Flow path cassette, cell culture kit and cell culture system

Abstract

Provided are a flow path cassette, a cell culture kit, and a cell culture system that enable greater flexibility in flow path layout. A flow path cassette (10) that aggregates flow paths (44A, 44B) includes a first cassette body (40A) in which the flow path (44A) is formed between resin sheets (42), a second cassette body (40B) in which the flow path (44B) is formed, and a frame (50) that accommodates these. The frame (50) accommodates the first cassette body (40A) in a first accommodation space (52A) formed in a first direction, and the second cassette body (40B) in a second accommodation space (52B) formed in a second direction opposite the first direction.

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 resin 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 a flow path cassette cannot form a flow path across layers, the layout of the flow path is limited. Therefore, it may be necessary to provide a flow path that makes a large detour around other flow paths, which may result in an increase in size. In addition, there is a problem that the arrangement of connectors is restricted, making the layout of surrounding piping (tubes) 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 flat cover portion disposed between the first cassette body and the second cassette body, a first side portion protruding from an outer edge of the cover portion in a first direction perpendicular to the cover portion, and a second side portion protruding from the outer edge of the cover portion in a second direction opposite to the first direction, and the flow path cassette accommodates the first cassette body in a first accommodating space defined by a concave shape formed by the first side portion and the cover portion, and accommodates the second cassette body in a second accommodating space defined by the concave shape formed by the second side portion and the cover portion.

[0009] Another 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; and a second cassette body in which a flow path is formed between a pair of overlapping resin sheets, the first cassette body being accommodated therein and comprising a first storage space having a bottomed shape with an opening that is open on one side; and the second cassette body being accommodated therein and comprising a second storage space having a bottomed shape with an opening that is open on one side; the first storage space and the second storage space are integrally formed so as to face each other with their bottoms in contact.

[0010] 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.

[0011] 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.

[0012] 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 side by side in the thickness direction, it becomes possible to arrange a flow path across layers, thereby increasing the freedom of layout of the flow path. [Brief description of the drawings]

[0013] [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 the circuit configuration of the cell culture kit of FIG. 1. [Figure 8] FIG. 8A is a cross-sectional view of the vicinity of an ultrasonic sensor in a cell culture system, and FIG. 8B is a cross-sectional view of a capacitance sensor in the cell culture system. [Figure 9] FIG. 9A is an enlarged perspective view showing the flow path opened / closed part and the clamp in an open state, and FIG. 9B is an enlarged perspective view showing the flow path opened / closed part and the clamp in a closed state. [Figure 10] FIG. 10A is an enlarged perspective view showing a multiple flow path open / closed portion and a switching clamp in an open state, and FIG. 10B is an enlarged perspective view showing a state in which one of the flow paths is closed by the switching clamp of FIG. 10A. [Figure 11] FIG. 4 is a cross-sectional view of a part to be subjected to pressure detection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will now be described in detail with reference

[0015] 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.

[0016] 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 members constituting a plurality of 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 through 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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. Instead of the bags 18 formed of a flexible resin sheet 42, 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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).

[0025] 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.

[0026] 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.

[0027] 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;

[0028] 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 and 16W for connecting the flow path cassette 10 to ports 29c and 29d of the gas exchanger 29. Interlayer connection tubes 16Q, 16R, 16S for connecting the flow path 44A of the first cassette body 40A and the flow path 44B of the second cassette body 40B.

[0029] 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).

[0030] 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 path for circulating 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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 has a notch 99 formed by cutting out a rectangular sheet to avoid a holder 53 (described later), and is formed into a substantially L-shape. The flow path 44B of the second cassette body 40B is connected to the tube 16 extending from the bag 18 and the bioreactor 21. The flow path 44B of the second cassette body 40B is connected to the flow path 44A of the first cassette body 40A via interlayer connection tubes 16Q, 16R, and 16S.

[0035] Although not particularly limited, the second cassette body 40B is a collection of auxiliary flow paths 44B to increase the freedom of layout of the first cassette body 40A, and does not have any locations for attaching sensors, clamps, etc., which will be described later.

