Cell Culture Systems

Abstract

The cell culture system (10) includes a reactor (12), a flow path (16) through which a culture medium flows in and out of the reactor (12), an optical measurement unit (54) that optically measures cells contained in the culture medium, and a control unit (32) that processes the measurement results of the optical measurement unit (54). The optical measurement unit (54) includes a first measurement unit (56) that performs optical measurement based on emission of a first measurement light (L1) and a second measurement unit (58) that performs optical measurement based on emission of a second measurement light (L2), which are located at different positions in the culture medium flow direction. A first optical path length (OP1) of the first measurement light (L1) is set longer than a second optical path length (OP2) of the second measurement light (L2).

Description

[Technical Field]

[0001] The present invention relates to a cell culture system for measuring the number of cells in a liquid flowing out of a reactor. [Background technology]

[0002] In regenerative medicine, cells from a living body are collected, cultured, and then administered to a patient. For example, a cell culture system using a cell culture vessel (reactor) with hollow fibers inside a case is used in the cell culture process, as disclosed in Japanese Patent Application Laid-Open No. 2017-143775. This cell culture system cultures the cells by seeding cells into the hollow fibers of the reactor and then feeding a culture medium into the reactor through a distribution channel connected to the reactor.

[0003] In this type of cell culture system, it is important to accurately monitor the number of cells grown during cultivation to understand the cell culture status and determine the timing of cell harvest. While it is common to aseptically collect the medium inside the cell culture system through a sterile filter, cells cannot pass through a sterile filter. Therefore, conventional methods involve aseptically collecting the medium and measuring the concentrations of cell metabolic substances, such as lactate, glucose, and oxygen, in the medium to indirectly calculate the number of grown cells. Another method for aseptically collecting cells directly involves sealing a certain length of the circuit tubing of the cell culture system and collecting a piece of tubing containing cell-containing medium. The disconnected circuit of the cell culture system is then reconnected using a sterile connector. Summary of the Invention

[0004] However, when the cell number is calculated indirectly as described above, changes in the culture environment cause changes in cell metabolism, making it impossible to accurately count the cell number. Furthermore, when sampling a tube containing the cell-containing medium as described above, the procedure becomes complicated, and there are problems such as temporary interruption of the culture operation and loss of cells during sampling.

[0005] The present invention has been made in consideration of the above-mentioned problems, and aims to provide a cell culture system that can continuously measure the number of cells in a reactor while ensuring sterility and that can obtain sufficient measurement accuracy.

[0006] In order to achieve the above-mentioned object, the cell culture system of the present invention comprises a reactor for culturing cells based on the flow of a culture medium, a flow path through which the culture medium flows in and out of the reactor, an optical measurement unit that is provided on the flow path downstream of the reactor in the flow direction of the culture medium and that irradiates measurement light onto the culture medium to optically measure the state of the culture medium, and a processing unit that processes the measurement results of the optical measurement unit, wherein the optical measurement unit has a first measurement unit and a second measurement unit that are arranged at different positions in the flow direction of the culture medium, the first measurement unit has a first optical path portion through which the culture medium flows and through which the first measurement light passes, and the second measurement unit has a second optical path portion through which the culture medium flows and through which the second measurement light passes, and the first optical path length of the first optical path portion is longer than the second optical path length of the second optical path portion.

[0007] The above cell culture system makes it possible to continuously measure the number of cells in the reactor while ensuring sterility, and also provides sufficient measurement accuracy. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a perspective view showing the overall configuration of a cell culture system according to one embodiment of the present invention. [Figure 2] FIG. 10 is a circuit diagram showing a flow path between a culture medium reservoir and a reactor, and a flow path control mechanism. [Figure 3] Fig. 3A is a perspective view showing a measurement container of an optical measurement unit, Fig. 3B is a cross-sectional view taken along line IIIB-IIIB in Fig. 3A, and Fig. 3C is a cross-sectional view taken along line IIIC-IIIC in Fig. 3A. [Figure 4] FIG. 10 is a partial cross-sectional explanatory view schematically showing the optical measurement unit during culture medium flow. [Figure 5] 10 is a flowchart showing the operation of a control unit. [Figure 6] FIG. 10 is a partial cross-sectional explanatory view schematically showing an optical measurement unit according to a modified example. DETAILED DESCRIPTION OF THE INVENTION

[0009] 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.

[0010] As shown in Fig. 1, a cell culture system 10 according to one embodiment of the present invention is configured as a stationary device installed in a sterile room or the like, and performs a culture process for culturing living cells in regenerative medicine. To this end, the cell culture system 10 includes a reactor 12, which is a cell culture container. The cell culture system 10 continues cell culture for an extended period of time by supplying culture medium and oxygen to the reactor 12 while discharging lactic acid, carbon dioxide, and the like (including unused culture medium and oxygen) generated during cell culture from the reactor 12.

[0011] The biological cells are not particularly limited, but examples thereof include cells contained in blood (T cells, etc.) and stem cells (ES cells, iPS cells, mesenchymal stem cells, etc.). The medium may also be selected appropriately depending on the biological cells, and examples thereof include those prepared by adding various amino acids, vitamins, serum, etc. to a balanced salt solution (BSS) as a base solution.

[0012] The cell culture system 10 also has a culture medium reservoir 14 that stores a culture medium, a distribution channel 16 provided between the reactor 12 and the culture medium reservoir 14, a plurality of medical bags 18 connected to the distribution channel 16, and a waste liquid section 20 that stores liquid discharged from the distribution channel 16. Although Fig. 1 illustrates the cell culture system 10 having one reactor 12, the cell culture system 10 may also have a configuration including a plurality of reactors 12.

[0013] A rigid tank capable of storing a large amount of culture medium is used as the culture medium reservoir 14 in order to supply the culture medium to the reactor 12. For example, the tank preferably has a volume of about 5 L to 30 L, which reduces the workload of frequently replacing the culture medium reservoir 14 during the culture treatment. Alternatively, a flexible medical bag or the like may be used as the culture medium reservoir 14.

