Priming method and biological component processing system

Abstract

The biological component processing system (22) includes an IC route (44A) that allows liquid to flow through the lumen (24a) of the hollow fibers (24) and an EC route (44B) that allows liquid to flow through the main space (26a) outside the hollow fibers (24). The biological component processing system (22) also includes a control unit (136) that controls the flow state of liquid through the IC route (44A) and the EC route (44B). During priming, the control unit (136) generates a pressure difference between the liquid flowing through the lumen (24a) and the liquid flowing through the main space (26a) while allowing liquid to flow through both the IC route (44A) and the EC route (44B), thereby causing gas to flow out of the hollow fibers (24).

Description

[Technical Field]

[0001] The present invention relates to a priming method and a biological component processing system for discharging gas from a processing section by circulating a liquid through the processing section. [Background technology]

[0002] In regenerative medicine, cells (biological components) from living organisms are collected, cultured, and then administered to patients. For cell proliferation treatments, a cell culture system (biological component processing system) using a cell culture vessel (processing unit) with hollow fibers inside a case is used, as disclosed in, for example, Japanese Patent Application Laid-Open No. 2017-143775. The biological component processing system supplies a liquid containing cells into the lumen of the hollow fibers to seed the cells on the inner surface of the hollow fibers, and then cultures the cells by supplying a culture medium to the processing unit (inside and outside the hollow fibers).

[0003] In this type of biological component processing system, prior to cell proliferation, priming is performed by circulating a specific liquid (a priming liquid such as physiological saline or a culture medium) through the treatment section, including the hollow fibers, to remove air (gas) from the treatment section. Conventional priming involves, for example, circulating a liquid through the lumen of the hollow fibers and then circulating the liquid outside the hollow fibers. Summary of the Invention

[0004] However, when hollow fibers having a coating formed on the inner surface of the lumen are used in the treatment section, fluid tends to accumulate in the membrane structure surrounding the lumen, which makes it difficult to remove air from the membrane structure even after the priming.

[0005] The present invention relates to the above-mentioned technology, and aims to provide a priming method and a biological component processing system that can easily and reliably discharge gas from hollow fibers.

[0006] In order to achieve the above-mentioned object, a first aspect of the present invention is a priming method for discharging gas from inside a hollow fiber by introducing a liquid into a processing unit having hollow fibers with a coating formed on the inner circumferential surface that forms the lumen and a container that houses the hollow fiber in an internal space, wherein the processing unit is connected to an internal route that allows the liquid to circulate through the lumen and an external route that allows the liquid to circulate through the internal space outside the hollow fiber, and during priming, while circulating the liquid through both the internal route and the external route, a differential pressure is generated between the liquid circulating through the lumen and the liquid circulating through the internal space, thereby discharging the gas from the hollow fiber.

[0007] In order to achieve the above-mentioned object, a second aspect of the present invention is a biological component processing system having a processing section including hollow fibers having a coating formed on the inner circumferential surface that forms a lumen, and a container that houses the hollow fibers in an internal space, the system comprising: an internal route connected to the processing section and capable of circulating a liquid through the lumen; an external route connected to the processing section and capable of circulating the liquid through the internal space outside the hollow fibers; and a control section that controls the flow state of the liquid through the internal route and the external route, wherein during priming, the control section circulates the liquid through both the internal route and the external route, while generating a differential pressure between the liquid flowing through the lumen and the liquid flowing through the internal space, thereby causing the gas to flow out of the hollow fibers.

[0008] The above-described priming method and biological component processing system can easily and reliably discharge gas from the hollow fibers. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is an explanatory diagram showing a biological component processing system to which a biological component cassette and a biological component kit according to a first embodiment of the present invention are applied. [Figure 2] FIG. 2 is an enlarged cross-sectional perspective view showing the structure of a hollow fiber. [Figure 3] FIG. 2 is an exploded perspective view of a biological component cassette. [Figure 4] FIG. 1 is an explanatory diagram showing the liquid pathway of a biological component kit. [Figure 5] FIG. 2 is an explanatory diagram schematically showing a path of a liquid during priming. [Figure 6] 6A and 6B are flowcharts showing the process flow of the biological component processing system and the priming step, respectively. [Figure 7] FIG. 10 is an explanatory diagram showing the operation of the IC priming step. [Figure 8] FIG. 10 is an explanatory diagram showing the operation of the EC priming step. [Figure 9] FIG. 10 is an explanatory diagram showing the operation of a differential pressure generating step. [Figure 10] FIG. 10 is an explanatory diagram showing the differential pressure generation step of the cell proliferation system according to the second embodiment. [Figure 11] FIG. 10 is an explanatory diagram showing the differential pressure generation step of the cell proliferation system according to the third embodiment. [Figure 12] FIG. 10 is an explanatory diagram showing the differential pressure generation step of the cell proliferation system according to the fourth embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0010] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to preferred embodiments thereof and the accompanying drawings.

[0011] [First embodiment] 1, a biological component processing system 22 according to a first embodiment of the present invention comprises a biological component kit 12 (hereinafter simply referred to as kit 12) capable of carrying a liquid containing a biological component and a liquid for processing the biological component, and a biological component processing device 14 into which the kit 12 is set. The kit 12 also has a biological component cassette 10 (hereinafter simply referred to as cassette 10) that aggregates multiple liquid paths and is set in the biological component processing device 14.

[0012] In addition to the cassette 10, the kit 12 also includes a plurality of tubes 16, a plurality of medical bags 18, and a processing unit 20 as components that form a plurality of pathways. The kit 12 is configured so that, under the operation of the biological component processing device 14, multiple types of liquids contained in the medical bags 18 are circulated through the cassette 10 and each tube 16, and the liquids are processed in the processing unit 20 to obtain the desired product.

