JP2019092393A - Container for cell suspension preparation and preparation method of cell suspension - Google Patents

Abstract

To provide a method for preparing cell suspension easily at a high cell recovery rate, from a biological tissue, and a container used therefor.SOLUTION: There are provided a container for cell suspension preparation comprising: a container body 10 including therein, an inner space 13 capable of holding a liquid; a mesh sheet 40 for partitioning the inner space 13 into a first space 11 and a second space 12; an input part 21 provided on the container body 10 and on which an input port 20 of a liquid into the first space 11 is formed; and a discharge part 31 provided on the container body 10 and on which a discharge port 30 of the liquid from the second space 12 is formed, in which when an angle of a narrow angle formed by a direction F of flow of a liquid discharged from the discharge port 30 and a normal line L in a surface along the mesh sheet 40 is θ(°), 90-θ is less than 50, and a method for preparing cell suspension from a cell-containing sample using such a container.SELECTED DRAWING: Figure 1-1

Description

  The present invention relates to a method of enzymatically treating a cell-containing sample such as collected biological tissue and removing contaminants to prepare a cell suspension, and a container for preparing the cell suspension for use in the method.

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

  For example, Patent Document 1 discloses a blood processing filter including a flexible container having a blood inlet and an outlet, and a sheet-like blood processing filter material disposed so as to separate the inside of the container on the inlet side and the outlet side. It is disclosed. Further, in Patent Document 2, a spiral-shaped primary side flow path from the membrane peripheral portion to the membrane center portion, which connects the primary side inlet and the primary side outlet, and a filtrate which permeates through the filtration membrane are There is disclosed a plate type membrane module for blood sampling for measurement of low molecular weight substance concentration, in which a secondary flow passage leading to the next side outlet is formed.

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

JP, 2011-72816, A Japanese Patent Application Laid-Open No. 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 white blood cells from liquid tissue such as blood, and a part of the separation is performed inside the filter. It is intended to leave. However, for example, in the case of removing contaminants such as undigested substances from the enzyme-treated matter of adipose tissue, if the separation operation using a filter is performed, clogging of the filter occurs 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, which leads to high cost, and further, the available facilities are limited. Also, when washing the cells, the supernatant separated from the cells is removed by centrifugation, but at that time the cells come in contact with the atmosphere and there is a risk of contamination or the like. In the first place, the cells pelleted by centrifugation are often damaged and die.

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

  Therefore, the present invention aims to provide a method for preparing a cell suspension from cell-containing samples such as biological tissue easily and at a high cell recovery rate, and a container used therefor.

  In view of the problems as described above, the present inventors can carry out an enzyme treatment of a cell-containing sample such as a living tissue internally, and further, a mesh sheet for removing contaminants from the obtained enzyme-treated solution And a container having a constitution capable of preparing a cell suspension without causing clogging and the like, and a method for preparing a cell suspension using the same. The gist of the present invention is as follows.

(1) A container for preparing a cell suspension,
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;
An inlet provided in the container body and having an inlet for liquid into the first space;
A discharge unit provided in the container body and formed with a discharge port of the liquid from the second space;
Equipped with
90-θ is less than 50, where θ (°) is the narrow angle formed by the flow direction of the liquid discharged from the discharge port and the normal to the surface along the mesh sheet .
(2) The container as described in (1) whose pore diameter of the said mesh sheet is 50-300 micrometers.
(3) The container according to (1) or (2), wherein the container body is flexible.

(4) A container for preparing a cell suspension,
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;
An inlet provided in the container body and having an inlet for liquid into the first space;
A discharge unit provided in the container body and formed with a discharge port of the liquid from the second space;
Equipped with
The container body includes a first side wall and a second side wall opposite to 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 input 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 with the outside through the input port.
The discharge portion is disposed between the peripheral portion of the second side wall and the peripheral portion of the mesh sheet so as to communicate the second space with the outside through the discharge port.
The container, wherein the first side wall, the second side wall, and the mesh sheet are integrated at the periphery.
(5) The input unit and the discharge unit are disposed at opposing positions of the container body,
A width W1 of an end on the first space side of the input portion along a direction in which the first side wall and the second side wall face each other.
The width of the end of the discharge portion on the side of the second space along the direction in which the first side wall and the second side wall oppose each other is W2,
W, the average value of W1 and W2,
The distance between the end on the first space side of the input part and the end on the second space side of the discharge part is D
And when
The container as described in (4) whose D / W is 12 or more.
(6) The container according to (4) or (5), wherein the first side wall and the second side wall are flexible.

(7) 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;
An inlet provided in the container body and having an inlet for liquid into the first space;
A discharge unit provided in the container body and formed with a discharge port of the liquid from the second space;
Placing a cell-containing sample and an enzyme through the input port, and performing an enzyme reaction in the first space to produce an enzyme-treated solution;
(B) The enzyme treatment liquid is passed through the mesh sheet to remove contaminants, a cell suspension is produced on the second space side, and the cell suspension produced is discharged through the outlet. Process,
Said method.
(8) In the step (b), when the narrow angle formed by the flow direction of the cell suspension discharged through the discharge port and the normal to the surface along the mesh sheet is θ (°) , The method according to (7), wherein the cell suspension is drained so that 90-θ is less than 50.
(9) The method according to (7) or (8), wherein the container is a container according to any one of (1) to (6).
(10) The outlet is connected to a cell suspension processor capable of washing and concentrating the cell suspension in a closed system;
(C) washing and concentrating the cell suspension discharged from the outlet in a closed system by the device, further comprising the step of obtaining a cell concentrate, according to any one of (7) to (9) the method of.

  According to the method of the present invention, a cell suspension can be prepared from a cell-containing sample such as a collected biological tissue in a closed system without undergoing an open system operation such as centrifugation. In addition, by using the cell suspension preparation container of the present invention in the method, the cell suspension can be prepared with excellent throughput and cell recovery rate without causing clogging of mesh and the like. .

