Cell culture device

Abstract

This cell culture apparatus (100) comprises: a container (101) for accommodating a culture solution containing a culture medium and cells; a stirrer (111) for stirring the culture solution, the stirrer being provided in the container (101); a discharge piping (125) for discharging the culture solution in the container (101) to the outside of the container (101); and an oxygen intake piping (121) for introducing oxygen into the container (101). The end (125a) of the discharge piping (125) is located below the stirrer (111) in the vertical direction.

Description

Technical Field

[0001] The present disclosure relates to a cell culture device.

Background Art

[0002] As disclosed in Patent Document 1, there is known a device for culturing cells such as microorganisms by adjusting the dissolved oxygen concentration while stirring the culture solution in a container.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the device disclosed in Patent Document 1, due to the influence of oxygen bubbling generated by supplying oxygen, the ratio of gas in the culture solution to be aspirated varies, and there is a possibility that the sample cannot be accurately aspirated and separated.

[0005] The present disclosure has been made to solve such problems, and an object thereof is to provide a cell culture device capable of accurately aspirating and separating a sample while keeping the amount of dissolved gas in the culture solution constant.

Means for Solving the Problems

[0006] The present disclosure relates to a cell culture device for culturing cells. The cell culture device includes a container for storing a culture solution containing a medium and cells, a stirrer provided in the container for stirring the culture solution, a discharge pipe for discharging the culture solution in the container to the outside of the container, and an oxygen intake pipe for taking in oxygen into the container. The end of the discharge pipe is located below the stirrer in the vertical direction.

Effects of the Invention

[0007] According to the present disclosure, it is possible to accurately aspirate and collect a sample while keeping the dissolved gas amount in the culture solution constant.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

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Figure 12

Figure 13

Modes for Carrying Out the Invention

[0009] This embodiment will be described in detail with reference to the drawings. For the same or corresponding parts in the drawings, the same reference numerals are given, and the description thereof will not be repeated in principle. <Schematic Configuration of Automatic Pretreatment System> FIG. 1 is a block diagram showing a schematic configuration of an automatic pretreatment system 10. The automatic pretreatment system 10 is a device for automatically performing pretreatment on an analyte. In the present embodiment, the analyte is, for example, cultured cells, and more specifically, bacterial cells.

[0010] The automatic pretreatment system 10 includes a sampling device 1 and a pretreatment device 2. After pretreatment is performed by the automatic pretreatment system 10, metabolites of the cells are extracted from the cells. The extracted metabolites are supplied to a liquid chromatograph mass spectrometer 3. The liquid chromatograph mass spectrometer 3 is merely an example of an analyzer for analyzing an analyte. It is also possible to analyze the analyte using other analyzers.

[0011] The sampling device 1 is a device for sampling a liquid from a container (culture container). For example, microorganisms and plant cells are cultured in a culture solution containing a medium in a container called a bioreactor. In the bioreactor, for example, a stirring member rotated using magnetic force, an oxygen concentration sensor for detecting the concentration of dissolved oxygen, and the like are provided. By adjusting the dissolved oxygen concentration while stirring the culture solution containing the medium and cells in the bioreactor, cells are cultured in the sampling device 1. A detailed description of the bioreactor functioning as a cell culture device will be given later.

[0012] The pretreatment device 2 performs pretreatment on the cells contained in the culture solution (culture sample) sampled from the bioreactor. In the sampling device 1, the culture solution containing the cells is accommodated in a test tube as a container (sampling container). The pretreatment device 2 includes a centrifugation mechanism 4, a liquid removal mechanism 5, a reagent supply mechanism 6, a stirring mechanism 7, an extraction mechanism 8, and the like. These mechanisms sequentially perform pretreatment on the cells contained in the culture solution in the test tube.

[0013] The centrifugal separation mechanism 4 applies centrifugal force to the culture solution in the test tube. As a result, the culture solution in the test tube separates into a solid component that sinks to the bottom of the test tube with the solid-liquid interface as the boundary and a liquid component that floats on top of the solid component. The solid component is a culture, for example, cultured cells. The liquid component that floats on top of the solid component is the supernatant separated from the culture solution.

