Flow path cassette, cell culture kit and cell culture system

Abstract

The present invention provides a flow path cassette, a cell culture kit, and a cell culture system that enable greater flexibility in flow path layout. The flow path cassette (10), which aggregates flow paths (44) for carrying a cell-containing liquid, includes a first cassette body (40A) and a second cassette body (40B), each having the flow path (44) formed between resin sheets (42), and a frame (50) that houses these. The frame (50) houses the first cassette body (40A) and the second cassette body (40B) in a housing space (52) defined by a recessed shape.

Description

[Technical field]

[0001] The present invention relates to a flow path cassette, a cell culture kit, and a cell culture system for processing cells. [Background technology]

[0002] In regenerative medicine, therapeutic cells such as ES cells, iPS cells, and mesenchymal stem cells are cultured in large quantities and administered to patients. As a cell culture system for efficiently culturing therapeutic cells, for example, a cell culture system for culturing cells in a bioreactor using hollow fibers has been proposed in JP 2017-143775 A.

[0003] In a conventional cell culture system, cells are cultured while supplying various nutrients such as oxygen and glucose and growth factors while circulating a liquid culture medium in a disposable cell culture kit that constitutes a closed circuit. For this purpose, a number of bags are connected to the cell culture system, such as a cell bag that contains cells to be seeded, a culture medium bag that contains culture medium, a waste liquid bag that collects waste liquid, and a detachment liquid bag that detaches cells from the flow path when harvesting cells. Summary of the Invention

[0004] In conventional cell culture systems, multiple bags are connected by multiple flow paths to form complex circuits, which means that it takes time for workers to assemble the cell culture kit into the cell culture device, and it is easy for errors to occur when assembling the various flow paths.

[0005] Therefore, it is conceivable to use a flow path cassette in which a flow path is formed between a pair of flexible sheets that are overlapped and welded in the thickness direction. This flow path cassette consolidates the complex circuits of the cell culture kit, and the cell culture kit can be assembled simply by connecting the piping extending from various bags and hollow fiber modules to the connectors provided on the outer edge of the flow path cassette.

[0006] However, since the flow channel cassette cannot form a flow channel across layers, the layout of the flow channel is limited. Therefore, it may be necessary to provide a flow channel that largely detours around other flow channels, which may result in an increase in size. In addition, there is a problem that the arrangement of connectors is restricted, and the layout of surrounding piping becomes complicated. In addition, the same problem occurs in various cell-containing liquid processing systems, including a cell washing system that washes cultured cells.

[0007] Therefore, an object of one aspect of the present invention is to provide a flow path cassette, a cell culture kit, and a cell culture system that can increase the degree of freedom in the layout of flow paths.

[0008] One aspect of the present invention is a flow path cassette that aggregates flow paths, comprising: a first cassette body in which a flow path is formed between a pair of overlapping resin sheets; a second cassette body in which a flow path is formed between a pair of overlapping resin sheets; and a frame that is formed harder than the first cassette body and the second cassette body and supports the first cassette body and the second cassette body, wherein the frame has a plate-shaped cover portion that covers the first cassette body and the second cassette body, and a wall-shaped side portion that protrudes from an outer edge of the cover portion and surrounds the outer edges of the first cassette body and the second cassette body, and the first cassette body and the second cassette body are accommodated in an accommodation space defined by a concave shape formed by the side portion and the cover portion.

[0009] Another aspect of the present invention is a cell culture kit including the flow path cassette of the above aspect, a plurality of bags connected to the flow path cassette, a bioreactor using hollow fibers connected to the flow path cassette, and a gas exchanger connected to the flow path cassette.

[0010] Yet another aspect of the present invention is a cell culture system including the cell culture kit of the above aspect and a cell culture device in which the cell culture kit is set.

[0011] According to the flow path cassette, cell culture kit, and cell culture system of the above-mentioned aspects, by providing a first cassette body and a second cassette body arranged on top of each other, it is possible to arrange a flow path across layers, thereby increasing the freedom of layout of the flow path. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic configuration diagram of a cell culture system according to a first embodiment. [Diagram 2] FIG. 2 is an exploded perspective view of the flow path cassette of FIG. [Diagram 3] FIG. 13 is a partially enlarged perspective view of a locking mechanism of the flow path cassette. [Figure 4] FIG. 2 is a perspective view of the bottom side of the frame. [Diagram 5] FIG. 2 is a plan view showing the layout of the flow path cassette of FIG. 1 in a state where it is mounted on the cell culture device. [Figure 6] FIG. 6 is a cross-sectional view taken along line VI-VI in FIG. 5. [Figure 7] FIG. 2 is a block diagram showing a circuit configuration of the cell culture kit of FIG. 1. [Figure 8] FIG. 8A is a cross-sectional view of the cell culture system near an ultrasonic sensor, and FIG. 8B is a cross-sectional view of the cell culture system near a capacitance sensor. [Figure 9] FIG. 9A is an enlarged perspective view showing the flow path openable / closable part and the clamp in an open state, and FIG. 9B is an enlarged perspective view showing the flow path openable / closable part and the clamp in a closed state. [Figure 10] FIG. 10 is a cross-sectional view of the pressure detection portion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0014] 1, a flow path cassette 10 according to an embodiment of the present invention constitutes a part of a cell culture kit 12, and is set in a cell culture device 14. This flow path cassette 10 aggregates a plurality of paths of the cell culture kit 12, and is used as a structure capable of passing a liquid containing cells to be cultured and a liquid for processing the cells.

[0015] In addition to the flow path cassette 10, the cell culture kit 12 includes a plurality of tubes 16 and a plurality of bags 18 as components constituting a plurality of paths (flow paths), and a processing chamber 20 set in the cell culture device 14. The cell culture kit 12 is configured to obtain a target product by circulating a plurality of types of liquids contained in each bag 18 via the flow path cassette 10 and each tube 16 and passing through the processing chamber 20 under the operation of the cell culture device 14.