It is a perspective view which shows one Embodiment of the container 1 for cell suspension preparation of this invention. It is the sectional view on the AA line cutting part of container 1 for cell suspension preparation of Drawing 1-1. It is a BB line cutting part sectional view of container 1 for cell suspension preparation of Drawing 1-1. It is a perspective view showing the state which put the liquid into the internal space 13 of the container 1 for cell suspension preparation of this invention shown to FIGS. 1-1. It is CC sectional view cut | disconnected part of the container 1 for cell suspension preparation of FIGS. 2-1. It is a schematic end elevation of the discharge part 31 vicinity part of the CC line cutting part of the container 1 for cell suspension preparation of FIGS. 2-1. FIG. 1 is a schematic view of an embodiment of a cell suspension processing apparatus connected to the cell suspension preparation container of the present invention. A graph showing the relationship between 90-θ and the throughput of cell suspension, where θ (°) is the narrow angle formed by the flow direction of the cell suspension and the normal to the surface along the mesh sheet. It is. When the narrow angle formed by the flow direction of the cell suspension and the normal to the surface along the mesh sheet is θ (°), the graph shows the relationship between 90-θ and the cell recovery rate. It is the graph which showed the pore diameter of a mesh sheet, and the relation of cell recovery. It is a graph which shows the result in Example 6 and Comparative Example 3.

(Container for preparing cell suspension)
1-1 to 1-3 are schematic views showing an embodiment of a container for preparing a cell suspension of the present invention. The cell suspension preparation container 1 of the present embodiment is disposed so as to divide the container body 10 containing the internal space 13 capable of holding liquid and the internal space 13 into the first space 11 and the second space 12 Provided in the container main body 10, and the liquid from the second space 12 provided in the container main body 10 and the liquid inlet port 20 formed in the first space 11; And a discharge unit 31 having a discharge port 30 formed therein. FIGS. 1-1 to 1-3 schematically show examples of the shape of the cell suspension preparation container 1 of the present embodiment when the contents are not accommodated in the internal space 13. FIGS. 2A and 2B show an example of the shape when the internal space 13 of the cell suspension preparation container 1 of the present embodiment is filled with a liquid. However, in FIGS. 2A and 2B, the depiction of the liquid is omitted for the sake of simplicity. Moreover, in order to fill the internal space 13 of the cell suspension preparation container 1 with liquid, the outlet 30 needs to be sealed, but the depiction of means for sealing is also omitted.

(Container body)
The container main body 10 constituting the outer shell of the internal space 13 capable of holding a liquid can be provided with the mesh sheet 40 in the mode to be described later inside, and should not affect cells etc. held inside. For example, it may be formed of any material and in any manner. The container body 10 and the mesh sheet 40 include, for example, the first side wall 15 and the second side wall 16 opposed to each other with the mesh sheet 40 interposed 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. 1-1 to 1-3 show the embodiment. 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 portion 150, the second side wall peripheral portion 160 and the mesh sheet peripheral portion 41.

  It is preferable that the container body 10 be at least partially made of a transparent or translucent material, since the appearance of the cell suspension inside can be visually observed. In addition, it is preferable to make at least a part, preferably the whole, flexible because if necessary, the internal cell suspension can be physically separated by clamping. It is preferable that the container body 10 does not contain an adhesive in the portion exposed to the internal space 13. When the container 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 is one that can be adhered by heat fusion without using an adhesive. It is preferable because it does not have to consider the influence of the adhesive on 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 copolymer or a hydrogenated product thereof, and mixtures of thermoplastic elastomers with polyolefins and softeners such as ethylene-ethyl acrylate. The inner surface which defines the internal space 13 of the container body 10 is preferably processed to reduce residual liquid when discharging the cell suspension.

  When the container body 10 has flexibility, it is usual that the shape changes when the liquid is contained in the internal space 13. In the embodiment of the container 1 for preparing a cell suspension shown in FIGS. 1-1 to 1-3, when the container body 10 in which the first wall 15 and the second wall 16 are combined has flexibility, the inside When the liquid is contained in the space 13, as shown in FIGS. 2-1 and 2-2, the central portion of the first wall portion 15 and the second wall portion 16 bulge outward, and the internal space The volume of 13 increases. Each process such as an enzyme process to be described later can be performed in the internal space 13 whose volume is increased.

(Mesh sheet)
The mesh sheet 40 is arranged to divide the inner space 13 into a first space 11 and a second space 12. As the mesh sheet, one that can permeate a liquid such as water, and preferably can pass necessary cells is used. The mesh sheet 40 preferably has a pore diameter in the range of 50 to 300 μm. If the pore diameter is in such a range, contaminants such as undigested tissue lumps 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. It can be passed. In order to further exhibit this effect, the pore size 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, so clogging is less likely to occur.

  In the present specification, a “mesh” is a material having a planar spread, in which a plurality of pores penetrating from one surface to the other are two-dimensionally arranged to form a network structure. Means Examples of the mesh include, but not limited to, those in which fibers are woven to form a network structure, and membranes in which a plurality of pores penetrated in the thickness direction are formed. Meshes also include what are commonly referred to as "screens". A sheet composed of mesh is called "mesh sheet". In the present specification, “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, and does not have the network structure and three-dimensional It is a concept different from the material having a pore). 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 safety and stability of the material and availability. And synthetic polymers selected from at least one of the foregoing, hydroxyapatite, inorganic materials such as glass, alumina, and titania, and metals such as stainless steel, titanium, and aluminum. When two or more kinds of materials are combined, the combination is not particularly limited, but polyolefin, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, synthetic polymers such as 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 preferred. The mesh sheet 40 is provided in the container main body 10 so that the mesh sheet surface is substantially flat at least when the liquid is introduced into the internal space 13. FIGS. 2-1 and 2-2 show the state when 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 is capable of transmitting liquid, not only the first space 11 but also the liquid medium in the liquid charged through the inlet 20, the component having a size that can permeate the dissolved component and the mesh sheet, is The mesh sheet 40 is also transmitted to the second space 12, and a component of a size that can not be transmitted through the mesh sheet 40 remains in the first space 11.

  The pore diameter of the mesh sheet is the maximum inscribed in the outline of the inner circumference of the pores, 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 value of the diameter of the inscribed circle. The average value is preferably calculated from, for example, a value obtained by observing 50 or more, preferably 100 or more pores. If the mesh sheet is such that the fibers are woven to form a network structure, the pore size is equal to the average opening of the mesh. The mean opening can be calculated based on the number of fibers per unit length (for example, 1 inch) measured using, for example, a luminometer.

(Inlet and outlet)
An inlet 20 for the liquid into the first space 11 of the inner space 13 is formed in the inlet 21, and an outlet 30 for the liquid from the second space 12 in the inner space 13 is formed in the outlet 31. There is. Only one each of the inlet 20 and the outlet 30 may be provided, or two or more may be provided as needed. From the input port 20, a cell-containing sample such as a living tissue and an enzyme for separating cells into a cell suspension are input, and an enzyme reaction is performed in the first space 11. When the enzyme-treated solution after the enzyme reaction 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.

