the range of 50 to 300 μm. If the pore size is in such a range, contaminants such as undigested tissue clumps and debris contained in the enzyme-treated product of living tissue are removed without causing clogging of the mesh, and only necessary cells are removed. Can be passed. In order to further manifest this effect, the pore diameter is preferably 50 μm or more, more preferably 95 μm or more, preferably 300 μm or less, more preferably 200 μm or less. The mesh sheet 40 preferably has a hole area ratio of 40% or more, particularly 45% or more because clogging is difficult to occur.

In this specification, “mesh” refers to a material having a network structure in which a plurality of pores penetrating from one surface to the other surface are two-dimensionally arranged in a planar shape. Means. Examples of the mesh include, but are not limited to, one in which fibers are woven so as to form a network structure, and a membrane in which a plurality of pores penetrating in the thickness direction are formed. The mesh also includes what is generally called a “screen”. A sheet made of mesh is referred to as a “mesh sheet”. In this specification, the “mesh” is a material that does not have the network structure and has three-dimensional pores (for example, a fibrous porous medium or a sponge-like structure, does not have the network structure, and has a three-dimensional structure. This is a different concept from a material having pores. The material of the mesh sheet 40 is a synthetic resin material such as nylon, polyester, rayon, polyolefin, polystyrene, acrylic resin, polycarbonate, polyacrylamide, polyurethane, vinyl chloride, etc. from the viewpoint of material safety, stability, and availability. A synthetic polymer selected from at least one of the above, an inorganic material such as hydroxyapatite, glass, alumina, and titania, and a metal such as stainless steel, titanium, and aluminum. When two or more materials are combined, the combination is not particularly limited, but synthetic polymers such as polyolefin, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, vinyl chloride, hydroxyapatite, glass, A combination of two or more materials selected from the group consisting of inorganic materials such as alumina and titania, and metals such as stainless steel, titanium, and aluminum is preferable. The mesh sheet 40 is provided in the container body 10 so that the surface of the mesh sheet is substantially flat when the liquid is put into at least the internal space 13. FIGS. 2-1 and 2-2 show a state in which a liquid (not shown) is put in the internal space 13 of the container 1 shown in FIGS. 1-1 to 1-3. Since the mesh sheet 40 can transmit the liquid, the liquid medium, the dissolved component, and the component having a size capable of transmitting the mesh sheet in the liquid input through the input port 20 are not limited to the first space 11. The components that pass through the mesh sheet 40 and move to the second space 12 and cannot pass through the mesh sheet 40 remain in the first space 11.

The pore size of the mesh sheet is the maximum inscribed in the contour of the inner circumference of the pore, which is specified by observing the pores penetrating from one surface of the mesh sheet to the other surface along the penetration direction. Means the average diameter of the inscribed circle. The average value is preferably calculated from, for example, a value obtained by observing 50 or more pores, preferably 100 or more pores. When the mesh sheet is woven so that the fibers form a network structure, the pore diameter is equal to the average opening of the mesh. The average opening can be calculated based on the number of fibers per unit length (for example, 1 inch) measured using, for example, a urnometer.

The opening rate of the mesh sheet refers to the ratio of the total area of the opening portions to the entire area when the mesh sheet is viewed in plan.

(Input section and discharge section)
The inlet 21 is provided with a liquid inlet 20 into the first space 11 of the internal space 13, and the outlet 31 is provided with a liquid outlet 30 from the second space 12 of the internal space 13. Yes. Only one input port 20 and one discharge port 30 may be provided, or two or more of one or both of the input ports 20 and the discharge ports 30 may be provided as necessary. In an example of a method for using the cell suspension preparation container 1, a cell-containing sample such as a living tissue and an enzyme for separating cells into a cell suspension are input from the input port 20. The enzyme reaction is carried out in 11. In another example of the method of using the cell suspension preparation container 1, an enzyme-treated solution obtained by subjecting a cell-containing sample such as a living tissue to an enzyme reaction treatment with an enzyme is introduced from the inlet 20 and accommodated in the first space 11. Is done. When the enzyme treatment liquid after the enzyme reaction or the charged enzyme treatment liquid passes through the mesh sheet 40, a cell suspension is generated on the second space 12 side. The cell suspension is discharged from the discharge port 30.

The input unit 21 and the discharge unit 31 may be provided in any way. For example, the relative relationship between the container body 10 and the mesh sheet 40 is the configuration shown in FIGS. When including the 1st side wall 15 and the 2nd side wall 16 which oppose through the sheet | seat 40 in between, the injection | throwing-in part 21 is between the peripheral part 150 of the 1st side wall 15, and the peripheral part 41 of the mesh sheet 40. In addition, the first space 11 and the outside are arranged to communicate with each other through the insertion port 20, and the discharge portion 31 is disposed between the peripheral portion 160 of the second side wall 16 and the peripheral portion 41 of the mesh sheet 40. It arrange | positions so that the 2nd space 12 and the exterior may be connected via the exit 30. FIG. More specifically, the input portion 21 and the discharge portion 31 are made of a hollow cylindrical material having an arbitrary cross-sectional shape, for example, a resin tube, and the peripheral portion 150 of the first side wall 15 and the peripheral portion 41 of the mesh sheet 40. Or between the peripheral edge portion 160 of the second side wall 16 and the peripheral edge portion 41 of the mesh sheet 40. Specific examples of the material constituting the input unit 21 and the discharge unit 31 include vinyl chloride, soft vinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polyolefin such as polyethylene and polypropylene, styrene-butadiene-styrene copolymer. Examples thereof include hydrogenated polymers, thermoplastic elastomers such as styrene-isoprene-styrene copolymers or hydrogenated products thereof, and mixtures of thermoplastic elastomers with softeners such as polyolefin and ethylene-ethyl acrylate. .

The inlet 20 and outlet 30 are closed by at least a part of the inlet 21 and outlet 31 that communicate with each other by silicon rubber or the like that allows a sample to be introduced using an injection needle so that the internal space 13 can be closed. It is preferable that a structure such as a needleless port can be directly inserted. In addition, for example, the portion of the input portion 21 that communicates with the input port 20 has a plurality of ports for inputting the sample by branching in the middle, and the like so that the cell-containing sample and the enzyme can be input from separate ports. It may be. The port may be provided with a filter as necessary.

