Cell culture system and method for determining life span of enzyme sensor

Abstract

A cell culture system (22) includes an enzyme sensor (124) that detects the concentration of a predetermined component in a culture medium used for cell culture, a sensor flow path (46B2) in which the enzyme sensor (124) is provided, and a bioreactor (20) that cultures cells while flowing the culture medium through the sensor flow path (46B2). The cell culture system (22) also includes a deterioration index calculation unit (138) that calculates a deterioration index of the enzyme sensor (124) based on a concentration detection value of the enzyme sensor (124) and a detection period for the concentration detection value, and a life determination unit (140) that determines the life of the enzyme sensor (124) by detecting whether an integrated value of the deterioration index has reached a threshold value.

Description

[Technical field]

[0001] The present invention relates to a cell culture system. Mu and and a method for determining the lifespan of an enzyme sensor. [Background technology]

[0002] In regenerative medicine, therapeutic cells (also called biological components) are cultured and administered to patients. For culturing therapeutic cells, a cell culture system for culturing cells in hollow fibers has been proposed, for example, in JP 2017-143775 A. This cell culture system has an inner circulation flow path that communicates with the inside of the hollow fibers and an outer circulation flow path that communicates with the outside of the hollow fibers. A liquid (culture medium) containing cells is supplied to the inner circulation flow path that communicates with the inside of the hollow fibers, and nutrients and gas components are supplied and metabolites are discharged through the liquid (culture medium) that flows through the outer circulation flow path that communicates with the outside of the hollow fibers.

[0003] In a cell culture system, it is preferable to manage the state of the culture medium flowing through the circulation flow path and the cell growth in real time when culturing cells. The state of the culture medium can be understood by detecting the amount of dissolved oxygen, the amount of dissolved carbon dioxide, the pH, and the glucose concentration (energy supplied to the cells). The amount of cell growth can be understood by measuring the concentration of lactic acid, a metabolic product of the cells. Summary of the Invention

[0004] Enzyme sensors are used to measure glucose and lactate concentrations from the viewpoint of selectivity. However, the enzymes used in enzyme sensors are unstable and have a relatively short lifespan, and the enzyme sensor may reach the end of its life during the cell culture period, resulting in a decrease in measurement sensitivity. Therefore, it is difficult to determine whether the decrease in the concentration measurement value during the cell culture period is due to the state of the medium or the amount of cell growth, or due to the lifespan of the enzyme sensor, making it difficult to manage the cell culture process.

[0005] Japanese Patent Application Laid-Open No. 62-240848 describes that when targeting urine or blood components in which the substrate concentration is relatively stable, the sensitivity of an enzyme sensor is detected and corrected according to the number of times or frequency of use of the enzyme sensor.

[0006] However, in a cell culture system in which the glucose concentration and lactic acid concentration fluctuate greatly between the beginning and end of cell culture, the method of JP-A-62-240848 has the problem that it is not possible to fully grasp the timing at which the sensitivity of the enzyme sensor decreases.

[0007] Therefore, the present invention provides a cell culture system capable of determining the end of the life of an enzyme sensor even during the culture of cells. Mu and The object of the present invention is to provide a method for determining the lifespan of an enzyme sensor.

[0008] One aspect of the present invention is a cell culture system comprising an enzyme sensor that detects the concentration of a specific component in a culture medium used for cell culture, a sensor flow path in which the enzyme sensor is provided, and a bioreactor that cultures cells while flowing culture medium through the sensor flow path, the cell culture system having a deterioration index calculation unit that calculates a deterioration index of the enzyme sensor based on the concentration detection value of the enzyme sensor and the detection period of the concentration detection value, and a life determination unit that detects whether the deterioration index has reached a threshold value and determines the life of the enzyme sensor.

[0010] The present invention Other One aspect of the present invention is a method for determining the lifespan of an enzyme sensor in a cell culture system in which cells are cultured while detecting the concentration of a specific component in a culture medium using an enzyme sensor, the method comprising: a step in which a control unit detects a concentration detection value of the enzyme sensor and a detection period for the concentration detection value; a deterioration index detection step in which a deterioration index calculation unit calculates a deterioration index of the enzyme sensor based on the concentration detection value of the enzyme sensor and the detection period; and a lifespan determination step in which a lifespan determination unit determines whether an integrated value of the deterioration index has reached a threshold value.

[0011] The cell culture system according to the above aspects Mu and According to the method for determining the lifespan of an enzyme sensor, the lifespan of the enzyme sensor can be determined even during the cell culture period, thereby enabling appropriate management in the cell culture system. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is an explanatory diagram showing a cell culture system according to a first embodiment of the present invention. [Diagram 2] FIG. 2 is an exploded perspective view of a flow path cassette. [Diagram 3] FIG. 2 is a plan view showing a cassette body and its peripheral portion. [Figure 4] 4 is an explanatory diagram illustrating a schematic diagram of a liquid path in a cell culture system during cell culture. Note that sensors other than the enzyme sensor are not shown in FIG. [Diagram 5] FIG. 5A is a plan view showing an example of an enzyme sensor, and FIG. 5B is an explanatory diagram showing a method for attaching the enzyme sensor. [Figure 6] Fig. 6A is a flow chart showing the process of the cell culture system, and Fig. 6B is a flow chart showing the method of determining the life of the enzyme sensor in the culture process. [Figure 7] FIG. 4 is an explanatory diagram showing the operation of a normal distribution step in the first embodiment. [Figure 8] FIG. 8A is a graph showing the relationship between the substrate concentration in the medium and the time course of the decrease in sensitivity of the enzyme sensor, and FIG. 8B is a graph showing the relationship between the change in substrate concentration in the medium of the cell culture system and the detection value of the enzyme sensor. [Figure 9] FIG. 11 is an explanatory diagram illustrating a schematic diagram of liquid paths in a cell culture system according to a second embodiment. [Figure 10] FIG. 11 is an explanatory diagram illustrating a schematic diagram of liquid paths in a cell culture system according to a third embodiment. [Figure 11] 11 is a flowchart showing a method for determining the life span of an enzyme sensor and a switching operation of the enzyme sensor in the cell culture system of FIG. [Figure 12]FIG. 11 is an explanatory diagram showing the liquid paths of the cell culture system of FIG. [Figure 13] FIG. 13 is an explanatory diagram illustrating a schematic diagram of liquid paths in a cell culture system according to a fourth embodiment. [Figure 14] FIG. 14 is a block diagram of a deterioration index calculation unit of the cell culture system of FIG. 13. [Figure 15] FIG. 15A is a graph showing the stability of the enzyme in the lactate sensor versus pH, with the pH of the culture medium on the horizontal axis and the measurement results of the residual activity of the enzyme in the enzyme sensor after contact with the culture medium for a certain period of time on the vertical axis. FIG. 15B is a graph showing the stability of the enzyme in the glucose sensor versus pH. [Figure 16] FIG. 16A is a graph showing the stability of the enzyme in the lactate sensor against temperature, with the temperature of the culture medium on the horizontal axis and the residual activity of the enzyme in the enzyme sensor after contact with the culture medium for a certain period of time on the vertical axis. FIG. 16B is a graph showing the stability of the enzyme in the glucose sensor against temperature. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to preferred embodiments thereof and accompanying drawings.

[0014] [First embodiment] 1, a cell culture system 22 according to a first embodiment of the present invention includes a cell culture kit 12 capable of flowing a liquid containing a biological component and a liquid for processing the biological component, and a cell culture device 14 in which the cell culture kit 12 is set. The cell culture kit 12 also includes a flow path cassette 10 that aggregates multiple liquid paths and is set in the cell culture device 14.

[0015] In addition to the flow path cassette 10, the cell culture kit 12 includes a plurality of tubes 16, a plurality of bags 18 (medical bags), and a bioreactor 20 as components constituting a plurality of paths. The cell culture kit 12 cultures cells in the bioreactor 20 by circulating a plurality of types of liquids contained in each bag 18 through the flow path cassette 10 and each tube 16 under the operation of the cell culture device 14.

[0016] The cell culture system 22 according to this embodiment performs a culture process for growing cells for treatment in regenerative medicine. Examples of liquids flowing within the cell culture kit 12 include a solution containing cells (hereinafter referred to as a cell liquid), a medium (culture liquid) supplied for growing cells, 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 system 22 performs a culture process in which, in a set state of the cell culture kit 12, cells are seeded in the bioreactor 20, a medium is further supplied to culture the cells, and the grown cells are detached and collected from the bioreactor 20, thereby recovering the cells.