[0036] 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. The frame 50 is formed in a thin plate shape in which a first storage space 52A and a second storage space 52B for storing the first cassette body 40A and the second cassette body 40B are formed. The second storage space 52B for storing the second cassette body 40B is formed on the upper surface 50b side of the frame 50, and the first storage space 52A for storing the first cassette body 40A is formed on the bottom surface 50a side of the frame 50 as shown in FIG. The material for forming the frame 50 is not particularly limited, and it is preferable to apply a thermoplastic resin such as polypropylene, polycarbonate, polyamide, polysulfone, polyarylate, or methacrylate-butylene-styrene copolymer.

[0037] The frame 50 has a rectangular, flat cover portion 54 that is slightly larger than the first cassette body 40A, and a side portion 56 that protrudes in a wall shape from the outer periphery of the cover portion 54 in a direction perpendicular to the cover portion 54. The side portion 56 has a first side portion 56a that protrudes toward the bottom surface 50a of the frame 50, and a second side portion 56b (see FIG. 2) that protrudes toward the front surface of the frame 50. The first side portion 56a and the second side portion 56b protrude in opposite directions from the cover portion 54, and go around the entire outer periphery of the cover portion 54. The first side portion 56a and the second side portion 56b are integrally formed to constitute the side portion 56.

[0038] 4, the frame 50 opens the first storage space 52A through an opening 52a surrounded by a first side portion 56a on the bottom surface 50a side of the cover portion 54. One surface of the first cassette body 40A is exposed through the opening 52a. Locking portions 70 for arranging and holding each connector 60 are provided in the first side portion 56a at locations corresponding to each connector 60 of the first cassette body 40A.

[0039] 2, the frame 50 opens the second storage space 52B through an opening 52b surrounded by a second side portion 56b on the upper surface 50b side of the cover portion 54. The second side portion 56b is also provided with locking portions 70 that hold the connectors 60 of the second cassette body 40B. The connectors 60 and the locking portions 70 constitute a locking mechanism 68 that locks the first cassette body 40A and the second cassette body 40B to the frame 50.

[0040] 3, the connector 60 of the first cassette body 40A has a first cylindrical portion 62 sealed to the first cassette body 40A, 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.

[0041] The first cylindrical portion 62 is welded to the first cassette body 40A with the communication hole 60a communicating 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 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. 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.

[0042] On the other hand, the locking portion 70 of the frame 50 has a locking recess 72 formed by cutting out the first side portion 56a, 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 it can firmly engage with the accommodated tube 16 and connector 60. 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.

[0043] 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.

[0044] The flange portion 66 is disposed in the fixed space 74a, so that the movement of the connector 60 along the axial direction is restricted. 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.

[0045] The structure of each connector 60 of the second cassette body 40B is similar to the structure of each connector 60 of the first cassette body 40A. In addition, the structure of the locking portion 70 formed on the second side portion 56b is similar to the structure of the locking portion 70 formed on the first side portion 56a.

[0046] In this manner, the locking mechanism 68 holds the connectors 60 of the first cassette body 40A and the second cassette body 40B at the locking portions 70 of the frame 50. This results in the flow path cassette 10 being in a state in which the first cassette body 40A, the second cassette body 40B and the frame 50 are integrated together (in a state in which they can be handled as a whole).

[0047] As shown in FIG. 2, the above-mentioned locking mechanisms 68 are provided on the four sides of the rectangular flow path cassette 10. That is, the first cassette body 40A (see FIG. 2) has a connector 60 on each of the four outer edges 41A, and the frame 50 has a locking portion 70 on each of the four first side portions 56a. Also, as shown in FIG. 2, the second cassette body 40B has a connector 60 on three outer edges 41B, and the frame 50 has a locking portion 70 on each of the three second side portions 56b. As a result, the frame 50 can hold the sheet-like first cassette body 40A and second cassette body 40B in a taut state, as shown in FIG. 6, and can satisfactorily extend the respective flow paths 44A, 44B along the surface direction.

[0048] 2, a holder 53 for holding the gas exchanger 29 is provided on the upper surface 50b 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 50b 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.