The input unit 21 and the discharge unit 31 may be provided at any position of the container body 10 as long as the relative position with respect to the mesh sheet 40 is as described above. However, if the input part 21 and the discharge part 31 are provided in positions opposite to each other in the container body 10, from the input of the cell-containing sample and the enzyme or the input of the enzyme-treated solution of the cell-containing sample to the discharge of the cell suspension. This is preferable because the processing can be performed smoothly. Note that “providing at positions facing each other” means that, for example, as shown in FIG. 1-1, when the container body 10 has a quadrangular shape or a shape similar thereto, the insertion portion 21 is provided on one of the opposed sides, and the other It means that the discharge part 31 is provided in the side of

(Preferred first embodiment of container for cell suspension preparation)
In a particularly preferred first embodiment (hereinafter sometimes referred to as “first embodiment”) of the cell suspension preparation container 1 of the present invention, the mesh sheet 40 flows in the flow direction of the liquid discharged from the discharge port 30. When the narrow angle formed by F and the normal line L of the surface along the mesh sheet 40 is θ (°), 90−θ is set to be less than 50. FIG. 2-3 shows the discharge part 31 of the CC line cutting part of the cell suspension preparation container 1 in a state where a liquid (not shown) is accommodated in the internal space 13 shown in FIG. FIG. 4 is a schematic end view of the vicinity, showing a relationship of θ (°) as a narrow angle formed by the flow direction F of the liquid discharged from the discharge port 30 and the normal line L of the surface along the mesh sheet 40. ing. In the first embodiment, since 90-θ is in the above range, positions on the mesh sheet 40 having different distances from the discharge port 30 along the direction F are included, and an undigested tissue mass It is difficult for the mesh sheet 40 to be clogged with impurities such as debris and debris, and the cell suspension can be taken out from the outlet 30 smoothly. Although the mechanism of this phenomenon is not necessarily clear, even when the contaminants clog the mesh sheet 40 at a position where the distance along the direction F from the discharge port 30 is relatively small, the mesh sheet 40 It is estimated that the clogging is less at a position where the distance is relatively larger. 90-θ is preferably less than 50, more preferably 45 or less, further preferably 30 or less, and particularly preferably 5 or less. The smaller the value of 90-θ, the higher the effect of the first embodiment. The lower limit of 90-0 is not particularly limited, and may be 0.

In the first embodiment, the portion exposed to the internal space 13 of the mesh sheet 40 is preferably 50% or more, more preferably 60% or more, more preferably with respect to the area when the portion is viewed in plan. Is 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and more preferably 100% of the area so that the 90-θ is in the above range. It is done. If the region in which the 90-θ is in the above range in the mesh sheet 40 is in the above range, the above effect of the first embodiment is sufficiently exhibited.

(Second preferred embodiment of container for cell suspension preparation)
In the particularly preferred second embodiment (hereinafter sometimes referred to as “second embodiment”) of the cell suspension preparation container 1 of the present invention, among the above structural features,
The container body 10 includes a first side wall 15 and a second side wall 16 that face each other with the mesh sheet 40 therebetween,
The first side wall 15 and the mesh sheet 40 surround the first space 11,
The second side wall 16 and the mesh sheet 40 surround the second space 12,
The insertion portion 21 is disposed between the peripheral portion 150 of the first side wall 15 and the peripheral portion 41 of the mesh sheet 40 so as to communicate the first space 21 and the outside via the input port 20.
The discharge part 31 is arrange | positioned so that the 2nd space 12 and the exterior may be connected via the discharge port 30 between the peripheral part 160 of the 2nd side wall 16, and the peripheral part 41 of the mesh sheet 40,
The first side wall 15, the second side wall 16, and the mesh sheet 40 are integrated at the

peripheral edge portions

150, 160, and 41.

In the cell suspension preparation container 1 of the second embodiment, the two members constituting the first side wall 15 and the second side wall 16, the mesh sheet material, and the material for the input part and the discharge part are integrated. This makes it relatively easy to manufacture.

The first side wall 15, the second side wall 16, and the mesh sheet 40 are integrated as long as these three members cannot be separated and need to be directly coupled to each other. Instead, any one or more of these three members or other members may be combined. For example, in the peripheral portion, the first side wall 15 and the second side wall 16 are not directly connected to each other and are connected and integrated via the mesh sheet 40 therebetween. Even when the side wall 15 and the second side wall 16 are not directly coupled to each other and are coupled and integrated via the input unit 21 (or the discharge unit 31) and the mesh sheet 40 therebetween, the first The side wall 15, the second side wall 16, and the mesh sheet 40 are an example “integrated at the peripheral edge”.

Further, according to the second embodiment, the angle θ (°) of the mesh sheet 40 can be adjusted by the dimensions and relative distances of the input portion 21 and the discharge portion 31 in the container body 10, thereby The cell suspension preparation container 1 having a 90-θ value of less than 50 can be easily produced. Specifically, when the input unit 21 and the discharge unit 31 are disposed at positions facing the container body 10, (i) the first side wall of the end 22 on the first space 11 side of the input unit 21. 15 and the width along the direction in which the second side wall 16 faces W1, and (ii) the first side wall 15 and the second side wall 16 of the end 32 on the second space 12 side of the discharge part 31 face each other. And (iii) the distance between the end 22 on the first space 11 side of the input portion 21 and the end 32 on the second space 12 side of the discharge portion 31. In the case of D, the angle θ (°) can be adjusted by the ratio (D / W) of D, which is the average value of D, W1, and W2. For example, if D / W is 12 or more, 15 or more, or 20 or more, the value of 90−θ can be 5 or less. As D / W is larger, 90-θ is smaller. The upper limit of D / W is not particularly limited, but is usually 50 or less, 40 or less, or 30 or less. The ratio of W1 and W2 is not particularly limited, but W1 / W2 is more preferably 0.2 to 5, and more preferably 0.5 to 2, from the viewpoint of facilitating the production of the cell suspension preparation container 1. 0.9 to 1.1 is more preferable, and 1 is particularly preferable. In this embodiment, it is preferable that the input part 21 and the discharge part 31 are arranged so that the input path by the input port 20 and the input path by the discharge port 30 are located on the same axis.

(Method for preparing cell suspension from cell-containing sample)
In another aspect, the present invention also relates to a method for preparing a cell suspension from a cell-containing sample such as a living tissue. The method is
(A) To the first space provided in the container body, the container main body containing the internal space capable of holding the liquid, the mesh sheet arranged to divide the internal space into the first space and the second space, A cell-containing sample and an enzyme through the input port in a container having an input unit in which the liquid input port is formed and a discharge unit provided in the container body and formed with a liquid discharge port from the second space. And an enzyme reaction is performed in the first space to generate an enzyme treatment solution and / or a cell-containing sample enzyme treatment solution is introduced through the inlet, and (b) the enzyme treatment solution is passed through the mesh sheet. At least a step of removing impurities, generating a cell suspension on the second space side, and discharging the generated cell suspension through a discharge port.

As an enzyme used for enzyme treatment of a cell-containing sample, an enzyme that can be used for separation of cells from a cell-containing sample such as a biological tissue such as collagenase, trypsin, dispase, collagenase, pepsin can be appropriately used.

In the container used in step (a), the mesh sheet is permeable to liquids such as water, and is selected from the range described for the mesh sheet used in the embodiment of the cell suspension preparation container. be able to. In the container used in the step (a), the pore size of the mesh sheet is, for example, in the range of 50 to 300 μm, preferably 50 μm or more, more preferably 95 μm or more, preferably 300 μm or less, more preferably 200 μm or less. The container is preferably sterilized before use. As the sterilization method, sterilization methods generally used for sterilization of medical devices such as γ-ray sterilization, electron beam sterilization, EOG sterilization, and high-pressure steam sterilization can be suitably used. The container for step (a) may be any container as long as it has the structure described above, but preparation of a cell suspension having a structure as shown in FIGS. 1-1 to 1-3. It is preferable to use a container for a cell because a cell suspension can be smoothly prepared without clogging or the like.