  Although the input unit 21 and the output unit 31 may be provided in any manner, for example, the configuration shown in FIGS. 1-1 to 1-3 as described above in relation to the relative relationship between the container body 10 and the mesh sheet 40, that is, the mesh When including the first side wall 15 and the second side wall 16 opposed to each other with the sheet 40 interposed therebetween, the insertion portion 21 is between the peripheral portion 150 of the first side wall 15 and the peripheral portion 41 of the mesh sheet 40. 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 so that the first space 11 communicates with the outside through the inlet 20. The second space 12 is placed in communication with the outside via the outlet 30. More specifically, the input unit 21 and the discharge unit 31 are hollow cylindrical materials having an arbitrary cross-sectional shape, for example, a resin tube, 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 160 of the second side wall 16 and the peripheral edge 41 of the mesh sheet 40. Specific examples of materials for forming the charging portion 21 and the discharging portion 31 include vinyl chloride, soft vinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polyolefin such as polyethylene and polypropylene, styrene-butadiene-styrene copolymer Polymeric hydrogenated products, thermoplastic elastomers such as styrene-isoprene-styrene copolymer or hydrogenated products thereof, and mixtures of thermoplastic elastomers and softeners such as polyolefin and ethylene-ethyl acrylate .

  At least a part of the input unit 21 and the discharge unit 31 communicating with each other is closed by a silicone rubber or the like which can input a sample using an injection needle so that the inner space 13 can be closed. Preferably, the structure is formed to allow direct insertion of a syringe, such as a needleless port. In addition, for example, the portion of the input unit 21 in communication with the input port 20 has a plurality of ports for inputting the sample by branching in the middle etc., so that the cell-containing sample and the enzyme can be input from different ports. It may be The port may be provided with a filter or the like as required.

  The input unit 21 and the discharge unit 31 may be provided at any position of the container main body 10 as long as the relative position to the mesh sheet 40 is as described above. However, providing the input unit 21 and the discharge unit 31 at mutually opposing positions in the container main body 10 is preferable because the processing from the input of the cell-containing sample and the enzyme to the discharge of the cell suspension can be smoothly performed. Note that “to provide at mutually opposing positions” means, for example, as shown in FIG. 1-1, when the container main body 10 has a quadrangular shape or a shape similar thereto, the feeding portion 21 is placed on one of the opposing sides. It means that the discharge part 31 is provided on the side of the.

(First Preferred Embodiment of Cell Suspension Preparation Container)
In the 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 has a flow direction of liquid discharged from the discharge port 30. When the narrow angle formed by F and the normal L of the surface along the mesh sheet 40 is θ (°), 90 − θ is set to be less than 50. 2-3 shows the discharge part 31 of the C-C line cutting part of the container 1 for cell suspension preparation in the state which accommodated the liquid (not shown) in the interior space 13 shown in FIG. 2-2. FIG. 10 is a schematic end view of the vicinity, showing the relationship of θ (°) between the narrow angle formed by the flow direction F of the liquid discharged from the discharge port 30 and the normal L of the surface along the mesh sheet 40 ing. In the first embodiment, by setting 90-θ in the above range, the mesh sheet 40 includes a position at which the distance from the discharge port 30 along the direction F is different, and undigested tissue lumps And clogging of the mesh sheet 40 by foreign substances such as debris are less likely to occur, and the cell suspension can be smoothly taken out from the discharge port 30. Although the mechanism of this phenomenon is not always clear, even when contaminants are clogged on 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 is It is presumed that clogging is less at positions where the distance is relatively larger. 90-θ is preferably less than 50, more preferably 45 or less, still more preferably 30 or less, particularly preferably 5 or less. The above effect of the first embodiment is higher as the value of 90-θ is smaller. The lower limit value of 90-0 is not particularly limited, and may be zero.

  In the first embodiment, the portion of the mesh sheet 40 exposed to the internal space 13 is preferably 50% or more, more preferably 60% or more, more preferably 50% or more, with respect to the area when the portion is planarly viewed. The area of the area of 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 100% is provided such that the 90-.theta. Be If the region where the 90-θ is in the above range in the mesh sheet 40 is the above range, the above effect of the first embodiment is sufficiently exhibited.

(Second Preferred Embodiment of Cell Suspension Preparation Container)
In the particularly preferred second embodiment (hereinafter sometimes referred to as “second embodiment”) of the container 1 for preparing a cell suspension of the present invention, among the structural features described above, in particular,
The container body 10 includes a first side wall 15 and a second side wall 16 opposed to each other with the mesh sheet 40 interposed 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 with the outside through the insertion port 20,
A 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 so as to communicate the second space 12 with the outside via the discharge port 30.
The first side wall 15, the second side wall 16 and the mesh sheet 40 are integrated at the peripheral portions 150, 160, 41.
The cell suspension preparation container 1 of the second embodiment integrates the two members constituting the first side wall 15 and the second side wall 16, the mesh sheet material, and the material for the inlet and outlet. Can be manufactured relatively easily.

  The integration of the first side wall 15, the second side wall 16 and the mesh sheet 40 is sufficient as long as these three members can not be separated, and it is necessary that they be directly coupled to each other. Alternatively, they may be connected via any one or more of these three members or other members. For example, in the peripheral portion, the first side wall 15 and the second side wall 16 are not directly coupled, but are integrally coupled via the mesh sheet 40 between them, or Also in the case where the side wall 15 and the second side wall 16 are not directly coupled, and are coupled and integrated via the input portion 21 (or the discharge portion 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 of "integrated at the peripheral portion".

  Further, according to the second embodiment, the angle θ (°) of the mesh sheet 40 can be adjusted by the dimensions and relative distance of the input portion 21 and the output portion 31 in the container main body 10. The cell suspension preparation container 1 in which the value of 90-θ is less than 50 can be easily manufactured. Specifically, when the input portion 21 and the discharge portion 31 are disposed at opposing positions of the container body 10, (i) the first side wall of the end 22 on the first space 11 side of the input portion 21 The width along the direction in which the second side wall 16 and the second side wall 16 face each other is 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 portion 31 face each other And (iii) the distance between the end 22 on the side of the first space 11 of the input portion 21 and the end 32 on the side of the second space 12 of the discharge portion 31 In the case of D, the angle θ (°) can be adjusted by the ratio (D / W) of D to W, which is the average value of W1 and W2. For example, when 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 becomes a smaller value. 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 to W2 is not particularly limited, but from the viewpoint of facilitating the production of the cell suspension preparation container 1, W1 / W2 is more preferably 0.2 to 5 and more preferably 0.5 to 2 0.9 to 1.1 is more preferable, and 1 is particularly preferable. In this embodiment, it is preferable that the feeding unit 21 and the discharging unit 31 be disposed such that the feeding path by the feeding port 20 and the feeding path by the discharge port 30 are coaxially positioned.