[0014] The liquid removal mechanism 5 sucks out the supernatant from the test tube. Thereby, the liquid in the test tube is removed, and cells remain in the test tube. The reagent supply mechanism 6 supplies a reagent for extracting metabolites in the cells to the cells in the test tube. Thereby, a mixture of cells and the reagent is generated in the test tube. The stirring mechanism 7 stirs the mixture. By stirring the mixture, a suspension in which metabolites have been extracted from the cells is obtained.

[0015] The extraction mechanism 8 extracts a part of the suspension as an extract. The extract is supplied to the liquid chromatography mass spectrometer 3. <Schematic Configuration of Sampling Device> Figure 2 is a flow path diagram showing the flow path configuration of the sampling device 1. In the sampling device 1, a culture solution containing cells in a cell culture device 100 called a bioreactor is sampled. Inside the cell culture device 100, a stir bar 111 is provided as a stirring member that is rotated using magnetic force.

[0016] The cell culture device 100 is held by a holding part 12 provided inside the sampling device 1. In this embodiment, three cell culture devices 100 can be held by one holding part 12, and a plurality (for example, four) of such holding parts 12 are provided. The holding part 12 may be configured to have only one. The holding part 12 may also be configured to be able to hold two or fewer or four or more cell culture devices 100.

[0017] The cell culture device 100 can perform culturing while being heated by a heater (not shown) provided in the holding part 12. A motor 13 for rotating a magnet (not shown) is connected to the holding part 12. By rotating this motor 13, the magnet can be rotated, and the stirrer 111 in each cell culture device 100 can be rotated by the magnetic force.

[0018] In the sampling device 1, culturing can be performed by stirring the culture solution with the stirrer 111 while controlling the temperature of the culture solution in the cell culture device 100. In the sampling device 1, the culture solution containing the cultured cells is sampled into the test tube 14 at an arbitrary timing.

[0019] The sampling device 1 is provided with a culture solution sampling mechanism 20 for sampling the culture solution into the test tube 14 and a reagent sampling mechanism 30 for sampling the reagent into the test tube 14. The test tube 14 contains a mixed solution of the culture solution and the reagent, is sealed with a cap (not shown), and is then conveyed to the pretreatment device 2.

[0020] The culture solution sampling mechanism 20 is provided with a pump 21 and a plurality of valves 22, 23. The valve 23 has, for example, a pair of common ports and five pairs (ten in total) of selection ports, and the flow path can be switched by arbitrarily selecting any one pair of selection ports and connecting them to the pair of common ports.

[0021] The pump 21 and the valve 22 are provided in a flow path 41 connecting between a pair of common ports. The valve 22 constitutes a flow path switching part (first flow path switching part) for switching whether to guide the liquid in the flow path 41 to a branch flow path 42 that branches from the flow path 41. That is, the valve 22 can be switched to either a state where the liquid flows between a pair of common ports via the flow path 41 or a state where the liquid in the flow path 41 is guided to the branch flow path 42.

[0022] Of the five pairs of selection ports, one pair of selection ports is respectively connected to a leading-out path 43 and a leading-in path 44 that communicate with one cell culture device 100. The leading-out path 43 is a flow path for leading out the culture solution in the cell culture device 100. On the other hand, the leading-in path 44 is a flow path for introducing again into the cell culture device 100 the culture solution that is led out from the cell culture device 100 via the leading-out path 43 and circulates via the flow path 41. Another pair of selection ports is respectively connected to a leading-out path 45 and a leading-in path 46 that communicate with another cell culture device 100. Still another pair of selection ports is connected to a leading-out path 47 and a leading-in path 48 that communicate with yet another cell culture device 100.

[0023] In the sampling device 1, any one of the leading-out paths 43, 45, 47 and the corresponding leading-in path 44, 46, 48 are made to communicate via the flow path 41, and in this state, by driving the pump 21, the culture solution in each cell culture device 100 can be circulated. That is, the flow path 41, each of the leading-out paths 43, 45, 47, and each of the leading-in paths 44, 46, 48 constitute a circulation flow path (first circulation flow path) for circulating the culture solution in each cell culture device 100.

[0024] The pump 21 constitutes a circulation mechanism (first circulation mechanism) for circulating the culture solution in each cell culture device 100 via the first circulation flow path by leading out the culture solution from each cell culture device 100 into the first circulation flow path and introducing the culture solution from the first circulation flow path into each cell culture device 100.

