Cell culture apparatus and calibration method

Abstract

To provide a cell culture apparatus and a calibration method in which efficiency of a calibration operation for creating a calibration curve can be improved, and discard of cell solution and culture medium can be eliminated.SOLUTION: A cell culture apparatus (10) comprises: a cell culture circuit (16); a sensor part (34) which is arranged in cell culture circuit, and measures the turbidity of cell solution; and a calculation part (130) having a calibration part, where the calibration part calculates the cell concentration of a first culture medium in which cell solution of a first cell mass is supplied to a culture medium as first concentration (C1), calculates the cell concentration of a second culture medium in which cell solution of a second cell mass is supplied to the first culture medium as second concentration (C2), and creates a calibration curve by a measured value of a sensor and a cell concentration by associating the first concentration with the first measured value, and associating the second concentration with the second measured value.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a cell culture device having a sensor configuration function and a method for calibrating the sensor.

Background Art

[0002] Patent Document 1 discloses a cell culture device for culturing cells. This cell culture device uses a turbidity sensor as a sensor for measuring the cell concentration in a culture medium. The turbidity sensor measures, for example, transmitted light, scattered light, etc. by irradiating light onto a solution. As the cell concentration increases, the number of cells in the culture medium increases. Then, the turbidity increases.

[0003] Turbidity is affected by the size of cells. For example, when comparing culture medium A containing small cells and culture medium B containing large cells, if the number of cells in both is the same, the light transmittance of culture medium A is higher than that of culture medium B. That is, while the concentrations of culture medium A and culture medium B are the same, the turbidity of culture medium A is smaller than that of culture medium B. The relationship between turbidity and cell concentration varies depending on the type of cells. Therefore, when measuring cell concentration with a turbidity sensor, it is necessary to set in advance the relationship between cell concentration and turbidity. For example, a calibration curve is set as the relationship between cell concentration and turbidity.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Calibration work is required to set a calibration curve. In conventional calibration work, for example, it was necessary to prepare two culture media with different cell concentrations. Further, after individually measuring the turbidity of the two culture media with a turbidity sensor, the turbidity sensor was attached to the cell culture device.

[0006] Traditional calibration procedures resulted in the waste of cell solution and culture medium, as two types of culture media were discarded after the calibration process. Furthermore, these procedures required work outside the cell culture apparatus, making them inconvenient.

[0007] The present invention aims to solve the above-mentioned problems. [Means for solving the problem]

[0008] A first aspect of the present invention is a cell culture apparatus comprising: a cell culture circuit capable of circulating a predetermined amount of culture medium supplied with cell saturation; a sensor disposed in the cell culture circuit for measuring the turbidity of the cell saturation; and a calibration unit for calibrating the measurement value of the sensor to the cell concentration of the culture medium, wherein the sensor measures the turbidity of a first culture medium supplied with a first cell volume of the cell saturation as a first measurement value, and further measures the turbidity of a second culture medium supplied with a second cell volume of the cell saturation as a second measurement value; the calibration unit calculates the cell concentration of the first culture medium as a first concentration, calculates the cell concentration of the second culture medium as a second concentration, and creates a calibration curve between the measurement value of the sensor and the cell concentration by relating the first concentration and the first measurement value, and relating the second concentration and the second measurement value.

[0009] A second aspect of the present invention is a calibration method using a cell culture circuit capable of circulating a predetermined amount of culture medium supplied with cell saturation, a sensor disposed in the cell culture circuit for measuring the turbidity of the cell saturation, and a calibration unit for calibrating the measurement value of the sensor to the cell concentration of the culture medium, wherein the sensor measures the turbidity of a first culture medium supplied with a first cell volume of the cell saturation as a first measurement value, and further measures the turbidity of a second culture medium supplied with a second cell volume of the cell saturation as a second measurement value, the calibration unit calculates the cell concentration of the first culture medium as a first concentration, calculates the cell concentration of the second culture medium as a second concentration, and associates the first concentration with the first measurement value, and the second concentration with the second measurement value, thereby creating a calibration curve between the measurement value of the sensor and the cell concentration. [Effects of the Invention]