[0014] The flow path 16 is composed of a plurality of tubes 22 (only the tubes 22 connected to the culture medium reservoir 14 are shown in FIG. 1). The plurality of tubes 22 are connected to the culture medium reservoir 14 and a plurality of medical bags 18, and are also connected to a plurality of reactors 12. The cell culture system 10 supplies and discharges the culture medium in the culture medium reservoir 14 and the liquid in the medical bags 18 to and from the reactor 12 via the plurality of tubes 22.

[0015] The multiple medical bags 18 include, for example, a cell fluid bag 18A that stores a fluid containing cells (cell fluid), a cleaning fluid bag 18B that stores a cleaning fluid, a detachment fluid bag 18C that stores a detachment fluid, and a recovery bag (not shown) that recovers the cultured cells. The cleaning fluid is a liquid used when priming the reactor 12 and the distribution channel 16. Examples of this cleaning fluid include buffer solutions such as PBS (Phosphate Buffered Salts) and TBS (Tris-Buffered Saline), or physiological saline. The detachment fluid is a liquid that detaches cells cultured by a culture process. Examples of the detachment fluid that can be used include trypsin and EDTA solution.

[0016] When the cell culture system 10 is constructed, the flow path 16 is set to pass through a flow path control mechanism 24. The flow path control mechanism 24 includes a housing 26 that houses a portion of the flow path 16. The flow path control mechanism 24 also includes, within the housing 26, a clamp 28 that opens and closes a predetermined tube 22, a pump 30 that circulates the liquid in the tube 22, and a control unit 32 (processing unit) that controls the operation of the clamp 28 and the pump 30 (see FIG. 2). That is, within the housing 26, the flow path control mechanism 24 selectively switches the tube 22 through which the liquid flows by opening and closing the clamp 28, while circulating the liquid in the flow path 16 under the operation of the pump 30.

[0017] In addition to the plurality of tubes 22, the flow path 16 may also include a cassette (not shown) having a plurality of liquid flow paths and to which some of the tubes 22 are connected. In this case, the cassette is placed in a clamp 28 of the flow path control mechanism 24 when set in the housing 26, and the clamp 28 opens, closes, switches, etc. the flow paths in the cassette.

[0018] In order to ensure a large culture area, the reactor 12 connected to the flow path 16 preferably has a structure including, for example, hollow fibers 34. Specifically, the reactor 12 includes a plurality of hollow fibers 34 (for example, 10,000 or more) and a case 36 that houses the plurality of hollow fibers 34 along the axial direction.

[0019] Each hollow fiber 34 has a lumen (not shown) that penetrates along the extension direction, and cells are seeded on the inner circumferential surface that constitutes the lumen. Each hollow fiber 34 also has a plurality of pores (not shown) that communicate between the outside and the lumen, and each pore is impermeable to cells and proteins but permeable to solutions and low-molecular-weight substances. Therefore, culture medium, predetermined gas components, etc. are supplied to the cells on the inner circumferential surface of the hollow fiber 34 through the pores. Hereinafter, a configuration in which liquid is circulated mainly through the lumen of the hollow fiber 34 will be referred to as an IC (intra capillary), and a configuration in which liquid is circulated mainly outside the hollow fiber 34 will be referred to as an EC (extra capillary).

[0020] The material constituting the hollow fibers 34 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] The case 36 is cylindrical and rigid. The case 36 includes a first IC terminal 36a, a second IC terminal 36b, a first EC terminal 36c, and a second EC terminal 36d, which are connected to the plurality of tubes 22. The first IC terminal 36a is provided at one axial end of the case 36 and is connected to the lumen of the hollow fibers 34. The second IC terminal 36b is provided at the other axial end of the case 36 and is connected to the lumen of the hollow fibers 34. The first EC terminal 36c is provided near the other end of a side surface of the case 36 and is connected to the space outside the hollow fibers 34 within the case 36. The second EC terminal 36d is provided near one end of a side surface of the case 36 and is connected to the space outside the hollow fibers 34 within the case 36.

[0022] Hereinafter, the configuration of the flow path 16 between the reactor 12 and the culture medium reservoir 14, and the flow path control mechanism 24 will be specifically described with reference to FIG.

[0023] The flow path 16 has a culture medium delivery route 40 connected to the culture medium reservoir 14, and an IC route 42 (internal route) and an EC route 44 (external route) branching off from the culture medium delivery route 40. The IC route 42 is a route for supplying liquid to the lumen of the hollow fibers 34. The EC route 44 is a route for supplying liquid to the inside of the case 36 outside the hollow fibers 34.

[0024] The culture medium delivery route 40 is provided with a first clamp 40a that opens or closes the supply of culture medium from the culture medium reservoir 14.

[0025] The IC route 42 includes an IC circulation circuit 42a capable of circulating liquid between the reactor 12 and the IC route 42, and an IC supply circuit 42b capable of distributing liquid from the culture medium delivery route 40 to the IC circulation circuit 42a. The IC circulation circuit 42a is provided with an IC circulation pump 30a for circulating the liquid. The IC supply circuit 42b is provided with an IC supply pump 30b for distributing liquid from the culture medium delivery route 40 to the IC circulation circuit 42a. Although not shown, in addition to the culture medium reservoir 14, the IC supply circuit 42b is also connected to multiple medical bags 18 (cell solution bag 18A, cleaning solution bag 18B, detachment solution bag 18C) via multiple tubes 22. The medical bag 18 may be replaced with a collection bag or the like using a sterile joining device that aseptically joins bags depending on the application.