[0025] The leading ends of each of the leading-out paths 43, 45, 47 are immersed in the culture solution in the corresponding cell culture device 100. On the other hand, the leading ends of each of the leading-in paths 44, 46, 48 are located at positions spaced upward from the culture solution in the corresponding cell culture device 100. The culture solution led out from the cell culture device 100 via each of the leading-out paths 43, 45, 47 and circulated via the flow path 41 is dropped from the leading ends of each of the leading-in paths 44, 46, 48 and introduced into the cell culture device 100.

[0026] In the sampling device 1, at least a portion of the flow path 41 connecting between a pair of common ports where the pump 21 is provided is constituted by a flexible tube. The pump 21 is, for example, a tubing pump, and can feed the liquid in the tube by deforming (compressing and relaxing) the flexible tube.

[0027] By switching the valve 22 as the first flow path switching portion provided in the middle of the flow path 41, the culture solution circulating into each cell culture device 100 via the flow path 41 can be made to flow out into the branch flow path 42. At this time, the tip of the branch flow path 42 is disposed in the test tube 14, and the culture solution is sampled into the test tube 14 via the branch flow path 42.

[0028] Of the two pairs of selection ports other than the three pairs of selection ports to which the respective outlet paths 43, 45, 47 and the respective inlet paths 44, 46, 48 are connected, one pair of selection ports are respectively connected to the cleaning liquid tank 26 and the waste liquid tank 27. The remaining one pair of selection ports are respectively connected to the filter 25 and the waste liquid tank 27. The cleaning liquid tank 26 stores a cleaning liquid for cleaning the flow path of the culture solution.

[0029] After sampling the culture solution from any of the cell culture devices 100 into the test tube 14, if the valve 23 is switched to connect the cleaning liquid tank 26 and the waste liquid tank 27 to the flow path 41 and the pump 21 is driven in that state, the cleaning liquid in the cleaning liquid tank 26 will be discharged as waste liquid to the waste liquid tank 27 via the flow path 41. Thereby, the flow path 41, the valve 22, etc. can be cleaned with the cleaning liquid.

[0030] After cleaning with the cleaning liquid, if the valve 23 is switched to connect the filter 25 and the waste liquid tank 27 to the flow path 41 and the pump 21 is driven in that state, air is introduced into the flow path 41 via the filter 25 and is discharged to the waste liquid tank 27 together with the moisture remaining in the flow path 41. Thereby, moisture can be removed from the flow path 41, the valve 22, etc.

[0031] The reagent sampling mechanism 30 is equipped with a pump 31 and multiple valves 32 and 33. The valve 33 has, for example, one common port and multiple selection ports, and can switch the flow path by arbitrarily selecting any of the selection ports and connecting it to the common port.

[0032] The pump 31 and the valve 32 are provided in a flow path 49 whose both ends communicate with the reagent tank 34. The reagent tank 34 contains a reagent to be mixed with the culture fluid sampled in the test tube 14. The flow path 49 constitutes a circulation flow path (second circulation flow path) for circulating the reagent in the reagent tank 34. The pump 31 constitutes a circulation mechanism (second circulation mechanism) for circulating the reagent in the reagent tank 34 via the second circulation flow path by drawing out the reagent from the reagent tank 34 into the second circulation flow path and introducing the reagent from the second circulation flow path into the reagent tank 34.

[0033] In the sampling device 1, at least a portion of the flow path 49, in which the pump 31 is provided, is configured with a flexible tube, the both ends of which are connected to the reagent tank 34. The pump 31 is, for example, a tubing pump, and can pump the reagent in the flexible tube by deforming (compressing and relaxing) the tube.

[0034] The valve 32 constitutes a flow path switching unit (second flow path switching unit) for switching whether or not the liquid in the flow path 49 is guided to a branch flow path 50 branching off from the flow path 49. In other words, the valve 32 can switch between a state in which the reagent in the reagent tank 34 is circulated via the flow path 49 and a state in which the reagent in the flow path 49 is guided to the branch flow path 50.