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

[0018] The multiple bags 18 of the cell culture kit 12 include a cell liquid bag 18A containing a cell liquid, a cleaning liquid bag 18B containing a cleaning liquid, and a culture medium bag 18C containing a culture medium. Furthermore, the cell culture kit 12 has empty bags 18 as the multiple bags 18. The empty bags 18 include a waste liquid bag 18D into which liquid discarded in the culture process flows, and a collection bag 18E for collecting cells (and other liquids) obtained in the culture process. In addition, a detachment liquid bag 18F containing a detachment liquid is prepared separately for the bag 18. The detachment liquid bag 18F is replaced with a previously connected bag 18 (e.g., the cell liquid bag 18A) by an operator during the culture process.

[0019] 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 commercially available aseptic joining device. The tube 16 is a thermoplastic tube made of a thermoplastic resin, and by bringing it into a molten state, it is possible to join thermoplastic tubes of the same diameter. In addition, each bag 18 may be fixed to the end of each tube 16 in an inseparable manner, so as 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 between the tube 16 and each bag 18.

[0020] The bioreactor 20 of the cell culture kit 12 is not particularly limited, but it is preferable to use a culture substrate with a large surface area, and in this embodiment, hollow fibers 24 are applied. Specifically, the bioreactor 20 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) for accommodating the plurality of hollow fibers 24.

[0021] The hollow fibers 24 extend along the axial direction of the container 26, and both ends of the hollow fibers 24 are held by a retaining wall (not shown) inside the container 26. Each hollow fiber 24 has an inner cavity (not shown) that penetrates the hollow fiber 24 along the extension direction. The diameter of the inner cavity is, for example, about 200 μm, and the inner cavity communicates with end spaces 26b on both axial sides defined by the retaining wall.

[0022] The membrane structure of the hollow fibers 24 surrounding the lumen is made of a porous body having many pores. Each pore communicates between the outside (main space 26a) of the hollow fibers 24 and the lumen, and is formed to a size that allows the passage of solutions and low molecular weight substances while restricting the permeation of cells and proteins. The diameter of the pores is set to, for example, about 0.005 μm to 10 μm.

[0023] The material constituting the membrane structure of the hollow fiber 24 is not particularly limited, and examples thereof include polymer materials such as polyolefin resins such as polypropylene and polyethylene, polysulfone, polyethersulfone, polyacrylonitrile, polytetrafluoroethylene, polystyrene, polymethyl methacrylate, cellulose acetate, cellulose triacetate, and regenerated cellulose.

[0024] In the above hollow fibers 24, cells are seeded (attached) to the inner peripheral surface of the lumen, and then culture medium, a predetermined gas component, and the like can be supplied to the cells through the lumen and pores. Hereinafter, in the cell culture system 22, a circulation flow path that mainly circulates liquid in the lumen of the hollow fibers 24 is referred to as an inner circulation flow path or IC (intra capillary). Also, a circulation flow path that mainly circulates liquid outside the hollow fibers 24 is referred to as an outer circulation flow path or EC (extra capillary).

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

[0026] The multiple tubes 16 of the cell culture kit 12 include a cell liquid tube 16A connecting the cell liquid bag 18A and the flow path cassette 10, a washing liquid tube 16B connecting the washing liquid 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 liquid tube 16D connecting the waste liquid 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 16F connecting the first IC terminal 28a of the bioreactor 20 and the flow path cassette 10, a second IC tube 16G connecting the second IC terminal 28b of the bioreactor 20 and the flow path cassette 10, a first EC tube 16H connecting the first EC terminal 28c of the bioreactor 20 and the flow path cassette 10, and a second EC tube 16I connecting the second EC terminal 28d of the bioreactor 20 and the flow path cassette 10.

[0027] A gas exchanger 29 that mixes a predetermined gas component into the liquid (culture medium) is provided at a midpoint of the first EC tube 16H. For example, the gas component to be mixed may be a component (nitrogen N 2 : 75% oxygen O 2 : 20% carbon dioxide CO 2 :5%) can be cited.

[0028] 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 20. 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 the movement inside the hollow fibers 29b with the liquid through the pores of the hollow fibers 29b.

[0029] The flow path cassette 10, which is one component of the cell culture kit 12, has the above-mentioned tubes 16 joined in advance, and functions as a relay section of the liquid path that circulates the cell fluid, washing fluid, culture medium, and stripping fluid in each bag 18 to another bag 18 or the bioreactor 20. 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 during the culture process.

[0030] As shown in FIG. 2, the flow path cassette 10 is composed of a flexible cassette body 40 to which multiple tubes 16 are directly connected, and a rigid frame 50 that holds the cassette body 40 and is fixed to the cell culture device 14.

[0031] The cassette body 40 is formed in a thin, flexible sheet shape with a substantially rectangular shape. The cassette body 40 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 each become a flow path wall with a semicircular cross section and a flow path 44 is formed inside the flow path wall. 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 cassette body 40 may be embossed to form minute irregularities. A plurality of connectors 60 that connect between the plurality of tubes 16 and the flow path 44 are provided on the outer edge 41 of the cassette body 40.

[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 constituting the frame 50 is also not particularly limited, and it is preferable to use a thermoplastic resin such as polypropylene, polycarbonate, polyamide, polysulfone, polyarylate, or a methacrylate-butylene-styrene copolymer.

[0033] The frame 50 has a substantially rectangular cover portion 54 that is slightly larger than the cassette body 40, and a side portion 56 that protrudes from the outer periphery of the cover portion 54 in a direction perpendicular to the cover portion 54. The side portion 56 goes around the entire outer periphery of the cover portion 54. The frame 50 opens the storage space 52 through an opening 52a surrounded by the side portion 56 on the opposite side to the cover portion 54, exposing one side of the cassette body 40. The frame 50 also has a holding frame 58 that extends from the upper side and the right side of the side portion 56 and holds the tubes 16 at a predetermined distance from the side portion 56. The side portion 56 has locking portions 70 that position and hold the connectors 60 at locations corresponding to the connectors 60 of the cassette body 40.

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

[0035] As shown in Fig. 3, the flow path cassette 10 is set in the cell culture device 14 with the cassette body 40 and the frame 50 integrated together, and with the surface direction of the cassette body 40 in an upright position along the direction of gravity (up and down direction). Note that the flow path cassette 10 in Fig. 3 is in a position as viewed from the cover part 54 side (the touch panel 134 side of the cell culture device 14) in a state in which it is attached to the cell culture device 14, and for convenience of explanation, only the cassette body 40 is shown without the frame 50.

[0036] Specifically, the outer edge 41 of the cassette body 40 is composed of a first short side 41a (left side in the figure), a second short side 41b (right side in the figure), a first long side 41c (upper side in the figure), and a second long side 41d (lower side in the figure). The cell liquid tube 16A, the washing liquid tube 16B, and the culture medium tube 16C are connected to the first long side 41c. The waste liquid tube 16D, the recovery tube 16E, the first IC tube 16F, the second IC tube 16G, the first EC tube 16H, and the second EC tube 16I are connected to the second short side 41b.

[0037] In addition, in the cell culture system 22, in the set state, four pumps 30 are disposed near the sides of the flow path cassette 10. That is, in the set state, the cell culture device 14 has a first pump 30a disposed near the first short side 41a, a second pump 30b and a third pump 30c disposed near the first long side 41c, and a fourth pump 30d disposed near the second long side 41d. The first to fourth pumps 30a to 30d are wound with the first to fourth pump tubes 16J to 16M protruding from the flow path cassette 10, and rotate in a manner that squeezes the wound first to fourth pump tubes 16J to 16M, thereby causing the liquid inside to flow.

[0038] The first pump 30a guides liquid to an IC route 44A, which will be described later, and the second pump 30b guides liquid to an EC route 44B, which will be described later. Meanwhile, the third pump 30c circulates the liquid in the EC route 44B, and the fourth pump 30d circulates the liquid in the IC route 44A.