[0010] According to the present invention, a cell solution with a known cell count can be prepared in advance, and calibration can be performed using the cell culture device without any work being done outside the cell culture device. Therefore, the efficiency of the work can be improved, and the waste of cell solution and culture medium can be eliminated. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the configuration of a cell culture apparatus. [Figure 2] Figure 2 shows an example of a supply unit. [Figure 3] Figure 3 shows an example of a supply unit. [Figure 4] Figure 4 shows an example of a supply unit. [Figure 5] Figure 5 shows the functional blocks of the arithmetic unit. [Figure 6] Figure 6 is a flowchart of the calibration process performed by the calculation unit. [Figure 7] Figure 7 is a graph showing the calibration curve. [Modes for carrying out the invention]

[0012] [1. Configuration of cell culture apparatus 10] Figure 1 shows the configuration of the cell culture apparatus 10 (calibration apparatus 12). In this embodiment, the calibration apparatus 12 uses the cell culture apparatus 10, which cultures cells, to calibrate the measured values ​​of the sensor unit 34. First, the basic configuration of the cell culture apparatus 10 will be explained.

[0013] The cell culture device 10 cultures cells isolated from living tissue in a culture medium. Examples of cells used in the cell culture device 10 include adherent cells and suspension cells. Specifically, examples of cells used in the cell culture device 10 include ES cells, iPS cells, and mesenchymal stem cells. The cells used in the cell culture device 10 are not limited to those mentioned above.

[0014] The cell culture apparatus 10 comprises a cell culture circuit 16, a support device 18, and a controller 20. A liquid flows through the cell culture circuit 16. The liquid includes at least one of cell saturation, culture medium, washing solution, and detachment solution. For example, culture medium supplied with cell saturation flows through the cell culture circuit 16. Culture medium containing cultured and enlarged cells flows through the cell culture circuit 16.

[0015] Cell saturation is a solution containing cells. Culture medium is a culture medium for growing cells. The culture medium is selected according to the cells being cultured. For example, MEM (Minimum Essential Media) is used as a culture medium. Washing solution is used to wash the inside of the cell culture circuit 16. For example, water, buffer solution, or physiological saline can be used as a washing solution. Examples of buffer solutions include PBS (Phosphate Buffered Salts) and TBS (Tris-Buffered Saline). Detachment solution is used to detach cells from the bioreactor 30 of the cell culture circuit 16. For example, trypsin or EDTA solution can be used as a detachment solution. The culture medium, washing solution, and detachment solution are not limited to the liquids described above.

[0016] [1-1 Cell culture circuit 16] The cell culture circuit 16 is discarded after a single use. In other words, the cell culture circuit 16 is discarded each time a predetermined number of cells are cultured. That is, the cell culture circuit 16 is a disposable item. The cell culture circuit 16 comprises a supply unit 22, a collection container 24, a waste liquid containment unit 26, and a culture body 28.

[0017] The supply unit 22 supplies cell fluid, a culture medium, a cleaning solution, and a detachment solution to the culture main body 28. FIG. 2 is a diagram showing an example of the supply unit 22. The supply unit 22 has a liquid holding unit 122. The liquid holding unit 122 is, for example, a medical bag formed by molding a soft resin material into a bag shape. The inside of the liquid holding unit 122 is filled with liquid. The liquid holding unit 122 is provided for each type of liquid. Below the liquid holding unit 122, a housing 120 that houses the culture main body 28, the support device 18, etc. is arranged. Each liquid holding unit 122 is suspended from a stand 124 attached to the housing 120. The liquid holding unit 122 has an output flow path 126 for discharging the liquid to the outside. The output flow path 126 is connected to the culture main body 28 (the first supply flow path 56 or the second supply flow path 60). The liquid holding unit 122 may be a tank or the like made of a hard material.

[0018] Returning to FIG. 1, the description of the cell culture circuit 16 will be continued. The recovery container 24 recovers the cells cultured in the culture main body 28. The waste liquid storage unit 26 stores the waste liquid generated in the culture main body 28. Each of the recovery container 24 and the waste liquid storage unit 26 is, for example, a medical bag. Each of the recovery container 24 and the waste liquid storage unit 26 may be a tank or the like made of a hard material.