[0035] Thus, by switching the valve 32 as the second flow path switching unit provided in the middle of the flow path 49, the reagent circulating into the reagent tank 34 via the flow path 49 can be made to flow out into the branch flow path 50. The branch flow path 50 is connected to the common port of the valve 33, and any one of the selection ports of the valve 33 is connected into the test tube 14. Therefore, by communicating the selection port connected into the test tube 14 with the common port, the reagent flowing out from the flow path 49 into the branch flow path 50 can be sampled into the test tube 14. <Schematic configuration of the control device> FIG. 3 is a block diagram showing the schematic configuration of the control device 60. The sampling device 1 includes the control device 60. The control device 60 includes, for example, a CPU (Central Processing Unit) 61 and a memory 62. The memory 62 is constituted by, for example, a ROM (Read Only Memory) and a RAM (Random Access Memory), and can store various data in addition to the control program. The CPU 61 can control the operations of the motor 13, the pumps 21, 31, and the valves 22, 23, 32, 33, etc. by executing the control program stored in the memory 62.

[0036] The control device 60 can circulate the culture solution in any one of the cell culture devices 100 by driving the pump 21 at a constant liquid feeding speed in a state where any one of the leading-out paths 43, 45, 47 and the corresponding leading-in paths 44, 46, 48 communicate with each other via the flow path 41. The control device 60 can sample the culture solution in the flow path 41 into the test tube 14 by switching the valve 22 for a predetermined time based on the control program and communicating the flow path 41 with the branch flow path 42.

[0037] The control device 60 can control the sampling amount of the culture solution by controlling the time for switching the flow path with the valve 22. That is, if the liquid feeding speed of the pump 21 is known in advance, by adjusting the time for communicating the flow path 41 with the branch flow path 42, a desired amount of the culture solution can be accurately sampled into the test tube 14.

[0038] <When the flow path 49 is in a state of communicating from one end to the other end, the control device 60 can circulate the reagent in the reagent tank 34 by driving the pump 31 at a constant liquid feeding speed. The control device 60 switches the valve 32 for a predetermined time based on a control program, communicates the flow path 49 with the branch flow path 50, and switches the valve 33 to communicate the branch flow path 50 with the test tube 14, so that the reagent in the flow path 49 can be sampled into the test tube 14.

[0039] <The control device 60 can control the sampling amount of the reagent by controlling the time for switching the flow path with the valve 32. That is, if the liquid feeding speed of the pump 31 is known in advance, by adjusting the time for communicating the flow path 49 with the branch flow path 50, a desired amount of reagent can be accurately sampled into the test tube 14. <Configuration of the cell culture device> <The structure of the cell culture device 100, which is a cell culture device, will be described with reference to FIGS. 4 to 10. FIG. 4 is a perspective view of the cell culture device 100, FIG. 5 is a plan view of the cell culture device 100 with some parts removed, FIG. 6 is a partial cross-sectional view taken along line VI-VI of FIG. 5, FIG. 7 is a partial cross-sectional view taken along line VII-VII of FIG. 5, FIG. 8 is a view showing the internal structure of the cell culture device 100, FIG. 9 is a view showing the stirrer 111, and FIG. 10 is a view showing a state where the stirrer 111 is removed from the shaft portion 110.

[0040] <As shown in FIG. 4, the cell culture device 100 includes a container 101, a lid portion 102, a DO (Dissolved Oxygen) sensor 103 connected to the lid portion 102, a pH sensor 104, a cap portion 105, and a shaft portion 110.

[0041] The container 101 is a transparent vessel into which a culture solution containing microorganisms, plant cells, etc. is placed. The lid portion 102 is for sealing the container 101 and has various components attached to it. The DO sensor 103 is a sensor for measuring the dissolved oxygen concentration inside the cell culture device 100. The pH sensor 104 is a sensor for measuring the hydrogen ion concentration in the culture solution. The cap portion 105 is a lid for the opening protruding from the upper part of the cell culture device 100. The shaft portion 110 is a shaft of the stirrer 111, and is a member that enables the stirrer 111 attached to its tip to rotate.

[0042] Figures 5 to 7 show diagrams in a state where some components are removed in the DO sensor 103 and the pH sensor 104. In the plan view of Figure 5, five pipes connected to a flexible tube are provided between the DO sensor 103 and the pH sensor 104. The five pipes include an oxygen intake pipe 121, an oxygen exhaust pipe 122, a sample addition pipe 123, a suction pipe 124, and a discharge pipe 125.

[0043] The oxygen intake pipe 121 is a pipe for supplying oxygen to the culture solution, and as shown in Figures 6 and 7, it is a pipe that extends to the stirrer 111 located below the container 101. The oxygen exhaust pipe 122 is a pipe for exhausting excess oxygen from the cell culture device 100. The sample addition pipe 123 is a pipe for adding a sample as needed. The suction pipe 124 is connected to any one of the above-described introduction paths 44, 46, 48, and is a pipe for introducing the culture solution into the cell culture device 100. The discharge pipe 125 is connected to any one of the above-described discharge paths 43, 45, 47, and is a pipe for discharging the culture solution inside the cell culture device 100 to the outside of the cell culture device 100.