[0013] The biological component processing system 22 according to this embodiment performs a proliferation process for proliferating living cells (biological components) in regenerative medicine, and a bioreactor 21 for cell proliferation is used as the processing unit 20 of the kit 12. Fluids flowing within the kit 12 include a solution containing cells (hereinafter referred to as a cell fluid), a culture medium (culture fluid) supplied for cell proliferation, a cleaning fluid for cleaning the interior of the kit 12, and a detachment fluid for detaching cells. In other words, the biological component processing device 14 performs a proliferation process in which, with the kit 12 set up, the cell fluid is seeded in the bioreactor 21, a culture medium is supplied to culture the cells, and the proliferated cells are detached and collected from the bioreactor 21. Hereinafter, the biological component processing device 14 is also referred to as a cell proliferation device 15, and the biological component processing system 22 is also referred to as a cell proliferation system 23.

[0014] The biological cells are not particularly limited, but examples thereof include blood cells (e.g., T cells) and stem cells (e.g., ES cells, iPS cells, mesenchymal stem cells). The culture medium may be selected appropriately depending on the biological 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. Examples of the detachment solution that can be used include trypsin and EDTA solution.

[0015] The multiple medical bags 18 of the kit 12 include a cell fluid bag 18A containing a cell fluid, a cleaning fluid bag 18B containing a cleaning fluid, and a culture medium bag 18C containing a culture medium. The kit 12 also includes empty bags as the multiple medical bags 18, including a waste fluid bag 18D into which fluid discarded during the proliferation process flows, and a collection bag 18E for collecting the cells (and other fluids) obtained during the proliferation process. A detachment fluid bag 18F containing a detachment fluid is also provided separately from the medical bag 18. The detachment fluid bag 18F is replaced by a previously connected medical bag 18 (e.g., the cell fluid bag 18A) by the operator during the proliferation process.

[0016] The cell solution bag 18A, the washing solution bag 18B, the culture medium bag 18C, etc. are aseptically joined to the end of each tube 16 using a sterile joining device (not shown). Alternatively, each medical bag 18 may be permanently attached to the end of each tube 16, ensuring sterility within the kit 12. Alternatively, the kit 12 may employ a connection structure (not shown) that detachably connects the tube 16 and each medical bag 18.

[0017] The bioreactor 21 of the kit 12 is not particularly limited, but preferably uses a culture substrate with a large surface area, and in this embodiment, hollow fibers 24 are used. 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 (internal space) that accommodates the plurality of hollow fibers 24.

[0018] A plurality of hollow fibers 24 are housed in a container 26 along the axial direction, with both ends held by a retaining wall (not shown) of the container 26. As shown in Fig. 2, each hollow fiber 24 has a lumen 24a surrounded by a membrane structure 25 (hollow fiber membrane). The lumen 24a penetrates the hollow fiber 24 along its extension direction. The diameter of the lumen 24a is, for example, about 200 µm, and the lumen 24a communicates with end spaces 26b on both axial sides of the retaining wall.

[0019] The membrane structure 25 is made of a porous body having a large number of pores 25a extending in the thickness direction. Each pore 25a communicates between the outside (main space 26a) of the hollow fiber 24 and the lumen 24a, and is formed to a size that allows the permeation of solutions and low-molecular-weight substances while restricting the permeation of cells and proteins. The diameter of the pores 25a is set to, for example, about 0.005 μm to 10 μm.

[0020] The material constituting the membrane structure 25 of the hollow fiber 24 is not particularly limited, and examples thereof 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.

[0021] Furthermore, hollow fibers 24 have a coating 27 on the inner circumferential surface of membrane structure 25 surrounding lumen 24a. Coating 27 has the function of promoting seeding of cells introduced into hollow fibers 24, and is applied to the entire inner circumferential surface of membrane structure 25 (or to a portion of pores 25a). Coating 27 has multiple pores corresponding to pores 25a of membrane structure 25, ensuring communication between lumen 24a of hollow fibers 24 and the outside. The material constituting coating 27 is not particularly limited, but examples thereof include fibronectin, laminin, vitronectin, collagen, tenascin, fibrinogen, gelatin, polylysine, etc.

[0022] The hollow fibers 24 described above supply culture medium, predetermined gas components, and the like to cells adhered to the coating 27 through the pores 25a. Hereinafter, in the cell proliferation system 23, a configuration in which a liquid is circulated mainly through the lumen 24a of the hollow fibers 24 is also referred to as an IC (intra capillary), and a configuration in which a liquid is circulated mainly outside the hollow fibers 24 is also referred to as an EC (extra capillary).

[0023] 1, the container 26 has an axial length sufficient to accommodate each hollow fiber 24 extending substantially linearly. 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 on the one end side. The second IC terminal 28b is provided at the other end of the container 26 and communicates with the end space 26b on the other end side. 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 one end and communicates with the main space 26a near the one end.

[0024] When set in the cell proliferation device 15, the bioreactor 21 is preferably fixed by the cell proliferation device 15 so that it can rotate vertically (direction along a vertical plane: longitudinal direction), horizontally (direction along a horizontal plane: lateral direction), or around the axis of the container 26. This makes it possible to easily guide the air inside the bioreactor 21 to any of the four terminals 28.

[0025] The multiple tubes 16 of the kit 12 include a cell solution tube 16A connecting the cell solution bag 18A and the cassette 10, a washing solution tube 16B connecting the washing solution bag 18B and the cassette 10, a culture medium tube 16C connecting the culture medium bag 18C and the cassette 10, a waste solution tube 16D connecting the waste solution bag 18D and the cassette 10, a recovery tube 16E connecting the recovery bag 18E and the cassette 10, a first IC tube 16F connecting the first IC terminal 28a of the bioreactor 21 and the cassette 10, a second IC tube 16G connecting the second IC terminal 28b of the bioreactor 21 and the cassette 10, a first EC tube 16H connecting the first EC terminal 28c of the bioreactor 21 and the cassette 10, and a second EC tube 16I connecting the second EC terminal 28d of the bioreactor 21 and the cassette 10.

[0026] A gas exchanger 29 is provided midway along the first EC tube 16H to mix predetermined gas components into the liquid (culture medium). For example, the gas components to be mixed may be similar to the mixture ratio of air in nature (nitrogen N2: 75%, oxygen O2: 20%, carbon dioxide CO2: 5%).