[0019] The culture main body 28 has a bioreactor 30, a flow path 32, a sensor unit 34, and a gas exchange unit 36.

[0020] The bioreactor 30 has a plurality of hollow fiber membranes 40 and a cylindrical housing 42. The plurality of hollow fiber membranes 40 are housed inside the housing 42. One end of each hollow fiber membrane 40 is fixed to one end of the housing 42. The other end of each hollow fiber membrane 40 is fixed to the other end of the housing 42. Each hollow fiber membrane 40 is made of, for example, a polymer material.

[0021] The bioreactor 30 comprises a first region 44 and a second region 46. The first region 44 is the inner pore of a plurality of hollow fiber membranes 40. The second region 46 is the space between the inner circumferential surface of the housing 42 and the outer circumferential surfaces of the plurality of hollow fiber membranes 40. Each hollow fiber membrane 40 has a plurality of pores (not shown). The first region 44 and the second region 46 are in communication with each other through the plurality of pores of each hollow fiber membrane 40. The diameter of each pore is such that it blocks the passage of macromolecules (cells, etc.) while allowing the passage of low molecular weight molecules (e.g., water, ions, oxygen, lactate, etc.). The diameter of each pore is set, for example, to between 0.005 [μm] and 10 [μm].

[0022] The housing 42 is fitted with a first inlet port 48, a first outlet port 50, a second inlet port 52, and a second outlet port 54. The first inlet port 48 is attached to one end of the housing 42. The first inlet port 48 communicates with the first region 44 via an inlet located at one end of the plurality of hollow fiber membranes 40. The first outlet port 50 is attached to the other end of the housing 42. The first outlet port 50 communicates with the first region 44 via an outlet located at the other end of the plurality of hollow fiber membranes 40.

[0023] The second inlet port 52 and the second outlet port 54 are mounted on the outer circumferential surface of the housing 42. The second inlet port 52 is located in the longitudinal direction of the housing 42 between the center of the housing 42 and the first inlet port 48. The second outlet port 54 is located in the longitudinal direction of the housing 42 between the center of the housing 42 and the first outlet port 50. Each of the second inlet port 52 and the second outlet port 54 communicates with the second region 46.

[0024] The flow path 32 includes multiple tubes through which liquid flows. Each tube is made of a flexible resin material. The flow path 32 comprises a first supply flow path 56, a first circulation flow path 58, a second supply flow path 60, a second circulation flow path 62, a recovery flow path 64, and a waste liquid flow path 66. One end of the first supply flow path 56 is connected to the supply unit 22. The other end of the first supply flow path 56 is connected to the first junction 68 of the first circulation flow path 58.

[0025] The first confluence section 68 is located in the middle portion of the extension direction of the first circulation channel 58. One end of the first circulation channel 58 is connected to the first inlet port 48. The other end of the first circulation channel 58 is connected to the first outlet port 50. The first circulation channel 58 communicates with the inner pores (first region 44) of a plurality of hollow fiber membranes 40.

[0026] One end of the second supply channel 60 is connected to the supply unit 22. The supply unit 22 supplies one type of culture medium and one type of washing solution to the second supply channel 60 at predetermined timings. The other end of the second supply channel 60 is connected to the second junction 70 of the second circulation channel 62.

[0027] The second confluence section 70 is located in the middle portion of the second circulation channel 62 in the direction of extension. Second circulation channel 62 One end is connected to the second inlet port 52. Second circulation channel 62 The other end is connected to the second outlet port 54. The second circulation channel 62 communicates with the space (second region 46) between the multiple hollow fiber membranes 40 and the housing 42.

[0028] The supply unit 22 supplies cell solution, culture medium, washing solution, and detachment solution one at a time at predetermined timings to the first circulation channel 58 via the first supply channel 56. The supply unit 22 also supplies culture medium to the second circulation channel 62 via the second supply channel 60 at predetermined timings.