[0026] The IC circulation circuit 42a is connected to the first IC terminal 36a and the second IC terminal 36b of the reactor 12. Therefore, the liquid circulating through the IC circulation circuit 42a flows through the lumen of the hollow fibers 34 under the operation of the IC circulation pump 30a. An IC waste liquid circuit 46 is connected to the IC circulation circuit 42a downstream of the reactor 12. A second clamp 46a is provided in the IC waste liquid circuit 46 to open or close the discharge of liquid from the IC circulation circuit 42a. The IC waste liquid circuit 46 is connected to a confluence route 50, and the culture medium that has flowed through the IC waste liquid circuit 46 is discharged to the waste liquid section 20 via the tube 22 of the confluence route 50.

[0027] The IC circulation circuit 42a has an optical measurement unit 54 downstream of the reactor 12 in the direction of liquid flow (between the reactor 12 and the IC waste liquid circuit 46). The optical measurement unit 54 performs optical measurement on the culture medium flowing out from the lumen of the hollow fibers 34 and transmits the measurement results to the control unit 32. The control unit 32 calculates the number of cells contained in the culture medium based on the measurement results, thereby managing the number of cells being cultured. The specific configuration of the optical measurement unit 54 will be described in detail later.

[0028] On the other hand, the EC route 44 has an EC circulation circuit 44a that can circulate liquid between the reactor 12 and the EC route 44, and an EC supply circuit 44b that can circulate liquid from the culture medium delivery route 40 to the EC circulation circuit 44a. The EC circulation circuit 44a is provided with an EC circulation pump 30c that circulates the liquid. The EC supply circuit 44b is provided with an EC supply pump 30d that circulates liquid from the culture medium delivery route 40 to the EC circulation circuit 44a. Although not shown, in addition to the culture medium reservoir 14, the EC supply circuit 44b is also connected to a plurality of medical bags 18 (cleaning solution bag 18B, stripping solution bag 18C) via a plurality of tubes 22.

[0029] The EC circulation circuit 44a is connected to the first EC terminal 36c and the second EC terminal 36d of the reactor 12. Therefore, the liquid circulating through the EC circulation circuit 44a flows through the case 36 under the operation of the EC circulation pump 30c. A gas exchanger 52 is provided upstream of the reactor 12 in the EC circulation circuit 44a. The gas exchanger 52 discharges carbon dioxide mixed in the culture medium while mixing predetermined gas components (nitrogen N2: 75%, oxygen O2: 20%, carbon dioxide CO2: 5%) into the culture medium. The structure of the gas exchanger 52 is not particularly limited, and for example, a gas exchanger having a plurality of hollow fibers inside a case, similar to the reactor 12, can be used.

[0030] An EC waste liquid circuit 48 is connected to the EC circulation circuit 44a downstream of the reactor 12. A third clamp 48a that opens or blocks the discharge of liquid from the EC circulation circuit 44a is provided in the EC waste liquid circuit 48. The EC waste liquid circuit 48 is connected to a confluence route 50, and the culture medium that has flowed through the EC waste liquid circuit 48 is discharged to the waste liquid section 20 via the tube 22 of the confluence route 50.

[0031] Furthermore, as described above, when a plurality of (five) reactors 12 are provided, the cell culture system 10 may be configured to include a plurality of IC circulation circuits 42a and EC circulation circuits 44a corresponding to each reactor 12. That is, another IC circulation circuit and an EC circulation circuit (not shown) that circulate liquid to another reactor 12 are connected in parallel to a branch point X between the IC supply pump 30b and the IC circulation circuit 42a, and to a branch point Y between the EC supply pump 30d and the EC circulation circuit 44a.

[0032] Next, the optical measurement unit 54 provided in the IC circulation circuit 42a will be described. The optical measurement unit 54 measures the number of cells contained in the culture medium by a turbidity method downstream of the reactor 12. The optical measurement unit 54 also has a first measurement unit 56 that performs optical measurement based on the emission of the first measurement light L1 and a second measurement unit 58 that performs optical measurement based on the emission of the second measurement light L2, which are located at different positions in the flow direction of the culture medium.

[0033] The flow path 16 has measurement containers 60 arranged in the first measurement unit 56 and the second measurement unit 58 as components of the optical measurement section 54. On the other hand, the flow path control mechanism 24 has a first measurement structure 70 provided in the first measurement unit 56 and a second measurement structure 71 provided in the second measurement unit 58 as components of the optical measurement section 54.

[0034] As shown in Figures 3A to 3C, the measurement container 60 is connected to the tubes 22 (upstream tube 22a, downstream tube 22b) that constitute the IC circulation circuit 42a. The measurement container 60 extends linearly and is formed in a rectangular tube shape that is thicker than the tubes 22. A passage 62 through which the culture medium flows is provided inside the measurement container 60. The passage 62 extends along the axial direction of the measurement container 60 (the direction of arrow A in Figure 3A) and communicates with the flow path in the upstream tube 22a and the flow path in the downstream tube 22b.

[0035] The measurement container 60 is formed to be colorless, transparent, or translucent so that the first measurement light L1 and the second measurement light L2 can pass through. The measurement container 60 is formed to be harder (higher elastic modulus) than the flexible tube 22, and is configured not to be elastically deformed when placed in the optical measurement unit 54. Note that the measurement container 60 may be soft enough to be elastically deformed so as to come into close contact with the first measurement structure 70 and the second measurement structure 71. In this case, the shape of the measurement container 60 is not limited to a rectangular tube, and may be, for example, a cylindrical shape, a disk shape, or the like.

[0036] The measurement container 60 has a plurality of measurement cells (first cell 64, second cell 66) along the axial direction. The first cell 64 is formed in a range from approximately the middle position in the axial direction of the measurement container 60 to one end side (arrow A1 side), and the second cell 66 is formed in a range from approximately the middle position in the axial direction of the measurement container 60 to the other end side (arrow A2 side). The axial length of the first cell 64 and the axial length of the second cell 66 are set to be approximately the same.

[0037] The first cell 64 is formed to have a relatively large flow path cross-sectional area and volume. The first cell 64 has a substantially square shape in a cross section perpendicular to the axial direction of the measurement container 60, and has a thickness T1 along the direction of arrow B in Fig. 3B (thickness direction) and a height H1 along the direction of arrow C in Fig. 3B (height direction) that are substantially equal.