[0039] In addition, in the set state of the cell culture system 22, the air bubble sensor 32 is disposed relative to the sensor tube 16N of the cassette body 40. Furthermore, in the set state, outer clamps 34 for opening and closing the internal flow paths are disposed in the cell liquid tube 16A, the washing liquid tube 16B, and the culture medium tube 16C. Specifically, a first outer clamp 34a is disposed in the cell liquid tube 16A, a second outer clamp 34b is disposed in the washing liquid tube 16B, and a third outer clamp 34c is disposed in the culture medium tube 16C.

[0040] As described above, the flow path 44 having a predetermined shape extending along the surface direction is formed inside the cassette body 40. Furthermore, inside the cassette body 40, there are provided a plurality of pressure detection parts 48 communicating with the flow path 44, a liquid level detection part 80, a check valve part 90, a plurality of flow path opening / closing parts 100 configured together with the flow path 44, and a plurality of parameter detection parts 110. Note that some of the parameter detection parts 110 are provided outside the flow path cassette 10, at midway positions of the tubes 16 (the second IC tube 16G, the second EC tube 16I, etc.).

[0041] Although detailed description is omitted, the flow path 44 constitutes an IC route 44A that supplies liquid to the inner cavity of the hollow fiber 24 together with the first and second IC tubes 16F, 16G, and an EC route 44B that supplies liquid to the outside (main space 26a) of the hollow fiber 24 together with the first and second EC tubes 16H, 16I. In particular, in the culture step of supplying a culture medium to the bioreactor 20, the outer clamps 34 and the inner clamps 35 of the cell culture device 14 are appropriately opened or closed, so that the IC route 44A and the EC route 44B take the form diagrammatically shown in FIG.

[0042] 3 and 4, the IC route 44A has a first IC port passage 44A1 (see FIG. 3) communicating with the first IC tube 16F and a second IC port passage 44A2 (see FIG. 3) communicating with the second IC tube 16G in the cassette body 40. The IC route 44A has an IC supply circuit 45A that draws out the liquids of the cell solution bag 18A, the washing solution bag 18B, and the culture medium bag 18C and supplies them to the downstream side. The IC route 44A also has an IC circulation circuit 46A that is provided downstream of the IC supply circuit 45A and circulates liquids between the IC route 44A and the bioreactor 20. The IC circulation circuit 46A forms an IC inflow path 46A1, which allows liquid to flow into the lumen of the hollow fiber 24, by the first IC tube 16F and the first IC port path 44A1, and forms an IC outflow path 46A2, which allows liquid to flow out of the lumen of the hollow fiber 24, by the second IC tube 16G and the second IC port path 44A2.

[0043] The EC route 44B has a first EC port passage 44B1 communicating with the first EC tube 16H and a second EC port passage 44B2 communicating with the second EC tube 16I in the cassette body 40. The EC route 44B has an EC supply circuit 45B that draws out the liquids from the cell solution bag 18A, the washing solution bag 18B, and the culture medium bag 18C and supplies them to the downstream side. The EC route 44B also has an EC circulation circuit 46B that circulates liquid between the EC route 44B and the bioreactor 20 downstream of the EC supply circuit 45B. The EC circulation circuit 46B forms an EC inflow path 46B1 that allows liquid to flow into the main space 26a via the first EC tube 16H and the first EC port passage 44B1, and forms an EC outflow path 46B2 that allows liquid to flow out of the main space 26a via the second EC tube 16I and the second EC port passage 44B2.

[0044] The pressure detection target 48 of the cassette body 40 is provided downstream of each pump 30 in the IC route 44A and the EC route 44B. In the set state, the pressure detection target 48 is disposed opposite the pressure sensor 36 of the cell culture device 14 to detect the pressure of the flow path 44. The liquid level detection target 80 is provided in the IC route 44A, temporarily stores the flowing liquid in a storage space 80a, and discharges the liquid to the IC route 44A, while discharging gas to a route separate from the liquid. This liquid level detection target 80 is disposed opposite the liquid level sensor 37 of the cell culture device 14 to detect the level of the liquid stored therein. The check valve 90 is provided in a route for discharging gas from the liquid level detection target 80, and regulates the inflow of gas or liquid from this route to the liquid level detection target 80.

[0045] The flow path openable / closable part 100 is composed of a plurality of notches 102 and is disposed in each of the inner clamps 35 of the cell culture device 14. The inner clamp 35 has a displaceable body 35a and a fixed body 35b that are inserted into the notches 102, and closes a predetermined flow path 44 by bringing the displaceable body 35a close to the fixed body 35b, and opens the predetermined flow path 44 by moving the displaceable body 35a away from the fixed body 35b. The flow path openable / closable part 100 is provided in the gas outflow route, the waste liquid route 44C from the IC route 44A or the EC route 44B to the waste liquid bag 18D, the recovery route 44D from the IC route 44A to the recovery bag 18E, the second IC port path 44A2, and the like. Hereinafter, the inner clamp 35 extending from the IC route 44A toward the waste liquid bag 18D will be referred to as an IC waste liquid clamp 35A, and the inner clamp 35 extending from the EC route 44B toward the waste liquid bag 18D will be referred to as an EC waste liquid clamp 35B.

[0046] The parameter detection section 110 is capable of detecting parameters related to cell culture in the liquid flowing through the bioreactor 20. Examples of parameters related to cell culture include the amount of dissolved oxygen, pH, amount of glucose, amount of dissolved carbon dioxide, and amount of lactic acid in the culture medium (liquid) flowing through the flow path 44. The amount of dissolved oxygen, pH, and amount of glucose correspond to parameters of the culture environment supplied to the cells. The pH, amount of dissolved carbon dioxide, and amount of lactic acid correspond to parameters generated by the metabolism of the cells. Note that the pH also varies depending on the concentration of metabolites, and therefore varies depending on the metabolism and metabolites of the cells. One parameter detection section 110 is capable of detecting any one of these five types of parameters.

[0047] That is, the culture parameter detection unit 122 includes the parameter detection unit 110, the cell culture device 14, and the enzyme sensor 124, and detects parameters related to the cells during cell culture. The cell culture device 14 records and adjusts (feeds back) the amounts of medium and gas components supplied to the bioreactor 20 based on the detection results of the culture parameter detection unit 122.

[0048] In this embodiment, the parameter detection unit 110 of the flow path cassette 10 shown in FIG. 3 is provided in a plurality (three units) on the second EC port path 44B2 to detect different types of parameters among the amount of dissolved oxygen, pH, amount of glucose, amount of dissolved carbon dioxide, and amount of lactic acid. Note that the parameter detection unit 110 may be provided in one unit for detecting one type of parameter, or in five units for detecting all five types of parameters. Furthermore, the parameters detected by the parameter detection unit 110 (culture parameter detection unit 122) are not limited to the above five types, and may be applied to any components required for culture. For example, other parameters include the number of cells, glutamine, ammonium ions, and proteins. The parameter detection unit 110 (culture parameter detection unit 122) may be provided in an appropriate number according to the type of component to be detected. Furthermore, the parameter detection unit 110 is not limited to being provided on the second EC port path 44B2, and may be provided on another flow path 44 (for example, the second IC port path 44A2).

[0049] The parameter detection portion 110 has a fluorescent chip (not shown) inside a bulge that communicates with the flow path 44 and expands the cross-sectional area of ​​the flow path. When set, each parameter detection portion 110 is disposed at a position facing the sensor 120 of the cell culture device 14. The fluorescent chip is configured in a layered structure that enables optical measurement of a predetermined parameter related to cell culture. One layer of the layered structure is a detection layer that changes color in response to a predetermined substance contained in the culture medium.

[0050] The sensor 120 of the cell culture device 14 may be an optical sensor having a light-emitting unit that emits measurement light and a light-receiving unit that receives excitation light generated from the fluorescent chip (both not shown). Each sensor 120 emits measurement light of a wavelength according to the characteristics of the fluorescent chip to each fluorescent chip and receives excitation light generated from the fluorescent chip under the control of the cell culture device 14. As a result, the sensor 120 transmits a detection signal based on the degree of color development of the fluorescent chip to the control unit 136.