(Method for preparing cell suspension from cell-containing sample)
The invention, in another aspect, also relates to a method for preparing a cell suspension from a cell-containing sample, such as a biological tissue. The method is
(A) 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, and a first space provided in the container body The cell-containing sample and the enzyme are provided through the inlet, and the container is provided with the inlet in which the inlet for the liquid is formed, and the outlet provided in the container body and the outlet for the liquid from the second space. Adding an enzyme reaction in the first space to produce an enzyme-treated solution, and (b) passing the enzyme-treated solution through a mesh sheet to remove contaminants, and a cell suspension on the second space side. At least the step of generating and discharging the generated cell suspension through the outlet.
As the enzyme to be introduced together with the cell-containing sample, enzymes that can be used for separation of cells from cell-containing samples such as living tissues, such as collagenase, trypsin, dispase, collagenase, pepsin, etc. can be used appropriately.

  In the container used in step (a), the mesh sheet is capable of transmitting a liquid such as water, and is selected from the ranges described for the mesh sheet used in the embodiment of the container for cell suspension preparation. be able to. In the container used in step (a), the pore diameter 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 pre-sterilized prior to use. As a sterilization method, sterilization methods widely used for sterilization of medical devices such as gamma ray sterilization, electron beam sterilization, EOG sterilization, high pressure steam sterilization and the like can be suitably used. The container of step (a) may be any container as long as it has the above-mentioned structure, 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 because the cell suspension can be smoothly prepared without clogging and the like.

  A cell-containing sample as a raw material of the cell suspension and an enzyme for separating the cells and suspending the cell are put into the first space of the container through the inlet, and the enzyme reaction is carried out there. The reaction mixture containing the enzyme and the cell-containing sample, which is formed at this time, is preferably in the form of a liquid, and for this purpose, the enzyme and the cell-containing sample are suitably dispersed in the presence of a liquid medium such as water as a dispersing medium. It is preferable to accommodate in a space. The enzyme can be added as an aqueous enzyme solution as needed. Since the mesh sheet can permeate the liquid, in step (a), the liquid medium, the dissolved components, and the size which can permeate the mesh sheet are contained in the reaction mixture contained in the first space. The component moves not only to the first space but also to the second space, and the component whose size can not be transmitted through the mesh sheet remains in the first space. In the enzyme reaction of the step (a), a component which can not permeate the mesh sheet in the first space is subjected to an enzyme treatment to form an enzyme treatment solution in which target cells are dispersed in a liquid medium. By passing the enzyme-treated solution after the enzyme reaction through the mesh sheet, contaminants such as undigested tissue lumps and debris are removed, and a cell suspension is generated in the second space, and then outside the container through the discharge port. Discharged into Preferably step (a) is carried out with the inlet and outlet of the container closed. For this purpose, the portions of the inlet and outlet communicating with the inlet and outlet of the container are sealed in a manner that allows the sample to be injected, such as silicone rubber or a needleless port, thereby the inside of the container Is a closed system, and it is preferable that the enzyme reaction can be performed without being exposed to the open air. In addition, when carrying out the enzyme reaction, the container can be placed on a shaker or placed in a constant temperature bath so that appropriate reaction conditions can be obtained. When the container is placed so that the first space is below the second space in the vertical direction when the cell-containing sample and the enzyme are put in, or when the enzyme reaction is carried out, the mesh sheet eyes It is preferable because clogging can be prevented.

Also in discharging the cell suspension in step (b), a closed system in which the outlet and the vessel for containing the cell suspension or the device for further processing steps are connected by a closed flow path. It is preferable from the viewpoint of suppressing contamination.
When discharging the cell suspension in step (b), the narrow angle formed by the flow direction of the cell suspension discharged through the outlet and the normal to the surface along the mesh sheet is θ (°) When 90-.theta. Is less than 50, preferably 45 or less, more preferably 30 or less, particularly preferably 5 or less, clogging of the mesh sheet is less likely to occur, which is preferable. In addition, when the container is held in a state where the discharge port is at the lowermost part, it is preferable because discharge of the cell suspension proceeds smoothly. Therefore, while the container is held with the discharge port at the bottom, the value of 90-θ based on the above angle θ (°) is less than 50, preferably 45 or less, more preferably 30 or less, particularly preferably 5 It is preferable to have the following structure, more specifically, the structure as shown in FIGS. The flow rate at which the enzyme-treated solution passes through the mesh sheet can be appropriately adjusted by applying a negative pressure from the discharge port or the like, or may be a flow rate allowed to fall naturally by gravity. In order for the enzyme treatment solution to smoothly pass through the mesh sheet, the flow rate through the mesh sheet may 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 the present embodiment, the step (b) may be performed after the step (a) is completed, or the steps (a) and (b) may be performed in parallel.

(Connection with cell suspension processor)
The outlet of the container may be connected to a separately prepared cell suspension processor, whereby the cell suspension obtained from the outlet is washed and concentrated in a closed system to obtain a cell concentrate You may do so. The washing of the cell suspension refers to a fluid containing a body fluid, an enzyme, etc. contained in the tissue treatment solution, such as physiological saline, infusion solution, culture medium, distilled water, inorganic salts, saccharides, serum, proteins, etc. , Buffer, medium, and plasma etc. are meant. In one embodiment, the present invention
(A) A container main body including 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, and the container main body The cell-containing sample and the enzyme are introduced through the inlet port, and the container is provided with the inlet portion in which the inlet port for the liquid is formed, and the outlet portion provided in the container body and the outlet port for the liquid from the second space. Carrying out an enzyme reaction in the first space to produce an enzyme-treated solution,
(B) passing the enzyme-treated solution through a mesh sheet to remove contaminants, generating a cell suspension on the second space side, and discharging the generated cell suspension through an outlet; A step of washing and concentrating the cell suspension discharged from the outlet in a closed system using a cell suspension processing apparatus capable of washing and concentrating the cell suspension in a closed system to obtain a cell concentrate And a method of obtaining a cell concentrate from living tissue. According to the method, cell concentrates having concentrations that can be directly administered to a subject can all be prepared directly from living tissue in a closed system, and high cell recovery can be achieved.