[0027] 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 first EC tube 16H 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 movement inside the hollow fibers 29b with the liquid through the pores of the hollow fibers 29b.

[0028] The cassette 10, which is one component of the kit 12, has the above-mentioned tubes 16 joined in advance, and functions as a relay section for the liquid pathway that circulates the cell fluid, cleaning fluid, culture medium, and detachment solution in each medical bag 18 to another medical bag 18 or to the bioreactor 21. When the kit 12 is set in the cell proliferation device 15, this cassette 10 is attached to a cassette placement section (not shown) in the cell proliferation device 15, simplifying the wiring work of the tubes 16 during the proliferation process.

[0029] As shown in Figure 3, the cassette 10 is composed of a soft cassette body 40 to which multiple tubes 16 are directly connected, and a hard frame 50 that holds the cassette body 40 and is fixed to the cell growth device 15.

[0030] The cassette body 40 is formed as a thin, flexible, rectangular sheet. The cassette body 40 is formed by stacking two resin sheets 42 made of a resin material in the thickness direction and welding them together. When welding the pair of resin sheets 42, gas is supplied and exhausted between the pair of resin sheets 42 along grooves formed in a welding mold. This causes each resin sheet 42 to become a channel wall with a semicircular cross section, forming a channel 44 within the wall. The material constituting the resin sheet 42 is not particularly limited as long as it is flexible enough to be deformed by the pressure of the liquid. Examples of suitable materials for the resin sheet 42 include vinyl chloride resin, polyolefin resin, and polyurethane resin. The surface of the cassette body 40 may be embossed to form minute irregularities. A plurality of connectors 60 are provided on the outer edge 41 of the cassette body 40, connecting the plurality of tubes 16 to the channel 44.

[0031] Also provided within the cassette body 40 are a plurality of pressure detection portions 48, liquid level detection portions 80, and check valve portions 90, which are in communication with the flow paths 44. Furthermore, the cassette body 40 is provided with flow path opening / closing portions 100 (a plurality of notches 102) at a plurality of locations, which enable predetermined flow paths 44 to be opened and closed.

[0032] On the other hand, the frame 50 is made of a resin material that is harder (has a higher elastic modulus) than the cassette body 40, and is formed into a thin, concave shape having an accommodation space 52 that accommodates the cassette body 40. The material that constitutes the frame 50 is not particularly limited, and it is preferable to use a thermoplastic resin such as polypropylene, polycarbonate, polyamide, polysulfone, polyarylate, or methacrylate-butylene-styrene copolymer.

[0033] The frame 50 has a generally rectangular cover portion 54 that is slightly larger than the cassette body 40, and side portions 56 that protrude slightly from the outer periphery of the cover portion 54 in a direction perpendicular to the cover portion 54. The side portions 56 surround 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 portions 56 on the side opposite the cover portion 54, exposing one side of the cassette body 40. The frame 50 also has holding frames 58 that extend from the upper and right sides of the side portions 56 and hold the tubes 16 a predetermined distance away from the side portions 56. Locking portions 70 that position and hold each connector 60 are provided on the side portions 56 at locations that correspond to each connector 60 on the cassette body 40.

[0034] The connectors 60 and the locking portions 70 are provided on each of the four sides of the substantially rectangular cassette 10. As a result, the frame 50 holds the sheet-like cassette body 40 in a taut state, allowing the flow path 44 to extend smoothly along the surface direction.

[0035] When the kit 12 (including the cassette 10) is set in the cell growth device 15, the cell growth system 23 forms a path for liquids (cell liquid, cleaning liquid, culture medium, etc.) as shown in Figure 4. In addition, when set, four pumps 30 (first to fourth pumps 30a to 30d) are arranged near the sides of the cassette 10. The first to fourth pumps 30a to 30d rotate in a manner that squeezes the pump tubes 16J to 16M protruding from the cassette 10, thereby applying a flow force to the liquid inside.

[0036] Furthermore, when the cell proliferation system 23 is set up, the sensor tube 16N is positioned opposite the air bubble sensor 32. Outer clamps 34 that open and close the internal flow paths are disposed on the cell solution tube 16A, the washing solution tube 16B, and the culture medium tube 16C. Specifically, a first outer clamp 34a is disposed on the cell solution tube 16A, a second outer clamp 34b is disposed on the washing solution tube 16B, and a third outer clamp 34c is disposed on the culture medium tube 16C.

[0037] In addition, in the cassette 10 in the set state, each pressure sensor 36 faces each pressure detection portion 48, and the liquid level sensor 37 faces each liquid level detection portion 80. An inner clamp 35 is disposed in each flow path opening / closing portion 100 (notch 102).

[0038] Although detailed description will be omitted, the flow path 44 within the cassette 10 (cassette body 40) constitutes an IC route 44A (internal route) that supplies liquid to the lumen 24a of the hollow fibers 24 together with the first and second IC tubes 16F, 16G, and an EC route 44B (external route) that supplies liquid to the outside (main space 26a) of the hollow fibers 24 together with the first and second EC tubes 16H, 16I. In particular, during priming of the bioreactor 21, the outer clamp 34 and the inner clamp 35 are each appropriately opened or closed, so that the IC route 44A and EC route 44B of the entire kit 12 assume a shape generally as shown in FIG.

[0039] In detail, the IC route 44A has an IC supply circuit 44A1 capable of supplying liquid from the cleaning liquid bag 18B to a predetermined position α (see also Figure 4) downstream of the first pump 30a, and an IC circulation circuit 44A2 capable of circulating liquid between the cleaning liquid bag 18B and the bioreactor 21 downstream of the predetermined position α.

[0040] That is, the IC supply circuit 44A1 supplies the liquid (cleaning liquid) in the cleaning liquid bag 18B to the IC circulation circuit 44A2 under the drive of the first pump 30a. In addition to the first pump 30a, the IC supply circuit 44A1 is also provided with an air bubble sensor 32, a pressure sensor 36, a liquid level detection section 80, and the like (see also FIG. 4).