[0051] In this embodiment, as shown in FIG. 5B, an enzyme sensor 124 for detecting the amount of glucose or lactic acid is attached to the second IC tube 16G, the second EC tube 16I, etc., as the culture parameter detection unit 122 of the flow path cassette 10. As shown in FIG. 5A, the enzyme sensor 124 is provided in a structure in which a sensor body 128 and a pair of tubes 125 are connected to a T-shaped joint 126. The detection unit of the sensor body 128 is exposed inside the joint 126. The detection unit has an electrode and an enzyme immobilization layer, and electrochemically detects a substance change caused by a reaction between a substance to be measured (e.g., lactic acid or glucose) in the culture medium flowing through the joint 126 and an enzyme as a current value using the electrode. The tube 125 connected to the joint 126 is stored in a sealed state by a welding part 125a.

[0052] 5B, when the enzyme sensor 124 is used, it is joined to the tube 16 (second IC tube 16G, second EC tube 16I, etc.) in which it is to be installed, using a commercially available sterile joining device. The enzyme sensor 124 can be replaced by connecting a new enzyme sensor 124 to the tube 16 in which it is to be installed, in place of the deteriorated enzyme sensor 124, using a commercially available sterile joining device.

[0053] The above cell culture system 22 performs a process of culturing cells by circulating the culture medium in the bioreactor 20 for a long period of time (e.g., several days), during which the state (parameters) of the culture medium is continuously detected by the culture parameter detection unit 122. Furthermore, in order to stabilize the detection accuracy of the culture parameter detection unit 122 during the culture, the cell culture system 22 performs a lifespan determination of the culture parameter detection unit 122 (enzyme sensor 124) at an appropriate timing during the culture. A method of determining the lifespan of the enzyme sensor 124 will be described later.

[0054] The cell culture kit 12 includes a bypass path 47 on the EC route 44B where the culture parameter detection unit 122 is provided. The bypass path 47 is configured by a bypass tube 16O that connects between the first EC tube 16H and the second EC tube 16I. One end of the bypass tube 16O is connected to a connection point α of the first EC tube 16H between the gas exchanger 29 and the bioreactor 20 (first EC terminal 28c). On the other hand, the other end of the bypass tube 16O is connected to a connection point β of the second EC tube 16I that connects between the bioreactor 20 and the parameter detection unit 110. For example, the first or second EC tube 16H, 16I and the bypass tube 16O are connected via a three-port connector 142 or the like.

[0055] Furthermore, in the cell culture system 22, with the cell culture kit 12 set in the cell culture device 14, a plurality of outer clamps 34 are disposed near a connection point β between the EC outflow path 46B2 and the bypass path 47. In detail, the cell culture device 14 has a main clamp 34d disposed on the second EC tube 16I upstream (bioreactor 20) of the connection point β, and a sub-clamp 34e disposed on the downstream (culture parameter detection unit 122) side of the connection point β. The cell culture device 14 also has a sub-clamp 34f disposed on the bypass tube 16O.

[0056] During cell culture, the main clamp 34d, the sub clamp 34e, and the sub clamp 34f each open and close the internal flow path of each tube under the operation of the cell culture device 14. For example, when supplying culture medium to the bioreactor 20 during cell culture, the cell culture system 22 opens the main clamp 34d and the sub clamp 34e while closing the sub clamp 34f. This allows the culture medium in the first EC tube 16H to be smoothly introduced into the bioreactor 20. On the other hand, when replacing the enzyme sensor 124, the cell culture system 22 closes the sub clamp 34e and stops the third pump 30c.

[0057] The configuration for changing the medium flow path is not limited to the above-mentioned main clamp 34d, sub clamp 34e, and sub clamp 34f. For example, the main clamp 34d may be provided in the EC inflow path 46B1 between the connection point α and the bioreactor 20. Also, for example, the cell culture system 22 may be configured without the sub clamp 34e.

[0058] 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 displaying during the culture process is provided on the outer surface of the device body 130. Furthermore, a cassette placement unit (not shown) for fixing the flow path cassette 10 in an upright position and for holding the bioreactor 20 at an appropriate height position, and a control unit 136 for controlling the operation of the cell culture system 22 are provided inside the device body 130.

[0059] The control unit 136 has a processor, memory, and input / output interface (not shown), and the processor executes a program stored in the memory to appropriately operate the pump 30, the outer clamp 34, the inner clamp 35, and the like in the culture process. In particular, the control unit 136 receives detection signals from each sensor 120 during cell culture (supply of culture medium), and adjusts (feedback control) the driving of the pump 30 based on the detection result. As shown in FIG. 4, the control unit 136 also includes a deterioration index calculation unit 138 that accumulates a deterioration index reflecting how long the enzyme sensor 124 has been in contact with the culture medium and the concentration of the medium based on the concentration detection value of the enzyme sensor 124, and a lifespan determination unit 140 that determines the lifespan of the enzyme sensor 124 by comparing the accumulated value of the deterioration index with a predetermined threshold value. The lifespan determination unit 140 performs lifespan determination of the culture parameter detection unit 122 (enzyme sensor 124) at an appropriate timing during cell culture.

[0060] For example, when the control unit 136 receives the detection result of the enzyme sensor 124 that detects the amount of lactic acid, it calculates the amount of lactic acid using an appropriate calculation formula or calibration curve information stored in advance. Then, the deterioration index calculation unit 138 of the control unit 136 calculates a concentration-time value by multiplying the concentration detection value by the elapsed time (detection period) from the previous measurement timing, and updates the deterioration index by adding this concentration-time value to the deterioration index, which is an integrated value of the past concentration-time values. Furthermore, the life determination unit 140 of the control unit 136 compares the deterioration index threshold value at which the detection sensitivity of the enzyme sensor 124 begins to decrease, which is experimentally obtained in advance, with the deterioration index calculated by the deterioration index calculation unit 138 to determine the life of the enzyme sensor 124.

[0061] Furthermore, the control unit 136 calculates the number of cells based on the calculated amount of lactic acid and the calibration information it holds, and adjusts the supply amount of culture medium (supply rate: rotation speed of pump 30) to be supplied to the bioreactor 20 based on the calculated number of cells.

[0062] The cell culture kit 12 and the cell culture system 22 according to this embodiment are basically configured as described above, and their operation will be described below.

[0063] As shown in FIG. 1, in the proliferation process of the cell culture system 22, an operator inserts a part of the cell culture kit 12 including the flow path cassette 10 into the cell culture device 14. The operator also places appropriate tubes 16 of the cell culture kit 12 on the pump 30, the air bubble sensor 32, and the outer clamp 34 of the cell culture device 14. When placing the flow path cassette 10, the inner clamp 35 is placed on the flow path opening and closing part 100, and the sensor 120 is placed on the parameter detection part 110. As a result, the flow path cassette 10 is set in the cell culture device 14 with its surface direction aligned with the gravity direction, as shown in FIG. 3. Furthermore, each bag 18 of the cell culture kit 12 is also hung from the stand 132 by the operator.

[0064] After the above setting, the proliferation process sequentially includes a priming step (S10), a medium replacement step (S20), a seeding step (S30), a culture step (S40), a detachment step (S50), and a recovery step (S60), as shown in FIG. 6A.

[0065] In the priming step (S10), the cleaning solution in the cleaning solution bag 18B is circulated through two routes (the IC route 44A and the EC route 44B) in the flow path cassette 10 to remove gas present in each route of the designated tube 16, the bioreactor 20, and the cassette body 40. The gas to be removed is also led to the waste solution bag 18D.

[0066] In the medium replacement step (S20), similarly to the priming step (S10), the medium in the medium bag 18C is introduced into each route of the predetermined tube 16, the bioreactor 20, and the cassette body 40, and the routes are filled with the medium. In the seeding step (S30) after the medium replacement step (S20), the cell solution in the cell solution bag 18A is supplied to the inner cavity of the hollow fiber 24 of the bioreactor 20 via the IC route 44A, while the medium present in the EC route 44B is circulated to supply gas components to the bioreactor 20.

[0067] Then, in the culture step (S40) after the seeding step (S30), as shown in Fig. 4, the culture medium is supplied from both the IC route 44A and the EC route 44B to culture the cells seeded in the bioreactor 20. Note that the cell culture system 22 may perform an operation of supplying the culture medium from the EC route 44B in the culture step (S40) without using the IC route 44A.

[0068] This culturing step (S40) is carried out for a longer period of time than other steps, thereby growing cells on the inner surface of the hollow fibers 24. In the culturing step (S40), the cell culture system 22 performs a life determination of the enzyme sensor 124 based on the process flow shown in FIG.