In the step (a), when a cell-containing sample and an enzyme are put through an inlet and an enzyme reaction is performed in the first space to generate an enzyme-treated solution, a cell-containing sample that is a raw material for the cell suspension; The enzyme for separating the cells and preparing the cell suspension is put into the first space of the container through the inlet, where the enzyme reaction is carried out. The reaction mixture containing the enzyme and the cell-containing sample formed at this time is preferably in a liquid state. For this purpose, the enzyme and the cell-containing sample are first mixed in the presence of a liquid medium such as water as a dispersion medium. It is preferable to accommodate in a space. The enzyme can be added as an aqueous enzyme solution as necessary. Since the mesh sheet can permeate the liquid, in step (a), the liquid medium contained in the reaction mixture placed in the first space, the dissolved component, and a size that can permeate the mesh sheet. The component moves not only in the first space but also in the second space, and the component having a size that cannot pass through the mesh sheet remains in the first space. In the enzyme reaction of the step (a), the component that cannot permeate the mesh sheet in the first space is enzyme-treated, and the target cell becomes an enzyme-treated solution dispersed in a liquid medium. By passing the enzyme-treated solution after the enzyme reaction through the mesh sheet, undigested tissue clumps and debris are removed, and a cell suspension is generated in the second space. To be discharged. The step (a) is preferably carried out in a state where the inlet and outlet of the container are closed. For this purpose, the inlet and outlet portions communicating with the inlet and outlet of the container are sealed in such a manner that a sample can be input, such as silicon rubber and a needleless port. Is a closed system, and it is preferable that an enzymatic reaction can be performed without touching the outside air. Moreover, when implementing an enzyme reaction, it can put on suitable reaction conditions by putting the container on a shaker or putting it in a thermostat. When placing the cell-containing sample and enzyme, or when carrying out the enzyme reaction, placing the container so that the first space is vertically below the second space, This is preferable because clogging can be prevented.

In the step (a), when the enzyme-treated solution of the cell-containing sample is introduced through the inlet, the enzyme-treated enzyme is used to separate the cells and prepare the cell suspension of the cell-containing sample as a raw material of the cell suspension. Is performed in another container, and the enzyme treatment liquid produced in the other container is accommodated in the first space of the container through the inlet. The enzyme treatment liquid accommodated in the first space includes a liquid medium such as water, a dissolved component, and a component (such as a target cell) having a size that can permeate the mesh sheet. In the step (b) described later, the enzyme-treated solution passes through the mesh sheet, thereby removing undigested tissue masses and debris and other contaminants, generating a cell suspension in the second space, and then the outlet. Through the container. When putting the enzyme treatment liquid, it is preferable to place the container so that the first space is located below the second space in the vertical direction because clogging of the mesh sheet can be prevented. Also in this case, it is preferable that the inlet of the container inlet and / or the outlet of the outlet are sealed in such a manner that a sample can be introduced, such as silicon rubber or a needleless port.

When the cell suspension is discharged in the step (b), a closed system in which the discharge port is connected to a container for containing the cell suspension or a device for a further processing step by a closed channel. It is preferable from the viewpoint of suppressing contamination.

When discharging the cell suspension in the step (b), the narrow angle formed by the flow direction of the cell suspension discharged through the discharge port and the normal of the surface along the mesh sheet is θ (°). When 90-θ is less than 50, preferably 45 or less, more preferably 30 or less, and particularly preferably 5 or less, clogging of the mesh sheet is less likely to occur. In addition, it is preferable to hold the container in a state where the discharge port is at the lowermost end, since the discharge of the cell suspension proceeds smoothly. Therefore, with the container held so that the discharge port is at the bottom, the value of 90-θ based on the angle θ (°) is less than 50, preferably 45 or less, more preferably 30 or less, and particularly preferably 5 It is preferable to have the following structure, more specifically, a structure as shown in FIGS. 1-1 to 1-3. The flow rate at which the enzyme treatment liquid passes through the mesh sheet can be appropriately adjusted by applying a negative pressure from the discharge port, but may be a flow rate that is allowed to fall naturally due to gravity. In order for the enzyme treatment solution to smoothly pass through the mesh sheet, the flow rate through the mesh sheet should be in the range of 1 to 600 mL / min, particularly in the range of 10 to 500 mL / min, especially in the range of 20 to 350 mL / min. preferable.

In this embodiment, the step (b) may be performed after the step (a) is completed, or the step (a) and the step (b) may be performed in parallel.

In an example of the method of the present invention including steps (a) and (b), the cell suspension preparation container 1 shown in FIGS. 1-1 to 1-3 is used.

In the step (a), an enzyme treatment with an enzyme for separating the cells and preparing the cell suspension of the cell-containing sample that is the raw material of the cell suspension is performed in another container, FIG. 8 shows an example of a cell suspension preparation system suitable for storing the generated enzyme treatment liquid in the first space 11 of the cell suspension preparation container 1 through the inlet 20.

A cell suspension preparation system 800 shown in FIG. 8 contains a cell-containing sample and an enzyme, performs an enzyme treatment, and prepares an enzyme treatment liquid, and an enzyme treatment liquid prepared. A container for cell suspension preparation 1 according to an embodiment of the present invention that accommodates and generates a cell suspension, and a recovery container 820 for recovering the generated cell suspension, and a container for enzyme treatment 810 And the cell suspension preparation container 1 are connected by a pipe 850, and the cell suspension preparation container 1 and the collection container 820 are connected by a pipe 850. As the pipe 850, a general plastic tube can be used, and among them, a vinyl chloride tube can be preferably used from the viewpoint of safety and durability.

In the cell suspension preparation system 800 shown in FIG. 8, the enzyme processing container 810 includes an enzyme processing container main body 811, a sample input part 812 in which an input port for inputting a cell-containing sample is formed, an enzyme An enzyme input unit 813 formed with an input port for supplying an enzyme solution containing, and a filter sterilization filter 814 disposed on the upstream side of the enzyme input unit 813 for filter sterilizing the enzyme solution to be input. The enzyme input part 813 and the enzyme processing container main body 811 are connected by a pipe 850 as necessary, and a clamp 854 for sealing the pipe 850 of this part as necessary is provided. The container body 811 for enzyme treatment is preferably a flexible plastic container. Moreover, it is preferable that the inner surface is pear-finished because residual liquid can be reduced. The cell-containing sample input through the sample input unit 812 and the enzyme solution input through the filter sterilization filter 814 and the enzyme input unit 813 are mixed in the enzyme treatment container body 811 to form a reaction mixture. The reaction mixture is preferably in a liquid form, and for that purpose, a liquid medium such as water is appropriately included as a dispersion medium. The enzyme can be added as an aqueous enzyme solution as necessary. An enzyme reaction is performed in the enzyme processing container main body 811 to generate an enzyme processing solution for the cell-containing sample. When carrying out the enzyme reaction, the enzyme treatment container 810 can be placed on a shaker or placed in a thermostatic bath, so that appropriate reaction conditions can be obtained.