  As a cell suspension processing apparatus, the apparatus disclosed by Unexamined-Japanese-Patent No. 2015-42167 can be utilized, for example. FIG. 3 shows a schematic view of an embodiment of the apparatus (cell suspension processing apparatus 900). In the following description, numbers in parentheses indicate corresponding parts in FIG. The cell suspension processing apparatus (900) of the document includes a cell suspension reservoir (903) having a solution inlet port (915), a circulation outlet port (916) and a circulation inlet port (917), and A hollow fiber separation membrane is filled in a container having a suspension inlet (918), a cell suspension outlet (919) and an outlet for filtrate (920), and the liquid is filtered and concentrated from the cell suspension Cell suspension processor (906) for performing the reaction, the circulation inlet port (917) of the reservoir (903), and the cell suspension inlet (918) of the cell suspension processor (906) An introductory communication tube (928) in communication with the cell suspension outlet (920) of the cell suspension processor (906) and a circulation passage (929) in communication with the circulation outlet port (916) of the storage container Storage capacity) (903) and a circulation circuit (930) for performing concentration while circulating cell suspension between the cell suspension processor (906), and a collection container (905) for collecting the concentrated cell concentrate And a recovery channel (931) for transferring the cell concentrate in the storage container (903), in the cell suspension processor (906) and in the circulation circuit (930) to the recovery container (905). ), An injection passage (927) for injecting a solution into the solution inlet port (915) of the storage container (903), and a communicating pipe (928) for introducing the inside of the storage container (903) or the circulation circuit (930). And detecting means (911) for detecting the liquid volume of the cell concentrate in the cell, and detecting that the cell concentrate has been concentrated to a predetermined liquid volume by the detection means (911); Cell suspension processor from (903) 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 a 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). It is connected via an injection channel (927) and a cell suspension preparation container connection (901). Although the device of the above-mentioned document can automatically carry out washing and concentration of the cell suspension, the cell suspension processing device (900) connected to the outlet of the container in the present invention is a cell suspension. The washing and concentration of the solution may be performed manually.

(Cell suspension processing device: Reservoir container)
The storage container (903) used in the cell suspension processing apparatus (900) stores the cell suspension for processing, and passes the cell suspension to the cell suspension processing unit as described later. It is a container which circulates and stores the obtained cell concentrate. The reservoir has a solution inlet port, a circulation outlet port and a circulation inlet port. The solution inlet port refers to a port for supplying a cell suspension into the storage container. The circulation inlet port is a port for passing the storage container to the cell suspension processor, and the circulation outlet port is for passing the cell concentrate concentrated by the cell suspension processor to the storage container To say the port to do. These ports are preferably installed at the bottom 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 stirred with the liquid circulating in the reservoir and circulation circuit. Concentration and dilution can be performed.

  The number of the solution inlet port, the circulation outlet port, and the circulation inlet port may be one each, but may be increased to plural as needed. For example, although the solution inlet port can be used not only as a cell suspension but also as a port for dilution fluid or priming solution, in addition to the port for cell suspension, an inlet port for dilution fluid or priming solution May be provided. Moreover, you may provide the port for the purpose different from said 3 types of ports. For example, a recovery port for passing the cell concentrate into a recovery container can be mentioned. By directly connecting the collection port of the storage container and the collection container, it is possible to quickly collect the cell concentrate in the storage container.

  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 good handleability. In addition, it is advantageous that the inner surface of the storage container is processed by nasi, because residual liquid when discharging the suspension from the storage container can be reduced. The volume of the storage container can be used without particular limitation. However, if it is too large, a large amount of dilution liquid to be used for dilution is required. 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 treated, and there is no particular limitation.

  In addition, a vent may be provided at the top of the storage container. By providing this vent, it becomes an open system to which the gas in the storage container can be exchanged with the atmosphere, and for example, when discharging the liquid in the storage container, the inside of the storage container is decompressed and the storage container is crushed. There is an advantage to prevent. An air filter (908) may be provided in this vent to prevent the mixing of unnecessary components into the storage container from the outside.

(Cell suspension processor: Cell suspension processor)
The cell suspension processor (906) is a device for filtering and concentrating liquid from the cell suspension, and is connected so as to allow the cell suspension to flow from the storage container. In the cell suspension processor, a hollow fiber separation membrane is filled in a container having a cell suspension inlet, a cell suspension outlet and an outlet for filtrate. The cell suspension inlet is an inlet for introducing a 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 this cell suspension outlet with the circulation outlet port of the storage container, it is possible to circulate and concentrate the cell suspension between the storage container and the cell suspension processor. The filtrate outlet is an outlet for removing the liquid filtered from the cell suspension.

  It is preferable that the hollow fiber separation membrane used in the cell suspension processing device is a tubular container in which several tens to several thousands of hollow fibers are bundled. The arrangement of the hollow fiber separation membrane may be linear, bent, or spiral, and both ends of the hollow fiber separation membrane may be between the cell suspension inlet and the cell suspension outlet. The shape is not particularly limited as long as

  The hollow fiber separation membrane used in the cell suspension processor can use a synthetic polymer material in terms of the safety and stability of the material. Among these, polysulfone-based, polyolefin-based or cellulose-based polymer materials can be preferably used. Further, the pore diameter of the hollow fiber separation membrane should be such that cells do not leak to the outside, and in order to be able to efficiently filter unnecessary components, it is preferable that the pore diameter be as large as possible. Specifically, those having an average pore diameter of 0.01 μm or more and 1.0 μm or less can be suitably used. The hollow fiber preferably has an inner diameter of 400 μm to 1000 μm.

  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 prepare a cell concentrate. As for the structure of the cell suspension processor, for example, a hollow fiber separation membrane is filled in a cylindrical container, and the hollow fiber end is in close contact with the cylindrical container end with an adhesive or the like and is opened at the end In order to allow the cell suspension to flow into and out of the hollow fiber membrane, the end of the cylindrical container is provided with a header portion serving as a cell suspension inlet and a cell suspension outlet. The cylindrical container may be provided with one or more filtrate outlet, and the filtrate filtered from the inside of the hollow fiber is discharged from the filtrate outlet. The cell suspension processor needs to be a structure in which the hollow fiber separation membrane is packed in a sealed container, but the cell suspension inlet and the cell suspension outlet are hollow fibers from the filtrate outlet. If it is provided with the structure separated by the wall material which comprises a separation membrane, it is possible to take various structures. For example, the dialyzer used for hemodialysis etc. can be illustrated as a similar structure.