[0038] A first space 64a that is wide in the thickness direction and height direction is formed in the first cell 64 according to the container shape of the first cell 64. The first space 64a constitutes a part of the passage 62 of the measurement container 60.

[0039] One end of the first cell 64 has a cylindrical first cell connecting portion 65a to which the upstream tube 22a is connected, and a first cell tapered portion 65b (end tapered portion) that gradually narrows from the cubic main body portion toward the first cell connecting portion 65a (upstream tube 22a). The first space 64a of the first cell 64 also has a tapered shape inside the first cell tapered portion 65b.

[0040] The second cell 66 is formed to have a smaller flow path cross-sectional area and volume than the first cell 64. For example, the flow path cross-sectional area of ​​the second cell 66 is set to be in the range of approximately 1 / 3 to 2 / 3 of the flow path cross-sectional area of ​​the first cell 64. The second cell 66 has a substantially rectangular shape in a cross section perpendicular to the axial direction of the measurement container 60, and its thickness T2 along the direction of arrow B in FIG. 3C is shorter than the thickness T1 of the first cell 64, while its height H2 along the direction of arrow C in FIG. 3C is equal to the height H1 of the first cell 64. In other words, the cross-sectional shape of the second cell 66 is smaller than the cross-sectional shape of the first cell 64 only in the thickness direction.

[0041] A second space 66a that is short in the thickness direction but wide in the height direction is provided in the second cell 66 according to the container shape of the second cell 66. The second space 66a forms a part of the passage 62 of the measurement container 60.

[0042] The other end of the second cell 66 has a cylindrical second cell connecting portion 67a to which the downstream tube 22b is connected, and a second cell tapered portion 67b (end tapered portion) that gradually narrows from the rectangular main body portion toward the second cell connecting portion 67a (downstream tube 22b). The second space 66a of the second cell 66 also has a tapered shape inside the second cell tapered portion 67b.

[0043] The first cell 64 and the second cell 66 are continuous with each other via a flow path transition portion 68 located at the axial center position of the measurement container 60. A pair of inclined wall portions 69 in the thickness direction of the measurement container 60 that constitute the flow path transition portion 68 are inclined so as to approach each other from the first cell 64 toward the second cell 66. In other words, the thickness (not shown) of the flow path transition portion 68 gradually changes between the thickness T1 of the pair of first cells 64 and the thickness T2 of the second cell 66. The height (not shown) of the flow path transition portion 68 coincides with the height H1 of the first cell 64 and the height H2 of the second cell 66.

[0044] According to the shape of the flow path transition section 68, a transition space 68a that narrows from the first cell 64 side toward the second cell 66 side is provided inside the flow path transition section 68. The transition space 68a forms part of the passage 62 of the measurement container 60.

[0045] 4, the first measurement structure 70 and the second measurement structure 71 are provided with a series of holders 72 that sandwich the measurement container 60 in the thickness direction, as shown in Fig. 4. The holder 72 has an arrangement space 72a extending along the direction of arrow A. The arrangement space 72a is open at both ends of the holder 72 in the direction of arrow A to expose the measurement container 60 (or the tube 22 connected to the measurement container 60).

[0046] The holder 72 has a pair of first holding walls 74 that form the first measurement structure 70 and hold the first cell 64, and a pair of second holding walls 76 that form the second measurement structure 71 and hold the second cell 66.

[0047] The pair of first retaining walls 74 are walls that stand upright in the thickness direction of the arrangement space 72a in the first measurement structure 70. The distance between the pair of first retaining walls 74 is equal to the thickness T1 of the first cell 64. Therefore, the pair of first retaining walls 74 sandwich the first cell 64 with an appropriate frictional force when the first cell 64 is arranged. The first measurement unit 56 forms a first optical path section 75, through which the culture medium flows and through which the first measurement light L1 passes, by the pair of first retaining walls 74 and the first cell 64 arranged between the pair of first retaining walls 74.

[0048] The pair of second retaining walls 76 are walls that stand upright in the thickness direction of the arrangement space 72a in the second measurement structure 71. The distance between the pair of second retaining walls 76 is equal to the thickness T2 of the second cell 66. Therefore, the pair of second retaining walls 76 sandwich the second cell 66 with an appropriate frictional force when the second cell 66 is arranged. The second measurement unit 58 forms a second optical path section 77, through which the culture medium flows and through which the second measurement light L2 passes, by the pair of second retaining walls 76 and the second cell 66 arranged between the pair of second retaining walls 76.

[0049] The first measurement structure 70 has a first light-emitting unit 78 that emits a first measurement light L1 toward the first cell 64, and a first light-receiving unit 80 that receives the light from the first cell 64.

[0050] The first light-emitting unit 78 is provided on one of the pair of first holding walls 74 and faces the arrangement space 72a via a lens (not shown). The first light-emitting unit 78 emits first measurement light L1 having a wavelength of, for example, 660 nm or more, preferably 860 nm or more. The first light-emitting unit 78 is composed of one or more light-emitting elements such as an LED, an organic EL element, an inorganic EL element, or an LD element.

[0051] The first light receiving unit 80 is configured to include one or more transmitted light elements 80a that receive transmitted light and one or more scattered light elements 80b that receive scattered light. The transmitted light elements 80a are provided on the first holding wall 74 directly opposite the first light emitter 78 across the arrangement space 72a. The scattered light elements 80b are provided, for example, on the same side of the first holding wall 74 as the first light emitter 78. The transmitted light elements 80a and scattered light elements 80b are configured using PD, CMOS, etc.

[0052] Similarly, the second measurement structure 71 has a second light-emitting unit 82 that emits a second measurement light L2 toward the second cell 66 and a second light-receiving unit 84 that receives the light from the second cell 66.