[0016] The cell culture kit 12 according to this embodiment is a disposable kit used for culturing therapeutic cells in regenerative medicine, and a bioreactor 21 in which cells are seeded and grown is applied to the processing chamber 20. Examples of liquids flowing in the cell culture kit 12 include a solution containing cells (hereinafter referred to as a cell liquid), a medium (culture liquid) supplied for cell growth, a cleaning liquid for cleaning the inside of the cell culture kit 12, and a detachment liquid for detaching cells. That is, the cell culture kit 12 and the cell culture device 14 constitute a cell culture system 22 that seeds the cell liquid in the bioreactor 21, supplies the medium to culture the cells, and then detaches and recovers the grown cells from the bioreactor 21.

[0017] The living body cells are not particularly limited, and examples thereof include blood cells (T cells, etc.) and stem cells (ES cells, iPS cells, mesenchymal stem cells, etc.). The culture medium may be selected appropriately according to the living body cells, and examples thereof include a medium prepared by adding various amino acids, vitamins, serum, etc. to a balanced salt solution (BSS) as a base solution. The washing solution is also not particularly limited, and examples thereof include buffer solutions such as PBS (Phosphate Buffered Salts) and TBS (Tris-Buffered Saline), or physiological saline. The detachment solution may be, for example, trypsin or EDTA solution.

[0018] The multiple bags 18 of the cell culture kit 12 include filled bags that are filled with liquid before use, and empty bags that are empty when first used. The filled bags include a cell liquid bag 18A that contains a cell liquid, a washing liquid bag 18B that contains a washing liquid, a culture medium bag 18C that contains a culture medium, and a detachment liquid bag 18F that contains a detachment liquid. The empty bags include a waste liquid bag 18D into which liquid discarded in the culture process flows, and a collection bag 18E that collects the cells (and other liquids) obtained in the culture process.

[0019] The cell culture device 14 of the cell culture system 22 can set a plurality of cell culture kits 12 and simultaneously perform culture using the plurality of cell culture kits 12. Therefore, some of the plurality of bags 18 may be enlarged and provided in common to the plurality of cell culture kits 12. Examples of bags 18 that can be used in common to the plurality of cell culture kits 12 include four types of bags: a culture medium bag 18C, a cleaning solution bag 18B, a stripping solution bag 18F, and a waste liquid bag 18D. In this case, the culture medium bag 18C, the cleaning solution bag 18B, the stripping solution bag 18F, and the waste liquid bag 18D may be configured as rigid tanks instead of the bags 18 formed of flexible sheets.

[0020] Each bag 18 is aseptically joined to an end of each tube 16 using a sterile joining device (not shown). Also, each bag 18 may be fixed in an inseparable manner to an end of each tube 16 to ensure sterility within the cell culture kit 12. Alternatively, the cell culture kit 12 may apply a connection structure (not shown) that detachably connects the tube 16 and each bag 18.

[0021] The bioreactor 21 of the cell culture kit 12 is not particularly limited, but it is preferable to use a culture substrate with a large surface area, for example, a structure having hollow fibers 24. Specifically, the bioreactor 21 includes a plurality of hollow fibers 24 (e.g., 10,000 or more) and a cylindrical container 26 having a main space 26a that accommodates the plurality of hollow fibers 24.

[0022] The hollow fibers 24 each have a lumen (not shown) that penetrates along the direction in which they extend, and cells are attached to and cultured on the inner peripheral surface of the hollow fibers 24 that form the lumen. Each hollow fiber 24 is housed in the axial direction of the container 26, and both ends are held by a holding wall (not shown). The lumen has a diameter of, for example, about 200 μm, and communicates with end spaces 26b on both axial sides of the holding wall.

[0023] Each hollow fiber 24 has a plurality of pores (not shown) that communicate between the outside (main space 26a) of the hollow fiber 24 and the lumen. Each pore is formed to a size that does not allow cells or proteins to pass through, but allows solutions and low molecular weight substances to pass through. The diameter of the pore is set to, for example, about 0.005 μm to 10 μm. As a result, the cells attached to the inner circumferential surface of the hollow fiber 24 are supplied with culture medium, predetermined gas components, etc. through the pores. Hereinafter, a configuration that mainly distributes liquids in the lumen of the hollow fiber 24 is also referred to as IC (intra capillary), and a configuration that mainly distributes liquids outside the hollow fiber 24 is also referred to as EC (extra capillary).

[0024] The material constituting the hollow fibers 24 is not particularly limited, and examples of the material include polymeric materials such as polyolefin resins such as polypropylene and polyethylene, polysulfone, polyethersulfone, polyacrylonitrile, polytetrafluoroethylene, polystyrene, polymethyl methacrylate, cellulose acetate, cellulose triacetate, and regenerated cellulose.

[0025] The container 26 has an axial length that allows the hollow fibers 24 to be housed in a substantially linear state. The container 26 includes four terminals 28 (a first IC terminal 28a, a second IC terminal 28b, a first EC terminal 28c, and a second EC terminal 28d) that are connected to the tubes 16, respectively. The first IC terminal 28a is provided at one end of the container 26 and communicates with the end space 26b at the one end. The second IC terminal 28b is provided at the other end of the container 26 and communicates with the end space 26b at the other end. The first EC terminal 28c is provided on the outer peripheral surface of the container 26 near the other end and communicates with the main space 26a near the other end. The second EC terminal 28d is provided on the outer peripheral surface of the container 26 near the one end and communicates with the main space 26a near the one end.

[0026] The cell culture kit 12 includes a plurality of tubes 16. Among these, the tubes 16 connecting the flow path cassette 10 to the bag 18 and the bioreactor 21 include the following: A cell fluid tube 16A connecting the cell fluid bag 18A and the flow path cassette 10. A washing fluid tube 16B connecting the washing fluid bag 18B and the flow path cassette 10. A culture medium tube 16C connecting the culture medium bag 18C and the flow path cassette 10. A waste fluid tube 16D connecting the waste fluid bag 18D and the flow path cassette 10. A recovery tube 16E connecting the recovery bag 18E and the flow path cassette 10. A first IC tube 16P connecting the first IC terminal 28a of the bioreactor 21 and the flow path cassette 10. A second IC tube 16G connecting the second IC terminal 28b of the bioreactor 21 and the flow path cassette 10. a first EC tube 16H connecting the first EC terminal 28c of the bioreactor 21 and the flow path cassette 10, and a second EC tube 16I connecting the second EC terminal 28d of the bioreactor 21 and the flow path cassette 10;