The container body 811 for enzyme treatment and the cell suspension preparation container 1 according to one embodiment of the present invention are connected by a tube 850. The enzyme treatment solution generated in the enzyme treatment container 810 passes through the tube 850 and enters the cell suspension preparation container from the input port 20 formed in the input part 21 of the cell suspension preparation container 1. 1 to the first space 11. This step corresponds to the above step (a). The portion of the tube 850 that connects the enzyme treatment container 810 and the cell suspension preparation container 1 can be configured to be separable by a pair of

luer locks

851 and 852. One of the pair of

luer locks

851 and 852 is a male luer lock and the other is a female luer lock, which can be connected in a liquid-tight manner. A clamp 856 that can close the tube 850 may be disposed in the portion of the tube 850 between the enzyme treatment container 810 and the upstream luer lock 851 as necessary. The cell suspension preparation system 800 is installed so that the enzyme treatment container 810 containing the enzyme treatment solution is located vertically above and the cell suspension preparation container 1 is located below vertically, The enzyme treatment liquid may be moved from the enzyme treatment container 810 to the cell suspension preparation container 1, or by applying negative pressure from the discharge port 30 side of the cell suspension preparation container 1 to perform the enzyme treatment. The liquid may be moved from the enzyme treatment container 810 to the cell suspension preparation container 1.

In the cell suspension preparation system 800 shown in FIG. 8, when the enzyme treatment liquid stored in the first space 11 of the cell suspension preparation container 1 passes through the mesh sheet 40, impurities are removed, and the second A cell suspension is generated on the space 12 side, and the generated cell suspension is accommodated in the collection container 820 through the discharge port 30 formed in the discharge unit 31 and the pipe 850 connected to the discharge unit 31. The The discharge part 31 of the cell suspension preparation container 1 and the collection container 820 are connected by a tube 850, and a clamp 857 for sealing the tube 850 of this portion as necessary is provided. The collection container 820 is preferably a flexible plastic container. Moreover, it is preferable that the inner surface is pear-finished because residual liquid can be reduced. A pipe 850 is connected to the downstream side of the collection container 820, and the cell suspension collected in the collection container 820 is discharged through the pipe 850. A luer lock 853 is provided at the downstream end of this portion of the tube 850 as necessary, and the luer lock 853 is attached to an apparatus for further processing of the cell suspension, for example, a cell suspension processing apparatus described later. Can be connected through. The luer lock 853 may be either a female lock or a male luer lock depending on the luer lock provided in the further downstream device. The cell suspension preparation system 800 is installed so that the cell suspension preparation container 1 is positioned above the vertical direction and the collection container 820 is positioned below the vertical direction. The suspension preparation container 1 may be moved from the second space 12 to the collection container 820, or a negative pressure is applied from the tube 850 downstream of the collection container 820 to apply the cell suspension. The cell suspension preparation container 1 may be moved from the second space 12 to the collection container 820.

(Connection with cell suspension treatment equipment)
The discharge port of the container may be connected to a separately prepared cell suspension processing apparatus, whereby the cell suspension obtained from the discharge port is washed and concentrated in a closed system to obtain a cell concentrate. You may do it. At this time, it is preferable that the discharge port of the container and the cell suspension treatment apparatus are connected by a closed flow path, and the collection container 820 as described above may be interposed therebetween. The washing of the cell suspension includes body fluids and enzymes contained in the cell suspension, including dilute solutions such as physiological saline, infusion, medium, distilled water, inorganic salts, saccharides, serum, and proteins. It means replacing with liquid, buffer, medium, plasma and the like. In one embodiment, the present invention provides:
(A) A container main body that encloses an internal space capable of holding a liquid, a mesh sheet that is disposed so as to divide the internal space into a first space and a second space, and the container main body. A cell-containing sample and an enzyme are placed through the inlet in a container provided with an inlet part formed with a liquid inlet and a discharge part provided in the container body and formed with a liquid outlet from the second space. Performing an enzyme reaction in the first space to generate an enzyme treatment solution, and / or adding an enzyme treatment solution of a cell-containing sample through an inlet,
(B) passing the enzyme treatment solution through a mesh sheet to remove impurities, generating a cell suspension on the second space side, and discharging the generated cell suspension through a discharge port; and (c) ) A step of washing and concentrating the cell suspension discharged from the discharge port in a closed system with a cell suspension treatment apparatus capable of washing and concentrating the cell suspension in a closed system to obtain a cell concentrate. The present invention relates to a method for obtaining a cell concentrate from a living tissue. According to this method, a cell concentrate having a concentration that can be directly administered to a subject can be prepared directly from a living tissue in a closed system, and a high cell recovery rate can be realized.

As the cell suspension treatment apparatus, for example, an apparatus disclosed in Japanese Patent Application Laid-Open No. 2015-42167 can be used. FIG. 3 shows a schematic view of one embodiment of the apparatus (cell suspension treatment apparatus 900). In the following description, the numbers in parentheses indicate corresponding portions in FIG. The cell suspension treatment apparatus (900) of the document includes a cell suspension storage container (903) having a solution inlet port (915), a circulation outlet port (916) and a circulation inlet port (917), a cell A container having a suspension inlet (918), a cell suspension outlet (919) and a filtrate outlet (920) is filled with a hollow fiber separation membrane, and the liquid is filtered from the cell suspension and concentrated. A cell suspension treatment device (906), a circulation inlet port (917) of the storage container (903), and a cell suspension introduction port (918) of the cell suspension treatment device (906). An introductory communication pipe (928) and an outflow communication pipe (929) communicating with the cell suspension outlet (919) of the cell suspension processor (906) and the circulation outlet port (916) of the storage container. Storage capacity (903) and a cell suspension processor (906), a circulation circuit (930) for performing concentration while circulating the cell suspension, and a collection container (905) for collecting the concentrated cell concentrate And a collection path (931) for feeding the cell concentrate in the storage container (903), the cell suspension treatment unit (906), and the circulation circuit (930) to the collection container (905). ), An injection path (927) for injecting a solution into the solution inlet port (915) of the storage container (903), and a communication pipe (928) for introducing the storage container (903) or the circulation circuit (930). ) Detecting means (911) for detecting the amount of the cell concentrate in the inside, and detecting that the cell concentrate has been concentrated to a predetermined amount by the detecting means (911), (903) to cell suspension processor And a control means for stopping the liquid feed cell concentrate to 906), characterized in that it comprises a. When the apparatus of the above document is used as the cell suspension processing apparatus (900), the outlet of the container of the present invention is for injecting a solution into the solution inlet port (915) of the cell suspension storage container (903). The injection path (927) is connected to the cell suspension preparation container connection section (901). At this time, it is preferable that the discharge port of the container of the present invention and the cell suspension treatment apparatus are connected by a closed channel, and the collection container 820 as described above is interposed therebetween. Also good. Although the apparatus of the above document can automatically perform washing and concentration of the cell suspension, the cell suspension treatment apparatus (900) connected to the discharge port of the container in the present invention is a cell suspension. The liquid may be manually washed and concentrated.

(Cell suspension treatment device: storage container)
A storage container (903) used in the cell suspension processing apparatus (900) stores a cell suspension for processing, and passes the cell suspension through a cell suspension processing device as described later. A container for circulating and storing the obtained cell concentrate. The storage container has a solution inlet port, a circulation outlet port, and a circulation inlet port. The solution inlet port refers to a port for supplying the cell suspension into the storage container. The circulation inlet port refers to a port for passing the liquid from the storage container to the cell suspension treatment device, and the circulation outlet port refers to the flow of the cell concentrate concentrated by the cell suspension treatment device through the storage container. The port to do. These ports are preferably installed in the lower part of the storage container. By installing all these ports at the bottom, the cell suspension supplied through the solution inlet port and the solution for dilution are efficiently agitated with the liquid circulating in the storage container and the circulation circuit. Can be concentrated and diluted.