[0053] The second light-emitting unit 82 is also configured with a light-emitting element similar to the first light-emitting unit 78, is provided on one of the pair of second holding walls 76, and faces the arrangement space 72a via a lens (not shown). The second light-emitting unit 82 also emits second measurement light L2 having a wavelength of, for example, 660 nm or longer, preferably 860 nm or longer. The wavelength of the second measurement light L2 may be the same as or different from the wavelength of the first measurement light L1.

[0054] Similar to the first light receiving unit 80, the second light receiving unit 84 is configured to include one or more transmitted light elements 84a that receive transmitted light and one or more scattered light elements 84b that receive scattered light. The transmitted light elements 84a are provided on the second holding wall 76 directly opposite the second light emitter 82 with the arrangement space 72a in between. The scattered light elements 84b are provided on, for example, the second holding wall 76 on the same side as the second light emitter 82.

[0055] That is, the optical measurement unit 54 employs a transmitted light / scattered light method for receiving and measuring the transmitted light (light absorbed by the cells contained in the culture medium) from each of the first cell 64 and the second cell 66 and the scattered light (light reflected by the cells contained in the culture medium) from each of the first cell 64 and the second cell 66. The optical measurement unit 54 is not limited to the transmitted light / scattered light method, and may employ a transmitted light method for receiving and measuring only the transmitted light from each of the first cell 64 and the second cell 66. Alternatively, the optical measurement unit 54 may employ a scattered light method for receiving and measuring only the scattered light from each of the first cell 64 and the second cell 66.

[0056] The first measurement structure 70 configured as described above emits a first measurement light L1 to the arranged first cell 64 and receives transmitted light and scattered light from the first cell 64. Therefore, the first optical path section 75 has a first optical path length OP1 along the traveling direction of the first measurement light L1 defined by the spacing between the pair of first holding walls 74. This first optical path length OP1 is equal to the thickness T1 of the first cell 64 sandwiched between the pair of first holding walls 74.

[0057] The second measurement structure 71 also emits a second measurement light L2 to the second cell 66 in the arranged state and receives the transmitted light and scattered light of the second cell 66. Therefore, the second optical path section 77 has a second optical path length OP2 along the traveling direction of the second measurement light L2 defined by the spacing between the pair of second holding walls 76. This second optical path length OP2 coincides with the thickness T2 of the second cell 66 sandwiched between the pair of second holding walls 76.

[0058] In the optical measurement unit 54, the first optical path length OP1 of the first optical path section 75 is set longer than the second optical path length OP2 of the second optical path section 77 (OP1>OP2). The first optical path length OP1 may be set, for example, in the range of 6 mm to 20 mm. The second optical path length OP2 may be set, for example, in the range of 0.5 mm to 3 mm. In other words, the first optical path length OP1 is preferably set in the range of 2 to 40 times.

[0059] The first measurement unit 56 and the second measurement unit 58 are set so that the measurement range of the first light receiving unit 80 that receives the transmitted light or scattered light of the first measurement light L1 is different from the measurement range of the second light receiving unit 84 that receives the transmitted light or scattered light of the second measurement light L2. That is, the first light receiving unit 80 provided in the long first optical path length OP1 corresponds to a state in which there are few cells contained in the culture medium (a state of few cells: a state of low concentration: a state of low turbidity). For example, the measurement range of the cell number of the first light receiving unit 80 is 10 5 ~10 7 On the other hand, the second light receiving unit 84 provided at the second optical path length OP2 corresponds to a state where a large number of cells are contained in the culture medium (a large number of cells: a high concentration state: a high turbidity state). For example, the measurement range of the cell number of the second light receiving unit 84 is 10 6 ~10 8 The cell culture system 10 uses the first measurement result of the first light receiving unit 80 when the cell count is low and the cell concentration is high, and uses the second measurement result of the second light receiving unit 84 when the cell count is high and the cell concentration is low. This ensures that the cell count measurement range of the optical measurement unit 54 as a whole is sufficiently wide. Note that the optical measurement unit 54 may be provided with three or more measurement units, and in this case, the measurement ranges may be set to differ in stages.

[0060] In the optical measurement section 54, the first measurement light L1 from the first light-emitting section 78 and the second measurement light L2 from the second light-emitting section 82 may be emitted in different emission states (intensity, wavelength, etc.). Specifically, the first light-emitting section 78 emits the first measurement light L1 at a low intensity to measure a low concentration. This allows the first measurement unit 56 to irradiate the culture medium and cells with the first measurement light L1 at an appropriate intensity. On the other hand, the second light-emitting section 82 emits the second measurement light L2 at an intensity stronger than the first measurement light L1 to measure a high concentration. This allows the second measurement unit 58 to irradiate the culture medium and cells with the second measurement light L2 at an appropriate intensity.

[0061] The control unit 32 of the flow path control mechanism unit 24 is configured by a computer having one or more processors, a memory, and an input / output interface. The control unit 32 is connected to an operation unit 90 and a display unit 92 provided in the flow path control mechanism unit 24, and sets the operation details of the flow path control mechanism unit 24 based on the operation of the operation unit 90 by the user, and also displays the operation details, errors, etc. on the display unit 92.

[0062] The control unit 32 then periodically controls the optical measurement unit 54 and receives the measurement results of the optical measurement unit 54 to calculate the number of cells being cultured in the reactor 12. For example, in optical measurement using the transmitted light / scattered light method, the control unit 32 calculates the turbidity of the culture medium based on the ratio of the detection value of the transmitted light elements 80a, 84a to the detection value of the scattered light elements 80b, 84b. Furthermore, the control unit 32 previously stores map information or a function (not shown) that indicates the relationship between the turbidity of the culture medium and the number of cells in the reactor 12, and calculates the number of cells in the reactor 12 from the calculated turbidity of the culture medium. The control unit 32 displays the calculated number of cells on the display unit 92.