[0069] As shown in Fig. 7, the culture step (S40) basically involves a normal flow step in which the culture medium is supplied to the bioreactor 20 to grow the cells in the hollow fibers 24. In the normal flow step, the control unit 136 opens the main clamp 34d and the sub-clamp 34e while closing the sub-clamp 34f. That is, in the EC circulation circuit 46B, the EC inflow path 46B1 and the EC outflow path 46B2 are in a flow state while the bypass path 47 is in a flow blocking state. In addition, the control unit 136 opens the IC waste liquid clamp 35A while closing the EC waste liquid clamp 35B. This causes the culture medium to flow out of the IC route 44A into the waste liquid bag 18D, and the culture medium circulates in the EC route 44B.

[0070] As a result, the IC route 44A circulates the culture medium to the bioreactor 20 (the lumen of the hollow fiber 24) under the driving of the fourth pump 30d. Also, the EC route 44B supplies the culture medium to the main space 26a of the bioreactor 20 (container 26) from the EC inflow path 46B1 under the driving of the third pump 30c. The culture medium of the IC route 44A and the EC route 44B is provided to the cells of the hollow fiber 24, and a part of the culture medium flows out of the bioreactor 20 through the main space 26a to the EC outflow path 46B2. The outflowed culture medium passes through the parameter detection part 110 (culture parameter detection part 122) provided in the cassette body 40 while flowing through the EC outflow path 46B2. Therefore, the control part 136 detects the state of the culture medium (parameters such as the amount of dissolved oxygen, pH, and amount of dissolved carbon dioxide) by the multiple sensors 120, and controls the supply amount (supply rate) of the culture medium based on the detection result.

[0071] During the normal circulation step, the control unit 136 performs a lifespan determination process for the enzyme sensor 124, which is made up of steps S72 to S84 shown in Fig. 6B. In the lifespan determination process, first, in an initialization step (S72), the control unit 136 performs a process of resetting the deterioration index of the deterioration index calculation unit 138 to an initial value of "0". Next, the control unit 136 performs a concentration measurement step (S74) in which the enzyme sensor 124 is used to measure the concentration of a predetermined component (e.g., glucose or lactic acid) in the medium.

[0072] Thereafter, the deterioration index calculation unit 138 of the control unit 136 performs a deterioration index update step (S76). In the deterioration index update step (S76), the deterioration index calculation unit 138 calculates a concentration-time value by multiplying the elapsed time (detection period) from the previous concentration measurement step (S74) to the current concentration measurement step (S74) by the concentration detection value of the current concentration measurement step (S74). Then, the deterioration index calculation unit 138 adds the concentration-time value to the past deterioration index (integrated value of the concentration-time value) to update the deterioration index.

[0073] Next, a lifespan determination step (S78) is performed in which the lifespan determination unit 140 of the control unit 136 determines the lifespan of the enzyme sensor 124 based on whether the updated deterioration index exceeds a predetermined threshold. If the lifespan determination unit 140 detects that the deterioration index exceeds the threshold (YES) in the lifespan determination step (S78), a sensor deterioration notification step (S80) is performed, and the control unit 136 displays a message on the touch panel 134 encouraging replacement of the enzyme sensor 124. Then, the process proceeds to an end determination step (S82).

[0074] In addition, in the lifespan determination step (S78), if the lifespan determination unit 140 does not detect that the deterioration index exceeds the threshold (NO), the process proceeds to an end determination step (S82). In the end determination step (S82), if the control unit 136 determines that the culture step (S40) has ended (YES), the lifespan determination process is ended.

[0075] On the other hand, if the control unit 136 determines in the end determination step (S82) that the culture step (S40) has not ended (NO), the process proceeds to a sensor replacement detection step (S84). In the sensor replacement detection step (S84), the control unit 136 determines whether the enzyme sensor 124 has been replaced. If the control unit 136 determines that the sensor has not been replaced (NO), the process proceeds to a concentration measurement step (S74), where the concentration of the medium is measured again using the enzyme sensor 124 after a predetermined period of time has elapsed. If the control unit 136 determines in the sensor replacement detection step (S84) that the enzyme sensor 124 has been replaced (YES), the process proceeds to an initialization step (S72), where the integrated value of the deterioration index is initialized.

[0076] By carrying out the above process, the life of the enzyme sensor 124 is detected. As shown in FIG. 8B, in the cell culture system 22, the lactic acid concentration in the culture medium gradually increases with cell proliferation. The detection value of the enzyme sensor 124 that measures the lactic acid concentration follows the change in the lactic acid concentration in the culture medium, but as the lactic acid concentration increases, deterioration gradually progresses faster, and when the deterioration index exceeds a threshold, the detection sensitivity of the enzyme sensor 124 decreases and a detection value lower than the actual concentration is output. The cell culture system 22 detects the life of the enzyme sensor 124 and displays a message on the touch panel 134 urging replacement, thereby enabling appropriate management of the culture process (S40).

[0077] Here, the method of determining the threshold value in the life determination step (S78) will be described with reference to FIG. 8A, taking the measurement of lactate concentration as an example. The threshold value is determined experimentally in advance using the enzyme sensor 124 to be used. As shown in the example, test solutions with lactate concentrations of 2, 8, 16, and 20 mM (millimola: 0.001 mol / L) are prepared. Then, while each test solution is flowed through the enzyme sensor 124, the detection value of the enzyme sensor 124 is measured at regular intervals. As shown in the figure, the detection sensitivity of the enzyme sensor 124 tends to start to decrease after a certain time has elapsed, but the higher the lactate concentration, the earlier the detection sensitivity starts to decrease. In this embodiment, based on the above experimental results, the threshold value is determined based on the lactate concentration and the time it takes for the detection sensitivity to fall below an acceptable detection sensitivity (for example, 80% of the initial value). For example, when the lactate concentration is 2 mM and the detection sensitivity falls below the acceptable detection sensitivity on the 18th day from the start of measurement, the threshold value is calculated as 864 mM·hr by 2 mM×18×24 hr.

[0078] Next, the operation of the cell culture system 22 when replacing the enzyme sensor 124 in the culture step (S40) will be described. In this case, the user issues an instruction to perform a sensor replacement operation through the touch panel 134. This causes the cell culture system 22 to close the sub-clamp 34e in FIG. 7 to stop the flow of culture medium to the enzyme sensor 124. This allows the user to perform the operation of connecting a new enzyme sensor 124 using a sterile joining device in the manner described with reference to FIG. 5B. After replacing the enzyme sensor 124, the user issues an instruction to return to the normal flow step through the touch panel 134, thereby returning to the operation of the normal flow step in FIG. 7.

[0079] The cell culture system 22 judges whether or not to end the culture step (S40). The control unit 136 ends the culture step (S40) if the duration of the culture step (S40) exceeds a predetermined threshold. Note that the judgment of the end of the culture step (S40) is not limited to this, and for example, the culture step (S40) may be ended when the number of cells calculated from the detection result of the culture parameter detection unit 122 becomes equal to or greater than a predetermined value.

[0080] In a detachment step (S50) after the culture step (S40), a detachment liquid is supplied from the IC route 44A to detach the cells cultured (grown) in the bioreactor 20. In a recovery step (S60) after the detachment step (S50), a culture medium is supplied to the IC route 44A, causing the cells detached in the detachment step (S50) to flow out of the bioreactor 20 and be guided to a recovery bag 18E. At this time, the culture medium and gas components are also supplied to the EC route 44B.

[0081] Through the above steps, the cell culture system 22 can effectively store the cells cultured in the bioreactor 20 in the collection bag 18E. In particular, the cell culture system 22 notifies the timing of replacing the enzyme sensor 124 in the culture step (S40), thereby preventing errors in the concentration detection value due to deterioration of the enzyme sensor 124. As a result, cell proliferation can be stably managed, and the amount of culture medium used can be reduced according to the proliferation of the cells.

[0082] The present invention is not limited to the above-mentioned embodiment, and various modifications are possible in accordance with the gist of the invention. For example, the cell culture system 22 is configured to include a flexible cassette body 40 (flow path cassette 10) in the cell culture kit 12, but the cell culture kit 12 may not include the flow path cassette 10, and a rigid flow path cassette 10 may be applied.