The number of solution inlet ports, circulation outlet ports, and circulation inlet ports may be one each, but may be increased to a plurality as necessary. For example, the solution inlet port can be used not only as a cell suspension but also as a port for a diluting solution or a priming solution. In addition to a port for a cell suspension, an inlet port for a diluting solution or a priming solution May be provided. In addition, a target port different from the above three types of ports may be provided. For example, a collection port for passing the cell concentrate through the collection container can be mentioned. By directly connecting the recovery port of the storage container and the recovery container, the cell concentrate in the storage container can be recovered quickly.

The material of the storage container is preferably one that does not affect the cells in the cell suspension or cell concentrate, and a flexible resin material is particularly preferable from the viewpoint of easy handling. Moreover, it is advantageous that the inner surface of the storage container is pear-finished because the remaining liquid when the suspension is discharged from the storage container can be reduced. The volume of the storage container can be used without any particular limitation. However, if it is too large, a large amount of diluent used for dilution is required, and therefore it is preferably 1000 mL or less. Further, the shape of the storage container, the structure of each port, the material, and the like may be appropriately determined depending on the type and volume of the cell suspension to be processed, and are not particularly limited.

In addition, a ventilation hole may be provided in the upper part of the storage container. By providing this vent, the atmosphere in the storage container can be exchanged with the atmosphere and the atmosphere can be exchanged. For example, when the liquid in the storage container is discharged, the storage container is decompressed and the storage container is crushed. There is an advantage to prevent. This vent may be provided with an air filter (908), which can prevent unwanted components from entering the storage container from the outside.

(Cell suspension treatment device: Cell suspension treatment device)
The cell suspension treatment device (906) is an apparatus for filtering and concentrating liquid from the cell suspension, and is connected so that the cell suspension can be passed from the storage container. In the cell suspension treatment device, a hollow fiber separation membrane is filled in a container having a cell suspension inlet, a cell suspension outlet, and a filtrate outlet. The cell suspension introduction port is an inlet for introducing the cell suspension from the storage container into the cell suspension processor, and is connected to a circulation inlet port of the storage container.

The cell suspension outlet is an outlet for taking out the concentrated cell suspension (cell concentrate). By connecting the cell suspension outlet to the circulation outlet port of the storage container, the cell suspension can be circulated between the storage container and the cell suspension treatment device to perform concentration. The filtrate outlet is an outlet for taking out the filtered liquid from the cell suspension.

The hollow fiber separation membrane used in the cell suspension treatment apparatus preferably has a cylindrical container filled with several tens to thousands of hollow fibers bundled. The arrangement of the hollow fiber separation membrane may be linear, bent, or spiral, and the hollow fiber separation membrane is arranged at both ends of the hollow fiber separation membrane between the cell suspension inlet and the cell suspension outlet. If is held, the shape is not particularly limited.

A synthetic polymer material can be used for the hollow fiber separation membrane used in the cell suspension treatment device from the viewpoint of the safety and stability of the material. Among these, polysulfone-based, polyolefin-based, or cellulose-based polymer materials can be preferably used. In addition, the pore diameter of the hollow fiber separation membrane is not limited as long as cells do not leak to the outside, and is preferably as large as possible so that unnecessary components can be efficiently filtered. Specifically, those having an average pore diameter of 0.01 μm or more and 1.0 μm or less can be suitably used. In addition, a hollow fiber having an inner diameter of 400 μm or more and 1000 μm or less is preferably used.

When the cell suspension supplied from the cell suspension inlet passes through the inside of the hollow fiber separation membrane, the liquid is filtered to the outside of the hollow fiber separation membrane to produce a cell concentrate. As a structure of the cell suspension treatment device, for example, a hollow container is filled with a hollow fiber separation membrane, and the end of the hollow fiber is in close contact with the end of the tubular container with an adhesive or the like, and is opened at the end. It is mentioned that a header portion serving as a cell suspension inlet or a cell suspension outlet is provided at the end of the cylindrical container so that the cell suspension can flow into and out of the hollow fiber membrane. The cylindrical container only needs to have one or more outlets for filtrate, and the filtrate filtered from the inside of the hollow fiber is discharged from the outlet for filtrate. The cell suspension treatment device generally needs to have a structure in which a hollow fiber separation membrane is packed in a sealed container, but the cell suspension inlet and the cell suspension outlet are outlets for filtrate. As long as it has a structure separated from the wall material constituting the hollow fiber separation membrane, various structures can be adopted. For example, a dialyzer used for hemodialysis can be exemplified as a similar structure.

At the filtrate outlet (920) of the cell suspension treatment device, a circuit is installed for the filtrate separated by the hollow fiber separation membrane to flow out. It is preferable to connect the circuit for the filtrate and the waste liquid container (904) because the concern that the filtrate leaks to the outside can be reduced. The filtrate taken out from the outlet for filtrate can be collected by passing it through a waste liquid container or the like. The waste liquid container can be used without particular limitation as long as it does not leak waste liquid.

A pump (914) for feeding the filtrate may or may not be installed between the filtrate outlet of the cell suspension treatment device and the waste container. For example, by providing a pump in the path connecting the filtrate outlet and the waste liquid container, the filtrate can be discharged at a constant flow rate, so that the treatment time can be made constant and the filtration efficiency in the cell suspension treatment device can be increased. It becomes possible to control. That is, by driving the pump, the discharge of the filtrate from the cell suspension treatment device is promoted, and as a result, the concentration treatment in the cell suspension treatment device can be promoted. In addition, when the cell concentrate concentrated to a predetermined concentration is passed through the collection container and collected, rapid recovery can be achieved by stopping the pump. Moreover, the collected filtrate may be discarded as it is, or may be reused by performing reprocessing such as sterilization. In addition to the cell suspension inlet, the cell suspension outlet, and the filtrate outlet, the cell suspension processor sends the cell concentrate in the cell suspension processor to a collection container. For this purpose, a cell concentrate recovery port may be provided. This cell concentrate recovery port can be connected to a recovery container (905).

(Cell suspension treatment equipment: Circulation circuit)
The circulation circuit (930) includes a communication pipe for introduction communicating with a circulation inlet port of the storage container and a cell suspension introduction port of the cell suspension treatment device, and a cell suspension of the cell suspension treatment device. It is a circuit comprising a lead-out port and a lead-out communicating tube that communicates with the circulation outlet port of the storage container. Through this circulation circuit, a cell concentrate can be produced by performing concentration while circulating the cell suspension between the storage container and the cell suspension treatment device. As a tube constituting the circulation circuit, a general plastic tube can be suitably used. Vinyl chloride can be suitably used in terms of safety and durability. The circulation circuit is preferably provided with a pump (913) from the viewpoint of facilitating control of circulation of the cell suspension or cell concentrate. There is no particular limitation on the number of pumps, but one is sufficient from the viewpoint of easy control. As for the position of the pump, it may be installed in either the introduction communication pipe or the extraction communication pipe, but if it is installed in the introduction communication pipe, the cell suspension introduction port of the cell suspension treatment device It is preferable because a solution having a high pressure can be introduced into the liquid and the liquid can be efficiently separated. A branch (not shown) is provided between the pump (913) and the cell suspension inlet (918), an air chamber is provided at the branch, and the tip of the branch is connected to a pressure gauge. It is preferable.