[0027] The multiple tubes 16 also include closed tubes 16 that protrude from the flow path cassette 10 and are folded back to be reconnected to the flow path cassette 10. The closed tubes 16 include the following: a first pump tube 16J, a second pump tube 16K, a third pump tube 16L, and a fourth pump tube 16M that are set to multiple (four) pumps 30 (see FIG. 5) of the cell culture device 14; an air bubble sensor tube 16N that is set to an air bubble sensor 32 (see FIG. 5) of the cell culture device 14; a sampling tube 16T for sampling the liquid in the flow path; a sensor tube 16U for detecting components by various sensors; and a pair of gas exchange tubes 16V, 16W for connecting the flow path cassette 10 to ports 29c, 29d (see FIG. 5) of the gas exchanger 29. Interlayer connection tubes 16Q, 16R, 16S for connecting the flow path 44A (see FIG. 5) of the first cassette body 40A and the flow path 44B (see FIG. 5) of the second cassette body 40B.

[0028] A gas exchanger 29 that mixes a predetermined gas component with the liquid (culture medium) is provided in the flow path cassette 10. For example, the gas components to be mixed include components (nitrogen N2: 75%, oxygen O2: 20%, carbon dioxide CO2: 5%) close to the mixture ratio of air (carbon dioxide concentration in the living body).

[0029] The structure of the gas exchanger 29 is not particularly limited, and may be one in which a plurality of hollow fibers 29b are provided in a container 29a, similar to the bioreactor 21. That is, the gas exchanger 29 guides the liquid flowing through the EC loop, which is a flow path circulating on the EC side of the bioreactor 21, into the lumen of the hollow fibers 29b, and mixes the gas components supplied to the container 29a (the space outside the hollow fibers 29b) during the movement inside the hollow fibers 29b with the liquid through the pores of the hollow fibers 29b. At the same time, the gas exchanger 29 removes excess gas components from the liquid.

[0030] The flow path cassette 10, which is one component of the cell culture kit 12, is previously joined to each of the tubes 16, and functions as a relay section for distributing the cell fluid, washing fluid, culture medium, and detachment fluid in each bag 18 to another bag 18 or the bioreactor 21. When the cell culture kit 12 is set in the cell culture device 14, this flow path cassette 10 is attached to a cassette placement section (not shown) in the cell culture device 14, and simplifies the wiring work of the tubes 16 in the culture process.

[0031] As shown in FIG. 2, the flow path cassette 10 of this embodiment is composed of a soft first cassette body 40A and a second cassette body 40B to which multiple tubes 16 are directly connected, and a hard frame 50 that holds the first cassette body 40A and the second cassette body 40B and is fixed to the cell culture device 14.

[0032] The first cassette body 40A is formed as a thin rectangular sheet having flexibility. The first cassette body 40A is formed by stacking two resin sheets 42 made of a resin material in the thickness direction and joining (welding). In joining the pair of resin sheets 42, gas is supplied and exhausted between the pair of resin sheets 42 along a groove formed in a metal mold for welding, so that the resin sheets 42 become flow path walls each having a semicircular cross section and a flow path 44A is formed inside the walls. The material constituting the resin sheet 42 is not particularly limited as long as it has flexibility that allows it to be deformed by the pressure of the liquid, and for example, vinyl chloride resin, polyolefin resin, polyurethane resin, etc. may be applied. The surface of the first cassette body 40A may be embossed to form minute irregularities. A plurality of connectors 60 that connect between the plurality of tubes 16 and the flow path 44A are provided on the outer edge 41A of the first cassette body 40A.

[0033] The second cassette body 40B is formed as a sheet having a structure basically similar to that of the first cassette body 40A. The second cassette body 40B is formed into a substantially L-shape by cutting out a large cutout portion 102C provided near two diagonal corners from a rectangular sheet. In addition, the second cassette body 40B has a cutout window 102B (cutout portion) having the same shape as the cutout window 102 (described later) of the first cassette body 40A at a portion overlapping the cutout window 102. The flow path 44B of the second cassette body 40B is arranged so as to avoid the cutout window 102B. This second cassette body 40B is an aggregate of auxiliary flow paths 44B for increasing the degree of freedom of layout of the first cassette body 40A, and does not have a portion for attaching a sensor, a clamp, or the like described later.

[0034] The shape of the second cassette body 40B is not limited to a substantially L-shape, and may be a rectangle having the same dimensions as the first cassette body 40A. In this case, the window-shaped cutout portion 102C may be formed only in the portions corresponding to the pressure detection portion 48 and the liquid level detection portion 80 of the first cassette body 40A.

[0035] On the other hand, the frame 50 is made of a resin material that is harder (has a larger elastic modulus) than the first cassette body 40A and the second cassette body 40B. As shown in Fig. 4, the bottom side of the frame 50 is formed in a thin concave shape having an accommodation space 52 that accommodates the first cassette body 40A and the second cassette body 40B. The material constituting the frame 50 is also not particularly limited, and may be, for example, a thermoplastic resin such as polypropylene, polycarbonate, polyamide, polysulfone, polyarylate, or methacrylate-butylene-styrene copolymer.

[0036] The frame 50 has a substantially rectangular cover portion 54 that is slightly larger than the first cassette body 40A, and a side portion 56 that protrudes from the outer periphery of the cover portion 54 in a direction perpendicular to the cover portion 54. The side portion 56 goes around the entire outer periphery of the cover portion 54. The frame 50 opens the storage space 52 through an opening 52a surrounded by the side portion 56 on the opposite side to the cover portion 54. As shown in FIG. 2, one surface of the second cassette body 40B is exposed through the opening 52a. The side portion 56 is provided with a locking portion 70 that positions and holds each connector 60 at a position corresponding to each connector 60 of the first cassette body 40A and the second cassette body 40B. The connector 60 and the locking portion 70 constitute a locking mechanism 68 that locks the first cassette body 40A and the second cassette body 40B.