[0029] The recovery channel 64 extends from the first circulation channel 58. One end of the recovery channel 64 is connected to the recovery branch section 74 of the first circulation channel 58. The recovery branch section 74 is located between the first junction section 68 and the first outlet port 50 in the first circulation channel 58. The other end of the recovery channel 64 is connected to the recovery container 24.

[0030] The waste liquid channel 66 carries the waste liquid from the first circulation channel 58 and the second circulation channel 62. The waste liquid channel 66 has a first waste liquid channel 76, a second waste liquid channel 78, and a third waste liquid channel 80. The first waste liquid channel 76 extends from the first circulation channel 58. One end of the first waste liquid channel 76 is connected to the first branch 82 of the first circulation channel 58. The first branch 82 is located between the first outlet port 50 and the recovery branch 74 of the first circulation channel 58. The second waste liquid channel 78 extends from the second circulation channel 62. One end of the second waste liquid channel 78 is connected to the second branch 84 of the second circulation channel 62. The second branch 84 is located between the second junction 70 and the second outlet port 54 of the second circulation channel 62. The other end of the first wastewater channel 76 and the other end of the second wastewater channel 78 are connected to each other at an intermediate junction 86. One end of the third wastewater channel 80 is connected to the first wastewater channel 76 and the second wastewater channel 78 at the intermediate junction 86. The other end of the third wastewater channel 80 is connected to the wastewater storage section 26.

[0031] The sensor unit 34 is installed between the first confluence 68 and the first inlet port 48 of the first circulation channel 58. The sensor unit 34 has a turbidity sensor. The relationship between the measured value of the turbidity sensor and the true value of turbidity is preset. The measured value of turbidity measured by the sensor unit 34 is converted into cell concentration by the calibration unit 148, which will be described later.

[0032] The gas exchange unit 36 ​​is installed between the second junction 70 and the second inlet port 52 of the second circulation channel 62. The gas exchange unit 36 ​​passes a gas of predetermined components through the liquid (culture medium) flowing through the second circulation channel 62. The gas used in the gas exchange unit 36 ​​has a composition similar to that of natural air. In other words, the gas contains nitrogen, oxygen, and carbon dioxide. Specifically, the gas contains, for example, 75% nitrogen, 20% oxygen, and 5% carbon dioxide by volume.

[0033] [1-2 Support device 18] The cell culture circuit 16 described above is set in a support device 18. The support device 18 has a cassette that supports the cell culture circuit 16. The support device 18 is a reusable item that can be used multiple times.

[0034] The support device 18 comprises a plurality of pumps 98 and a plurality of clamps 100. Each of the plurality of pumps 98 imparts fluid force to the liquid in the flow path 32 by squeezing the wall of the flow path 32. Each of the plurality of pumps 98 has a pressing member (not shown). The pressing member includes, for example, a rotating member and a plurality of pressing rollers. The plurality of pressing rollers are attached to the outer circumference of the rotating member. The plurality of pressing rollers are spaced apart in the circumferential direction of the rotating member. Each pressing roller rubs against the outer surface of the wall of the flow path 32.

[0035] The multiple pumps 98 include a first supply pump 102, a first circulation pump 104, a second supply pump 106, and a second circulation pump 108. Note that the state in which the cell culture circuit 16 is set on the support device 18, as shown in Figure 1, is simply referred to as the "set state."

[0036] In the set state, a portion of the first supply channel 56 is attached to the first supply pump 102. The first supply pump 102 imparts a fluid force to the liquid in the first supply channel 56 in the direction from the supply unit 22 toward the first circulation channel 58.

[0037] In the set state, a portion of the first circulation channel 58 is attached to the first circulation pump 104. The first circulation pump 104 imparts a fluid force to the liquid in the first circulation channel 58 in the direction from the first outlet port 50 to the first inlet port 48. The first circulation pump 104 can also impart a fluid force to the liquid in the first circulation channel 58 in the direction from the first inlet port 48 to the first outlet port 50.

[0038] In the set state, a portion of the second supply channel 60 is attached to the second supply pump 106. The second supply pump 106 imparts a fluid force to the liquid in the second supply channel 60 in the direction from the supply unit 22 toward the second circulation channel 62.