A branching portion may be provided on either of the circulation circuits. By connecting the branch portion to the recovery container (905) by piping, the path from the branch portion to the recovery container is used as the final recovery path (931), and the cell suspension The cell concentrate in the liquid processor and the circulation circuit can be quickly recovered in the recovery container. It is preferable that the branching section be installed at a position as close as possible to the circulation outlet port because the amount of residual liquid remaining in the circulation circuit from the branching section to the outlet port can be reduced in the recovery step.

(Cell suspension treatment device: collection container)
The recovery container (905) is a container for recovering the cell concentrate concentrated to a predetermined concentration. The collection container is preferably a flexible plastic container. Moreover, if pear processing is given to the inner surface, there exists an advantage which can reduce the residual liquid after collect | recovering cell concentrate. Further, the collection container may have a connection port to which a needle, a syringe, or the like can be connected. By adopting such a configuration, it can be suitably used when the cell concentrate recovered in the recovery container is transferred to another container. The shape of the collection container is not particularly limited. For example, when the volume increases, the inner area of the container with which the collected cells come into contact increases, and the attached cells may remain in the container and lead to cell loss. Therefore, the capacity inside the container is preferably small.

(Cell suspension treatment device: recovery path)
The collection path (931) is a path for sending the cell concentrate in the storage container, the cell suspension treatment device, and the circulation circuit to the collection container. A general plastic tube can be used as the tube constituting the recovery path, and among these, a vinyl chloride tube can be preferably used from the viewpoint of safety and durability.

Examples of the recovery path include the following three aspects.
1) When a branch portion is provided on the circulation circuit, a path connecting the branch portion and the recovery container is a recovery path.
2) When the recovery port of the storage container and the recovery container are connected, the connected path becomes the recovery path.
3) When the cell concentrate recovery port of the cell suspension treatment device and the recovery container are connected, this connected path becomes the recovery path.

Regarding the recovery path, any one of the above embodiments is preferable, and two or more may be used in combination.

(Cell suspension treatment device: injection path)
The injection path (927) is a path for injecting the solution into the solution inlet port of the storage container. As the pipe constituting the injection path, a general plastic tube can be used, and among them, a vinyl chloride tube can be preferably used from the viewpoint of safety and durability. As the solution, in addition to the cell suspension, the whole cell suspension treatment apparatus (900) before the concentration treatment or a dilution solution for diluting the cell concentrate obtained by concentrating the cell suspension again. A priming solution for priming may be included. Diluents include physiological saline, infusion, distilled water, buffer, culture medium, plasma and inorganic salts, saccharides, serum, liquids containing proteins, etc. Especially from the viewpoint of safety, physiological saline and infusion are suitable. Can be used. Examples of the priming solution include physiological saline, infusion solution, distilled water, buffer solution, culture medium, plasma, inorganic salts, saccharides, serum, and liquids containing protein. It can be used suitably. The same solution may be used for the diluent and the priming solution, or different solutions may be used. When using a different solution, it is possible to install a circuit that is separated from the branch portion on the circuit connected to the solution inlet port of the storage container and connected to each connection portion.

It is preferable that a pump (912) for feeding a liquid is installed in the injection path. The pump can stably feed liquid to the storage container. In addition, the position where the pump is installed is a junction of circuits connected to the cell suspension preparation container connection part (901), the diluent container connection part (902), and the optionally provided priming liquid container connection part (926). And a solution inlet port are preferable because the number of pumps required for liquid feeding can be reduced. A branch (not shown) is provided between the pump (912) and the clamp (921 or 922), an air chamber is provided at the branch, and the tip of the branch is connected to a pressure gauge. Is preferred.

(Cell suspension treatment device: detection means)
The detection means is means for detecting the amount of the cell concentrate in the storage container or the communication pipe for introduction of the circulation circuit. Examples of the means for detecting the liquid amount in the storage container include using a bubble sensor that directly detects the liquid level of the storage liquid in the storage container. Further, as another embodiment, for example, a tube is installed so as to be parallel to the vertical direction with respect to the storage container, and a circuit in which the storage container and the tube communicate with each other below is used. The bubble sensor may be installed in this tube by adjusting it so that it is equal to the liquid level. With this means, the liquid level in the tube can be detected by the bubble sensor as the liquid level in the storage container. Moreover, you may install a chamber under the said tube installed in parallel with the storage container. In particular, by installing a chamber having an inner diameter larger than that of the tube, it is expected to reduce the occurrence of defects that are detected by the intrusion of bubbles in the tubes installed in parallel.

As a means for detecting the amount of the cell concentrate in the introduction communication pipe, a bubble sensor (911) is provided in the introduction communication pipe (928) communicating with the circulation inlet port of the storage container. If it is this form, since the cell suspension can be concentrated more than the means for detecting the amount of liquid in the storage container, the amount of the concentrated suspension (cell concentrate) can be reduced, There is an advantage that the amount of unnecessary components in the suspension can be reduced. Further, the position of the bubble sensor to be installed in the introduction communication pipe is not particularly limited, but by installing it at a position close to the storage container, the length of the circuit connecting the storage container and the bubble sensor can be increased. Can be shortened.

In the cell suspension treatment apparatus (900), the bubble sensor (910) may be installed in the injection path (927) connected to the solution inlet port of the storage container. When bubbles are detected by the bubble sensor, storage of a solution such as a cell suspension, a diluent, a priming solution in the storage container can be stopped. For example, even if the processing amount of the target cell suspension is not known, by installing the bubble sensor in the injection path, the entire amount of the target liquid can be processed without setting the pump driving time for each processing. There is an advantage that can be. Moreover, it can also be set as the process of adding a fixed quantity of solution from the drive time of a pump. For example, when it is necessary to process only a part of the cell suspension, the process can be performed by setting the driving time of the pump. This bubble sensor can be installed anywhere in the injection path, but it is more advantageous to install the bubble sensor as close to the storage container as possible to reduce the amount of liquid remaining in the injection path.

Further, another bubble sensor (909) may be provided on the upper part of the storage container. By installing this bubble sensor, it is possible to determine the presence or absence of a solution near the top in the storage container. That is, when a liquid exceeding the capacity of the storage container is supplied, the sensor can detect and notify the user as an alarm.

A commonly used pump or bubble sensor used in the cell suspension treatment apparatus (900) can be used. It is also possible to switch the flow path by providing a clamp at a desired position in the circuit. These pumps, bubble sensors, and clamps are not particularly limited, and for example, those used in dialysis machines and the like may be used.

(Cell-containing sample)
An example of a cell-containing sample used as a starting material in the method of the present invention or a cell-containing sample used for preparing an enzyme-treated solution is biological tissue. The biological tissue is not particularly limited as long as it is collected from animal tissue, for example, fat, skin, blood vessel, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle, prostate, intestine, amniotic membrane, placenta, The thing derived from an umbilical cord etc. is mentioned. The method of the present invention is particularly useful for removing stromal vascular fraction (SVF) cells from collected adipose tissue. In addition, another example of the cell-containing sample is a cell culture prepared in vitro.