[0037] 3, the connector 60 of the first cassette body 40A and the second cassette body 40B has a first cylindrical portion 62 sealed to the first cassette body 40A and the second cassette body 40B, a second cylindrical portion 64 connected to the tube 16, and a flange portion 66 protruding radially outward between the first cylindrical portion 62 and the second cylindrical portion 64. In addition, a communication hole 60a penetrating the first cylindrical portion 62, the second cylindrical portion 64, and the flange portion 66 is formed in the axis of the connector 60.

[0038] The first cylindrical portion 62 is welded to the cassette body 40 in a state where the communication hole 60a communicates with the flow path 44 of the flow path cassette 10 by being sealed together when the two resin sheets 42 of the first cassette body 40A and the second cassette body 40B are sealed. The outer peripheral surface of the first cylindrical portion 62 is formed to have a smaller diameter than the second cylindrical portion 64 in order to correspond to the flow path 44 of the flow path cassette 10. In addition, the tube 16 is inserted inside the second cylindrical portion 64 and is firmly fixed to the second cylindrical portion 64 by an appropriate fixing means. The flange portion 66 has a predetermined thickness in the axial direction of the connector 60 and is formed in a ring shape that goes around the entire outer peripheral surface of the connector 60.

[0039] On the other hand, the locking portion 70 of the frame 50 has a locking recess 72 formed by cutting out the side portion 56, and a movement restricting portion 74 protruding from the side portion 56 near the locking recess 72 toward the inside of the frame 50. The locking recess 72 is opened in the same direction as the opening 52a of the frame 50, and is formed in an arc shape (C-shape) capable of accommodating the tube 16 connected to the connector 60 (second cylindrical portion 64). The size of the locking recess 72 is set so that the accommodated tube 16 and connector 60 are firmly fitted to each other. The depth of the locking recess 72 for fixing the connector 60 of the first cassette body 40A is deeper than the locking recess 72 for fixing the connector 60 of the second cassette body 40B. This allows the first cassette body 40A and the second cassette body 40B to be fixed to the frame 50 in a state where they are stacked in the thickness direction.

[0040] The movement restricting portion 74 is configured with a pair of hook portions 76 that protrude inward from the inner surface of the side portion 56 and are bent in an orthogonal direction and in a direction approaching each other. The movement restricting portion 74 accommodates the flange portion 66 of the connector 60 in a fixing space 74a formed between the movement restricting portion 74 and the side portion 56.

[0041] The flange portion 66 is disposed in the fixed space 74a, so that the connector 60 is restricted from moving in the axial direction. Furthermore, the connector 60 is housed in the locking recess 72 together with the tube 16, so that the connector 60 is locked by the locking portion 70 (side portion 56) with an appropriate locking force, and thus the connector 60 is prevented from coming off the frame 50. The connectors 60 of the first cassette body 40A and the second cassette body 40B are held by the locking portions 70 of the frame 50, so that the flow path cassette 10 is in a state in which the first cassette body 40A and the second cassette body 40B and the frame 50 are integrated together (in a state in which they can be handled as a whole).

[0042] 2, the above-mentioned locking mechanisms 68 are provided on each of the four sides of the rectangular flow path cassette 10. That is, the first cassette body 40A and the second cassette body 40B are provided with connectors 60 on each of the four outer edges 41A, 41B, while the frame 50 is also provided with locking portions 70 on each of the four side portions 56. This allows the frame 50 to hold the sheet-like first cassette body 40A and the second cassette body 40B in a taut state, allowing the flow path 44 to be smoothly extended along the surface direction.

[0043] A holder 53 for holding the gas exchanger 29 is provided on the upper surface of the frame 50. The holder 53 is provided in the vicinity of the second side 51b to which the first EC tube 16H and the second EC tube 16I are connected. The holder 53 fixes the gas exchanger 29 to the upper surface of the frame 50 with the longitudinal direction of the gas exchanger 29 aligned with the second side 51b. Since the gas exchanger 29 is disposed in the vicinity of the connectors 60 of the first EC tube 16H and the second EC tube 16I, the ports 29c, 29d of the gas exchanger 29 can be connected to the first cassette body 40A by the relatively short gas exchange tubes 16V, 16W.

[0044] As shown in Fig. 4, the cover 54 of the frame 50 has a plurality (five) of windows 54a and a plurality (five) of protrusions 54b corresponding to the configuration of the first cassette body 40A and the second cassette body 40B to be held. The windows 54a are provided at positions facing the flow path openable / closable part 100 described below. The windows 54a are formed in a rectangular shape corresponding to the configuration of the flow path openable / closable part 100 (shape of the notched window 102). The protrusions 54b are provided at positions facing the pressure detection part 48 and the liquid level detection part 80 described below. One of the plurality of protrusions 54b, a protrusion 54b1, is formed in the same shape as the planar shape of the liquid level detection part 80.

[0045] The protrusions 54b of the frame 50 ensure contact between the first cassette body 40A and the second cassette body 40B and the frame 50. If the thickness of the cover portion 54 of the frame 50 or the shape of the frame 50 allows the first cassette body 40A and the second cassette body 40B to easily come into contact with the frame 50, the frame 50 does not need to be provided with the protrusions 54b.

[0046] The flow path cassette 10 is formed by integrating the first cassette body 40A, the second cassette body 40B, and the frame 50. As shown in FIG. 6, the first cassette body 40A is disposed on the frame 50 side, and the second cassette body 40B is disposed on the opening 52a side. The pressure detection target portion 48 of the first cassette body 40A is located in a portion where the second cassette body 40B is cut out, so that the pressure sensor 36 can be installed without being obstructed by the second cassette body 40B. In addition, the second cassette body 40B has a cutout window 102B at a position overlapping the flow path opening / closing target portion 100 of the first cassette body 40A, so that the second cassette body 40B does not obstruct the operation of the clamp 110. The flow path 44B of the second cassette body 40B is arranged to straddle the flow path 44A within the layer of the first cassette body 40A, and is connected to the flow path 44A of the first cassette body 40A via interlayer connection tubes 16R, 16S, and 16Q, as shown in Figure 2.