[0044] The lengths of the five pipes will be described. The oxygen exhaust pipe 122 is the shortest among the five pipes and extends to a position overlapping the lid portion 102 in the vertical direction (downward among the top and bottom of the paper). The sample addition pipe 123 and the suction pipe 124 are of substantially the same length, are longer than the oxygen exhaust pipe 122, and extend to the central position of the container 101 in the vertical direction.

[0045] The oxygen intake pipe 121 is longer than the sample addition pipe 123 and the inhalation pipe 124, and extends vertically to a position overlapping the stirrer 111. The discharge pipe 125 is longer than the oxygen intake pipe 121 and extends vertically to a position below the stirrer 111.

[0046] The five pipes are fixed to the pedestal portion 110c on the upper surface of the lid portion 102 together with the shaft portion 110. As shown in FIG. 8, three baffle plates 110b extend downward from the pedestal portion 110c. The ends of the baffle plates 110b are fixed to the annular portion 110a. Among the five pipes, the oxygen intake pipe 121 and the discharge pipe 125 are fixed to the annular portion 110a.

[0047] The baffle plate 110b is a member for forming a turbulent flow that generates a vertical flow with respect to the horizontal flow generated by the rotation of the stirrer 111. A magnet is disposed inside the magnet portion 111d of the stirrer 111. As shown in FIGS. 6 to 9, the end portion 121a of the oxygen intake pipe 121 is at a position overlapping the stirrer 111 in the vertical direction, and the end portion 125a of the discharge pipe 125 is below the stirrer 111 in the vertical direction.

[0048] In this way, since the end portion 121a of the oxygen intake pipe 121 is at a position overlapping the stirrer 111 and the end portion 125a of the discharge pipe 125 is below the stirrer 111, even if the culture solution is stirred by the stirrer 111, the culture solution can be discharged out of the container 101 at a position hardly affected by the oxygen bubbling caused by the oxygen supply. Therefore, the cell culture device 100 can accurately aspirate and collect the sample while keeping the dissolved gas amount of the culture solution constant.

[0049] Next, the stirrer 111 will be described in detail. As shown in FIG. 10, the stirrer 111 includes a main body 111c, a bearing portion 111a, and a locking portion 111b. The main body 111c is formed in a columnar shape with a hole portion 111f provided at the center, and rotating blade portions 111e are formed every 90°. The stirrer 111 includes two magnet portions 111d protruding from the main body 111c. The magnet portion 111d is covered with the same material as the main body 111c.

[0050] The main body 111c of the stirrer 111 is made of polyetheretherketone. Since polyetheretherketone is generally referred to as peek (abbreviation), it will be referred to as peek hereinafter. The material covering the rotating blade portion 111e and the magnet portion 111d integrally formed with the main body 111c is also made of peek. On the other hand, the bearing portion 111a and the locking portion 111b are made of polyacetal (abbreviation POM).

[0051] Both polyacetal and peek are resins, and there are differences in properties. Polyacetal is an engineering plastic with excellent strength, elastic modulus, and impact resistance because the amorphous part and the crystalline part are mixed. Polyacetal is also used as a bearing part because of its excellent sliding characteristics. Peek, on the other hand, is classified as a super engineering plastic with the highest performance. Peek is known as a resin with particularly excellent heat resistance and chemical resistance among super engineering plastics and is a very reliable resin.

[0052] The peek constituting the rotating blade portion 111e has higher strength and higher wear resistance than the polyacetal constituting the bearing portion 111a. The polyacetal constituting the bearing portion 111a has self-lubricity and a particularly low friction coefficient with metal. The inside of the shaft portion 110 is formed of a metal such as stainless steel material (for example, SUS316). The polyacetal constituting the bearing portion 111a is suitable as a bearing member because it has higher slidability with respect to the metal than the peek constituting the rotating blade portion 111e.

[0053] As shown in FIGS. 9 and 10, the bearing portion 111a is inserted into the hole portion 111f of the main body 111c made of peek of the stirrer 111. The bearing portion 111a is rotatably arranged with respect to the shaft formed of the metal inside the shaft portion 110, and the bottom surface is fixed by the locking portion 111b.