The cell suspension or cell concentrate obtained by the present invention is used for leukemia treatment, myocardial regeneration and blood vessel regeneration, stem cell exhaustion disease, bone disease, cartilage disease, ischemic disease, vascular disease, neurological disease, burn, chronic inflammation. It can be used for regenerative medicine such as heart disease, immune deficiency, coulomb disease, breast augmentation, wrinkle removal, cosmetic molding, tissue enlargement such as tissue depression. In addition, the obtained cells can be seeded and cultured in a structural material such as a scaffold and used for treatment. Alternatively, the resulting cells may be stored frozen for future therapeutic uses.

According to the method of the present invention, preparation of a cell suspension that has been subjected to enzyme treatment and separation treatment from a biological tissue, which has been performed in a conventional method through a plurality of steps in the conventional method, or from an enzyme treatment solution of biological tissue. The preparation of the cell suspension that has undergone the separation treatment can be carried out all in a closed system and in the same container. In addition, since the passage of the enzyme-treated solution through the mesh sheet smoothly proceeds inside the container, even an operator who does not have a special technique can easily prepare the cell suspension. Furthermore, since the cell suspension preparation container of the present invention does not have a particularly complicated structure, it provides a low-cost device capable of preparing a cell suspension in a short time without causing clogging of the mesh sheet. can do. The present invention is very useful for collecting an SVF fraction from adipose tissue, which has conventionally been troublesome.

Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to these examples. In the following description, “pore diameter” represents the mesh opening, and is calculated based on the result of measurement with a luminometer.

1. Preparation of cell suspension A rabbit or human adipose tissue was prepared, a 0.075% collagenase solution was added, and the mixture was reacted at 37 ° C for 30 minutes to 1 hour to prepare a cell suspension.

2. Examination of mesh sheet surface angle The following preliminary test was performed using a cell suspension derived from the same individual. The cell concentration of the cell suspension used for the test was 1.0 × 10 ⁶ cells / mL. A commercially available cell strainer having a nylon mesh sheet with a pore size of 100 μm, a porosity of 42%, and a membrane area of 3.14 cm ² is attached to the centrifuge tube, and the angle of the mesh sheet surface is changed to change the liquid flow direction (vertical direction ) And the mesh sheet surface were adjusted. The cell suspension is allowed to flow spontaneously through a mesh sheet set at a predetermined angle, and the number of cells contained in the cell suspension after passing through the mesh sheet is counted in the cell suspension before passing through the mesh sheet. The cell recovery rate was calculated by dividing by the number of cells contained in. The number of cells was measured with a modified Neubauer hemocytometer (Japanese blood association certified product).

Example 1 When the narrow angle formed by the flow direction of the cell suspension and the normal of the surface along the mesh sheet is θ (°), clogging occurs when 90-θ is set to 45. 30 mL of the cell suspension was allowed to pass through until the occurrence of (stopping the flow of the liquid passing through the mesh sheet). Cell recovery was 95%.

(Example 2) When the narrow angle formed by the flow direction of the cell suspension and the normal of the surface along the mesh sheet is θ (°), clogging occurs when 90-θ is set to 30 32 mL of cell suspension could be passed before the occurrence of. The cell recovery rate was 98%.

(Comparative Example 1) When the angle of the narrow angle formed by the flow direction of the cell suspension and the normal of the surface along the mesh sheet is θ (°), clogging occurs when 90-θ is set to 50 27 mL of cell suspension could be passed through before the occurrence of. The cell recovery rate was 90%.

(Comparative Example 2) When the narrow angle formed by the flow direction of the cell suspension and the normal of the surface along the mesh sheet is θ (°), clogging occurs when 90-θ is set to 90. 26 mL of cell suspension could be passed before the occurrence of. The cell recovery rate was 88%.

FIG. 4 shows the relationship between 90-θ and the throughput of the cell suspension, where θ (°) is the narrow angle formed by the flow direction of the cell suspension and the normal of the surface along the mesh sheet. FIG. 5 is a graph showing the relationship between 90-θ and the cell recovery rate. It was confirmed that by using a mesh sheet having a pore size of 95 μm and setting 90-θ to less than 50, a high volume cell suspension can be treated and a high cell recovery rate can be achieved.

3. Test Using Cell Suspension Preparation Container A test was conducted using a cell suspension preparation container 1 having the structure shown in FIGS. 1-1 to 2-2. In the cell suspension preparation container used for the test, the first side wall 15 and the second side wall 16 are made of a flexible resin sheet. Three types of containers having different pore diameters of the incorporated polyester mesh sheet 40 were prepared. The side wall of the container was manufactured using flexible polyvinyl chloride. In any of the containers, the outer diameter of the tube sandwiched between the container ends as the input part 21 and the discharge part 22 is 8 mm, and the container internal side end part 32 of the discharge part 31 from the container internal side end part 22 of the input part 21. The distance to was about 20 cm. For this reason, the value of D / W defined in this specification was about 25. The membrane area of the mesh sheet was 208 cm ² . The cell suspension preparation containers are suspended so that the input part 21 is at the top, and 50 mL of the cell suspension derived from the same individual is allowed to flow through each container by natural dropping and is taken out from the discharge port 30. It was. The cell concentration of each cell suspension was 1.4 × 10 ⁶ cells / mL. The mesh sheet in the container at the time of liquid flow has 90-θ of about 1 when the narrow angle formed by the normal L of the surface along the mesh sheet and the liquid flow direction F (vertical direction) is θ (°). Met.

(Example 3) When a container having a mesh sheet with a pore size of 95 μm and an open area ratio of 46% was used, clogging did not occur during liquid flow, and the cell recovery rate was 95%.

(Example 4) When a container having a mesh sheet with a pore size of 200 μm and an open area ratio of 61% was used, no clogging occurred during the flow of liquid, and the cell recovery rate was 100%.

(Example 5) When a container having a mesh sheet with a pore size of 300 μm and an open area ratio of 65% was used, clogging did not occur during liquid flow, and the cell recovery rate was 100%.

FIG. 6 is a graph showing the relationship between the pore size of the mesh sheet and the cell recovery rate. It was confirmed that when the angle of the mesh sheet surface with respect to the liquid passing direction was set to the same angle, the cells could be collected at a high recovery rate at least when the pore diameter of the mesh sheet was 95 μm to 300 μm.

4). Test using a cell suspension preparation container and a cell concentration washing system The cell suspension preparation container having the structure shown in FIGS. 1-1 and 2-2 and a cell concentration washing system (manufactured by Kaneka) were connected. The cell suspension was prepared in a closed system, and the results were compared with the results when a cell suspension was prepared in an open system by a conventional method from a sample obtained from the same individual. The cell concentration washing system (manufactured by Kaneka) is an example of “a cell suspension treatment apparatus capable of washing and concentrating a cell suspension in a closed system” disclosed in the present specification. In addition, the container for cell suspension preparation used what the pore diameter of the mesh sheet | seat used in Example 3 is 95 micrometers.