[0083] Furthermore, the application of the method for determining the life span of the enzyme sensor 124 according to the present invention is not limited to the cell culture system 22, but may be applied to various systems that measure substrate concentrations using the enzyme sensor 124. For example, the method for determining the life span of the enzyme sensor 124 according to the present invention may be applied to a blood processing system that processes blood.

[0084] Second Embodiment 9, the cell culture system 22A according to the second embodiment differs from the cell culture system 22 according to the first embodiment in that a branch path 46B3 is provided in the EC outflow path 46B2, and a culture parameter detection unit 122 (enzyme sensor 124) is arranged in the branch path 46B3. The culture parameter detection unit 122 (enzyme sensor 124) is not provided in the EC outflow path 46B2. In the cell culture system 22A, the configurations of the IC route 44A (IC supply circuit 45A, IC circulation circuit 46A) and the EC supply circuit 45B are the same as those of the above-mentioned cell culture system 22, and detailed description thereof will be omitted.

[0085] Specifically, one end of the branch path 46B3 is connected to a connection point β where the EC outflow path 46B2 and the bypass path 47 are connected, and the other end of the branch path 46B3 is connected to the EC outflow path 46B2 downstream of the secondary clamp 34e. The branch path 46B3 is formed of a tube 16 (not shown).

[0086] A switching clamp 34g capable of opening and closing the branch path 46B3 is provided in the vicinity of the connection point β of the branch path 46B3. Note that the main clamp 34d, the sub-clamp 34e, and the sub-clamp 34f are provided in the same positions as in the first embodiment. In addition, the culture parameter detection unit 122 is provided in series on the branch path 46B3 downstream of the switching clamp 34g.

[0087] The cell culture system 22A according to the second embodiment is basically configured as described above, and hereinafter, replacement of the enzyme sensor 124 during the culture process of this cell culture system 22A will be described.

[0088] In the cell culture system 22A, in a normal flow step of the culture process, the control unit 136A opens the main clamp 34d, the sub clamp 34e, and the switching clamp 34g, while closing the sub clamp 34f. As a result, in the EC route 44B, under the driving of the third pump 30c, the culture medium flows from the EC inflow path 46B1 into the main space 26a of the bioreactor 20, and flows out from the bioreactor 20 to the EC outflow path 46B2. In the process of flowing through the EC outflow path 46B2, the culture medium passes through the parameter detection unit 110 (culture parameter detection unit 122) provided in the cassette body 40. Since the sub clamp 34e and the switching clamp 34g are open, the culture medium flows through both the EC outflow path 46B2 and the branch path 46B3.

[0089] The amount of glucose or lactic acid in the medium flowing through the branch path 46B3 is detected by the culture parameter detection unit 122 (enzyme sensor 124). The control unit 136A controls the supply amount (supply rate) of the medium based on the detection result. Note that, in the normal flow step, the sub-clamp 34e may be closed to guide all of the medium flowing out from the bioreactor 20 to the branch path 46B3.

[0090] When replacing the enzyme sensor 124, the control unit 136A opens the main clamp 34d and the sub clamp 34e, while closing the sub clamp 34f and the switching clamp 34g. As a result, in the EC circulation circuit 46B, the medium flowing through the EC outflow path 46B2 does not flow through the branch path 46B3 when the third pump 30c is driven. Since the medium does not flow through the branch path 46B3, the deteriorated enzyme sensor 124 is replaced with a new enzyme sensor 124 using a sterile joining device in the manner described with reference to FIG. 5B.

[0091] After replacing the enzyme sensor 124, the control unit 136A continues to measure the concentration of the predetermined component by opening the switching clamp 34g to allow the medium to flow into the branch path 46B3. In this way, in the cell culture system 22A, the enzyme sensor 124 can be replaced without stopping the circulation of the medium in the EC circulation circuit 46B.

[0092] Third Embodiment The cell culture system 22B according to the third embodiment differs from the cell culture system 22A according to the second embodiment in that a culture parameter detector 122 (enzyme sensor 124) is disposed in each of the EC outflow path 46B2 and the branch path 46B3, as shown in Fig. 10. Note that in the cell culture system 22B, the configurations of the EC outflow path 46B2 and the branch path 46B3, as well as the sub clamp 34e and the switching clamp 34g provided thereon are the same as those in the above-described cell culture system 22A, and detailed description thereof will be omitted.

[0093] In the cell culture system 22B, the enzyme sensors 124 are attached in parallel to the EC outflow path 46B2 and the branch path 46B3. At the beginning of the culture step (S40), only one of the enzyme sensors 124 is used, and after the enzyme sensor 124 deteriorates, it is possible to switch to the other enzyme sensor 124.

[0094] The cell culture system 22B according to the third embodiment is basically configured as described above, and the switching operation of the enzyme sensor 124 in the culture step (S40) of this cell culture system 22B will be described below.

[0095] In the cell culture system 22B, in the normal flow step of the culture process, the control unit 136B opens the main clamp 34d and the sub clamp 34e, while closing the switching clamp 34g and the sub clamp 34f. This allows the culture medium to flow through the EC outflow path 46B2, but not into the branch path 46B3. The enzyme sensor 124 in the branch path 46B3 is kept out of contact with the predetermined components of the culture medium, preventing deterioration.

[0096] During the normal circulation step, the control unit 136B performs a switching determination process for the enzyme sensor 124, which is made up of steps S72 to S100 shown in Fig. 11. In the switching determination process in Fig. 11, the initialization (step S72) to lifespan determination (step S78) and culture end determination (step S82) are similar to the lifespan determination process in Fig. 6B, and therefore the description thereof will be omitted.

[0097] In the lifespan determination process (step S78), if the lifespan determination unit 140 detects that the deterioration index of the enzyme sensor 124 exceeds the threshold, a sensor flow path switching process (step S100) is performed. In the sensor flow path switching process (step S100), the control unit 136B closes the sub-clamp 34e and opens the switching clamp 34g, as shown in Fig. 12, to allow the medium to flow into the branch path 46B3. This allows the undeteriorated enzyme sensor 124 to continue measuring the concentration of a predetermined component in the medium.

[0098] In addition, the medium does not flow into the EC outflow path 46B2 due to the blocking of the sub clamp 34e. Therefore, while the concentration is being measured by the enzyme sensor 124 on the branch path 46B3 side, the enzyme sensor 124 on the EC outflow path 46B2 side may be replaced in preparation for the next sensor switching operation.

[0099] Thereafter, in order to perform a life determination of the new enzyme sensor 124 (branch path 46B3), the process proceeds to step S72 to perform a life determination of the enzyme sensor 124. As described above, according to the cell culture system 22B of the present embodiment, the concentration measurement of the medium can be continued without reconnecting the enzyme sensor 124, thereby enabling labor saving.

[0100] In the above description, one branch path 46B3 is provided in parallel to the EC outflow path 46B2, but the present embodiment is not limited to this. A plurality of branch paths 46B3 each equipped with the enzyme sensor 124 may be provided to protect against deterioration of the enzyme sensor 124.

[0101] The technical ideas and effects that can be understood from the above-described embodiments will be described below.

[0102] The first aspect is a cell culture system 22, 22A, 22B that includes an enzyme sensor 124 that detects the concentration of a specific component (substrate) in a culture medium used for cell culture, a sensor flow path (EC outflow path 46B2) in which the enzyme sensor 124 is provided, and a bioreactor 20 that cultures cells while flowing the culture medium through the sensor flow path, and the cell culture system 22, 22A, 22B has a deterioration index calculation unit 138 that calculates a deterioration index of the enzyme sensor 124 based on the concentration detection value and the detection period of the concentration detection value of the enzyme sensor 124, and a life determination unit 140 that detects whether the accumulated value of the deterioration index has reached a threshold value and determines the life of the enzyme sensor 124.

[0103] According to the above configuration, even if the concentration of a specific component (substrate) in the culture medium varies greatly between the early stage and the late stage of the culture, it is possible to grasp the lifespan of the enzyme sensor 124 by reflecting the progress of deterioration due to an increase in the substrate concentration.