[0054] The stirrer 111 is made of polyacetal in which the bearing portion 111a has self-lubricity and a low coefficient of friction with metals. Therefore, even if the bearing portion 111a slides on the shaft portion 110 for a long time, no shaving chips are produced. Since the rotating blade portion 111e of the stirrer 111 is composed of peaks, even if it rotates for a long time, no shaving chips are produced between the oxygen intake pipe 121 and the discharge pipe 125 at the contacting positions. As a result, the metabolite analysis of microorganisms can be stably performed.

[0055] Particularly in this embodiment, as shown in FIGS. 6 to 8, the end portion of the baffle plate 110b is fixed to the annular portion 110a, and the stirrer 111 rotates at a position below the annular portion 110a. As a result, since the baffle plate 110b does not slide on the portion covering the rotating blade portion 111e or the magnet portion 111d of the stirrer 111, it is possible to prevent the generation of shaving chips. As a result, the metabolite analysis of microorganisms can be stably performed. <Flow of Sampling Process> FIG. 11 is a flowchart of a process executed to sample the culture solution in the cell culture device 100 into the test tube 14 in the sampling device 1. In one implementation example, in the sampling device 1, the CPU 61 of the control device 60 executes a given program, and the process of FIG. 11 is implemented.

[0056] The control device 60 is an example of a controller that controls the operations of the stirrer (stirrer 111) and the flow path switching unit (valve 22). The control device 60 controls the operation of the stirrer 111 by controlling the operation of the motor 13.

[0057] The program may be stored in the memory 62. In this case, the memory 62 is an example of a recording medium that stores the program non-temporarily. The program may be stored in a recording medium that is accessible by the CPU 61 and is detachable from the control device 60. In this case, the recording medium is an example of a recording medium that stores the program non-temporarily.

[0058] The process of FIG. 11 is performed for each test tube 14. Hereinafter, for the sake of convenience of explanation, sampling from the leftmost of the three cell culture devices 100 shown in FIG. 2 will be described. That is, in this explanation, the flow path 41 constitutes a first circulation flow path together with the lead-out path 43 and the introduction path 44. Hereinafter, with reference to FIG. 11, the flow of the process will be described.

[0059] In step S10, the control device 60 causes the cell culture device 100 to perform a basic operation. The basic operation includes circulating the culture solution in the first circulation flow path and circulating the reagent in the second circulation flow path. Circulating the culture solution in the first circulation flow path includes rotating the stirrer 111 by rotating the motor 13 to stir the culture solution in the cell culture device 100.

[0060] In step S12, the control device 60 determines whether the timing for sampling the culture solution into the test tube 14 has arrived. The control device 60 repeats the determination in step S12 until it determines that the timing has arrived (NO in step S12). When the control device 60 determines that the timing has arrived (YES in step S12), it proceeds to step S14.

[0061] In step S14, the control device 60 stops the rotation of the stirrer 111 by stopping the rotation of the motor 13. Thereby, the stirring of the culture solution in the cell culture device 100 is stopped.

[0062] In step S16, the control device 60 determines whether a given time has elapsed since the stirring was stopped in step S14. The control device 60 continues the control in step S16 until it determines that the given time has elapsed (NO in step S16), and when it determines that the given time has elapsed (YES in step S16), it proceeds to step S18.

[0063] In step S18, the control device 60 causes the valve 22 to switch the flow path so as to guide the liquid in the flow path 41 to the branch flow path 42.

[0064] In step S20, after a specific time has elapsed since the control device 60 caused the valve 22 to switch the flow path in step S18, the control device 60 causes the valve 22 to switch the flow path so as to introduce the liquid in the flow path 41 into the introduction path 44. The specific time in step S20 means the time corresponding to a given amount of sampling.

[0065] In step S22, the control device 60 resumes the rotation of the motor 13, thereby resuming the stirring of the culture solution in the cell culture device 100. Thereafter, the control device 60 ends the process of FIG. 11.

[0066] In the process described above with reference to FIG. 11, in step S18, the flow path is switched by the valve 22 for sampling into the test tube 14. Note that, a given time before the switching of the flow path in step S18, the stirring in the cell culture device 100 is stopped (step S14). That is, after the stirring is stopped in step S14, after a given time has elapsed, the flow path is switched in step S18 and sampling is started.

