Cell culture device and cell count calculation method

Abstract

Provided is a cell count calculation method capable of accurately calculating the number of cells. A cell culture device (1) has a sensor (26) for measuring the lactic acid concentration in a culture medium. The sensor (26) measures, as a first physical quantity, the lactic acid concentration in the culture medium in the culture vessel (21) during cell culture. The sensor (26) measures, as an error output value, the output value of the lactic acid concentration in the culture medium in the supply bag (23), i.e., the output value of the lactic acid concentration in the culture medium supplied to the culture container (21). Furthermore, the cell culture device (1) is provided with a calculation unit (14) that calculates the number of cells in the culture vessel (21) on the basis of the value of a correction physical quantity calculated by subtracting the error output value from the value of the first physical quantity.

Description

Technical Field

[0001] The invention relates to a cell culture device and a cell number calculation method. Background Art

[0002] Conventional technology discloses a cell culture device for culturing cells in a culture container. For example, Patent Document 1 discloses a cell culture device having a culture container for accommodating cells and a liquid culture medium for cell culture (herein, refer to the second container of Patent Document 1).

[0003] In the cell culture apparatus of Patent Document 1, the number of cells in the culture container is appropriately calculated during the cell culture process, and the culture is terminated when the target number of cells is reached. As a method for calculating the number of cells, for example, there is a method of predicting the number of cells based on the lactic acid concentration in the culture medium in the culture container measured by a sensor. Lactic acid is a substance discharged by cell metabolism, and as the number of cells increases, the amount of lactic acid discharged also increases. Since the number of cells corresponding to the amount of lactic acid discharged is determined for each cell type or culture medium composition, the number of cells can be predicted by measuring the lactic acid concentration in the culture medium.

[0004] As a sensor for measuring lactic acid concentration, as shown in Patent Document 2, an enzyme sensor using an enzyme reaction (such as the biosensor in Patent Document 2) is generally used. The enzyme sensor disclosed in Patent Document 2 includes: an oxidoreductase that reacts with lactic acid, which is a substance to be measured and one of the metabolic products of cells; and an electron transfer substance that transfers the electrons obtained by the oxidation-reduction reaction of lactic acid to a measuring electrode. The enzyme sensor uses the electrons generated by the reaction between lactic acid in the culture medium and the oxidoreductase, and the lactic acid concentration in the culture medium can be quantified by measuring the electrons transferred to the measuring electrode.

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2021-087364

[0007] Patent Document 2: Japanese Patent Application Publication No. 2019-002738 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] However, when the enzyme sensor measures the lactic acid concentration in the culture medium in the culture container, a value greater than the lactic acid concentration actually discharged from the cells may be obtained. This is because in addition to the lactic acid discharged by cell metabolism, other interfering substances originally contained in the culture medium may also react with the enzyme sensor unintentionally. As a result, in addition to the output value derived from the lactic acid contained in the culture medium, the enzyme sensor also misjudges the output value derived from the interfering substances as the lactic acid concentration, resulting in an error in the measured value of the lactic acid concentration. Therefore, if the measured value of the lactic acid concentration is erroneous, the number of cells predicted based on the lactic acid concentration will also be biased, making it impossible to accurately calculate the number of cells in the culture container.

[0010] In addition, the above problem is not limited to the case where the number of cells is calculated by measuring the lactate concentration, and the same problem may also occur when a method is used to calculate the number of cells by measuring a physical quantity related to cell metabolites.

[0011] An object of the present invention is to provide a cell culture device and a cell number calculation method capable of accurately calculating the number of cells.

[0012] Solution to the problem

[0013] The cell culture device of the present invention includes: a culture container for accommodating cells and culture medium and culturing cells; a sensor for measuring a first physical quantity and an error output value of a physical quantity of a cell metabolite that increases with the proliferation of cells, wherein the first physical quantity is a physical quantity in the culture medium in the culture container during the cell culture process, and the error output value is an output value of a physical quantity in the culture medium supplied to the culture container; and a cell number calculation unit for calculating the number of cells in the culture container based on a correction physical quantity calculated by subtracting the error output value from the value of the first physical quantity.

[0014] According to the present invention, the correction physical quantity is calculated by subtracting the error output value derived from the interfering substance originally contained in the culture medium from the value of the first physical quantity in the culture medium in the culture container. Thus, a correction physical quantity related to the number of cells in the culture container can be obtained, and this correction physical quantity is a physical quantity of cell metabolites that is not affected by the interfering substance contained in the culture medium. Therefore, by deriving the number of cells based on the correction physical quantity, the number of cells in the culture container can be correctly calculated.