[0063] When calculating the number of cells, the control unit 32 selectively uses the first measurement result of the first measurement unit 56 and the second measurement result of the second measurement unit 58. For example, at the start of cell culture, the number of cells contained in the medium is small, and the medium has a low concentration (low turbidity). Therefore, the control unit 32 calculates the number of cells based on the first measurement result of the first measurement unit 56, which has a measurement range of low concentration. At this time, the control unit 32 may or may not perform measurement by the second measurement unit 58. If measurement is performed by the second measurement unit 58, the control unit 32 may correct the first measurement result using the second measurement result.

[0064] As the cell count increases during cell culture, the measurement error of the cell count increases within the measurement range of the first measurement unit 56. Therefore, when the control unit 32 determines that the cell count (turbidity) has reached or exceeded a preset switching threshold (not shown), it switches from using the first measurement result of the first measurement unit 56 to using the second measurement result of the second measurement unit 58. Note that switching between the first and second measurement results is not limited to using the calculated cell count, and may be performed, for example, based on the passage of a predetermined period of culture time. This predetermined period may be configured to vary depending on the amount and speed of medium supply, etc.

[0065] After switching, the control unit 32 calculates the number of cells based on the second measurement result of the second measurement unit 58, which has a measurement range of high concentration (high turbidity). At this time, the control unit 32 may or may not perform measurement using the first measurement unit 56. If measurement is performed using the first measurement unit 56, the control unit 32 may correct the second measurement result using the first measurement result.

[0066] The cell culture system 10 according to this embodiment is basically configured as described above, and its operation will be described below.

[0067] As shown in Fig. 1, before starting the culture treatment, the operator of the cell culture system 10 places the culture medium reservoir 14, multiple medical bags 18, and waste liquid portion 20 near the outside of the housing 26 of the flow path control mechanism 24. The operator also sets the reactor 12 and some of the multiple tubes 22 that make up the flow path 16 inside the housing 26. This creates the flow path 16 shown in Fig. 2 between the culture medium reservoir 14 and the reactor 12. When the cell culture system 10 is set up, the measurement container 60 is held in the holder 72 of the flow path control mechanism 24, as shown in Figs. 3A and 4.

[0068] After the above setup, the cell culture system 10 sequentially performs the following steps in the culture process: priming, medium replacement, seeding, culture, detachment, and recovery. In the priming step, cleaning solution stored in the cleaning solution bag 18B is supplied to the reactor 12 through the flow path 16, and air is removed from the reactor 12 and the flow path 16. In the medium replacement step, medium is supplied from the medium reservoir 14 to the reactor 12 through the primed flow path 16, filling the inside and outside of the hollow fibers 34 with medium. In the seeding step, the cell solution stored in the cell solution bag 18A is supplied into the hollow fibers 34 of the reactor 12 through the IC route 42, and cells are seeded on the inner surface of the hollow fibers 34.

[0069] As shown in FIG. 2, in the culture process, the cell culture system 10 supplies the culture medium from the culture medium reservoir 14 into the hollow fibers 34 through both the IC route 42 and the EC route 44, and cultures cells in the hollow fibers 34. During this process, oxygen is supplied to the culture medium and carbon dioxide is discharged from the culture medium by the gas exchanger 52. The culture process is carried out for a longer period (e.g., several days) than other processes, so that cells gradually grow on the inner surface of the hollow fibers 34. Note that the cell culture system 10 may be configured to supply the culture medium to the reactor 12 via the EC route 44 without passing through the IC supply circuit 42b. The culture medium that flows into the reactor 12 via the EC route 44 is supplied to the cells by seeping from the outside to the inside of the hollow fibers 34.

[0070] In the culturing process, the culture medium flows into the passage 62 of the measurement container 60 from the upstream tube 22a. This culture medium passes through the first cell side tapered section 65b, where the cross-sectional area of ​​the flow path gradually increases, and moves to the main body of the first cell 64. The first measurement unit 56 performs optical measurement in the main body of this first cell 64.

[0071] The culture medium then flows from the first cell 64 toward the transition space 68a of the flow path transition section 68, gradually gathers in the transition space 68a, and moves from the transition space 68a to the main body of the second cell 66. The second measurement unit 58 performs optical measurement in the main body of the second cell 66. The culture medium then flows from the second cell 66 through the second cell side tapered section 67b, where the cross-sectional area of ​​the flow path gradually decreases, and flows into the downstream tube 22b.

[0072] That is, the measurement container 60 has a first cell 64 and a second cell 66 whose flow path cross-sectional area decreases stepwise, but the culture medium flows while suppressing turbulence by the end-side tapered portions (first cell-side tapered portion 65b, second cell-side tapered portion 67b) and the flow path transition portion 68. The optical measurement unit 54 performs optical measurement on the culture medium that is in laminar flow in the first space 64a of the first cell 64 and the second space 66a of the second cell 66, thereby improving measurement accuracy.

[0073] 5, the control unit 32 of the flow path control mechanism 24 monitors and controls the culture state based on the measurement by the optical measurement unit 54. In detail, at the start of the culture step, the control unit 32 first emits the first measurement light L1 from the first light emitter 78 of the first measurement unit 56 to measure the low-concentration medium (step S1). As a result, the first measurement light L1 travels through the first optical path length OP1 of the first optical path unit 75, and during this time, it hits the cells in the first cell 64 and is absorbed or scattered.

[0074] Therefore, the first light receiving section 80 of the first measurement unit 56 receives the transmitted light and scattered light from the first cell 64 and transmits a first measurement result, which is a detected value. The control section 32 receives the first measurement result from the first measurement unit 56 (step S2). Based on the received first measurement result, the control section 32 calculates the number of cells in the reactor 12 and displays the calculated number of cells on the display section 92 (step S3). The operator can recognize the state of the cell culture by checking the number of cells displayed on the display section 92.