[0039] In the set state, a portion of the second circulation channel 62 is attached to the second circulation pump 108. The second circulation pump 108 imparts a fluid force to the liquid in the second circulation channel 62 in the direction from the second outlet port 54 to the second inlet port 52. The second circulation pump 108 can also impart a fluid force to the liquid in the second circulation channel 62 in the direction from the second inlet port 52 to the second outlet port 54.

[0040] The multiple clamps 100 close the flow path 32 by pressing the outer surface of the flow path 32 toward the inner surface. For example, the multiple clamps 100 are on-off valves. The multiple clamps 100 include a recovery clamp 110, a first waste liquid clamp 112, a second waste liquid clamp 114, and a third waste liquid clamp 116.

[0041] In the set state, a portion of the recovery channel 64 is attached to the recovery clamp 110. The recovery clamp 110 opens and closes the recovery channel 64. In the set state, a portion of the first waste liquid channel 76 is attached to the first waste liquid clamp 112. The first waste liquid clamp 112 opens and closes the first waste liquid channel 76. In the set state, a portion of the second waste liquid channel 78 is attached to the second waste liquid clamp 114. The second waste liquid clamp 114 opens and closes the second waste liquid channel 78. In the set state, a portion of the third waste liquid channel 80 is attached to the third waste liquid clamp 116. The third waste liquid clamp 116 opens and closes the third waste liquid channel 80.

[0042] [1-3 Controller 20] The controller 20 is, for example, a computer. The controller 20 includes an arithmetic unit 130, a storage unit 132, and various drive circuits (not shown).

[0043] The arithmetic unit 130 has a processing circuit. The processing circuit may be a processor such as a CPU. The processing circuit may be an integrated circuit such as an ASIC or FPGA. The processor can perform various processes by executing a program stored in the storage unit 132. At least a portion of the multiple processes may be performed by an electronic circuit including discrete devices.

[0044] The storage unit 132 includes volatile memory and non-volatile memory. Examples of volatile memory include RAM. The volatile memory is used as the working memory of the processor. The volatile memory temporarily stores data necessary for processing or calculation. Examples of non-volatile memory include ROM and flash memory. The non-volatile memory is used as storage memory. The non-volatile memory stores programs, tables, maps, etc. At least a part of the storage unit 132 may be provided in a processor, integrated circuit, etc. as described above.

[0045] [2 Configuration of Calibration Device 12] Here, we will describe the configuration required when the cell culture apparatus 10 is used as a calibration apparatus 12. The calibration apparatus 12 utilizes the following components of the cell culture apparatus 10: the supply unit 22, the first supply channel 56, the first supply pump 102, the first circulation channel 58, the first circulation pump 104, the bioreactor 30, the sensor unit 34, and the controller 20.

[0046] As shown in Figures 2 to 4, the supply unit 22 includes an agitator 134 and a flow velocity sensor 136. The agitator 134 agitates the cell solution inside the liquid holding unit 122 so that the concentration of the cell solution inside the liquid holding unit 122 becomes uniform during the calibration process described later. The agitator 134 may be one or more vibration motors 138 attached to the medical bag, as shown in Figure 2. Alternatively, the agitator 134 may be a shaker 140 that shakes the medical bag, as shown in Figure 3. Alternatively, the agitator 134 may be a cuff 142 attached to the medical bag, as shown in Figure 4. The flow velocity sensor 136 is provided in the output channel 126. The flow velocity sensor 136 detects the flow velocity of the cell solution flowing through the output channel 126. A flow rate sensor may be provided instead of the flow velocity sensor 136.

[0047] As shown in Figure 5, the calculation unit 130 of the controller 20 functions as a control unit 146 and a calibration unit 148. The control unit 146 controls the first supply pump 102, the first circulation pump 104, and the stirring unit 134. The control unit 146 acquires detection values ​​from the flow velocity sensor 136. The calibration unit 148 acquires turbidity measurements from the sensor unit 34 and creates a calibration curve 150 (Figure 7) between the turbidity measurements and cell concentration.