[0047] As shown in Fig. 5, the flow path cassette 10 is set in the cell culture device 14 in an upright position with the surface direction of the first cassette body 40A and the second cassette body 40B aligned along the gravity direction (vertical direction). That is, in the cell culture device 14, the flow path cassette 10 is fixed to the cassette placement part of the cell culture device 14 in the up-down direction shown in Fig. 5. Note that the flow path cassette 10 in Fig. 5 is in a position as viewed from the top side of the frame 50 in a state where it is attached to the cell culture device 14, and the flow path 44A of the first cassette body 40A is illustrated by a broken line for convenience of explanation. When multiple cell culture kits 12 are installed in the cell culture device 14, multiple flow path cassettes 10 are arranged so as to be stacked in the thickness direction of the frame 50.

[0048] Specifically, the outer edge 41A of the first cassette body 40A is composed of a first side 41a (left side in the figure), a second side 41b (right side in the figure), a third side 41c (upper side in the figure), and a fourth side 41d (lower side in the figure). The cell liquid tube 16A, the washing liquid tube 16B, and the recovery tube 16E are connected to the first side 41a. That is, the tubes 16 from the cell liquid bag 18A, the washing liquid bag 18B, and the recovery bag 18E that need to be provided individually for each cell culture kit 12 are arranged together on the first side 41a. In addition, the culture medium tube 16C, the waste liquid tube 16D, and the stripping liquid tube 16F are connected to the second side 41b. In this way, the tubes 16 connected to the culture medium bag 18C, the waste liquid bag 18D, and the stripping liquid bag 18F that can be used in common for a plurality of cell culture kits 12 are arranged together on the second side 41b. Furthermore, the first EC tube 16H, the second EC tube 16I, the gas exchange tubes 16V and 16W, and the sensor tube 16U are connected to the second side 41b.

[0049] In addition, in the flow path cassette 10, four pumps 30 are arranged at positions near the sides when the cassette is set in the cell culture device 14. The pumps 30 include a first pump 30a arranged near the first side 41a, a second pump 30b and a third pump 30c arranged near the third side 41c, and a fourth pump 30d arranged near the fourth side 41d. The first pump 30a pumps liquid into the IC loop, and the second pump 30b pumps liquid into the EC loop. The third pump 30c circulates the liquid in the EC loop, and the fourth pump 30d circulates the liquid in the IC loop.

[0050] For this reason, the cell culture kit 12 (flow path cassette 10) connects the first pump tube 16J to the first side 41a, the second pump tube 16K and the third pump tube 16L to the third side 41c, and the fourth pump tube 16M to the fourth side 41d. The first pump tube 16J to the fourth pump tube 16M are arranged so that their arc-shaped folded back portions wrap around the circular wrapped portions of the first pump 30a to the fourth pump 30d. The first pump 30a to the fourth pump 30d rotate in a manner that squeezes the first pump tube 16J to the fourth pump tube 16M that wrap around them, thereby applying a flow force to the liquid inside.

[0051] Furthermore, in the set state, the cell culture system 22 places the air bubble sensor 32 in a position near the fourth side 41d of the first cassette body 40A. For this reason, the cell culture kit 12 connects the air bubble sensor tube 16N to the fourth side 41d, and places the air bubble sensor tube 16N facing the air bubble sensor 32. The air bubble sensor 32 is not particularly limited, but for example, an ultrasonic sensor or the like can be applied in which the air bubble sensor tube 16N is sandwiched between a pair of inspection walls (not shown) and ultrasonic waves are transmitted between the inspection walls.

[0052] In the cell culture system 22, in the set state, a plurality of clamps 110 are arranged near the outside of the flow path cassette 10 and inside the flow path cassette 10. In detail, in the set state, clamps 110 are arranged near the cell liquid tube 16A, the cleaning liquid tube 16B, the culture medium tube 16C, the recovery tube 16E, and the stripping liquid tube 16F on the outside of the flow path cassette 10. These clamps 110 clamp each tube 16 under the control of the cell culture device 14 to block the flow path of each tube 16, while holding each tube 16 in an open state. In addition, a plurality of clamps 110 are provided inside the flow path cassette 10 to clamp the flow path 44A of the first cassette body 40A. The configuration of the clamps 110 will be described later.

[0053] The frame 50 of the flow path cassette 10 has holding frames 58 that extend from three sides of a side portion 56 and hold the tubes 16 at a predetermined distance from the side portion 56. The holding frames 58 provided on each side hold the tubes 16 near the clamps 110 in an extended state, thereby enabling the clamps 110 to smoothly open and close the flow paths of the tubes 16.

[0054] As shown in FIG. 2, the first cassette body 40A, in addition to having the above-mentioned flow path 44, also has a plurality of pressure detection parts 48 communicating with the flow path 44, a liquid level detection part 80, a check valve part 90, and a plurality of flow path opening / closing parts 100 formed together with the flow path 44.

[0055] 7, the flow path cassette 10 having the flow path 44 integrally includes an IC loop that supplies liquid to the inner cavity of the hollow fibers 24 in the culture step, and an EC loop that supplies liquid to the outside (main space 26a) of the hollow fibers 24. The IC loop is a circulation circuit that circulates liquid between the cassette body 40 and the inner cavity of the hollow fibers 24 in the bioreactor 21. On the other hand, the EC loop is a circulation circuit that circulates liquid between the cassette body 40 and the outside of the bioreactor 21.

[0056] 2, the first cassette body 40A is provided with a plurality of pressure detection targets 48. In a plan view of the first cassette body 40A, the pressure detection targets 48 are formed to extend in a substantially circular shape in a planar direction relative to the connected flow path 44.

[0057] 10, the pressure detection target portion 48 has first and second bulging portions 49a, 49b formed by bulging a pair of resin sheets 42 in the thickness direction. A flow chamber 48a is formed between the first and second bulging portions 49a, 49b. The first bulging portion 49a is supported in close contact with the convex portion 54b of the frame 50. Meanwhile, the second bulging portion 49b bulges and restores in the thickness direction of the resin sheet 42 (toward the bottom in the figure) based on the pressure of the liquid that has flowed into the flow chamber 48a.

[0058] A notch 102C is provided in the second cassette body 40B at a portion corresponding to the pressure detection target 48 of the first cassette body 40A. That is, the second bulge 49b of the pressure detection target 48 is not covered by the second cassette body 40B and is exposed to the cell culture device 14 side.