[0015] In the cell culture apparatus of the present invention, preferably, one sensor measures the first physical quantity and the error output value.

[0016] Even sensors of the same type may have different measurement performances due to different batches. Then, when different sensors are used to measure the physical quantity in the culture medium in the culture container and the physical quantity in the culture medium supplied to the culture container, measurement errors may occur between the sensors. According to the present invention, since one sensor is used to measure the physical quantity in the culture medium in the culture container and the physical quantity in the culture medium supplied to the culture container, the measurement errors between the sensors can be eliminated.

[0017] In the cell culture apparatus of the present invention, the culture medium in the culture container is replaced during the culture process, and the sensor preferably outputs a physical quantity value of the culture medium resupplied to the measurement culture container as an error output value when the culture medium is replaced.

[0018] The sensor measuring the physical quantity may deteriorate due to repeated measurements, resulting in a decrease in measurement capability. Therefore, the measured value of the physical quantity will deviate according to the measurement timing of the sensor. If the timing of the sensor measuring the first physical quantity and the timing of the sensor measuring the error output value have a large deviation, the first physical quantity and the error output value will be measured by sensors with different measurement capabilities. In this way, the first physical quantity measured by the sensor with a deviation in measurement capability and the corrected physical quantity calculated by the error output value may deviate from the actual value. According to the present invention, the sensor re-measures the output value of the physical quantity in the culture medium re-supplied to the culture container as the error output value when the culture medium is replaced. Therefore, the large deviation between the timing of the first physical quantity in the culture medium in the culture container under cultivation measured by the sensor and the timing of the measurement error output value can be suppressed. Thereby, the deviation of the corrected physical quantity from the actual value caused by the deviation of the measurement capability of the sensor can be suppressed. By deriving the number of cells based on the corrected physical quantity obtained in this way, the number of cells in the culture container can be calculated more accurately.

[0019] In the cell culture device of the present invention, the first physical quantity is preferably lactic acid concentration.

[0020] Generally speaking, the concentration of a substance with a large total amount can be measured with higher accuracy than the concentration of a substance with a small total amount. In the present invention, the concentration of lactic acid is measured as a physical quantity. Lactic acid is excreted in large quantities as a metabolite of cells, and the concentration of lactic acid can be measured more accurately than the concentration of other substances.

[0021] The cell number calculation method of the present invention contains cells and culture medium, calculates the number of cells in a culture container used to culture cells, and performs the following steps: a first measurement step, measuring a first physical quantity of a physical quantity of a cell metabolite that increases with the proliferation of the cell, the first physical quantity being a physical quantity in the culture medium in the culture container during the culture of the cell; a second measurement step, measuring an error output value, the error output value being an output value of a physical quantity in the culture medium supplied to the culture container; and a cell number calculation step, calculating the number of cells in the culture container based on a correction physical quantity calculated by subtracting the error output value from the value of the first physical quantity.

[0022] According to the present invention, the correction physical quantity is calculated by subtracting the error output value derived from the interfering substance originally contained in the culture medium from the value of the first physical quantity in the culture medium in the culture container. In this way, a correction physical quantity related to the number of cells in the culture container can be obtained, and this correction physical quantity is a physical quantity of cell metabolites that is not affected by the interfering substance contained in the culture medium. Therefore, by deriving the number of cells based on the correction physical quantity, the number of cells in the culture container can be correctly calculated.

[0023] In the cell count method of the present invention, the culture medium in the culture container is replaced during the culture process, and in the second measurement step, preferably when the culture medium is replaced, the output value of the physical quantity of the culture medium resupplied to the culture container is measured as the error output value.

[0024] The sensor measuring the physical quantity may deteriorate due to repeated measurements, resulting in a decrease in measurement capability. Therefore, the measured value of the physical quantity will deviate according to the measurement timing of the sensor. If the timing of the sensor measuring the first physical quantity and the timing of the sensor measuring the error output value have a large deviation, the first physical quantity and the error output value will be measured by sensors with different measurement capabilities. In this way, the first physical quantity measured by the sensor with a deviation in measurement capability and the corrected physical quantity calculated by the error output value may deviate from the actual value. According to the present invention, the sensor re-measures the output value of the physical quantity in the culture medium re-supplied to the culture container as the error output value at the timing of the culture medium replacement. Therefore, it is possible to suppress the large deviation between the timing of the sensor measuring the first physical quantity in the culture medium in the culture container during the culture and the timing of the measurement error output value. Thereby, the deviation between the corrected physical quantity and the actual value caused by the deviation of the measurement capability of the sensor can be suppressed. By deriving the number of cells based on the corrected physical quantity obtained in this way, the number of cells in the culture container can be calculated more accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1 is a front view schematically showing the structure of the cell culture apparatus of this example.