[0048] [3. Calibration Process] Figure 6 is a flowchart of the calibration process performed by the calculation unit 130. Prior to the calibration process shown in Figure 6, the following processes are performed: The supply unit 22 pre-supplies cell-free culture medium to each channel of the cell culture circuit 16. Meanwhile, the user counts the number of cells in the cell solution using a cell counter or the like. The user fills the liquid holding unit 122 with the cell solution whose cell count has been recorded and sets the liquid holding unit 122 on the supply unit 22. The user stores the volume of the culture medium in the first circulation channel 58, the counted number of cells, and the volume of the cell solution in the storage unit 132. The control unit 146 operates the stirring unit 134 from before the supply of cell solution to the first circulation channel 58 starts until the supply ends.

[0049] In step S1, the control unit 146 operates the first supply pump 102 and the first circulation pump 104. In response to the operation of the first supply pump 102 and the first circulation pump 104, the supply unit 22 starts supplying cell saturation to the first circulation channel 58.

[0050] In step S2, the control unit 146 calculates the amount of cell saturation supplied to the first circulation channel 58 based on the value detected by the flow velocity sensor 136. For example, the control unit 146 calculates the flow rate per unit time based on the value detected by the flow velocity sensor 136 and the cross-sectional area of ​​the output channel 126. Furthermore, the control unit 146 calculates the supply amount based on the calculated flow rate and supply time. The supply amount calculated here is the total amount of cell saturation supplied from the start of supply to the most recent point in time.

[0051] In step S3, the control unit 146 compares the amount of cell saturation supplied calculated in step S2 with half the volume of cell saturation stored in the memory unit 132 (volume / 2). Here, the control unit 146 determines whether or not half of the cell saturation (the first cell volume of cells) filled in the liquid holding unit 122 has been supplied. If the amount of cell saturation supplied is half or more of the volume of cell saturation, i.e., half of the cell saturation has been supplied (step S3: YES), the process proceeds to step S4. On the other hand, if the amount of cell saturation supplied is less than half of the volume of cell saturation, i.e., half of the cell saturation has not yet been supplied (step S3: NO), the process returns to step S2.

[0052] In step S4, the control unit 146 stops the first supply pump 102. In response to the stopping of the first supply pump 102, the supply unit 22 stops supplying cell saturation to the first circulation channel 58.

[0053] In step S5, the calibration unit 148 acquires a measurement value from the sensor unit 34. This measurement value is designated as the first measurement value M1. The storage unit 132 stores the first measurement value M1.

[0054] In step S6, the control unit 146 restarts the first supply pump 102. In response to the operation of the first supply pump 102, the supply unit 22 resumes supplying cell saturation to the first circulation channel 58.

[0055] In step S7, the control unit 146 calculates the amount of cell saturation supplied to the first circulation channel 58 based on the value detected by the flow velocity sensor 136. Similar to step S2, the amount calculated here is the total amount of cell saturation supplied from the start of supply to the most recent point in time.

[0056] In step S8, the control unit 146 compares the amount of cell saturation to be supplied, calculated in step S7, with the volume of cell saturation stored in the memory unit 132. Here, the control unit 146 determines whether all of the cell saturation (second cell volume of cells) filled in the liquid holding unit 122 has been supplied. If the amount of cell saturation to be supplied is greater than or equal to the volume of cell saturation, i.e., all of the cell saturation has been supplied (step S8: YES), the process proceeds to step S9. On the other hand, if the amount of cell saturation to be supplied is less than the volume of cell saturation, i.e., not all of the cell saturation has been supplied yet (step S8: NO), the process returns to step S7.

[0057] In step S9, the control unit 146 stops the first supply pump 102. In response to the stopping of the first supply pump 102, the supply unit 22 stops supplying cell saturation to the first circulation channel 58.

[0058] In step S10, the calibration unit 148 acquires a measurement value from the sensor unit 34. This measurement value is designated as the second measurement value M2. The storage unit 132 stores the second measurement value M2.