[0049] 4, a first convex portion 54a and a second convex portion 54b are formed on the bottom surface 50a of the frame 50. The first convex portion 54a is formed in the same shape as the planar shape of a liquid-level detection target portion 80 (described later) of the first cassette body 40A, and is provided at a position facing the liquid-level detection target portion 80. A plurality of second convex portions 54b are provided, and are arranged at positions facing pressure detection target portions 48 (described later).

[0050] The second protrusion 54b of the frame 50 ensures 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 second protrusion 54b.

[0051] As shown in FIG. 6, when the first cassette body 40A, the second cassette body 40B, and the frame 50 are integrated in the flow path cassette 10, the first cassette body 40A and the second cassette body 40B are arranged so as to overlap in the thickness direction with the cover part 54 sandwiched therebetween. The first cassette body 40A is arranged on the bottom surface 50a side of the cover part 54 of the frame 50, and the second cassette body 40B is arranged on the top surface 50b side of the cover part 54. Since the second cassette body 40B is separated from the first cassette body 40A by the cover part 54, it does not interfere with the pressure detection target part 48 and the flow path opening and closing part 100 attached to the first cassette body 40A. Therefore, the flow path 44 can be arranged in the second cassette body 40B even in a position facing the pressure detection target part 48 and the flow path opening and closing part 100.

[0052] 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 upper surface 50b side of the frame 50 in a state where it is attached to the cell culture device 14, and for convenience of explanation, the flow path 44A of the first cassette body 40A is illustrated by a broken line. 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.

[0053] The outer edge 51 of the frame 50 is composed of a first side 51a (left side in the figure), a second side 51b (right side in the figure), a third side 51c (upper side in the figure), and a fourth side 51d (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 51a. That is, the tubes 16 from the cell liquid bag 18A, the washing liquid bag 18B, and the recovery bag 18E individually provided in each cell culture kit 12 are connected together to the first side 51a. These cell liquid tube 16A, the washing liquid tube 16B, and the recovery tube 16E are connected to the second cassette body 40B. In addition, the interlayer connection tubes 16R and 16Q are connected to the first side 51a.

[0054] The culture medium tube 16C, the waste liquid tube 16D, and the stripping solution tube 16F are connected to the second side 51b. In this manner, the connection positions of the tubes 16 of the culture medium bag 18C, the waste liquid bag 18D, and the stripping solution bag 18F that can be used in common with a plurality of cell culture kits 12 are arranged together on the second side 51b. In addition, the tubes 16 constituting the EC loop, such as the first EC tube 16H, the second EC tube 16I, and the gas exchange tubes 16V and 16W, the sensor tube 16U, and the interlayer connection tube 16S are connected to the second side 51b. One side of the culture medium tube 16C, the stripping solution tube 16F, and the interlayer connection tube 16S are connected to the second cassette body 40B.

[0055] 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 51a, a second pump 30b and a third pump 30c arranged near the third side 51c, and a fourth pump 30d arranged near the fourth side 51d. 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.

[0056] For this reason, the cell culture kit 12 (flow path cassette 10) connects the first pump tube 16J to the first side 51a, the second pump tube 16K and the third pump tube 16L to the third side 51c, and the fourth pump tube 16M to the fourth side 51d. 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.

[0057] Furthermore, the first IC tube 16P and the second IC tube 16G constituting the IC loop of the bioreactor 21 and the sampling tube 16T are connected to the fourth side 51d of the frame 50. By providing the first IC tube 16P and the second IC tube 16G constituting the IC loop on a different side from the first EC tube 16H and the second EC tube 16I constituting the EC loop, connection errors can be prevented.

[0058] Furthermore, in the set state, the cell culture system 22 places the air bubble sensor 32 in a position near the fourth side 51d of the frame 50. For this reason, the cell culture kit 12 connects the air bubble sensor tube 16N to the fourth side 51d, 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.

[0059] In the cell culture system 22, in the set state, a plurality of clamps 110 are arranged in the vicinity of 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 in the vicinity of 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 close each tube 16 by clamping each tube 16 under the control of the cell culture device 14. In addition, a plurality of clamps 110 are provided inside the flow path cassette 10 to open and close the flow path 44A of the first cassette body 40A. The configuration of the clamps 110 will be described later.