(Example 6) A fat tissue sample and a 0.075% collagenase aqueous solution were prepared in a cell

suspension preparation container

1, and 102 mL of a cell suspension having a cell concentration of 1.6 × 10 ⁶ cells / mL was prepared in the container. The enzyme reaction was performed while shaking the container with a shaker. The enzyme reaction was performed with the inlet 20 and outlet 30 closed. After the enzyme reaction, the solution in the container was separated into two layers, an oil layer and an aqueous layer. The container was hung with the input part 21 facing upward, and the boundary between the two separated layers was clamped so that the components of the oil layer did not flow toward the discharge part 31. The mesh sheet 40 in the container had 90-θ of about 1 when the narrow angle formed by the normal L of the surface along the mesh sheet and the liquid flow direction F (vertical direction) was θ (°). . In that state, negative pressure is applied from the discharge port, the solution is discharged from the container at a rate of 300 mL / min from the discharge port, the cell suspension is transferred to the connected cell concentration washing system, and washing is performed. It was. The cell recovery rate in the cell suspension obtained after washing was 83%.

(Comparative Example 3) A cell suspension having a cell concentration of 1.6 × 10 ⁶ cells / mL was prepared in a centrifuge tube using the same adipose tissue sample and 0.075% collagenase solution as in Example 6. As usual, 20 mL of the resulting cell suspension was opened by opening the centrifuge tube lid, the supernatant liquid was discharged, washing liquid was added, the lid was closed, and centrifugation was repeated and concentrated and washed. Was 68%.

FIG. 7 is a graph showing the results in Example 6 and Comparative Example 3. By comparing the results of Example 6 and Comparative Example 3, by using a container capable of forming a closed system (container for cell suspension preparation) and an automated cell concentration washing system, centrifugation is a common method. It was confirmed that the cells could be recovered at a higher cell recovery rate.

DESCRIPTION OF SYMBOLS 1 ... Cell suspension preparation container, 10 ... Container main body, 11 ... First space, 12 ... Second space, 13 ... Internal space, 15 ... First side wall, 16 ... Second side wall, 20 ... Input port, DESCRIPTION OF SYMBOLS 21 ... Input part, 22 ... End of 1st space side of input part, 30 ... Discharge port, 31 ... Discharge part, 32 ... End of 2nd space side of discharge part, 40 ... Mesh sheet, 41 ... Perimeter of mesh sheet , 150 ... first side wall peripheral part, trough 160 ... second side wall peripheral part, 901 ... cell suspension preparation container connection part, 902 ... dilution liquid bag connection part, 903 ... storage container, 904 ... waste liquid container, 905 ... collection Container, 906 ... Cell suspension treatment device, 908 ... Air filter, 909 ... Bubble sensor, 910 ... Bubble sensor, 911 ... Bubble sensor, 912-14 ... Pump, 915 ... Solution inlet port, 916 ... Circulation outlet port, 917 ... circulation inlet port, 18 ... Cell suspension inlet, 919 ... Cell suspension outlet, 920 ... Filtrate outlet, 921-25 ... Clamp, 926 ... Priming solution bag connection, 927 ... Injection path, 928 ... Inlet communication tube, 929... Communication pipe for derivation, 930... Circulation circuit, 931.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

A cell suspension preparation container,
A container body containing an internal space capable of holding liquid;
A mesh sheet arranged to divide the internal space into a first space and a second space;
A charging unit provided in the container body, and formed with a liquid charging port into the first space;
A discharge part provided in the container body, and formed with a discharge port for liquid from the second space;
With
The container in which 90-θ is less than 50, where θ (°) is a narrow angle formed between the flow direction of the liquid discharged from the discharge port and the normal line of the surface along the mesh sheet .
The container according to claim 1, wherein the pore size of the mesh sheet is 50 to 300 µm.
The container according to claim 1 or 2, wherein the container body has flexibility.
The container according to any one of claims 1 to 3, wherein at least a part of the container body is made of a transparent or translucent material.
The container according to any one of claims 1 to 4, wherein the mesh sheet has a porosity of 40% or more.
2. A region having an area of 50% or more with respect to an area of the mesh sheet exposed to the internal space when viewed in plan is a region where 90−θ is less than 50. The container according to any one of 1 to 5.
A cell suspension preparation container,
A container body containing an internal space capable of holding liquid;
A mesh sheet arranged to divide the internal space into a first space and a second space;
A charging unit provided in the container body, and formed with a liquid charging port into the first space;
A discharge part provided in the container body, and formed with a discharge port for liquid from the second space;
With
The container body includes a first side wall and a second side wall facing each other with the mesh sheet interposed therebetween,
The first side wall and the mesh sheet surround the first space;
The second side wall and the mesh sheet surround the second space;
The insertion portion is disposed between the peripheral portion of the first side wall and the peripheral portion of the mesh sheet so as to communicate the first space and the outside via the input port,
The discharge part is arranged between the peripheral part of the second side wall and the peripheral part of the mesh sheet so as to communicate the second space and the outside through the discharge port,
The said container by which said 1st side wall, said 2nd side wall, and said mesh sheet are integrated in the peripheral part.
The input part and the discharge part are arranged at opposing positions of the container body,
W1 is the width along the direction in which the first side wall and the second side wall face each other at the end of the first space on the first space side,
The width of the end of the discharge portion on the second space side along the direction in which the first side wall and the second side wall face each other is W2,
The average value of W1 and W2 is W,
The distance between the end of the input portion on the first space side and the end of the discharge portion on the second space side is D
When
The container of Claim 7 whose D / W is 12 or more.
The container according to claim 7 or 8, wherein the first side wall and the second side wall have flexibility.
A method for preparing a cell suspension from a cell-containing sample comprising:
(A) a container body containing an internal space capable of holding a liquid;
A mesh sheet arranged to divide the internal space into a first space and a second space;
A charging unit provided in the container body, and formed with a liquid charging port into the first space;
A discharge part provided in the container body, and formed with a discharge port for liquid from the second space;
A cell-containing sample and an enzyme are introduced through the inlet, and an enzyme treatment solution is generated by performing an enzyme reaction in the first space, and / or an enzyme treatment of the cell-containing sample is conducted through the inlet. Adding liquid,
(B) The enzyme treatment liquid is passed through the mesh sheet to remove impurities, a cell suspension is generated on the second space side, and the generated cell suspension is discharged through the discharge port. A process comprising the steps of:
In step (b), when the narrow angle formed by the flow direction of the cell suspension discharged through the discharge port and the normal of the surface along the mesh sheet is θ (°), 90− The method according to claim 10, wherein the cell suspension is discharged so that θ is less than 50.
The method according to claim 10 or 11, wherein the container is the container according to any one of claims 1 to 9.
The cell-containing sample is derived from one or more selected from the group consisting of fat, skin, blood vessels, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle, prostate, intestine, amniotic membrane, placenta and umbilical cord; The method according to any one of claims 10 to 12.
The method according to any one of claims 10 to 13, wherein the input port and / or the discharge port are sealed with silicon rubber and / or a needleless port.
The outlet is connected to a cell suspension treatment device capable of washing and concentrating the cell suspension in a closed system;
The cell suspension according to any one of claims 10 to 14, further comprising a step (c) of washing and concentrating the cell suspension discharged from the discharge port in a closed system by the device to obtain a cell concentrate. the method of.