[0104] In the cell culture systems 22, 22A, and 22B of the above-mentioned aspects, the enzyme sensor 124 may detect a glucose concentration or a lactate concentration. This allows the enzyme sensor 124 to be used with excellent selectivity for measuring the glucose concentration or the lactate concentration, and allows the culture operation of the cell culture systems 22, 22A, and 22B to be more appropriately managed.

[0105] The cell culture systems 22, 22A, and 22B of the above-mentioned aspects may have a touch panel 134 (display operation unit) that displays the concentration of a predetermined component, and the touch panel 134 may be configured to display a message urging replacement of the enzyme sensor 124 when the life determination unit 140 determines that the enzyme sensor 124 has reached the end of its life. This allows replacement of the enzyme sensor 124 at an appropriate time without being confused by fluctuations in the detection value.

[0106] The cell culture system 22A from the above viewpoint may be configured to include an EC outflow path 46B2 provided in the branch path 46B3 (sensor flow path) and a switching clamp 34g capable of blocking the bioreactor 20 and the branch path 46B3, and the control unit 136 may be configured to close the switching clamp 34g when replacing the enzyme sensor 124. By configuring in this way, the enzyme sensor 124 can be replaced without stopping the circulation of the culture medium.

[0107] The cell culture system 22B of the above aspect may be provided with a plurality of sensor flow paths (EC outflow path 46B2 and branch path 46B3) arranged in parallel, each of which is provided with an enzyme sensor 124, clamps (secondary clamp 34e and switching clamp 34g) that switch and connect one of the plurality of sensor flow paths to the bioreactor 20, and a control unit 136B that operates the clamps (secondary clamp 34e and switching clamp 34g) to switch and connect to another sensor flow path (EC outflow path 46B2 and branch path 46B3) when the life determination unit 140 detects that the deterioration index of one enzyme sensor 124 has reached a threshold value. With this configuration, the enzyme sensor 124 can be switched to a new enzyme sensor 124 without replacement work, thereby saving labor.

[0108] The second aspect is a method for determining the lifespan of the enzyme sensor 124 in the cell culture system 22, 22A, 22B in which cells are cultured while detecting the concentration of a predetermined component of the culture medium using the enzyme sensor 124, the method comprising: a step (S74) in which the control unit 136 detects the concentration detection value and the detection period of the concentration detection value of the enzyme sensor 124; a deterioration index detection step (S76) in which the deterioration index calculation unit 138 calculates an integrated value of the deterioration index of the enzyme sensor 124 based on the concentration detection value and the detection period of the enzyme sensor 124; and a lifespan determination step (S78) in which the lifespan determination unit 140 determines the end of the lifespan based on whether the deterioration index has reached a threshold value. According to the above-mentioned lifespan determination method, even if the concentration of the predetermined component (substrate) of the culture medium varies greatly between the early stage and the end stage of the culture, it is possible to grasp the lifespan of the enzyme sensor 124 by reflecting the progress of deterioration due to an increase in the substrate concentration.

[0109] The third aspect is a sensor kit for use in cell culture systems 22, 22A, 22B, comprising an enzyme sensor 124 for detecting the concentration of a predetermined component in a culture medium used for cell culture, and a tube 125 (sensor flow path) extending from one end and the other end of the enzyme sensor 124, the tube 125 (sensor flow path) being made of a thermoplastic tube whose both ends are blocked by welded portions 125a and which can be aseptically joined to a flow path (second IC tube 16G or second EC tube 16I) through which the culture medium of the cell culture systems 22, 22A, 22B flows, and the enzyme sensor 124 is configured to detect the concentration of the predetermined component in the culture medium and send the detected value to the cell culture systems 22, 22A, 22B.

[0110] According to the above-mentioned sensor kit, the enzyme sensor 124 can be easily replaced by simply changing the connection of the tube 125, so that the management work of the cell culture systems 22, 22A, and 22B can be reduced.

[0111] [Fourth embodiment] As shown in Fig. 13, the cell culture system 22C according to this embodiment is different from the cell culture system 22 in Fig. 4 in that a temperature sensor 144 and a pH sensor 143 are added to the EC circulation circuit 46B as the culture parameter detection unit 122. In the cell culture system 22C, the influence of the temperature and pH of the EC medium is reflected in the calculation of the deterioration index by the deterioration index calculation unit 138A. In the cell culture system 22C of this embodiment, the same components as those in the cell culture system 22 in Fig. 4 are denoted by the same reference numerals and detailed description thereof will be omitted.

[0112] The temperature sensor 144 is provided in the EC outflow path 46B2, and is a sensor that detects the temperature of the EC medium flowing through the EC circulation circuit 46B and outputs the temperature data to the control unit 136. The installation position of the temperature sensor 144 is not limited to the EC outflow path 46B2, and may be provided at any position in the EC circulation circuit 46B.

[0113] The pH sensor 143 is provided in the EC outflow path 46B2, detects the pH (hydrogen ion concentration) of the EC medium flowing through the EC circulation circuit 46B, and outputs the pH data to the control unit 136. A commercially available pH measuring device using a glass electrode can be used as the pH sensor 143. The installation position of the pH sensor 143 is not limited to the EC outflow path 46B2, and may be provided at any position in the EC circulation circuit 46B.

[0114] 14, the deterioration index calculation unit 138A of the present embodiment includes a calculation unit 146, a correction coefficient determination unit 148, and a storage unit 150. The calculation unit 146 calculates a deterioration index based on concentration data, which is a detection value of the enzyme sensor 124, a pH correction coefficient, and a temperature correction coefficient.

[0115] The correction coefficient determination unit 148 determines a pH correction coefficient based on the pH data of the culture medium detected by the pH sensor 143. The correction coefficient determination unit 148 also determines a temperature correction coefficient based on the temperature data of the culture medium detected by the temperature sensor 144.

[0116] 15A and 15B show the stability of the enzyme against pH, with the pH of the culture medium on the horizontal axis and the measurement results of the enzyme activity (residual activity value) of the enzyme sensor 124 after contact with the culture medium for a certain period of time on the vertical axis. FIG. 15A shows the residual stability of the enzyme of the enzyme sensor 124 (lactate sensor) that detects the lactate concentration. As shown in the figure, the stability of the enzyme of the lactate sensor decreases when the pH of the culture medium falls below 8, and the lower the pH value, the faster the degradation progresses. Therefore, it can be seen that the degree of degradation of the enzyme sensor 124 (lactate sensor) varies with the pH.

[0117] 15B, in the case of an enzyme sensor 124 (glucose sensor) that detects a glucose concentration, the stability of the enzyme decreases when the pH is outside the range of 5 to 8. The degree of deterioration of the enzyme in the glucose sensor also changes according to the pH. In this embodiment, the degree of deterioration of the enzyme sensor 124, such as the lactate sensor or glucose sensor, depending on the pH is reflected in the calculation of the deterioration index as a pH correction coefficient.

[0118] 14 stores a pH correction coefficient map that indicates the relationship between the pH value of the culture medium and the pH correction coefficient of the enzyme sensor 124. The pH correction coefficient is a numerical value, for example, from 0.1 to 2, and is an experimentally determined value that increases as the degree of deterioration (deterioration rate) of the enzyme sensor 124 increases. The correction coefficient determination unit 148 determines the pH correction coefficient by referring to the pH correction coefficient map in the memory unit 150 based on the pH data, which is the detection value of the pH sensor 143.

[0119] On the other hand, the stability of the enzyme sensor 124 also changes depending on the temperature of the culture medium. Figures 16A and 16B show the stability of the enzyme, with the temperature of the culture medium on the horizontal axis and the residual activity value of the enzyme used in the enzyme sensor 124 after a certain time has passed on the vertical axis. As shown in Figure 16A, in the case of the enzyme sensor 124 (lactate sensor), the residual activity (stability) changes depending on the culture medium temperature, and it can be seen that degradation progresses rapidly especially when the temperature exceeds 50°C. Also, as shown in Figure 16B, in the case of the enzyme sensor 124 (glucose sensor), it can be seen that degradation also progresses rapidly when the culture medium temperature exceeds 50°C.

[0120] 14 stores a temperature correction coefficient map showing the relationship between the temperature of the culture medium and the temperature correction coefficient of the enzyme sensor 124. The temperature correction coefficient is a numerical value, for example, from 0.1 to 2, and an experimentally determined value is used so that the higher the degree of deterioration (deterioration rate) of the enzyme sensor 124, the higher the value becomes. The correction coefficient determination unit 148 determines the temperature correction coefficient by referring to the temperature correction coefficient map in the storage unit 150 based on the temperature data, which is the detection value of the temperature sensor 144.