[0060] The frame 50 of the flow path cassette 10 has holding frames 58 on three sides, i.e., a first side 51a, a second side 51b, and a fourth side 51d of a side portion 56. The holding frames 58 hold the tubes 16 at a position a predetermined distance away from the side portion 56. The holding frames 58 hold the tubes 16 in a stretched state near the side portion 56, thereby preventing blockage of the tubes 16. In addition, the holding frames 58 enable the tubes 16 to be opened and closed smoothly by clamps 110 provided inside the holding frames 58.

[0061] As shown in FIG. 2, the first cassette body 40A, in addition to having the above-mentioned flow path 44A, also has a plurality of pressure detection parts 48 communicating with the flow path 44A, 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 44A.

[0062] As shown in FIG. 2, the liquid level detection portion 80 is provided at one location in the first cassette body 40A, and is capable of detecting the level of the liquid flowing through the liquid level detection portion 80. The liquid level detection portion 80 has a storage space 80a (see FIG. 8A) inside (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 first cassette body 40A. The longitudinal direction of the liquid level detection portion 80 is aligned with the direction of gravity of the first cassette body 40A 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.

[0063] 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 as large) than the cross-sectional area of ​​the flow path 44A. 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.

[0064] 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).

[0065] 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 15b 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 15b and the cover portion 54 are configured to form a state in which the first cassette body 40A (each of the pair of resin sheets 42) is in contact with each other when the flow path cassette 10 is set.

[0066] 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 (first convex portion 54a) 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 15b 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 in FIG. 4.

[0067] 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.

[0068] 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.

[0069] The check valve section 90 of the first cassette body 40A in Fig. 2 has a function of allowing fluids (liquids and gases) to flow in a first direction (rightward in Fig. 5) while blocking the flow of fluids in a second direction (leftward in Fig. 5) 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 connecting portions are formed at both ends in the first and second directions) between a pair of resin sheets 42.

[0070] On the other hand, the multiple flow path opening and closing parts 100 are configured by forming cutout windows 102 at adjacent positions on both sides (width direction) perpendicular to the extension direction of the flow path 44A. That is, both sides of each flow path 44A are cut out in a rectangular shape. That is, in the flow path opening and closing part 100, each flow path 44A is separated from the resin sheet 42 by the cutout windows 102 and is in a floating state.

[0071] 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.

[0072] The rotating body 112 has a disk portion 112a facing both the flow path 44A 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.

[0073] 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 44A. 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 and closes the flow path 44A between the pin 112b and the fixed body 114. The clamp 110 attached to the tube 16 also has a structure similar to that described above.

[0074] 10A, the multiple flow path opening and closing unit 100A has a notched window 102A for selectively opening and closing two adjacent parallel flow paths 44A. In the notched window 102A, the two flow paths 44A are arranged parallel to each other, and the outer peripheral surface (cylindrical flow path wall) of the flow path 44A is separated from the resin sheet 42 over the entire circumference. The notched window 102A is formed in a rectangular shape and is provided so as to include each of the flow paths 44A.

[0075] On the other hand, in the cell culture device 14, a switching clamp 110A is placed on the multiple flow path openable / closable part 100A in a state where the flow path cassette 10 is set. The switching clamp 110A includes a rotating body 112A provided on the arrangement surface 15b and multiple (two) fixed bodies 114A protruding from the arrangement surface 15b.

[0076] The rotor 112A, like the rotor 112 described above, has a disk portion 112a and one pin 112b (displacement body) provided near the outer periphery of the disk portion 112a. The disk portion 112a is disposed opposite the two flow paths 44A. 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 farther away from the two fixed bodies 114A. When set, the pin 112b is disposed in the space between the two flow paths 44A. One of the two fixed bodies 114A is disposed outside one flow path 44A, and the other is disposed outside the other flow path 44A.

[0077] 10B, when the pin 112b is closest to one of the fixed bodies 114A, the switching clamp 110A crushes the flow path 44A between the fixed body 114A and the pin 112b, thereby closing the flow path 44A. At this time, the opposite flow path 44A is opened. When the pin 112b is closest to the other fixed body 114A, the switching clamp 110A closes the other flow path 44A and opens one of the flow paths 44A.

[0078] 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 the planar direction relative to the connected flow path 44A.