[0026] FIG. 2 is a schematic diagram showing various structural parts arranged inside the culture section.

[0027] FIG. 3 is a diagram showing the state of the culture unit when the cell suspension is transferred from the cell bag to the culture container.

[0028] FIG. 4 is a diagram showing the state of the culture unit when measuring the lactic acid concentration in the culture medium in the supply bag.

[0029] FIG. 5 is a diagram showing a state of the culture unit when the culture medium is supplied from the supply bag to the culture container.

[0030] FIG. 6 is a diagram showing the state of the culture unit when the lactic acid concentration in the culture medium in the culture container is measured.

[0031] FIG. 7 is a diagram showing a state of the culture unit when the culture medium is transferred from the culture container to the drainage bag.

[0032] FIG. 8 is a diagram showing the state of the culture unit when the cell suspension is transferred from the culture container to the recovery bag.

[0033] FIG. 9 shows a flow chart for culturing cells in a cell culture apparatus.

[0034] FIG. 10 is a flow chart showing the process of culturing cells in the cell culturing apparatus according to the first modification. DETAILED DESCRIPTION

[0035] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.

[0036] (Cell culture device 1)

[0037] The cell culture device 1 is a device that automatically cultures cells while adjusting the culture environment. As shown in FIG1 , the cell culture device 1 includes: a cold storage unit 11 for storing culture medium, stripping solution and other reagents; a culture unit 12, which has a culture container 21 inside and performs cell culture while adjusting the internal environment; a control unit 13, which controls the culture, subculture, replacement of the culture medium in the culture container 21, recovery of the cell suspension, etc. in the culture unit 12; and a calculation unit 14, which calculates the number of cells in the culture container 21 (the cell number calculation unit of the present invention).

[0038] The culture section 12 is internally configured with: a culture container 21, a cell bag 22, a supply bag 23, a drainage bag 24, and a recovery bag 25 (as shown in FIG. 2 ). The culture container 21 is a slightly rectangular container capable of culturing cells in a plurality of culture layers. When cell culture is performed, cells adhere to the bottom surface of each culture layer of the culture container 21. As the culture proceeds, the cells proliferate along the bottom surface of each culture layer of the culture container 21. The cell bag 22 contains a cell suspension supplied to the culture container 21. The cell suspension is a suspension of cells in a culture medium. The supply bag 23 contains a culture medium supplied to the culture container 21. In addition, the culture medium constituting the cell suspension contained in the cell bag 22 and the culture medium contained in the supply bag 23 are, for example, the same type of culture medium. When the culture medium is replaced, the drainage bag 24 contains the culture medium discharged from the culture container 21. The recovery bag 25 is used to recover the culture medium or cell suspension containing the cells that have completed the culture.

[0039] In addition, a sensor 26 is arranged inside the culture part 12, which is used to measure the lactic acid concentration in the culture medium in the culture container 21 and the lactic acid concentration in the culture medium in the supply bag 23. In this embodiment, the sensor 26 is a sensor that measures physical quantities using enzyme reactions, and is a sensor that measures the lactic acid concentration as a physical quantity. Cells absorb glucose contained in the culture medium and excrete lactic acid by metabolism. That is, lactic acid is a cell metabolite that increases as cells proliferate. The sensor 26 is, for example, an enzyme sensor manufactured by Sartorius. The structure of the sensor 26 will be described below. Although the sensor 26 is not shown, it includes: a container part that can accommodate a small amount of culture medium and a buffer solution; an electrode part that is immersed in the buffer solution accommodated in the container part; an enzyme coated on the electrode part; and an electron transfer substance for transferring generated electrons to the electrode part. The container part has a part for accommodating the culture medium and a part for accommodating the buffer solution, and each part is separated by a dialysis membrane. The components contained in the small amount of culture medium transported from the culture container 21 and the supply bag 23 to the container part of the sensor 26 flow into the buffer solution side through the dialysis membrane. The buffer solution into which the components contained in the culture medium flow is transported to the electrode portion of the sensor 26. The electrode portion is, for example, a platinum electrode. This enzyme is lactate oxidase. The steps of measuring the lactate concentration by the sensor 26 are as follows. First, the lactate contained in the culture medium is oxidized by lactate oxidase, and pyruvic acid and hydrogen peroxide are generated. Then, a voltage is applied using the electrode portion to decompose the hydrogen peroxide into oxygen and hydrogen ions and generate electrons. Subsequently, the generated electrons are measured as a current, and the lactate concentration is calculated based on the measured current.