[0041] The IC circulation circuit 44A2 is provided with a fourth pump 30d and is connected to the first IC terminal 28a and the second IC terminal 28b of the bioreactor 21. Therefore, the liquid in the IC circulation circuit 44A2 flows through the lumen 24a of the hollow fibers 24 when driven by the fourth pump 30d. A waste liquid route 44C leading to the waste liquid bag 18D is connected to a predetermined position β (see also FIG. 4) of the IC circulation circuit 44A2. Therefore, the liquid circulating through the IC circulation circuit 44A2 is discharged into the waste liquid bag 18D via the waste liquid route 44C when the IC waste liquid clamp 35a (inner clamp 35) provided on the waste liquid route 44C is open.

[0042] On the other hand, the EC route 44B has an EC supply circuit 44B1 capable of supplying liquid from the cleaning solution bag 18B to a predetermined position γ (see also Figure 4) downstream of the second pump 30b, and an EC circulation circuit 44B2 capable of circulating liquid between the cleaning solution bag 18B and the bioreactor 21 downstream of the predetermined position γ.

[0043] The EC supply circuit 44B1 supplies the liquid (cleaning liquid) in the cleaning liquid bag 18B to the EC circulation circuit 44B2 under the drive of the second pump 30b. Meanwhile, the EC circulation circuit 44B2 includes a third pump 30c and is connected to the first EC terminal 28c and the second EC terminal 28d of the bioreactor 21. Therefore, the liquid in the EC circulation circuit 44B2 flows through the main space 26a of the container 26 under the drive of the third pump 30c. A waste liquid route 44C is connected to a predetermined position δ of the EC circulation circuit 44B2. Therefore, the liquid circulating through the EC circulation circuit 44B2 is discharged to the waste liquid bag 18D via the waste liquid route 44C when the EC waste liquid clamp 35b (inner clamp 35) provided on the waste liquid route 44C is open.

[0044] The liquid used during priming (in other words, the medical bag 18 connected to the IC supply circuit 44A1 and the EC supply circuit 44B1) is not particularly limited in the cell proliferation system 23. For example, the cell proliferation system 23 may be connected to the culture medium bag 18C and use a culture medium as the priming liquid.

[0045] Returning to Figure 1, the cell proliferation device 15 to which the kit 12 is attached comprises a box-shaped device main body 130 and a stand 132 that holds each medical bag 18 of the kit 12. A touch panel 134 (display / operation unit) is provided on the exterior of the device main body 130 for operations and displays during proliferation processing. Furthermore, the interior of the device main body 130 is provided with a cassette placement unit (not shown) that fixes the cassette 10 in an upright position and holds the bioreactor 21 at an appropriate height, and a control unit 136 that controls the operation of the cell proliferation system 23.

[0046] The control unit 136 is configured as a computer having a processor, memory, and input / output interface (not shown). The control unit 136 appropriately operates the pump 30, outer clamp 34, inner clamp 35, etc. during cell proliferation processing by the processor executing a program (not shown) stored in the memory. In particular, the control unit 136 performs the processing flow described below during priming within the kit 12, thereby sufficiently discharging air from within the membrane structure 25 (pores 25a) of the hollow fiber 24.

[0047] The cell proliferation system 23 according to this embodiment is basically configured as described above, and the proliferation process operation and priming method will be described below.

[0048] As shown in Figure 1, in the proliferation process of cell proliferation system 23, an operator inserts part of kit 12, including cassette 10, into cell proliferation device 15, and hangs each medical bag 18 of kit 12 from stand 132. The operator also places appropriate tubing 16 of kit 12 on pump 30, air bubble sensor 32, and outer clamp 34 of cell proliferation device 15. Furthermore, in setting cassette 10, flow path openable / closable part 100 is placed on inner clamp 35, pressure detection part 48 is placed on pressure sensor 36, and liquid level detection part 80 is placed on liquid level sensor 37. As a result, the pathways shown in Figure 4 are established in cell proliferation system 23.

[0049] After the above setup, the proliferation process involves sequentially carrying out a priming step, a medium replacement step, a seeding step, a culture step, a detachment step, and a recovery step, as shown in Fig. 6A. In the priming step, the process flow (priming method) shown in Fig. 6B is carried out to sufficiently remove air from inside the kit 12 including the bioreactor 21.

[0050] In the priming step, the cell proliferation system 23 first circulates the cleaning solution through the IC route 44A (step S1: IC priming step). Specifically, as shown in FIG. 7, the control unit 136 opens the second outer clamp 34b and rotates the first pump 30a and the fourth pump 30d at an appropriate speed while stopping the second pump 30b and the third pump 30c. As a result, the cleaning solution in the cleaning solution bag 18B is supplied to the bioreactor 21 via the IC route 44A (IC supply circuit 44A1, IC circulation circuit 44A2). Within the bioreactor 21, the cleaning solution flowing through the IC route 44A flows into the lumen 24a of the hollow fibers 24, removing air from the hollow fibers 24 and discharging the air to the outside of the bioreactor 21 (IC circulation circuit 44A2). At this time, the cell proliferation system 23 may remove air from within the IC by rotating the bioreactor 21 vertically and positioning the second IC terminal 28b at the bottom end and the first IC terminal 28a at the top end.

[0051] Furthermore, in the IC priming step, the control unit 136 opens the IC waste liquid clamp 35a of the waste liquid route 44C, which causes the air present in the IC route 44A to be discharged together with the cleaning liquid from the IC circulation circuit 44A2 through the waste liquid route 44C into the waste liquid bag 18D.

[0052] Next, the cell proliferation system 23 circulates the cleaning solution through the EC route 44B (step S2: EC priming step). Specifically, as shown in FIG. 8, the control unit 136 rotates the second pump 30b and the third pump 30c at an appropriate speed while keeping the second outer clamp 34b open, while stopping the first pump 30a and the fourth pump 30d. This causes the cleaning solution in the cleaning solution bag 18B to be supplied to the bioreactor 21 via the EC route 44B (EC supply circuit 44B1, EC circulation circuit 44B2). Within the bioreactor 21, the cleaning solution circulating through the EC route 44B flows into the main space 26a of the container 26 to remove air, and then discharges the air to the outside of the bioreactor 21 (EC circulation circuit 44B2). At this time, the cell proliferation system 23 may rotate the bioreactor 21 horizontally to remove air from the EC. The cell growth system 23 may rotate the bioreactor 21 vertically and horizontally to facilitate de-airing.