  At the filtrate outlet (920) of the cell suspension processor, a circuit for flowing out the filtrate separated by the hollow fiber separation membrane is installed. It is preferable to connect the circuit for the filtrate and the waste liquid container (904) because the possibility of the filtrate leaking to the outside can be reduced. The filtrate removed from the filtrate outlet 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 the waste liquid does not leak.

  A pump (914) for feeding the filtrate may or may not be installed between the filtrate outlet and the waste liquid container of the cell suspension processor. For example, by providing a pump in the path connecting the filtrate outlet and the waste container, the filtrate can be discharged at a constant flow rate, so that the processing time can be made constant, and the filtration efficiency in the cell suspension processor can be increased. It becomes possible to control. That is, driving the pump promotes drainage of the filtrate from the cell suspension processor, and as a result, concentration processing in the cell suspension processor can be promoted. In addition, when the cell concentrate concentrated to a predetermined concentration is passed through a recovery container for recovery, the pump can be stopped to enable rapid recovery. Further, the collected filtrate may be discarded as it is, or may be reused by 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 recovery container. A cell concentrate recovery port may be provided. The cell concentrate recovery port can be connected to a recovery container (905).

(Cell suspension processing device: circulation circuit)
A circulation circuit (930) includes a communicating communication tube communicating the circulation inlet port of the storage container and the cell suspension inlet of the cell suspension processor, and the cell suspension of the cell suspension processor. It is a circuit which consists of an outlet communicating pipe which connects an outlet and a circulation outlet port of the storage container. A cell concentrate can be produced by performing concentration while circulating the cell suspension between the reservoir and the cell suspension processor through the circulation circuit. As a pipe which constitutes a circulation circuit, a general plastic tube can be used suitably. Vinyl chloride can be suitably used in terms of safety and durability. From the viewpoint of facilitating control of the circulation of the cell suspension or cell concentrate, a pump (913) is preferably installed in the circulation circuit. The number of pumps is not particularly limited, but may be one 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 discharge communication pipe, but if it is installed in the introduction communication pipe, the cell suspension inlet of the cell suspension processor It is preferable that a high pressure solution can be introduced to efficiently separate the liquid. 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 Is preferred.

  A branch portion may be provided on any of the circulation circuits. By connecting a pipe from this branch to the collection container (905) by piping, the route from the branch to the collection container is used as a final collection path (931) in the storage container, cell suspension The cell concentrate in the fluid processor and in the circulation circuit can be quickly recovered to a recovery container. It is preferable to install the branch portion as close to the circulation outlet port as possible because the amount of residual liquid remaining in the circulation circuit from the branch portion to the outlet port can be reduced in the recovery step.

(Cell suspension processing device: recovery container)
The collection container (905) is a container for collecting the cell concentrate concentrated to a predetermined concentration. The recovery container is preferably a flexible plastic container. In addition, if the inner surface is subjected to Nashiji processing, there is an advantage that the residual liquid after recovering the cell concentrate can be reduced. Further, the recovery container may have a connection port to which a needle, a syringe or the like can be connected. By setting it as such a structure, it can use suitably, when transferring the cell concentrate collect | recovered by the collection container to another container. The shape of the recovery container is not particularly limited. For example, when the volume increases, the inner area of the container with which the collected cells come in contact is large, and the attached cells may remain in the container and lead to the loss of cells. .

(Cell suspension processing device: recovery path)
The recovery path (931) is a path for transferring the cell concentrate in the storage container, in the cell suspension processor and in the circulation circuit to the recovery container. A general plastic tube can be used as a tube constituting the recovery passage, and among them, a vinyl chloride tube can be suitably used in terms of safety and durability.

The following three modes may be mentioned as the recovery path.
1) When a branch portion is provided on the circulation circuit, a path connecting the branch portion and the collection container is a collection path.
2) When the recovery port of the storage container and the recovery container are connected, the connected path becomes a recovery path.
3) When the cell concentrate recovery port of the cell suspension processor and the recovery container are connected, this connected path becomes a recovery path.
The recovery path is preferably any one of the modes described above, and two or more of them can be used in combination.

(Cell suspension processor: injection channel)
The injection channel (927) is a channel for injecting a solution into the solution inlet port of the reservoir. A general plastic tube can be used as the tube constituting the injection passage, and a vinyl chloride tube can be suitably used among them in terms of safety and durability. In addition, as a solution, in addition to the cell suspension, a dilution liquid for re-diluting a cell concentrate obtained by concentrating the cell suspension or the whole cell suspension processing apparatus (900) before concentration processing A priming solution for priming may be included. Dilutions 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 preferable. It can be used. The priming solution may also be physiological saline, infusion, distilled water, buffer, culture medium, plasma or liquid containing inorganic salts, saccharides, serum, protein, etc. Especially from the viewpoint of safety, physiological saline or infusion It can be used suitably. The same solution may be used for the dilution solution and the priming solution, or different solutions may be used. When different solutions are used, it is possible to separate circuits from branch parts on the circuit leading to the solution inlet port of the storage container and install circuits connected to the respective connections.

  It is preferable that a pump (912) for sending a liquid is installed in the injection path. The pump can stably send the liquid to the storage container. In addition, the position where the pump is installed is the junction point of the circuit connected to the cell suspension preparation container connection part (901), the dilution liquid container connection part (902) and the priming liquid container connection part (926) optionally provided. It is preferable to be on the circuit connecting the solution inlet port with the solution inlet port in that the number of pumps required for liquid transfer 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 the pressure gauge Is preferred.

(Cell suspension processing device: detection means)
The detection means is a means for detecting the amount of the cell concentrate in the storage container or in the communication tube for introduction of the circulation circuit. As a means to detect the liquid quantity in the said storage container, using the bubble sensor which directly detects the liquid level of the storage liquid in a storage container is mentioned. In another embodiment, for example, a tube is installed parallel to the storage container in the vertical direction, and a circuit in which the storage container and the tube communicate with each other in the lower part, and the liquid level in the tube is in the storage container The air bubble sensor may be installed in this tube so as to be equal to the liquid level of. With this means, the liquid level in the tube can be detected by the air bubble sensor as the liquid level in the storage container. In addition, a chamber may be installed below the tube installed parallel to the storage container. In particular, by installing a chamber whose inner diameter is larger than that of the tube, it is expected to reduce the occurrence of a defect that air bubbles infiltrate into the tubes installed in parallel to be detected.