[0067] By performing sampling after the stirring in the cell culture device 100 has been stopped for a "given time", a situation where the amount of dissolved gas in the sampled culture solution varies in multiple samplings is suppressed. If the "given time" is too short, the variation in the amount of dissolved gas for each sampling is not sufficiently suppressed. On the other hand, if the "given time" is too long, uneven culture is assumed to occur in the culture solution sampled for each sampling. In this sense, in one implementation example, the "given time" may be set between 2 minutes and 10 minutes. In another implementation example, the "given time" may be set between 3 minutes and 7 minutes.

[0068] Note that if the variation in the amount of dissolved gas per sampling can be suppressed, stirring does not have to be completely stopped. That is, in step S14, instead of stopping the rotation of the stirrer 111, the control device 60 may reduce the rotation speed of the stirrer 111 to be lower than the rotation speed in the basic operation in step S10. The control device 60 reduces the rotation speed of the stirrer 111 by reducing the rotation speed of the motor 13. The rotation speed of the stirrer 111 in step S10 is also referred to as the "basic speed" and is the speed set for the circulation of the culture solution.

[0069] In one implementation example, in step S14, the control device 60 reduces the rotation speed of the stirrer 111 to about 1 / 10 of the basic speed. In this case, after sampling is completed and the flow path is switched in step S20, the control device 60 returns the rotation speed of the stirrer 111 to the rotation speed in step S10. <Uniformity of the introduction amount into the test tube> FIG. 12 is a diagram showing an example of the result of the introduction amount of the liquid by the introduction of the liquid according to the process of FIG. 11. FIG. 13 is a diagram showing an example of the result of the introduction amount of the liquid by the introduction of the liquid according to the comparative example. Each result of FIGS. 12 and 13 is the result when pure water is adopted as the liquid introduced from the cell culture device 100 into the test tube 14. In each graph of FIGS. 12 and 13, the vertical axis represents the weight of the liquid introduced into the test tube 14.

[0070] The result shown in FIG. 12 is the result when the stirring by the stirrer 111 is stopped from a given time before the liquid is introduced from the cell culture device 100 into the test tube 14, as described with reference to FIG. 11. On the other hand, the result shown in FIG. 13 is the result when the stirring of the stirrer 111 is continued before and after the introduction of the liquid from the cell culture device 100 into the test tube 14.

[0071] In both FIGS. 12 and 13, for each of the 10 groups (A1 to E2), the range (maximum and minimum values) of the introduced amount in the six liquid introductions is shown. Note that the target introduction volume of the liquid is different for each group. The target introduction volumes of groups A1 and A2 are 2 mL, those of groups B1 and B2 are 1 mL, those of groups C1 and C2 are 0.5 mL, those of groups D1 and D2 are 0.2 mL, and those of groups E1 and E2 are 0.1 mL.

[0072] In the results of FIG. 13, in any group, the difference between the maximum and minimum values of the weight of the liquid introduced into test tube 14 is larger than that in the results of FIG. 12.

[0073] For example, in FIG. 13, in group A1, the maximum value is 1.65 g, the minimum value is 1.50 g, and the difference between the maximum and minimum values is 0.15 g. Since the median value is 1.575 g, the difference of 0.15 g between the maximum and minimum values is a relatively high value of about 10% with respect to the median value of 1.575 g.

[0074] Also, in FIG. 13, in group B2, the maximum value is 0.90 g, the minimum value is 0.60 g, and the difference between the maximum and minimum values is 0.30 g. Since the median value is 0.75 g, the difference of 0.30 g between the maximum and minimum values is a relatively high value of about 43% with respect to the median value of 0.75 g.

[0075] On the one hand, in the results of FIG. 12, in any of the groups, the difference between the maximum value and the minimum value of the weight of the liquid introduced into the test tube 14 is small. In other words, in the results of FIG. 12, in any of the groups, almost no variation in the weight of the liquid introduced into the test tube 14 is observed. That is, as described with reference to FIG. 11, when the stirring by the stirrer 111 is stopped from a given time before the liquid is introduced from the cell culture device 100 to the test tube 14, the variation in the ratio of the gas in the aspirated solution is suppressed, and thereby, the accuracy of the aspirated solution can be controlled. Note that such an effect can be expected not only when the stirring by the stirrer 111 is completely stopped, but also when the rotational speed for the stirring by the stirrer 111 is reduced.