[0053] Furthermore, in the EC priming step, the control unit 136 opens the EC waste fluid clamp 35b of the waste fluid route 44C, which causes the air present in the EC route 44B to be discharged together with the cleaning fluid from the EC circulation circuit 44B2 through the waste fluid route 44C into the waste fluid bag 18D.

[0054] Furthermore, the cell proliferation system 23 circulates the cleaning solution through both the IC route 44A and the EC route 44B, and generates a pressure difference between the two routes (step S3: pressure difference generation step). At this time, the control unit 136 stops the first pump 30a and the second pump 30b, as shown in Figure 9, while rotating the fourth pump 30d of the IC circulation circuit 44A2 and the third pump 30c of the EC circulation circuit 44B2.

[0055] The control unit 136 then controls the flow rate of the cleaning liquid in the EC circulation circuit 44B2 by the third pump 30c to be faster than the flow rate of the cleaning liquid in the IC circulation circuit 44A2 by the fourth pump 30d. For example, the control unit 136 sets the flow rate of the cleaning liquid in the IC circulation circuit 44A2 to 25 ml / min and the flow rate of the cleaning liquid in the EC circulation circuit 44B2 to 250 ml / min. This speed difference between the cleaning liquid flowing in the IC circulation circuit 44A2 and the cleaning liquid flowing in the EC circulation circuit 44B2 generates a pressure difference in the cleaning liquid at the interface (boundary) between the IC circulation circuit 44A2 and the EC circulation circuit 44B2.

[0056] As shown in FIG. 2, the coating 27 formed on the inner circumferential surface of the membrane structure 25 of the hollow fibers 24 prevents air from escaping from within the membrane structure 25 (pores 25a). However, in the cell growth system 23 of this embodiment, the pressure in the lumen 24a is lower than that in the main space 26a outside the hollow fibers 24 (a pressure differential is generated), which allows the cleaning solution to easily penetrate the pores 25a. Therefore, the pressure differential acts on the air within the membrane structure 25, causing it to move toward the lumen 24a. In particular, the pressure differential caused by the difference in the flow rate of the cleaning solution in the IC circulation circuit 44A2 and the flow rate of the cleaning solution in the EC circulation circuit 44B2 does not impart a force to the cleaning solution or air sufficient to peel off the coating 27 from the membrane structure 25, thereby maintaining the coating 27 in good condition.

[0057] The ratio (speed ratio) of the flow rate of the EC circulation circuit 44B2 to the flow rate of the IC circulation circuit 44A2 is not particularly limited, but may be set to, for example, approximately 5 to 15. If the speed ratio is less than 5, the pressure difference between the IC circulation circuit 44A2 and the EC circulation circuit 44B2 is small, and it takes time for air to move from the EC circulation circuit 44B2 to the IC circulation circuit 44A2. On the other hand, if the speed ratio is 15 or more, the pressure difference between the IC circulation circuit 44A2 and the EC circulation circuit 44B2 becomes too large, which may peel off the coating 27 in the membrane structure 25 when air moves from the EC circulation circuit 44B2 to the IC circulation circuit 44A2.

[0058] The cell proliferation system 23 then performs an operation to discharge the air discharged into the IC route 44A by performing step S3 from the IC circulation circuit 44A2 together with the cleaning fluid (step S4: discharge step). In step S4, similar to step S1 (see also FIG. 7), the controller 136 opens the second outer clamp 34b and rotates the first pump 30a and the fourth pump 30d at appropriate speeds while stopping the second pump 30b and the third pump 30c. The controller 136 also opens the IC waste liquid clamp 35a to open the waste liquid route 44C from the IC circulation circuit 44A2. At this time, the cell proliferation system 23 preferably removes air from the IC by rotating the bioreactor 21 vertically and positioning the second IC terminal 28b at the bottom and the first IC terminal 28a at the top.

[0059] As a result, the IC circulation circuit 44A2 can guide the air and cleaning liquid discharged from the membrane structure 25 of the hollow fibers 24 to the waste liquid route 44C while the cleaning liquid is supplied from the cleaning liquid bag 18B (IC supply circuit 44A1).The cleaning liquid and air circulating in the waste liquid route 44C are then smoothly discharged into the waste liquid bag 18D.

[0060] After step S4, the cell proliferation system 23 determines whether or not to terminate the priming step (step S5: determination step). In this determination step, for example, the control unit 136 counts the duration of the priming step and the number of times steps S3 and S4 have been performed. If the count is equal to or less than a predetermined threshold (step S5: NO), the control unit 136 returns to step S3 and repeats the subsequent processing flow. On the other hand, if the count is greater than the predetermined threshold (step S5: YES), it determines that air has been released from the bioreactor 21, and terminates the priming step.

[0061] Returning to FIG. 6A, after the priming step, the cell proliferation system 23 performs a medium replacement step, introducing the medium from the culture medium bag 18C into the designated tubes 16, the bioreactor 21, the flow path 44 of the cassette body 40, etc., and filling them with the medium. The cell proliferation system 23 then performs a seeding step after the medium replacement step. During this step, the cell proliferation system 23 supplies the cell solution from the cell solution bag 18A to the hollow fibers 24 (lumen 24a) of the bioreactor 21 via the IC route 44A, while circulating the medium present in the EC route 44B to supply gas components to the bioreactor 21. Because the coating 27 inside the hollow fibers 24 is well maintained during this seeding step, cells can adhere smoothly to the coating 27.