[0059] A plate member 122 made of a magnetic material is joined to the second bulging portion 49b. The plate member 122 is disposed at a position facing the load cell 120 of the pressure sensor 36 provided in the cell culture device 14. The load cell 120 has a magnet 104 at the tip of a pressure detection portion 126, and the plate member 122 is attracted to the magnet 104, thereby connecting the pressure detection portion 126 and the pressure detection target portion 48. When a positive pressure acts on the flow chamber 48a, a corresponding load acts on the pressure detection portion 126, and the load cell 120 detects the load. This makes it possible to detect the internal pressure of the flow path 44.

[0060] On the other hand, as shown in FIG. 2, the liquid-level detection portion 80 is provided at one location within the cassette body 40, and enables the detection of the level of the liquid flowing through the liquid-level detection portion 80. The liquid-level detection portion 80 has a storage space 80a therein (between the pair of resin sheets 42) for temporarily storing the liquid. The liquid-level detection portion 80 is formed in a rectangular shape with rounded corners when viewed from the front of the cassette body 40. The longitudinal direction of the liquid-level detection portion 80 is aligned with the direction of gravity of the cassette body 40 in the set state. It is preferable that the longitudinal length of the liquid-level detection portion 80 is formed to be at least twice the length of the lateral length of the liquid-level detection portion 80.

[0061] In the cross-sectional view shown in FIG. 8A, the liquid level detection target 80 has an outer peripheral portion 82 that smoothly rises from the welded portion of the pair of resin sheets 42, and a flat portion 84 that is formed flat inside the outer peripheral portion 82. The flat portion 84 is configured to maintain the parallelism of the pair of resin sheets 42, and is disposed opposite the liquid level sensor 37 in the set state. The cross-sectional area of ​​the portion where the liquid is stored in the storage space 80a is sufficiently larger (for example, more than twice) than the cross-sectional area of ​​the flow path 44. In the liquid level detection target 80 configured in this manner, the liquid level rises when the amount of liquid flowing into the storage space 80a exceeds the amount of liquid flowing out, and the liquid level drops when the amount of liquid flowing in from the storage space 80a falls below the amount of liquid flowing out.

[0062] The second cassette body 40B has a notch 102C provided in a portion corresponding to the liquid level detection portion 80, and the liquid level detection portion 80 is exposed on the side of the liquid level sensor 37 of the cell culture device 14. The liquid level sensor 37 of the cell culture device 14 abuts against the liquid level detection portion 80 through the notch 102C of the second cassette body 40B.

[0063] The liquid level sensor 37 provided in the cell culture device 14 includes an upper sensor that detects the upper limit position of the liquid level in the storage space 80a, and a lower sensor that detects the lower limit position. That is, the cell culture system 22 is configured to detect the upper limit position and the lower limit position when the liquid is stored, thereby adjusting the amount of liquid stored in the liquid level detection target portion 80 and removing air from the liquid. In addition, the upper sensor and the lower sensor face the flat portion 84 in the set state, and can stably detect the liquid level. The type of the liquid level sensor 37 is not particularly limited, but it is preferable to apply an ultrasonic sensor 38 (see FIG. 8A) or a capacitance sensor 39 (see FIG. 8B).

[0064] 8A, the ultrasonic sensor 38 includes an oscillator 38a that outputs ultrasonic waves and a receiver 38b that detects ultrasonic waves on the arrangement surface 15a on which the liquid-level detection target 80 is arranged. That is, the ultrasonic sensor 38 is of a reflective type that utilizes the frame 50 of the flow path cassette 10. In this case, the ultrasonic sensor 38 reflects ultrasonic waves oscillated from the oscillator 38a at the cover portion 54 (wall portion) and detects the reflected waves with the receiver 38b. The arrangement surface 15a and the cover portion 54 are configured to form a state in which the cassette body 40 (each of the pair of resin sheets 42) is in contact with each other when the flow path cassette 10 is set.

[0065] It is preferable that the flow path cassette 10 is in a state in which the liquid-level detection target portion 80 is in contact with the cover portion 54 (protruding portion 54b1) in advance. For example, the resin sheet 42 constituting the liquid-level detection target portion 80 may be fixed to the cover portion 54. The ultrasonic sensor 38 may be configured to protrude from the arrangement surface 15a in order to contact the liquid-level detection target portion 80. The cover portion 54 may protrude in the direction of the ultrasonic sensor 38 in order to contact the liquid-level detection target portion 80, as shown by the protruding portion 54b1 in FIG. 4.

[0066] Alternatively, as shown in Fig. 8B, the capacitance sensor 39 includes a flat plate 39a on the arrangement surface 15a on which the liquid-level detection target 80 is arranged, and an AC power source 39b electrically connected to the flat plate 39a. In other words, the liquid-level detection target 80 of the cassette body 40 can be said to be grounded via the frame 50 and the cell culture device 14, and therefore capacitance is generated between the flat plate 39a to which AC power is supplied from the AC power source 39b and the liquid-level detection target 80. The capacitance changes depending on whether there is liquid or gas in the storage space 80a facing the flat plate 39a, and the capacitance sensor 39 can detect this change to detect the level of the liquid stored in the storage space 80a.

[0067] When the capacitance sensor 39 is used as the liquid level sensor 37, the liquid level detection part 80 may be placed slightly away from the placement surface 15a without touching it. This is because the capacitance sensor 39 can detect the change in capacitance due to the difference between liquid and gas even if it is placed at a distance. Also, the surface (one side and the other side) of the liquid level detection part 80 may be embossed or the like to form minute irregularities.

[0068] 7, the check valve section 90 of the cassette body 40 has a function of allowing fluids (liquids and gases) to flow in a first direction (rightward in FIG. 7) while blocking the flow of fluids in a second direction (leftward in FIG. 7) opposite to the first direction. The configuration of the check valve section 90 is not particularly limited, but can be constructed by stacking a valve structure (for example, a structure in which two valve sheets narrowing toward the first direction are joined together and openings are formed at both ends in the first and second directions) between a pair of resin sheets 42.