[0040] In addition, the sensor 26 measures the lactic acid concentration in the culture medium in the culture container 21 by using an enzyme reaction according to the above principle. However, since an unexpected reaction may occur between a predetermined interfering substance in the culture medium and the enzyme, electrode portion, etc. of the sensor 26, the sensor 26 may mistakenly recognize the output value derived from the interfering substance as the lactic acid concentration. The "predetermined interfering substance" is one or more chemical substances contained in the culture medium, and is a substance that can generate electrons by a certain reaction with the enzyme, electrode portion, etc. of the sensor 26. The interfering substance in this embodiment is, for example, a substance that can chemically react with the lactate oxidase, platinum electrode, etc. of the sensor 26. Therefore, regarding the lactic acid concentration in the culture medium in the culture container 21 during cell culture, in addition to the actual lactic acid concentration, the lactic acid concentration measurement value measured by the sensor 26 will include an error output value caused by the interfering substance in the culture medium. Therefore, in this embodiment, the sensor 26 measures the lactic acid concentration in the culture medium in the culture container 21 during cell culture as the first physical quantity, and measures the output value of the lactic acid concentration in the culture medium in the supply bag 23 of the culture medium supplied to the culture container 21 as the error output value derived from the interfering substance. Then, the calculation unit 14 described later calculates the correction physical quantity by subtracting the error output value from the first physical quantity. The correction physical quantity can be said to be the true lactic acid concentration that is not affected by the output value of the interfering substance in the culture medium in the culture container 21. The calculation unit 14 calculates the number of cells in the culture container 21 based on the correction physical quantity, so that the number of cells can be calculated correctly. In this embodiment, a sensor 26 is used to measure both the lactic acid concentration in the culture medium in the culture container 21 as the first physical quantity and the output value of the lactic acid concentration in the culture medium supplied to the culture container 21 as the error output value.

[0041] The inside of the container portion for supplying the culture medium to the culture container 21, the cell bag 22, the supply bag 23, the drainage bag 24, the recovery bag 25, and the sensor 26 is in a sterile state.

[0042] The culture container 21, the cell bag 22, the supply bag 23, the drainage bag 24, the recovery bag 25 and the sensor 26 are connected by a connection path 27 (as shown in FIG. 2 ). The culture container 21, the cell bag 22, the supply bag 23, the drainage bag 24 and the recovery bag 25 can be loaded and unloaded relative to the connection path 27 described later while maintaining a sterile state. The connection path 27 is, for example, a resin tube. The connection path 27 has a main path 27a and a branch path 27b. The branch path 27b is a path branched from the main path 27a, and has six branch paths 27b1 to 27b6.

[0043] The branch path 27b1 is a path connecting the culture container 21 and the main path 27a. The branch path 27b2 is a path connecting the cell bag 22 and the main path 27a. The branch path 27b3 is a path connecting the supply bag 23 and the main path 27a. The branch path 27b4 is a path connecting the drainage bag 24 and the main path 27a. The branch path 27b5 is a path connecting the recovery bag 25 and the main path 27a. The branch path 27b6 is a path connecting the sensor 26 and the main path 27a. As shown in FIG. 2, the branch paths 27b1, 27b2, 27b3 and 27b6 are connected to the opposite side of the main path 27a separated from the branch paths 27b4 and 27b5 by the second pump 28b described later.

[0044] A valve V1 is provided in the branch path 27b2. When the valve V1 is open, liquid can pass through the branch path 27b2, and when the valve V1 is closed, the liquid cannot pass through the branch path 27b2. A valve V2 is provided in the branch path 27b3. When the valve V2 is open, liquid can pass through the branch path 27b3, and when the valve V2 is closed, the liquid cannot pass through the branch path 27b3. A valve V3 is provided in the branch path 27b6. When the valve V3 is open, liquid can pass through the branch path 27b6, and when the valve V3 is closed, the liquid cannot pass through the branch path 27b6. A valve V4 is provided in the branch path 27b4. When the valve V4 is open, liquid can pass through the branch path 27b4, and when the valve V4 is closed, the liquid cannot pass through the branch path 27b4. A valve V5 is provided in the branch path 27b5. When the valve V5 is open, liquid can pass through the branch path 27b5, and when the valve V5 is closed, the liquid cannot pass through the branch path 27b5. The opening and closing of each valve is controlled by the control unit 13.