[0076] [Aspect] It is understood by those skilled in the art that the above-described plurality of exemplary embodiments are specific examples of the following aspects.

[0077] (Item 1) A cell culture device according to one aspect is a cell culture device for culturing cells, including a container for storing a culture solution containing a culture medium and cells, a stirrer provided in the container for stirring the culture solution, a discharge pipe for discharging the culture solution in the container to the outside of the container, and an oxygen intake pipe for inhaling oxygen into the container. The end of the discharge pipe is located at a position below the stirrer in the vertical direction.

[0078] According to the cell culture device described in Item 1, since the discharge pipe is located at a position below the stirrer, even if the culture solution is stirred by the stirrer, the culture solution can be discharged to the outside of the container at a position less affected by the oxygen bubbling caused by oxygen supply. Therefore, the cell culture device described in Item 1 can accurately aspirate and separate a sample while keeping the dissolved gas amount of the culture solution constant.

[0079] (Item 2) The end of the oxygen intake pipe is located at a position overlapping the stirrer in the vertical direction. According to the cell culture device described in claim 2, the end of the oxygen intake pipe is located at a position overlapping the stirrer. Therefore, the cell culture device described in claim 2 can make the discharge pipe whose end is provided below the stirrer less affected by oxygen bubbling caused by the oxygen supplied from the oxygen intake pipe.

[0080] (Claim 3) The cell culture device further includes a drive device that rotationally drives the stirrer non - contact from outside the container using magnetic force, and a control device that controls the operation of the drive device. After the control device stops the drive device, the culture solution is discharged from the discharge pipe to the outside of the container.

[0081] According to the cell culture device described in claim 3, the culture solution is discharged to the outside of the container after the control device stops the drive device. Therefore, the cell culture device described in claim 3 can aspirate and collect the culture solution in a state where oxygen bubbling has subsided, and can accurately aspirate and collect the sample while keeping the dissolved gas amount of the culture solution constant.

[0082] The embodiments disclosed this time should be considered as illustrative in all respects and not restrictive. The scope of the present disclosure is shown by the claims rather than the description of the above - described embodiments, and it is intended that all modifications within the meaning and scope equivalent to the claims are included.

Explanation of reference numerals

[0083] 1 Sampling device, 2 Pretreatment device, 3 Liquid chromatograph mass spectrometer, 4 Centrifugal mechanism, 5 Liquid removal mechanism, 6 Reagent supply mechanism, 7 Stirring mechanism, 8 Extraction mechanism, 10 Pretreatment system, 12 Holding part, 13 Motor, 14 Test tube, 20 Culture solution sampling mechanism, 21, 31 Pump, 22, 23, 32, 33 Valve, 25 Filter, 26 Cleaning solution tank, 27 Waste liquid tank, 30 Reagent sampling mechanism, 34 Reagent tank, 41, 42, 49, 50 Flow path, 43, 45, 47 Outlet path, 44, 46, 48 Inlet path, 60 Control device, 61 CPU, 62 Memory, 100 Cell culture device, 101 Container, 102 Lid part, 103 DO sensor, 104 pH sensor, 105 Cap part, 110 Shaft part, 110a Annular part, 110b Baffle plate, 110c Pedestal part, 111 Stirrer, 111a Bearing part, 111b Locking part, 111c Body, 111d Magnet part, 111e Rotating blade part, 121 Oxygen intake pipe, 121a, 125a End part, 122 Oxygen exhaust pipe, 123 Sample addition pipe, 124 Suction pipe, 125 Discharge pipe.

Claims

1. A cell culture apparatus for culturing cells, comprising: a container for storing a culture medium and a culture solution containing cells; a stirrer provided in the container for stirring the culture solution; a discharge pipe for discharging the culture solution in the container to the outside of the container; an oxygen intake pipe for taking in oxygen into the container, wherein an end of the discharge pipe is located below the stirrer in the vertical direction; and an end of the oxygen intake pipe is located at a position overlapping the stirrer in the vertical direction. The cell culture apparatus.

2. The cell culture apparatus according to claim 1, further comprising: a driving device for rotationally driving the stirrer in a non-contact manner using magnetic force from outside the container; and a control device for controlling the operation of the driving device, wherein the culture solution is discharged from the discharge pipe to the outside of the container after the control device stops the driving device. ​

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