[0069] 2, the multiple flow path opening and closing parts 100 are configured by forming cutout windows 102 (sheet separating parts 101) at adjacent positions on both sides (width direction) perpendicular to the extension direction of the flow paths 44. That is, the cutout windows 102 are formed on both sides of each flow path 44. That is, in the flow path opening and closing part 100, each flow path 44 is separated from the resin sheet 42 by the cutout windows 102 and is in a floating state.

[0070] 5, when the flow path cassette 10 is set in the cell culture device 14, a clamp 110 is placed on each of the multiple flow path openable / closable parts 100. As shown in FIG 9A, the clamp 110 includes a rotating body 112 (rotating part) provided on the arrangement surface 15b and a fixed body 114 protruding slightly from the arrangement surface 15b.

[0071] The rotating body 112 has a disk portion 112a facing both the flow path 44 and the notched window 102, and one pin 112b (displacement body) provided near the outer periphery of the disk portion 112a. The disk portion 112a rotates about its center point under the control of the cell culture device 14, and moves the pin 112b closer to and away from the fixed body 114. When set, the pin 112b is inserted into the notched window 102 at a position spaced a certain distance from the fixed body 114.

[0072] The fixed body 114 is formed as a rectangular block that can be inserted into the notched window 102, and protrudes at a height shorter than the side portion 56 of the frame 50. The fixed body 114 cooperates with the rotating body 112 to open and close each of the flow paths 44. That is, when the pin 112b of the clamp 110 comes closest to the fixed body 114 as the rotating body 112 rotates, the clamp 110 crushes the flow path wall between the pin 112b and the fixed body 114, thereby closing the flow path 44.

[0073] The cell culture kit 12 attached to the cell culture device 14 constitutes a flow path circuit shown in FIG. 7. In the figure, the portion surrounded by a rectangular frame line is the configuration of the flow path 44 formed in the first cassette body 40A and the second cassette body 40B. Among them, the flow path 44A formed in the first cassette body 40A is indicated by a thick line. Note that each side of the rectangle in the figure does not correspond to the first side 41a to the fourth side 41d shown in FIG. 5. In this embodiment, the cell liquid bag 18A, the cleaning liquid bag 18B, the culture medium bag 18C, and the peeling liquid bag 18F are configured to be connected to the flow path 44B of the second cassette body 40B, so that the degree of freedom in arranging the connection position of the tube 16 connected to them to the flow path cassette 10 is increased. As a result, the tube 16 of the bag 18 common to a plurality of cell culture kits 12 and the tube 16 of the bag 18 specific to each cell culture kit 12 can be connected to different sides. As a result, the tube 16 can be easily routed and the occurrence of connection work errors can be prevented.

[0074] Also, the first IC tube 16P and the second IC tube 16G are connected to the flow path 44B of the second cassette body 40B. This allows the side where the tube 16 connected to the IC loop of the bioreactor 21 is connected to the frame 50 to be different from the side where the tube 16 connected to the EC loop is connected to the frame 50. This prevents the tube 16 from being connected incorrectly.

[0075] Returning to FIG. 1, the cell culture device 14 to which the cell culture kit 12 is attached includes a box-shaped device body 130 and a stand 132 for holding each bag 18 of the cell culture kit 12. A touch panel 134 (display operation unit) for performing operations and display during the culture process is provided on the outer surface of the device body 130. Furthermore, inside the device body 130, a cassette placement unit (not shown) for fixing the flow path cassette 10 in an upright position and for holding the bioreactor 21 at an appropriate height position, and a control unit 136 for controlling the operation of the cell culture system 22 are provided. Although not shown, the cell culture device 14 may of course include a functional unit for realizing various conditions required for cell culture. For example, the cell culture device 14 may include a temperature adjustment unit for performing temperature control to maintain the culture environment at 37°C.

[0076] The flow path cassette 10, the cell culture kit 12, and the cell culture system 22 of this embodiment have the following advantages.

[0077] The first aspect of this embodiment is a flow path cassette 10 that aggregates flow paths 44 for flowing a medium (cell-containing liquid) used for cell culture. The flow path cassette 10 includes a first cassette body 40A in which the flow paths 44 are formed between a pair of overlapping resin sheets 42 (flexible sheets), a second cassette body 40B in which the flow paths 44 are formed between a pair of overlapping resin sheets 42 (flexible sheets), and a frame 50 that is formed harder than the first cassette body 40A and the second cassette body 40B and supports the first cassette body 40A and the second cassette body 40B. The frame 50 includes a plate-shaped cover portion 54 that covers the first cassette body 40A and the second cassette body 40B, and a wall-shaped side portion 56 that protrudes from the outer edge of the cover portion 54 and surrounds the outer edges 41A and 41B of the first cassette body 40A and the second cassette body 40B. The first cassette body 40A and the second cassette body 40B are housed in an overlapping manner in the housing space 52 defined by the recessed shape formed by the side portion 56 and the cover portion .

[0078] According to the above configuration, by providing the second cassette body 40B, it is possible to arrange a flow path 44 that straddles the flow path 44 of the first cassette body 40A, so that the degree of freedom in the layout of the flow paths 44 within the flow path cassette 10 is improved.

[0079] In the above-mentioned flow path cassette 10, interlayer connection tubes 16R, 16S, and 16Q that connect the flow path 44 of the first cassette body 40A and the flow path 44 of the second cassette body 40B are provided on the outside of the side portion 56. This makes it possible to connect the flow paths of the first cassette body 40A and the second cassette body 40B to each other simply by connecting the interlayer connection tubes 16R, 16S, and 16Q from the outside of the flow path cassette 10.

[0080] In the above-described flow path cassette 10, a connector 60 communicating with the flow path 44 may be provided on the outer edges 41A, 41B of the first cassette body 40A and the second cassette body 40B, and a locking portion 70 for locking the connector 60 may be provided on the side portion 56. With this configuration, the first cassette body 40A and the second cassette body 40B can be held by the frame 50 in a non-slack state, and blockage of the flow path 44 due to slackness can be prevented.

[0081] In the above-described flow path cassette 10, the frame 50 is formed in a rectangular shape in a plan view, and among the connectors 60, a plurality of connectors 60 to which the bags 18 are connected may be arranged together on the same side of the side portion 56. This simplifies the routing of the tubes 16 and prevents connection errors by the user.