[0059] In step S11, the calibration unit 148 creates a calibration curve 150 (Figure 7). The calibration unit 148 calculates the cell concentration of the culture medium (first medium) in the first circulation channel 58 at the time of step S5. The calibration unit 148 calculates the cell concentration from the volume of the culture medium stored in the memory unit 132 and half the number of cells stored in the memory unit 132. This cell concentration is designated as the first concentration C1. The calibration unit 148 associates the first concentration C1 with the first measured value M1. Furthermore, the calibration unit 148 calculates the cell concentration of the culture medium (second medium) in the first circulation channel 58 at the time of step S10. The calibration unit 148 calculates the cell concentration from the volume of the culture medium stored in the memory unit 132 and the number of cells stored in the memory unit 132. This cell concentration is designated as the second concentration C2. The calibration unit 148 associates the second concentration C2 with the second measured value M2. Cell concentration and turbidity have a linear relationship. Therefore, the calibration unit 148 creates a calibration curve 150 between cell concentration and turbidity (measured value) from the first concentration C1, the first measured value M1, the second concentration C2, and the second measured value M2, as shown in Figure 7. The calibration unit 148 stores the calibration curve 150 in the storage unit 132.

[0060] In the calibration process described above, the calibration unit 148 creates a calibration curve 150 using two points determined by the cell concentration and the measured value. Alternatively, the calibration unit 148 may create a calibration curve 150 using three or more points determined by the cell concentration and the measured value. In this case, the calibration unit 148 creates the calibration curve 150 using the least squares method.

[0061] In the calibration process described above, the calibration unit 148 acquires the measurement value of the sensor unit 34 when half of the cell solution has been supplied to the culture medium, and the measurement value of the sensor unit 34 when all of the cell solution has been supplied to the culture medium. Alternatively, the calibration unit 148 may acquire the measurement value of the sensor unit 34 at other timings.

[0062] [4. Inventions obtained from the embodiments] The inventions that can be understood from the above embodiments are described below.

[0063] A first aspect of the present invention is a cell culture apparatus (10) comprising: a cell culture circuit (16) capable of circulating a predetermined amount of culture medium supplied with cell saturation; a sensor (34) disposed in the cell culture circuit for measuring the turbidity of the cell saturation; and a calibration unit (148) for calibrating the sensor's measured value to the cell concentration of the culture medium. The sensor measures the turbidity of a first culture medium supplied with a first cell volume of the cell saturation as a first measured value (M1), and further measures the turbidity of a second culture medium supplied with a second cell volume of the cell saturation as a second measured value (M2). The calibration unit calculates the cell concentration of the first culture medium as a first concentration (C1), calculates the cell concentration of the second culture medium as a second concentration (C2), and creates a calibration curve (150) between the sensor's measured value and the cell concentration by relating the first concentration to the first measured value and relating the second concentration to the second measured value.

[0064] According to the first embodiment, a cell solution with a known cell count can be prepared in advance, and calibration can be performed using the cell culture device without any work being done outside the cell culture device. This improves work efficiency and eliminates the need to discard cell solution and culture medium.

[0065] In the first embodiment, the system includes a supply unit (22) for supplying the cell saturation to the cell culture circuit, and a control unit (146) for controlling the amount of the cell saturation supplied by the supply unit, wherein the supply unit may, after supplying the first cell amount of the cell saturation to the cell culture medium of the cell culture circuit which does not contain cells, further supply the second cell amount of the cell saturation to the first culture medium of the cell culture circuit.

[0066] In the first embodiment, the supply unit may include a liquid holding unit (122) for holding the cell saturation and a stirring unit (134) for stirring the cell saturation held in the liquid holding unit.

[0067] According to the above configuration, the concentration of the cell saturation in the liquid-holding section can be made uniform. As a result, the concentration of the cell saturation supplied to the culture medium can be kept constant. Therefore, a more accurate calibration curve can be established.

[0068] In the first embodiment, the liquid holding unit may be a bag filled with the cell fluid, and the stirring unit may be a vibration motor (138) attached to the bag.

[0069] In the first embodiment, the stirring unit may be a shaker (140) that shakes the liquid holding unit.

[0070] In the first embodiment, the liquid holding section may be a bag filled with the cell fluid, and the stirring section may be a cuff (142) attached to the bag.