[0075] The control unit 32 also compares the calculated cell count with a stored switching threshold to determine whether or not to switch from measurement by the first measurement unit 56 to measurement by the second measurement unit 58 (step S4). If the cell count is below the switching threshold, the process returns to step S1 and continues the same processing flow. On the other hand, if the cell count is equal to or greater than the switching threshold, the process proceeds to step S5.

[0076] In step S5, in order to measure the high-concentration medium, the control unit 32 first emits the second measurement light L2 from the second light emitter 82 of the second measurement unit 58. As a result, the second measurement light L2 travels through the second optical path length OP2 of the second optical path section 77, and at this time hits the cells in the second cell 66 and is absorbed or scattered.

[0077] Therefore, the second light receiving section 84 of the second measurement unit 58 receives the transmitted light and scattered light from the second cell 66 and transmits the detected value, which is a second measurement result, and the control section 32 receives this second measurement result (step S6). The control section 32 calculates the number of cells in the reactor 12 based on this second measurement result and displays the calculated number of cells on the display section 92 (step S7).

[0078] Furthermore, the control unit 32 compares the calculated cell number with the stored recovery value to determine whether or not to terminate the culture process (step S8). If the cell number is below the recovery value, the process returns to step S5 and continues the same processing flow. On the other hand, if the cell number is equal to or greater than the recovery value, the culture process is terminated (step S9) and the process automatically proceeds to the detachment process. Note that the control unit 32 may notify the user that the detachment process will be performed when the cell number is equal to or greater than the recovery value, and may perform the detachment process under user operation.

[0079] In the detachment step, the control unit 32 temporarily stops the flow of the culture medium to the reactor 12 and guides the detachment liquid stored in the detachment liquid bag 18C into the hollow fibers 34 of the reactor 12 via the IC route 42 to detach the proliferated cells (step S10). Furthermore, in the recovery step after the detachment step, the control unit 32 supplies the culture medium to the IC route 42, thereby causing the cells detached in the detachment step to flow out of the reactor 12 and move to a recovery bag (not shown).

[0080] By the above process flow, the cell culture system 10 can accurately monitor in real time the number of cells being cultured in the reactor 12. Therefore, the cell culture system 10 can appropriately determine the timing to recover cells from the reactor 12, and can successfully store the desired number of cells in the recovery bag.

[0081] 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, in the above-described embodiment, the measurement container 60 is configured so that the culture medium flows through the first cell 64 and then the second cell 66. However, the culture medium may flow through the second cell 66 and then the first cell 64. Furthermore, for example, in the above-described embodiment, the first measurement unit 56 and the second measurement unit 58 are provided adjacent to each other. However, the first measurement unit 56 and the second measurement unit 58 may be located apart from each other. Therefore, the first cell 64 and the second cell 66 of the measurement container 60 do not have to be configured in a continuous series, but may be located apart from each other, for example, via a tube 22.

[0082] 6, the first measurement unit 56 and the second measurement unit 58 of the optical measurement section 54A may share the light-emitting unit 100 that emits the first measurement light L1 and the second measurement light L2. Even in this configuration, the first light-receiving unit 80 of the first measurement unit 56 and the second light-receiving unit 84 of the second measurement unit 58 are provided separately. Therefore, the measurement light emitted from the light-emitting unit 100 is received by the first light-receiving unit 80 as the first measurement light L1 of the first measurement unit 56 and by the second light-receiving unit 84 as the second measurement light L2 of the second measurement unit 58.

[0083] The optical measurement unit 54A may change the light intensity of the first measurement light L1 from the first measurement unit 56 and the light intensity of the second measurement light L2 from the second measurement unit 58 by appropriately adjusting the first optical path 102 and the second optical path 104 from the light emitter 100 to the arrangement space 72a. For example, as shown by the dotted line in FIG. 6, a lens 102a that reduces the light intensity may be provided in the first optical path 102 to make the light intensity of the first measurement light L1 smaller than the light intensity of the second measurement light L2.

[0084] The technical ideas and effects that can be understood from the above-described embodiments will be described below.

[0085] One aspect of the present invention comprises a reactor 12 for culturing cells based on the flow of culture medium, a flow path 16 through which the culture medium flows in and out of the reactor 12, an optical measurement unit 54 located downstream of the reactor 12 in the flow path 16 in the direction of culture medium flow and for optically measuring the state of the culture medium by irradiating measurement light onto the culture medium, and a processing unit (control unit 32) for processing the measurement results of the optical measurement units 54, 54A, wherein the optical measurement units 54, 54A have a first measurement unit 56 and a second measurement unit 58 arranged at different positions in the direction of culture medium flow, the first measurement unit 56 has a first optical path section 75 through which the culture medium flows and through which the first measurement light L1 passes, and the second measurement unit 58 has a second optical path section 77 through which the culture medium flows and through which the second measurement light L2 passes, and a first optical path length OP1 of the first optical path section 75 is longer than a second optical path length OP2 of the second optical path section 77.

[0086] As described above, the cell culture system 10 can continuously measure the number of cells in the reactor 12 while ensuring sterility by using the optical measurement units 54, 54A provided downstream of the reactor 12 in the flow path 16 in the direction of flow of the culture medium. Furthermore, the optical measurement units 54, 54A have a first optical path length OP1 of the first optical path unit 75 longer than a second optical path length OP2 of the second optical path unit 77, which allows the cell number measurement range of the first measurement unit 56 to differ from the cell number measurement range of the second measurement unit 58. Therefore, each of the first measurement unit 56 and the second measurement unit 58 can perform highly accurate measurements within a narrow measurement range, and the optical measurement units 54, 54A can achieve sufficient measurement accuracy overall.

[0087] The cell culture system 10 also includes a first cell 64 disposed in the first optical path section 75 and through which the culture medium flows, and a second cell 66 disposed in the second optical path section 77 and through which the culture medium flows, and the flow path 16 has a tube 22 connected to the first cell 64 and the second cell 66, and the first cell 64 and the second cell 66 are configured to be harder than the tube 22. The hard first cell 64 and second cell 66 allow the cell culture system 10 to stably form flow paths for the culture medium with a first optical path length OP1 and a second optical path length OP2.