[0079] 11, the pressure detection target portion 48 has first and second bulging portions 49a, 49b formed by bulging the 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 second protruding portion 54b of the frame 50. Meanwhile, the second bulging portion 49b bulges and restores in the thickness direction of the resin sheet 42 based on the pressure of the liquid that has flowed into the flow chamber 48a.

[0080] 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 126 at the tip of a pressure detection portion 128, and the plate member 122 is attracted to the magnet 126, thereby connecting the pressure detection portion 128 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 128, and the load cell 120 detects the load. This makes it possible to detect the internal pressure of the flow path 44A.

[0081] 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.

[0082] The above cell culture kit 12 is set in a cell culture device 14 to form a flow path circuit as 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 44 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 51a to the fourth side 51d of the frame 50 shown in Fig. 5.

[0083] The flow path cassette 10 having the flow path 44B has 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.

[0084] In this embodiment, the cell fluid bag 18A, the washing fluid bag 18B, the culture medium bag 18C, and the stripping fluid 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 positions of the tubes 16 connected to these to the flow path cassette 10 increases. This allows the tubes 16 of the bags 18 common to a plurality of cell culture kits 12 and the tubes 16 of the bags 18 specific to each cell culture kit 12 to be connected to different sides. This makes it easier to route the tubes 16 and prevents mistakes in the connection work.

[0085] 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.

[0086] The flow path cassette 10, the cell culture kit 12, and the cell culture system 22 of this embodiment have the following advantages.

[0087] The first aspect of this embodiment is a flow path cassette 10 in which flow paths 44A and 44B for flowing a medium (cell-containing liquid) used for cell culture are integrated. The flow path cassette 10 includes a first cassette body 40A in which the flow path 44A is formed between a pair of overlapping resin sheets 42, a second cassette body 40B in which the flow path 44B is formed between the pair of overlapping resin sheets 42, 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 flat cover portion 54 disposed between the first cassette body 40A and the second cassette body 40B, a first side portion 56a protruding from an outer edge 51 of the cover portion 54 in a first direction perpendicular to the cover portion 54, and a second side portion 56b protruding from the outer edge 51 of the cover portion 54 in a second direction opposite to the first direction. The first cassette body 40A is accommodated in the first storage space 52A defined by the concave shape formed by the first side portion 56a and the cover portion 54, and the second cassette body 40B is accommodated in the second storage space 52B defined by the concave shape formed by the second side portion 56b and the cover portion 54.

[0088] According to the above configuration, by providing the second cassette body 40B, the flow path 44B can be arranged to straddle the flow path 44A of the first cassette body 40A, improving the degree of freedom in the layout of the flow paths 44A and 44B in the flow path cassette 10. In addition, since the first cassette body 40A and the second cassette body 40B are separated by the cover part 54, the clamp 110 and sensors installed on the first cassette body 40A side do not interfere with the second cassette body 40B. This improves the degree of freedom in the layout of the flow path 44B of the second cassette body 40B. In addition, the clamp 110 and sensors can also be provided in the flow path 44B of the second cassette body 40B.

[0089] In the flow path cassette 10 of the above embodiment, interlayer connection tubes 16Q, 16R, and 16S that connect the flow path 44A of the first cassette body 40A and the flow path 44B of the second cassette body 40B may be connected to the outside of the first side portion 56a and the second side portion 56b. This allows the flow paths 44A and 44B of different layers to be connected to each other simply by connecting the interlayer connection tubes 16R, 16S, and 16Q from the outside of the flow path cassette 10.

[0090] In the flow path cassette 10 of the above aspect, a connector 60 communicating with the flow paths 44A, 44B 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 first side portion 56a and the second side portion 56b. With this configuration, the first cassette body 40A and the second cassette body 40B can be held in a taut state in the first accommodating space 52A and the second accommodating space 52B, respectively, and the flow paths 44A, 44B can be extended along the surface direction.

[0091] In the flow path cassette 10 of the above embodiment, the frame 50 is formed in a rectangular shape in a plan view, and among the multiple connectors 60, the connectors 60 to which the bag 18 is connected may be arranged together on the same side of the first side portion 56a and the second side portion 56b. This configuration simplifies the routing of the tube 16 and prevents connection errors by the user.