[0062] Furthermore, the cell proliferation system 23 performs a culture step in which cells are cultured after the seeding step. In the culture step, culture medium is supplied from both the IC route 44A and the EC route 44B to culture the cells inside the hollow fibers 24. This culture step is carried out for a longer period of time (e.g., several days) than other steps, thereby allowing the cells to grow on the coating 27. Note that the cell proliferation system 23 may also perform an operation in which culture medium is supplied from the EC route 44B during the culture step without using the IC route 44A.

[0063] In addition, in the detachment step after the culture step, a detachment liquid is supplied from the IC route 44A to detach the cells cultured (grown) in the bioreactor 21. In the recovery step after the detachment step, a culture medium is supplied to the IC route 44A, causing the cells detached in the detachment step to flow out of the bioreactor 21 and be guided to the recovery bag 18E. At this time, the culture medium and gas components are also supplied through the EC route 44B.

[0064] Through the above steps, cell proliferation system 23 can effectively store in collection bag 18E the cells cultured in bioreactor 21. In particular, cell proliferation system 23 can sufficiently remove air from membrane structure 25 of hollow fiber 24 during the priming step, thereby preventing poor cell proliferation due to remaining air.

[0065] The present invention is not limited to the above-described embodiment, and various modifications are possible within the spirit and scope of the invention. For example, the cell proliferation system 23 may be configured to simultaneously supply cleaning fluid to the IC circulation circuit 44A2 and the EC circulation circuit 44B2 without performing the IC priming step or the EC priming step, thereby generating a pressure difference between the IC circulation circuit 44A2 and the EC circulation circuit 44B2.

[0066] Furthermore, the above-mentioned biological component processing system 22 (cell proliferation system 23) was configured to include a flexible cassette body 40 (cassette 10) in the kit 12, but the kit 12 may be configured not to include the cassette 10, or a rigid cassette 10 may be used.

[0067] The priming method and biological component processing system 22 of the present invention are not limited to cell growth systems 23 that perform cell growth processing, but may be applied to various systems that perform priming on hollow fibers 24. For example, the priming method and biological component processing system 22 of the present invention may be applied to blood processing systems that process blood (such as filtration).

[0068] Second Embodiment As shown in Figure 10, the cell proliferation system 23A according to the second embodiment differs from the cell proliferation system 23 described above in that it continuously supplies liquid (cleaning liquid) from the EC supply circuit 44B1 to create a pressure difference between the IC route 44A and the EC route 44B. Note that the configuration for operating the liquid in the cell proliferation system 23A is the same as that in the cell proliferation system 23, with only the control by the control unit 136A being different. Therefore, the priming method of the second embodiment will be described below.

[0069] The cell proliferation system 23A performs an IC priming step and an EC priming step similar to the first embodiment, followed by a differential pressure generation step, which differs from the first embodiment. In the differential pressure generation step, the controller 136A opens the second outer clamp 34b and the IC waste liquid clamp 35a while closing the EC waste liquid clamp 35b. The controller 136A also stops the first pump 30a while rotating the fourth pump 30d of the IC circulation circuit 44A2, the second pump 30b of the EC supply circuit 44B1, and the third pump 30c of the EC circulation circuit 44B2.

[0070] As a result, the EC circulation circuit 44B2 receives cleaning fluid from the EC supply circuit 44B1 while the third pump 30c circulates it. This increases the internal pressure of the EC circulation circuit 44B2 relative to the IC circulation circuit 44A2. Therefore, in the cell growth system 23A, the pressure of the cleaning fluid in the lumen 24a is lower than the pressure of the cleaning fluid in the main space 26a (a pressure difference occurs) in the hollow fibers 24 where the IC circulation circuit 44A2 and the EC circulation circuit 44B2 interface. As a result, the cleaning fluid in the main space 26a moves toward the lumen 24a through the pores 25a (the movement of the liquid is indicated by dotted lines in FIG. 10), allowing air within the membrane structure 25 to be expelled into the lumen 24a. At this time, the cell growth system 23A may remove air from the IC by vertically rotating the bioreactor 21 and positioning the second IC terminal 28b at the bottom and the first IC terminal 28a at the top.

[0071] With the IC waste liquid clamp 35a open and the cleaning liquid circulating by the fourth pump 30d, the IC circulation circuit 44A2 can smoothly discharge the cleaning liquid into the waste liquid route 44C (waste liquid bag 18D) according to the amount of cleaning liquid moving from the EC circulation circuit 44B2.

[0072] In addition to supplying cleaning liquid from the EC supply circuit 44B1, the control unit 136A of the cell proliferation system 23A may also control the flow rate of the cleaning liquid in the EC circulation circuit 44B2 to be faster than the flow rate of the cleaning liquid in the IC circulation circuit 44A2. Alternatively, when cleaning liquid is supplied from the EC supply circuit 44B1, the flow rate of the cleaning liquid in the EC circulation circuit 44B2 may be slower than the flow rate of the cleaning liquid in the IC circulation circuit 44A2 (or the flow rates may be the same), thereby adjusting the differential pressure of the cleaning liquid.

[0073] Furthermore, in the cell proliferation system 23A according to the second embodiment, the IC circulation circuit 44A2 and the waste liquid route 44C are connected even during the differential pressure generation step, and the cleaning liquid containing air is discharged. Therefore, the priming method may be configured not to perform the discharge step (i.e., the differential pressure generation step and the discharge step are not repeated).

[0074] Third Embodiment As shown in Figure 11, the cell proliferation system 23B according to the third embodiment differs from the cell proliferation systems 23 and 23A according to the first and second embodiments in that a pressure differential is generated so that the internal pressure of the EC circulation circuit 44B2 is lower than the internal pressure of the IC circulation circuit 44A2. Specifically, after performing the IC priming step and the EC priming step as in the first embodiment, the control unit 136B of the cell proliferation system 23B stops the first pump 30a and the second pump 30b and controls the flow rate of the cleaning solution in the IC circulation circuit 44A2 by the fourth pump 30d to be faster than the flow rate of the cleaning solution in the EC circulation circuit 44B2 by the third pump 30c. The ratio (speed ratio) of the flow rate of the IC circulation circuit 44A2 to the flow rate of the EC circulation circuit 44B2 is not particularly limited, but is set to, for example, approximately 5 to 15 times.