  As a means for detecting the liquid concentration of the cell concentrate in the introductory communication pipe, a bubble sensor (911) may be provided in the introductory communication pipe (928) communicating with the circulation inlet port of the storage container. With this configuration, the cell suspension can be more concentrated than the means for detecting the liquid volume in the storage container, so the amount of concentrated suspension (cell concentrate) can be reduced. There is an advantage that the amount of unnecessary components in the suspension can be reduced. The position of the air bubble sensor installed in the introduction communication pipe is not particularly limited, but the length of the circuit connecting the storage container and the air bubble sensor is set by installing the air bubble sensor at a position close to the storage container. It can be shortened.

  In addition, in the cell suspension processing apparatus (900), a bubble sensor (910) may be installed in the injection path (927) connected to the solution inlet port of the storage container. When air bubbles are detected by the air bubble sensor, it is possible to stop the storage of solutions such as the cell suspension, the dilution liquid, and the priming solution in the storage container. For example, even if the throughput of the target cell suspension is not known, processing the entire volume of the target fluid without setting the driving time of the pump for each process by installing the bubble sensor in the injection channel. Has the advantage of being able to It is also possible to add a fixed amount of solution from the pump operation time. For example, when only a part of the cell suspension needs to be treated, the treatment can be carried out by setting the driving time of the pump. Although this bubble sensor can be installed anywhere in the injection channel, it is advantageous to install the bubble sensor as close as possible to the storage container in terms of reducing the amount of liquid remaining in the injection channel.

  In addition, another air bubble sensor (909) may be provided on the top of the storage container. By installing this bubble sensor, the presence or absence of the solution in the vicinity of the upper portion in the storage container can be determined. That is, when the liquid exceeding the capacity of the storage container is supplied, the sensor can detect and notify the user as an alarm.

  As a pump and a bubble sensor used for the cell suspension processing apparatus (900), those generally used can be used. Alternatively, a clamp may be provided at a desired position of the circuit to switch the flow path. There is no particular limitation on the pump, the bubble sensor and the clamp, and for example, those used in a dialysis device or 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 is a biological tissue. The biological tissue is not particularly limited as long as it is collected from animal tissue, and examples thereof include fat, skin, blood vessels, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, muscle, prostate, intestine, amniotic membrane, placenta, etc. Those derived from umbilical cord and the like can be mentioned. The methods of the invention are particularly useful for removing interstitial vascular fraction (SVF) cells from harvested adipose tissue. In addition, other examples of cell-containing samples include cultures of cells prepared in vitro.

  The cell suspension or cell concentrate obtained by the present invention is a leukemia treatment, myocardial regeneration and revascularization, stem cell exhaustion disease, bone disease, cartilage disease, ischemic disease, vascular disease, neurological disease, burn, chronic inflammation It can be used for diseases such as heart disease, immunodeficiency, Coulomb's disease, breast augmentation, wrinkle removal, cosmetic molding, and regenerative medicine such as tissue enlargement such as tissue sink disease. Alternatively, the obtained cells can be seeded on a scaffold or other structural material, cultured, and used for treatment. Alternatively, the resulting cells may be cryopreserved for future therapeutic applications.

  According to the method of the present invention, preparation of a cell suspension which has been subjected to an enzyme treatment and separation treatment from a living tissue which has been carried out in a plurality of steps in an open system in the conventional method is all closed and in the same container. Can be implemented in In addition, since the passage of the mesh sheet of the enzyme treatment liquid in the container also proceeds smoothly, even an operator who does not have a special technique can easily prepare a cell suspension. Furthermore, since the container for preparing a cell suspension of the present invention does not have a particularly complicated structure, an apparatus capable of preparing a cell suspension in a short time without causing clogging of a mesh sheet is provided at low cost. can do. The present invention is very useful for collecting SVF fraction from fat tissue, which has conventionally been complicated.

  Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. In the following description, "pore diameter" represents the mesh size of the mesh, and the one calculated based on the result measured by the luminometer is used.

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

2. Examination of Angle of Mesh Sheet Surface The following preliminary test was performed using cell suspensions derived from the same individual. The cell concentration of each cell suspension used in the test was 1.0 × 10 ⁶ cells / mL. A commercially available cell strainer having a nylon mesh sheet with a pore diameter of 100 μm, an opening ratio of 42%, and a membrane area of 3.14 cm ² is attached to the centrifuge tube, and the flow direction (vertical direction) ) And the angle of mesh sheet surface was 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 the mesh sheet is the cell suspension before passing the mesh sheet. The cell recovery rate was calculated by dividing by the number of cells contained in. In addition, the number of cells was measured by a modified Neubauer blood cell counter (product certified by the Japan Blood Association).

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

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

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

  (Comparative Example 2) When 90-θ is set to 90, the clogging occurs when the narrow angle formed by the flow direction of the cell suspension and the normal to the surface along the mesh sheet is θ (°). 26 mL of cell suspension could be passed through until 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 to the surface along the mesh sheet. And FIG. 5 is a graph showing the relationship between 90-.theta. And the cell recovery rate. By using a mesh sheet with a pore diameter of 95 μm and making 90-θ less than 50, it was confirmed that high-volume cell suspension can be processed and high cell recovery can be achieved.

3. Test Using Cell Suspension Preparation Container A test was performed using the cell suspension preparation container 1 having the structure shown in FIGS. 1-1 and 2-2. In the cell suspension preparation container used in 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 polyester mesh sheet 40 incorporated were prepared. The side walls of the container were manufactured using flexible polyvinyl chloride. In any of the containers, the outer diameter of the tube inserted into the container end as the inlet 21 and the outlet 22 is 8 mm, and the container inner side end 22 of the inlet 21 to the container inner side 32 of the outlet 31 The distance to reach was about 20 cm. For this reason, the value of the D / W defined herein was about 25. The membrane area of the mesh sheet was 208 cm ² . The container for preparing cell suspension is suspended so that the input part 21 is at the top, 50 mL of cell suspension derived from the same individual is allowed to flow into each container from the inlet 20 by spontaneous fall, and taken out from the outlet 30 The The cell concentration of each cell suspension was 1.4 × 10 ⁶ cells / mL. The mesh sheet in the container at the time of passing the liquid has a narrow angle of θ (°) formed by the normal line L of the surface along the mesh sheet and the passing direction F (vertical direction). Met.