[0088] The first cell 64 and the second cell 66 form a single container (measurement container 60) that is continuous with each other along the direction of culture medium flow, which allows the operator of the cell culture system 10 to easily set the first cell 64 and the second cell 66 in the device.

[0089] Furthermore, the container (measurement container 60) has a flow path transition section 68 between the first cell 64 and the second cell 66, which gradually narrows the cross-sectional area of ​​the medium flow path from the first cell 64 toward the second cell 66. This allows the cell culture system 10 to stabilize the flow state of the medium between the first cell 64 and the second cell 66, even when the first cell 64 and the second cell 66 are configured to be continuous.

[0090] Furthermore, the end of the first cell 64 and the end of the second cell 66 to which the tube 22 is connected have end-side tapered portions (first cell-side tapered portion 65b, second cell-side tapered portion 67b) in which the flow path cross-sectional area gradually decreases toward the tube 22. This makes it possible for the first cell 64 and the second cell 66 to suppress turbulence of the culture medium when the culture medium flows in and out. Therefore, the cell culture system 10 can further improve the accuracy of optical measurements on the first cell 64 and the second cell 66.

[0091] Furthermore, when cell culture starts, the processing unit (control unit 32) calculates the number of cells in the reactor 12 using the first measurement result of the first measurement unit 56, and when the calculated number of cells reaches or exceeds a predetermined switching threshold, calculates the number of cells in the reactor 12 using the second measurement result of the second measurement unit 58. This allows the cell culture system 10 to effectively track changes in the number of cells in the reactor 12.

[0092] Furthermore, when the number of cells reaches a predetermined recovery value based on the second measurement result, the processing unit (control unit 32) temporarily stops the flow of culture medium to the reactor 12 and supplies a detachment liquid to the reactor 12 for detaching the cells in the reactor 12. This allows the cell culture system 10 to smoothly recover the cells in the reactor 12 when the number of cells being cultured in the reactor 12 reaches a target cell number.

[0093] The first measurement unit 56 includes a first light receiving section 80 that receives the transmitted light of the first measurement light L1 and also receives the scattered light of the first measurement light L1 scattered by the culture medium, and the second measurement unit 58 includes a second light receiving section 84 that receives the transmitted light of the second measurement light L2 and also receives the scattered light of the second measurement light L2 scattered by the culture medium. This allows the cell culture system 10 to perform optical measurement using the transmitted light / scattered light method, thereby obtaining more accurate first and second measurement results.

[0094] The first measurement unit 56 and the second measurement unit 58 share the light-emitting unit 100 that emits the first measurement light L1 and the second measurement light L2. By sharing the light-emitting unit 100 in this way, the optical measurement unit 54A can have a simpler structure and lower costs.

[0095] Furthermore, the first measurement unit 56 emits the first measurement light L1 at a light intensity weaker than the light intensity of the second measurement light L2 emitted by the second measurement unit 58. This allows the cell culture system 10 to more accurately measure a low-concentration medium with the first measurement unit 56 and more accurately measure a high-concentration medium with the second measurement unit 58.

Claims

1. a reactor for culturing cells based on the flow of a culture medium; A flow path for introducing and discharging the culture medium into and from the reactor; an optical measurement unit that is provided in the flow path downstream of the reactor in the flow direction of the culture medium and that irradiates the culture medium with measurement light to optically measure the state of the culture medium; a processing unit that processes the measurement results of the optical measurement unit, the optical measurement unit has a first measurement unit and a second measurement unit arranged at different positions in the flow direction of the culture medium, the first measurement unit has a first optical path portion through which the culture medium flows and through which the first measurement light passes; the second measurement unit has a second optical path portion through which the culture medium flows and through which second measurement light passes, a first optical path length of the first optical path section is longer than a second optical path length of the second optical path section; The processing unit When starting the culture of the cells, calculate the number of cells in the reactor using the first measurement result of the first measurement unit; When the calculated number of cells is equal to or greater than a predetermined switching threshold, the number of cells in the reactor is calculated using the second measurement result of the second measurement unit; The first measurement unit emits the first measurement light with a light intensity weaker than the light intensity of the second measurement light emitted by the second measurement unit. Cell culture system.

2. The cell culture system according to claim 1, a first cell disposed in the first optical path section and through which the culture medium flows; and a second cell disposed in the second optical path section and through which the culture medium flows, the flow path includes a tube connected to the first cell and the second cell, The first cell and the second cell are configured to be harder than the tube. Cell culture system.

3. The cell culture system according to claim 2, The first cell and the second cell constitute a single container that is continuous with each other along the flow direction of the culture medium. Cell culture system.

4. The cell culture system according to claim 3, The container has a flow path transition portion between the first cell and the second cell, in which a flow path cross-sectional area of ​​the culture medium gradually narrows from the first cell toward the second cell. Cell culture system.

5. The cell culture system according to any one of claims 2 to 4, The end of the first cell and the end of the second cell to which the tube is connected have end-side tapered portions in which the flow path cross-sectional area gradually decreases toward the tube. Cell culture system.

6. The cell culture system according to claim 1, The processing unit When the number of cells reaches a predetermined recovery value based on the second measurement result, the flow of the culture medium to the reactor is temporarily stopped, and a detachment solution for detaching the cells in the reactor is supplied to the reactor. Cell culture system.

7. The cell culture system according to any one of claims 1 to 6, the first measurement unit includes a first light receiving unit that receives transmitted light of the first measurement light and receives scattered light of the first measurement light scattered by the culture medium; The second measurement unit includes a second light receiving section that receives transmitted light of the second measurement light and receives scattered light of the second measurement light scattered by the culture medium. Cell culture system.

8. The cell culture system according to any one of claims 1 to 7, The first measurement unit and the second measurement unit share a light emitting unit that emits the first measurement light and the second measurement light. Cell culture system.

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