[0075] As a result, the difference in speed between the IC circulation circuit 44A2 and the EC circulation circuit 44B2 generates a pressure difference in the hollow fibers 24, where the pressure in the IC circulation circuit 44A2 is higher than the pressure in the EC circulation circuit 44B2. This pressure difference causes the air in the membrane structure 25 to move toward the main space 26a. In particular, because the pressure in the IC circulation circuit 44A2 is higher than that in the EC circulation circuit 44B2, peeling of the coating 27 from the inner surface of the lumen 24a due to the movement of the cleaning liquid is suppressed, and the coating 27 can be maintained in good condition.

[0076] [Fourth embodiment] As shown in FIG. 12, the cell growth system 23C (controller 136C) according to the fourth embodiment differs from the cell growth systems 23, 23A, and 23B in that it continuously supplies cleaning fluid from the IC supply circuit 44A1 to generate a pressure differential. This configuration, in which cleaning fluid is continuously supplied from the IC supply circuit 44A1, facilitates increasing the internal pressure of the IC circulation circuit 44A2 relative to the EC circulation circuit 44B2. As a result, the cleaning fluid in the lumen 24a moves toward the main space 26a through the pores 25a (the movement of the liquid is indicated by dotted lines in FIG. 12), allowing air within the membrane structure 25 to be expelled into the main space 26a. Therefore, the cell growth system 23C also allows air to be expelled from the treatment unit 20 (membrane structure 25 of hollow fiber 24) quickly via the EC route 44B.

[0077] It goes without saying that the present invention allows for the priming methods of the cell proliferation systems 23, 23A to 23C described in the above embodiments to be partially or entirely extracted and incorporated into other embodiments.

[0078] The technical ideas and effects that can be understood from the above-described embodiments will be described below.

[0079] A first aspect of the present invention is a priming method for discharging gas from hollow fibers 24 by introducing a liquid into a processing unit 20 having hollow fibers 24 with a coating 27 formed on the inner surface constituting the lumen 24a and a container 26 containing the hollow fibers 24 in an internal space (main space 26a). The processing unit 20 is connected to an internal route (IC route 44A) that allows liquid to flow through the lumen 24a and an external route (EC route 44B) that allows liquid to flow through the internal space outside the hollow fibers 24. During priming, a pressure difference is generated between the liquid flowing through the lumen 24a and the liquid flowing through the internal space while flowing liquid through both the internal route and the external route, thereby discharging gas from the hollow fibers 24.

[0080] In the above-described priming method, a pressure difference is generated between the liquid flowing from the internal route (IC route 44A) to the lumen 24a in the hollow fibers 24 and the liquid flowing from the external route (EC route 44B) to the internal space (main space 26a) in the hollow fibers 24, thereby easily and reliably discharging gas from the hollow fibers 24. In particular, even in a configuration in which the coating 27 is formed on the inner circumferential surface of the hollow fibers 24, the pressure difference between the lumen 24a of the hollow fibers 24 and the internal space allows the liquid to permeate into the membrane structure 25, thereby enabling the gas to be efficiently discharged from the hollow fibers 24.

[0081] The internal route (IC route 44A) has an internal circulation circuit (IC circulation circuit 44A2) that circulates liquid through the processing section 20 and an internal supply circuit (IC supply circuit 44A1) that supplies liquid to the internal circulation circuit, and the external route (EC route 44B) has an external circulation circuit (EC circulation circuit 44B2) that circulates liquid through the processing section 20 and an external supply circuit (EC supply circuit 44B1) that supplies liquid to the external circulation circuit, and during priming, a pressure difference is generated between the liquid circulating in the internal circulation circuit and the liquid circulating in the external circulation circuit. This makes it possible to reduce the amount of liquid used for priming and to effectively discharge gas from the hollow fibers 24 in the priming method.

[0082] Furthermore, when generating a pressure difference, the supply of liquid from the internal supply circuit (IC supply circuit 44A1) and the external supply circuit (EC supply circuit 44B1) is stopped, while a difference is created between the flow rate of the liquid circulating in the internal circulation circuit (IC circulation circuit 44A2) and the flow rate of the liquid circulating in the external circulation circuit (EC circulation circuit 44B2). This makes it possible to easily generate a pressure difference in the IC circulation circuit 44A2 and the EC circulation circuit 44B2 that is capable of removing gas from the hollow fibers 24 while suppressing peeling of the coating 27.

[0083] During priming, a differential pressure generating step for generating a differential pressure and a discharge step for discharging liquid and gas from the internal circulation circuit (IC circulation circuit 44A2) or the external circulation circuit (EC circulation circuit 44B2) to the waste liquid route 44C are repeated. As a result, in the priming method, gas can be easily removed from the hollow fibers 24 in the differential pressure generating step, and the gas can be smoothly discharged together with liquid to the waste liquid route 44C in the subsequent discharge step.

[0084] Furthermore, the ratio of the flow rate of the liquid circulating through the internal circulation circuit (IC circulation circuit 44A2) to the flow rate of the liquid circulating through the external circulation circuit (EC circulation circuit 44B2) is set within a range of 5 to 15. This allows the priming method to generate an appropriate pressure difference between the internal pressure on the lumen 24a side of the hollow fibers 24 and the internal pressure outside the hollow fibers 24 (main space 26a). As a result, during priming, peeling of the coating 27 is further suppressed, and gas can be more reliably removed from the hollow fibers 24.

[0085] Furthermore, when generating a pressure difference, liquid is circulated in both the internal circulation circuit (IC circulation circuit 44A2) and the external circulation circuit (EC circulation circuit 44B2), while liquid is supplied from the internal supply circuit (IC supply circuit 44A1) to the internal circulation circuit, or liquid is supplied from the external supply circuit (EC supply circuit 44B1) to the external circulation circuit. In the priming method, by supplying liquid from the internal supply circuit to the internal circulation circuit (or supplying liquid from the external supply circuit to the external circulation circuit), the internal pressure of one of the internal route and the external route can be easily increased. Therefore, a pressure difference sufficient to remove gas from the hollow fibers 24 can be easily obtained.