[0082] In the above-mentioned flow path cassette 10, a connector 60 to be connected to an IC loop of a bioreactor 21 using hollow fibers 24 and a connector 60 to be connected to an EC loop of the bioreactor 21 may be provided on different sides of the side portion 56. This simplifies the routing of the tubes 16 and prevents connection errors by the user.

[0083] A second aspect of the present embodiment is a cell culture kit 12 including the flow path cassette 10 of the first aspect, a plurality of bags 18 connected to the flow path cassette 10, a bioreactor 21 using hollow fibers 24 connected to the flow path cassette 10, and a gas exchanger 29 connected to the flow path cassette 10. Since the cell culture kit 12 includes the flow path cassette 10 of the first aspect, the degree of freedom in the layout of the flow path 44 is increased, the routing of the tubes 16 can be simplified, and the user's mistake in connecting the tubes 16 can be prevented.

[0084] A third aspect of this embodiment is a cell culture system 22 having the cell culture kit 12 of the second aspect and a cell culture device 14 in which the cell culture kit 12 is set. This cell culture system 22 includes the flow path cassette 10 of the first aspect, which increases the degree of freedom in the layout of the flow path 44, simplifies the routing of the tubes 16, and prevents the user from making an incorrect connection of the tubes 16. In particular, when multiple cell culture kits 12 are set in the cell culture device 14 to perform culture, it is possible to provide some bags 18 (tanks) in common to the multiple cell culture kits 12. Even in such a case, the routing of the tubes 16 can be simplified, resulting in excellent scalability.

[0085] In the above, the present invention has been described by citing preferred embodiments, but the present invention is not limited to the above embodiments, and it goes without saying that various modifications are possible without departing from the spirit of the present invention.

Claims

1. A flow path cassette in which flow paths are aggregated, a first cassette body having a first flow path formed between a pair of overlapping resin sheets; a second cassette body having a second flow path formed between a pair of overlapping resin sheets; a frame that is formed to be harder than the first cassette body and the second cassette body and supports the first cassette body and the second cassette body, The frame is a plate-shaped cover portion that covers the first cassette body and the second cassette body; a wall-like side portion that protrudes from an outer edge of the cover portion and surrounds the outer edges of the first cassette body and the second cassette body, The first cassette body and the second cassette body are accommodated in an accommodation space defined by a concave shape formed by the side portion and the cover portion, A plurality of connectors communicating with the first flow path or the second flow path are provided in the side portion, A flow path cassette, wherein the connector to be connected to an IC loop of a bioreactor using hollow fibers and the connector to be connected to an EC loop of the bioreactor are each provided on different sides of the side portion.

2. 2. The flow path cassette of claim 1, The first cassette body and the second cassette body are disposed in the accommodation space in a stacked manner, in that order from the cover portion side, and The second cassette body is provided with a notch through which a specific portion of the first cassette body is exposed.

3. 3. The flow path cassette according to claim 2, a flow path cassette, wherein the specific portion of the first cassette body includes a flow path openable / closable portion for opening and closing the first flow path of the first cassette body.

4. 4. The flow path cassette according to claim 2 or 3, a flow path cassette, wherein the specific portion of the first cassette body includes a pressure detection portion for measuring pressure in the first flow path of the first cassette body.

5. 5. A flow path cassette according to claim 1, wherein each of the first cassette body and the second cassette body is formed by folding and stacking a single resin sheet.

6. A flow path cassette according to any one of claims 1 to 5, wherein an interlayer connection tube connecting the first flow path of the first cassette body and the second flow path of the second cassette body is connected to the outside of the side portion.

7. 7. The flow path cassette according to claim 6, wherein the side portion is provided with a locking portion for locking the connector.

8. 8. A flow path cassette according to claim 7, wherein the frame is formed in a rectangular shape when viewed in a plane, and among the plurality of connectors, the connectors connected to a bag are arranged together on the same side of the side portion.

9. a flow path cassette in which the flow paths are aggregated; A plurality of bags connected to the flow path cassette; A bioreactor using hollow fibers connected to the flow path cassette; a gas exchanger connected to the flow path cassette; The flow path cassette includes: a first cassette body having a first flow path formed between a pair of overlapping resin sheets; a second cassette body having a second flow path formed between a pair of overlapping resin sheets; a frame that is formed to be harder than the first cassette body and the second cassette body and supports the first cassette body and the second cassette body, The frame is a plate-shaped cover portion that covers the first cassette body and the second cassette body; a wall-like side portion that protrudes from an outer edge of the cover portion and surrounds the outer edges of the first cassette body and the second cassette body, The cell culture kit accommodates the first cassette body and the second cassette body in an accommodation space defined by a concave shape formed by the side portion and the cover portion.

10. a cell culture kit including a flow path cassette in which flow paths are aggregated, a plurality of bags connected to the flow path cassette, a bioreactor using hollow fibers connected to the flow path cassette, and a gas exchanger connected to the flow path cassette; A cell culture device in which the cell culture kit is set; A cell culture system having the flow path cassette, a first cassette body having a first flow path formed between a pair of overlapping resin sheets; a second cassette body having a second flow path formed between a pair of overlapping resin sheets; a frame that is formed to be harder than the first cassette body and the second cassette body and supports the first cassette body and the second cassette body, The frame is a plate-shaped cover portion that covers the first cassette body and the second cassette body; a wall-like side portion that protrudes from an outer edge of the cover portion and surrounds the outer edges of the first cassette body and the second cassette body, A cell culture system, comprising: a storage space defined by a concave shape formed by the side portion and the cover portion, and the first cassette body and the second cassette body are stored in the storage space.

11. The cell culture system according to claim 10, The first cassette body and the second cassette body are disposed in the accommodation space in a stacked manner, in that order from the cover portion side, and A cell culture system, wherein the second cassette body is provided with a cutout portion through which a specific portion of the first cassette body is exposed.

12. The cell culture system according to claim 11, A cell culture system, wherein the specific portion of the first cassette body includes a flow path openable / closable portion for opening and closing a flow path within the first cassette body.

13. The cell culture system according to claim 11 or 12, A cell culture system, wherein the specific portion of the first cassette body includes a pressure detection portion for measuring pressure in a flow path in the first cassette body.

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