[0092] In the flow path cassette 10 of the above embodiment, the connector 60 connected to the IC loop of the bioreactor 21 using the hollow fibers 24 and the connector 60 connected to the EC loop of the bioreactor 21 may be provided on different sides of the first and second side portions 56a and 56b, respectively. This simplifies the routing of the tube 16 and prevents the user from making a connection mistake.

[0093] 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 paths 44A and 44B is increased, the routing of the tubes 16 can be simplified, and the user's mistake in connecting the tubes 16 can be prevented.

[0094] 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 paths 44A and 44B, simplifies the routing of the tube 16, and prevents the user from making a mistake in connecting the tube 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 tube 16 can be simplified, resulting in excellent scalability.

Claims

1. A flow path cassette in which flow paths are aggregated, a first cassette body having a flow path formed between a pair of overlapping resin sheets; a second cassette body having a 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 flat cover portion disposed between the first cassette body and the second cassette body; A first side portion protruding from an outer edge of the cover portion in a first direction perpendicular to the cover portion; a second side portion protruding from an outer edge of the cover portion in a second direction opposite to the first direction, the first cassette body is accommodated in a first accommodating space defined by a concave shape formed by the first side portion and the cover portion, and the second cassette body is accommodated in a second accommodating space defined by a concave shape formed by the second side portion and the cover portion, the side portion having the first side portion and the second side portion includes a first side, a second side, a third side, and a fourth side that are provided on all four sides so as to go around an outer periphery of the rectangular cover portion, A flow path cassette, wherein a plurality of tubes through which a fluid flows are connected to the outer sides of each of the first side, the second side, the third side, and the fourth side.

2. 2. The flow path cassette according to claim 1, wherein the plurality of tubes include interlayer connection tubes that connect the flow paths of the first cassette body and the flow paths of the second cassette body.

3. 3. A flow path cassette as described in claim 2, wherein connectors communicating with the flow path are provided on the outer edges of the first cassette body and the second cassette body, and a locking portion for locking the connector is provided on the first side portion and the second side portion.

4. 4. A flow path cassette according to claim 3, wherein the frame is formed in a rectangular shape when viewed in a plane, and among the plurality of connectors, the connectors to which bags are connected are arranged together on the same side of the first side portion and the second side portion.

5. 5. The flow path cassette according to claim 4, 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 provided on different sides of the first and second side portions.

6. 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 flow path formed between a pair of overlapping resin sheets; a second cassette body having a flow path formed between a pair of overlapping resin sheets; 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, The frame is a flat cover portion disposed between the first cassette body and the second cassette body; A first side portion protruding from an outer edge of the cover portion in a first direction perpendicular to the cover portion; a second side portion protruding from an outer edge of the cover portion in a second direction opposite to the first direction, the first cassette body is accommodated in a first accommodating space defined by a concave shape formed by the first side portion and the cover portion, and the second cassette body is accommodated in a second accommodating space defined by a concave shape formed by the second side portion and the cover portion, the side portion having the first side portion and the second side portion includes a first side, a second side, a third side, and a fourth side that are provided on all four sides so as to go around an outer periphery of the rectangular cover portion, A cell culture kit, wherein a plurality of tubes through which fluid flows are connected to the outside of each of the first side, the second side, the third side, and the fourth side.

7. 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 comprising: The flow path cassette includes: a first cassette body having a flow path formed between a pair of overlapping resin sheets; a second cassette body having a 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 flat cover portion disposed between the first cassette body and the second cassette body; A first side portion protruding from an outer edge of the cover portion in a first direction perpendicular to the cover portion; a second side portion protruding from an outer edge of the cover portion in a second direction opposite to the first direction, the first cassette body is accommodated in a first accommodating space defined by a concave shape formed by the first side portion and the cover portion, and the second cassette body is accommodated in a second accommodating space defined by a concave shape formed by the second side portion and the cover portion, the side portion having the first side portion and the second side portion includes a first side, a second side, a third side, and a fourth side that are provided on all four sides so as to go around an outer periphery of the rectangular cover portion, A cell culture system, wherein a plurality of tubes through which fluid flows are connected to the outside of each of the first side, the second side, the third side, and the fourth side.

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