[0086] Furthermore, a pressure difference is generated by increasing the internal pressure of the external route (EC route 44B) relative to the internal pressure of the internal route (IC route 44A), causing gas to move from the hollow fibers 24 to the lumen 24a. This allows the priming method to easily move the gas in the hollow fibers 24 to the lumen 24a and discharge it via the internal route.

[0087] Furthermore, a pressure difference is generated by increasing the internal pressure of the internal route (IC route 44A) relative to the internal pressure of the external route (EC route 44B), causing gas to move from the hollow fibers 24 to the internal space (main space 26a). This allows the priming method to easily move the gas in the hollow fibers 24 to the internal space and discharge it via the external route.

[0088] Furthermore, the processing unit 20 is a bioreactor 21 that supplies a culture medium to the hollow fibers 24 after cells have been attached to the inner circumferential surface of the hollow fibers 24, thereby growing the cells on the inner circumferential surface of the hollow fibers 24, and the priming method is performed before supplying a liquid containing cells to the hollow fibers 24. As a result, the bioreactor 21 can favorably grow cells in the hollow fibers 24 from which gas has been discharged by performing the priming method.

[0089] A second aspect of the present invention is a biological component processing system (22) including a processing unit (20) having hollow fibers (24) with a coating (27) formed on the inner circumferential surface that forms the lumen (24a) and a container (26) containing the hollow fibers (24) in an internal space (main space (26a)). The system is equipped with an internal route (IC route (44A)) connected to the processing unit (20) for circulating a liquid through the lumen (24a), an external route (EC route (44B)) connected to the processing unit (20) for circulating a liquid through the internal space outside the hollow fibers (24), and a control unit (136) for controlling the flow of liquid through the internal route and the external route. During priming, the control unit (136) generates a pressure difference between the liquid flowing through the lumen (24a) and the liquid flowing through the internal space while circulating liquid through both the internal route and the external route, thereby discharging gas from the hollow fibers (24). The biological component processing system (22) can easily and reliably discharge gas from the hollow fibers (24).

Claims

1. A priming method for discharging gas from within a hollow fiber by introducing a liquid into a treatment section having a hollow fiber with a coating formed on an inner circumferential surface that constitutes an inner cavity and a container that contains the hollow fiber in an internal space, the method comprising: The processing unit includes: an internal route capable of passing the liquid through the lumen; an external route through which the liquid can flow is connected to the internal space outside the hollow fiber; The hollow fiber has a membrane structure surrounding the lumen, the membrane structure has a plurality of pores that connect the lumen to the outside of the hollow fiber, During priming, while the liquid is circulated through both the internal route and the external route, a pressure difference is generated between the liquid circulating through the lumen and the liquid circulating through the internal space, and the gas is caused to flow out from the pores of the hollow fibers to the lumen side or the internal space side by the pressure difference. Priming Method.

2. The priming method according to claim 1, the internal route includes an internal circulation circuit that circulates the liquid through the processing section, and an internal supply circuit that supplies the liquid to the internal circulation circuit, the external route includes an external circulation circuit that circulates the liquid through the processing section, and an external supply circuit that supplies the liquid to the external circulation circuit, During the priming, a pressure difference is generated between the liquid circulating in the internal circulation circuit and the liquid circulating in the external circulation circuit. Priming method.

3. The priming method according to claim 2, When the pressure difference is generated, the supply of the liquid from the internal supply circuit and the external supply circuit is stopped, while a difference is created between a flow rate of the liquid circulating in the internal circulation circuit and a flow rate of the liquid circulating in the external circulation circuit. Priming method.

4. The priming method according to claim 3, During the priming, a differential pressure generating step of generating the differential pressure and a discharge step of discharging the liquid and the gas from the internal circulation circuit or the external circulation circuit to a waste liquid route are repeated. Priming method.

5. The priming method according to claim 3 or 4, The flow rate ratio of the liquid flowing through the internal circulation circuit to the liquid flowing through the external circulation circuit is set within a range of 5 to 15 times. Priming Method.

6. The priming method according to any one of claims 2 to 5, When the pressure difference is generated, the liquid is circulated in both the internal circulation circuit and the external circulation circuit, while the liquid is supplied from the internal supply circuit to the internal circulation circuit, or the liquid is supplied from the external supply circuit to the external circulation circuit. Priming Method.

7. The priming method according to any one of claims 1 to 6, The internal pressure of the external route is increased relative to the internal pressure of the internal route to generate the pressure difference, and the gas is moved from the hollow fiber to the lumen. Priming Method.

8. The priming method according to any one of claims 1 to 6, The internal pressure of the internal route is increased relative to the internal pressure of the external route to generate the pressure difference, and the gas is moved from the hollow fiber to the internal space. Priming Method.

9. The priming method according to any one of claims 1 to 8, the processing section is a bioreactor that causes cells to adhere to an inner circumferential surface of the hollow fiber, and then supplies a culture medium to the hollow fiber to grow the cells on the inner circumferential surface of the hollow fiber; The priming method is carried out before supplying the liquid containing the cells to the hollow fibers. Priming Method.

10. A biological component processing system including a processing section having a hollow fiber having a coating formed on an inner peripheral surface that constitutes an inner cavity, and a container that contains the hollow fiber in an internal space, an internal route connected to the processing section and capable of passing a liquid through the lumen; an external route connected to the processing section and capable of circulating the liquid in the internal space outside the hollow fibers; a control unit for controlling a flow state of the liquid through the internal route and the external route, The hollow fiber has a membrane structure surrounding the lumen, the membrane structure has a plurality of pores that connect the lumen to the outside of the hollow fiber, The control unit, during priming, causes the liquid to flow through both the internal route and the external route, and generates a pressure difference between the liquid flowing through the lumen and the liquid flowing through the internal space, causing gas to flow out from the pores of the hollow fibers to the lumen side or the internal space side by the pressure difference. Biological component processing system.

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