  Example 3 When a container having a mesh sheet with a pore diameter of 95 μm and an open area ratio of 46% was used, no clogging occurred during the passage of liquid, and the cell recovery rate was 95%.

  Example 4 When a container having a mesh sheet with a pore diameter of 200 μm and an opening ratio of 61% was used, clogging did not occur during flow-through, and the cell recovery rate was 100%.

  Example 5 When a container having a mesh sheet with a pore diameter of 300 μm and an opening ratio of 65% was used, clogging did not occur during flow-through, 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. When the angle of the mesh sheet surface with respect to the flow direction was made the same angle, it was confirmed that cells can be recovered at a high recovery rate at least in the range of 95 μm to 300 μm in the pore diameter of the mesh sheet.

4. Test using the cell suspension preparation container and the cell concentration washing system Connect the cell suspension preparation container having the structure shown in Fig. 1-1 and 2-2 and the cell concentration washing system (manufactured by Kaneka). The cell suspension was prepared in a closed system and compared with the results obtained when preparing the cell suspension 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 the “cell suspension processing apparatus capable of washing and concentrating cell suspension in a closed system” disclosed herein. In addition, as a container for cell suspension preparation, the thing whose pore diameter of the mesh sheet used in Example 3 is 95 micrometers was used.

(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 vessel. The enzyme reaction was carried out while shaking the container on a shaker. The enzyme reaction was performed with the inlet 20 and the outlet 30 closed. After the enzyme reaction, the solution in the vessel separated into two layers, an oil layer and an aqueous layer. The container was suspended so that the input portion 21 was at the top, and the boundary between the separated two layers was clamped to prevent the component of the oil layer from flowing toward the discharge portion 31. The mesh sheet 40 in the container had a 90-θ of about 1 when the narrow angle formed by the normal L of the surface along the mesh sheet and the flow direction F (vertical direction) was θ (°). . In that state, negative pressure is applied from the discharge port, the solution is drained 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. The The cell recovery rate in the cell suspension obtained after washing was 83%.

Comparative Example 3 The same adipose tissue sample as in Example 6 and a 0.075% collagenase solution were used to prepare a cell suspension at a cell concentration of 1.6 × 10 ⁶ cells / mL in a centrifuge tube. 20 mL of the obtained cell suspension was opened the lid of the centrifuge tube, the supernatant was removed, the washing solution was added, the lid was closed and centrifugation was repeated to concentrate and wash the cells as in the conventional method. Was 68%.

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

DESCRIPTION OF SYMBOLS 1 ... Container for cell suspension preparation, 10 ... Container main body, 11 ... 1st space, 12 ... 2nd space, 13 ... Internal space, 15 ... 1st side wall, 16 ... 2nd side wall, 20 ... input port, DESCRIPTION OF SYMBOLS 21 ... Insertion part, 22 ... End of the 1st space side of an insertion part 30, 30 ... Discharge port, 31 ... Discharge part, 32 ... End of the 2nd space side of discharge part, 40 ... Mesh sheet, 41 ... Mesh sheet peripheral part 150: first side wall peripheral portion 160: second side wall peripheral portion 901: cell suspension preparation container connection portion 902 dilution liquid bag connection portion 903 storage container 904 waste container 905 recovery Container 906 Cell suspension processor 908 Air filter 909 Air bubble sensor 910 Air bubble sensor 911 Air bubble sensor 912-14 Pump 915 Solution inlet port 916 Circulation outlet port 917 ... circulation inlet port, 9 18: cell suspension inlet, 919: cell suspension outlet, 920: outlet for filtrate, 921-25: clamp, 926: priming solution bag connection, 927: injection channel, 928: communicating tube for introduction, 929 ... lead-out communication pipe, 930 ... circulation circuit, 931 ... recovery path

Claims (10)

A container for preparing a cell suspension,
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;
An inlet provided in the container body and having an inlet for liquid into the first space;
A discharge unit provided in the container body and formed with a discharge port of the liquid from the second space;
Equipped with
90-θ is less than 50, where θ (°) is the narrow angle formed by the flow direction of the liquid discharged from the discharge port and the normal to the surface along the mesh sheet .   The container according to claim 1, wherein the pore size of the mesh sheet is 50 to 300 m.   The container according to claim 1, wherein the container body is flexible. A container for preparing a cell suspension,
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;
An inlet provided in the container body and having an inlet for liquid into the first space;
A discharge unit provided in the container body and formed with a discharge port of the liquid from the second space;
Equipped with
The container body includes a first side wall and a second side wall opposite to 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 input 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 with the outside through the input port.
The discharge portion is disposed between the peripheral portion of the second side wall and the peripheral portion of the mesh sheet so as to communicate the second space with the outside through the discharge port.
The container, wherein the first side wall, the second side wall, and the mesh sheet are integrated at the periphery. The input unit and the discharge unit are disposed at opposite positions of the container body,
A width W1 of an end on the first space side of the input portion along a direction in which the first side wall and the second side wall face each other.
The width of the end of the discharge portion on the side of the second space along the direction in which the first side wall and the second side wall oppose each other is W2,
W, the average value of W1 and W2,
The distance between the end on the first space side of the input part and the end on the second space side of the discharge part is D
And when
The container according to claim 4, wherein D / W is 12 or more.   The container according to claim 4 or 5, wherein the first side wall and the second side wall are flexible. 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;
An inlet provided in the container body and having an inlet for liquid into the first space;
A discharge unit provided in the container body and formed with a discharge port of the liquid from the second space;
Placing a cell-containing sample and an enzyme through the input port, and performing an enzyme reaction in the first space to produce an enzyme-treated solution;
(B) The enzyme treatment liquid is passed through the mesh sheet to remove contaminants, a cell suspension is produced on the second space side, and the cell suspension produced is discharged through the outlet. Process,
Said method.   In the step (b), when the narrow angle formed by the flow direction of the cell suspension discharged through the discharge port and the normal to the surface along the mesh sheet is θ (°), 90- The method according to claim 7, wherein the cell suspension is drained so that θ is less than 50.   The method according to claim 7 or 8, wherein the container is the container according to any one of claims 1 to 6. The outlet is connected to a cell suspension processor capable of washing and concentrating the cell suspension in a closed system,
(C) The method according to any one of claims 7 to 9, further comprising the step of washing and concentrating the cell suspension discharged from the outlet in a closed system by the device to obtain a cell concentrate. the method of.

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