[0071] A second aspect of the present invention is a calibration method using a cell culture circuit capable of circulating a predetermined amount of culture medium supplied with cell saturation, a sensor disposed in the cell culture circuit for measuring the turbidity of the cell saturation, and a calibration unit for calibrating the sensor's measured value to the cell concentration of the culture medium. The sensor measures the turbidity of a first culture medium supplied with a first cell volume of the cell saturation as a first measured value, and further measures the turbidity of a second culture medium supplied with a second cell volume of the cell saturation as a second measured value. The calibration unit calculates the cell concentration of the first culture medium as a first concentration, calculates the cell concentration of the second culture medium as a second concentration, and creates a calibration curve between the sensor's measured value and the cell concentration by relating the first concentration to the first measured value and relating the second concentration to the second measured value. [Explanation of Symbols]

[0072] 10...Cell culture device 16...Cell culture circuit 22... Supply unit 34... Sensor unit (sensor) 122...Liquid holding section 134...Agitation section 138...Vibration motor 140...Shaker 142...Cuff 146...Control Unit 148...Calibration section 150...Calibration curve

Claims

1. A cell culture circuit capable of circulating a predetermined amount of culture medium supplied with cell saturation, A sensor is placed in the cell culture circuit to measure the turbidity of the cell solution, A calibration unit that calibrates the measurement value of the sensor to the cell concentration of the culture medium, A cell culture apparatus comprising, A supply unit that supplies the cell solution to the cell culture circuit, A control unit that controls the amount of cell sap supplied by the supply unit, Furthermore, The supply unit first converts the culture medium into a first medium by supplying a first cell volume of the cell solution to the culture medium of the cell culture circuit that does not contain cells, and then further converts the first medium into a second medium by supplying a second cell volume of the cell solution to the first culture medium of the cell culture circuit. The sensor measures the turbidity of the first culture medium as a first measurement value, and further measures the turbidity of the second culture medium as a second measurement value. A cell culture apparatus comprising a calibration unit which calculates the cell concentration of the first culture medium as a first concentration, calculates the cell concentration of the second culture medium as a second concentration, relates the first concentration to the first measured value, and relates the second concentration to the second measured value, thereby creating a calibration curve between the measured value of the sensor and the cell concentration.

2. A cell culture apparatus according to claim 1, The aforementioned supply unit is A liquid-holding section for holding the cell sap, A stirring unit for stirring the cell solution held in the liquid holding unit, A cell culture device equipped with the following features.

3. A cell culture apparatus according to claim 2, The liquid-holding section is a bag into which the cell fluid is filled. The stirring unit is a vibration motor attached to the bag, in this cell culture apparatus.

4. A cell culture apparatus according to claim 2, The cell culture apparatus is characterized in that the stirring section is a shaker that shakes the liquid holding section.

5. A cell culture apparatus according to claim 2, The liquid-holding section is a bag into which the cell fluid is filled. The stirring unit is a cuff attached to the bag, in a cell culture apparatus.

6. A cell culture circuit capable of circulating a predetermined amount of culture medium supplied with cell saturation, A sensor is placed in the cell culture circuit to measure the turbidity of the cell solution, A calibration unit that calibrates the measurement value of the sensor to the cell concentration of the culture medium, A calibration method that uses, A supply unit that supplies the cell solution to the cell culture circuit, A control unit that controls the amount of cell sap supplied by the supply unit, Further use The supply unit first converts the culture medium into a first medium by supplying a first cell volume of the cell solution to the culture medium of the cell culture circuit that does not contain cells, and then further converts the first medium into a second medium by supplying a second cell volume of the cell solution to the first culture medium of the cell culture circuit. The sensor measures the turbidity of the first culture medium as a first measurement value, and further measures the turbidity of the second culture medium as a second measurement value. Calibration method comprising: the calibration unit calculating the cell concentration of the first culture medium as the first concentration, calculating the cell concentration of the second culture medium as the second concentration, relating the first concentration to the first measured value, and relating the second concentration to the second measured value, thereby creating a calibration curve between the measured value of the sensor and the cell concentration.

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