[0121] The deterioration index calculation section 138A calculates the deterioration index in the arithmetic section 146 based on the concentration data, which is the detection value of the enzyme sensor 124, the pH correction coefficient, and the temperature correction coefficient, using the following formula. Deterioration index = (actual time x pH correction factor x temperature correction factor) x concentration data In the above formula, the actual time can be the difference between the previous concentration, pH, and temperature measurement time and the current concentration, pH, and temperature measurement time.

[0122] The cell culture system 22C of this embodiment is configured as described above, and basically performs the same operations as those described with reference to Figures 6A and 6B. However, in the deterioration index update (step S76) of Figure 6B, the deterioration index calculation unit 138A calculates a deterioration index based on a pH correction coefficient and a temperature correction coefficient, and updates the deterioration index.

[0123] That is, the deterioration index calculation unit 138A acquires pH data in the correction coefficient determination unit 148, and determines a pH correction coefficient according to the pH value by referring to the pH correction coefficient map in the storage unit 150. The correction coefficient determination unit 148 also acquires temperature data, and determines a temperature correction coefficient by referring to the temperature correction coefficient map in the storage unit 150. Thereafter, the deterioration index calculation unit 138A multiplies the time, the pH correction coefficient, the temperature correction coefficient, and the concentration data in the calculation unit 146 to calculate a deterioration index. The deterioration index is added to the accumulated value of the past deterioration index, thereby updating the accumulated value of the deterioration index.

[0124] As described above, the cell culture system 22C of this embodiment determines the lifespan of the enzyme sensor 124 by taking into consideration the pH and temperature of the medium in addition to the concentration and its detection period. This allows the lifespan of the enzyme sensor 124 to be determined more accurately.

[0125] Furthermore, based on this embodiment, the sensor kit may be configured as a sensor kit provided with a pH sensor 143 for detecting the hydrogen ion concentration (pH) of the culture medium together with the enzyme sensor 124. With this configuration, the pH sensor 143 can be replaced together with the enzyme sensor 124, and the management work of the cell culture systems 22, 22A, and 22B can be reduced.

[0126] In the above, the present invention has been described by citing preferred embodiments, but the present invention is not limited to the above embodiments, and it goes without saying that various modifications are possible without departing from the spirit of the present invention.

Claims

1. an enzyme sensor for detecting the concentration of a predetermined component in a medium used for cell culture; a sensor flow path in which the enzyme sensor is provided; A cell culture system comprising: a bioreactor that cultures cells while flowing the culture medium through the sensor flow path, a deterioration index calculation unit for calculating a deterioration index of the enzyme sensor; A lifespan determination unit that determines the lifespan of the enzyme sensor, the deterioration index calculation unit calculates a concentration-time value by multiplying the elapsed time from the previous concentration measurement timing to the current concentration measurement timing by the enzyme sensor and the concentration detection value at the current concentration measurement timing, and updates the deterioration index by adding the current concentration-time value to an integrated value of the concentration-time values ​​up to the previous time, A cell culture system, wherein the life determination unit determines the life of the enzyme sensor based on whether the updated deterioration index exceeds a predetermined threshold.

2. 2. The cell culture system according to claim 1, wherein the enzyme sensor is a sensor for detecting a glucose concentration or a lactate concentration.

3. The cell culture system according to claim 1 or 2, A display operation unit that displays the concentration of the predetermined component, The display operation unit displays a message encouraging replacement of the enzyme sensor when the life determination unit determines that the life of the enzyme sensor has arrived.

4. 2. The cell culture system according to claim 1, a flow path provided in parallel with the sensor flow path; a switching clamp capable of closing the sensor flow path; A control unit that closes the switching clamp when the enzyme sensor is replaced.

5. 2. The cell culture system according to claim 1, A plurality of the sensor channels are arranged in parallel, a clamp that selectively connects any one of the plurality of sensor flow paths to the bioreactor; A cell culture system comprising: a control unit that, when the life determination unit detects that the deterioration index of one enzyme sensor has reached a threshold value, operates the clamp to switch and connect to another sensor flow path.

6. An enzyme sensor for detecting the concentration of a predetermined component in a medium used for cell culture; a sensor flow path in which the enzyme sensor is provided; A cell culture system comprising: a bioreactor that cultures cells while flowing the culture medium through the sensor flow path, a deterioration index calculation unit for calculating a deterioration index of the enzyme sensor; A lifespan determination unit that determines the lifespan of the enzyme sensor; A pH sensor for detecting a hydrogen ion concentration (pH) of the culture medium; A memory unit storing a pH correction coefficient map showing the relationship between hydrogen ion concentration and the deterioration rate of the enzyme sensor; a correction coefficient determination unit that determines a pH correction coefficient by referring to the pH correction coefficient map based on the hydrogen ion concentration of the culture medium detected by the pH sensor; the deterioration index calculation unit calculates the deterioration index of the enzyme sensor based on the elapsed time, which is the time interval between multiple concentration measurements by the enzyme sensor, the concentration detection value of the enzyme sensor, and the pH correction coefficient, and the life determination unit determines the life of the enzyme sensor based on whether the deterioration index exceeds a predetermined threshold.

7. An enzyme sensor for detecting the concentration of a predetermined component in a medium used for cell culture; a sensor flow path in which the enzyme sensor is provided; A cell culture system comprising: a bioreactor that cultures cells while flowing the culture medium through the sensor flow path, a deterioration index calculation unit for calculating a deterioration index of the enzyme sensor; A lifespan determination unit that determines the lifespan of the enzyme sensor; A temperature sensor for detecting the temperature of the culture medium; A memory unit storing a temperature correction coefficient map showing the relationship between temperature and the deterioration rate of the enzyme sensor; a correction coefficient determination unit that determines a temperature correction coefficient by referring to the temperature correction coefficient map based on the temperature of the culture medium detected by the temperature sensor; the deterioration index calculation unit calculates the deterioration index of the enzyme sensor based on the elapsed time, which is the time interval between multiple concentration measurements by the enzyme sensor, the concentration detection value of the enzyme sensor, and the temperature correction coefficient, and the life determination unit determines the life of the enzyme sensor based on whether the deterioration index exceeds a predetermined threshold.

8. A method for determining the lifespan of an enzyme sensor in a cell culture system in which cells are cultured while detecting the concentration of a predetermined component in a culture medium using the enzyme sensor, comprising: A step in which a control unit detects a concentration detection value of the enzyme sensor and an elapsed time from a previous concentration measurement timing to a current concentration measurement timing by the enzyme sensor; a deterioration index detection step of determining a deterioration index of the enzyme sensor; a lifespan determination step in which a lifespan determination unit determines the lifespan of the enzyme sensor; having In the deterioration index detection step, the deterioration index calculation unit calculates a concentration-time value by multiplying the elapsed time by the concentration detection value at the current concentration measurement timing, and updates the deterioration index by adding the current concentration-time value to an integrated value of the concentration-time values ​​up to the previous time, A method for determining a lifespan of an enzyme sensor, wherein in the lifespan determination step, the lifespan determination unit determines the lifespan of the enzyme sensor based on whether or not the updated deterioration index exceeds a predetermined threshold value.

9. A method for determining the life span of an enzyme sensor in a cell culture system in which cells are cultured while detecting the concentration of a predetermined component in a culture medium using the enzyme sensor, comprising: A step in which a control unit detects a concentration detection value of the enzyme sensor and an elapsed time which is a time interval between multiple concentration measurement timings by the enzyme sensor; a deterioration index detection step of determining a deterioration index of the enzyme sensor; a lifespan determination step in which a lifespan determination unit determines the lifespan of the enzyme sensor; having The deterioration index detection step is a method for determining the lifespan of an enzyme sensor, in which a deterioration index calculation unit calculates the deterioration index by multiplying a pH correction coefficient based on the pH of the culture medium and a temperature correction coefficient based on the temperature of the culture medium by the concentration detection value of the enzyme sensor and the elapsed time, and the lifespan determination unit determines the lifespan of the enzyme sensor based on whether the deterioration index exceeds a predetermined threshold value.

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