[0045] In addition, as shown in FIG2 , a first pump 28a is provided in the branch path 27b1. A second pump 28b is provided in the main path 27a. A third pump 28c is mounted to the sensor 26. Each pump is, for example, a peristaltic pump including a motor for driving a rotor, the rotor clamping the pipe between metal rotors and rotating the rotor to suck and deliver the fluid. In addition, each pump may be a syringe pump including a cylindrical cylinder, a piston that sucks and delivers the fluid in a reciprocating motion in the cylinder, and a motor that drives the piston.

[0046] The control unit 13 is connected to the valves V1 to V5, the first pump 28a, the second pump 28b, the third pump 28c, etc. The control unit 13 controls the opening and closing of the valves V1 to V5 and the driving control of each pump. The connection between the control unit 13 and each valve and each pump can be a wireless connection or an electrical connection.

[0047] The calculation unit 14 is connected to the sensor 26, and calculates the number of cells based on the lactic acid concentration in the culture medium measured by the sensor 26. The method of calculating the number of cells by the calculation unit 14 will be described in detail later.

[0048] (Cell Culture Steps)

[0049] Next, the steps of culturing cells in the cell culturing apparatus 1 of this embodiment will be described with reference to Figures 3 to 9. In the cell culturing, a cell number counting method is performed to count the number of cells in the culture container 21. Figure 9 is a flow chart showing the cell culturing process.

[0050] First, when cell culture is performed, the culture section 12 is preset with: an empty culture container 21, a cell bag 22 containing a cell suspension containing primary cells and a culture medium, a supply bag 23 containing a culture medium, an empty drainage bag 24, and an empty recovery bag 25. That is, the culture container 21, the cell bag 22, the supply bag 23, the drainage bag 24, and the recovery bag 25 are pre-connected to the connection path 27. The connection of each bag to the connection path 27 is performed by the operator. At this time, valves V1 to V5 are closed. In FIGS. 3 to 8, black valves indicate a closed state, and white valves indicate an open state.

[0051] Next, as shown in FIG3 , the control unit 13 opens the valve V1 and drives the first pump 28 a. As a result, the cell suspension in the cell bag 22 is transported to the culture container 21 (step S1) (as shown by the solid arrow in FIG3 ). At this time, all the cell suspension in the cell bag 22 is transported to the culture container 21.

[0052] Afterwards, as shown in FIG. 4 , the control unit 13 closes the valve V1, opens the valves V2 and V3, and drives the third pump 28 c. As a result, a portion of the culture medium in the supply bag 23 is transported to the sensor 26 (as shown by the solid arrow in FIG. 4 ). At this time, the amount of culture medium transported from the supply bag 23 to the sensor 26 is a portion of the culture medium contained in the supply bag 23, for example, 40 ml. The sensor 26 measures the output value of the lactic acid concentration derived from the interfering substance in the culture medium transported from the supply bag 23 as an error output value (step S2). The output value of the lactic acid concentration here is a value output as a physical quantity similar to the lactic acid concentration, but is different from the actual lactic acid concentration. The step of measuring the error output value corresponds to the second measurement step of the present invention. In addition, the culture medium transported from the supply bag 23 to the sensor 26 is transported to the drainage bag 24 at a preset timing before executing the later-described step S5 after the sensor 26 measures the error output value. For example, after the sensor 26 measures the error output value and before executing step S3 described later, the culture medium transported from the supply bag 23 to the sensor 26 is transported to the drainage bag 24 .

[0053] Next, as shown in FIG5 , the control unit 13 closes the valve V3 and drives the first pump 28a. As a result, the culture medium contained in the supply bag 23 is transported to the culture container 21 (step S3) (as shown by the solid arrow in FIG5 ). At this time, the required amount of culture medium contained in the supply bag 23 is transported to the culture container 21. The required amount varies depending on the type of culture container 21, for example, one culture layer in the culture container 21 is 200 to 300 ml. That is, for example, for a culture container 21 having 5 culture layers, the amount of culture medium supplied from the supply bag 23 is 1000 ml to 1500 ml. In the present embodiment, the supply bag 23 contains, for example, an amount of culture medium that can supply the culture container 21 having 10 culture layers at one time.

[0054] In addition, when the amount of the culture medium contained in the supply bag 23 decreases as the culture medium is supplied from the supply bag 23 to the culture container 21, the culture medium stored in the cold storage unit 11 is resupplied to the supply bag 23. The supply bag 23 and the container (not shown) containing the culture medium in the cold storage unit 11 are connected by a pipe or the like (not shown). In one culture cycle from the start of cell culture to cell recovery, the same batch of culture medium is supplied from the cold storage unit 11 to the supply bag 23. If cells are grown in one culture cycle and then subcultured to another culture cycle, different batches of culture medium can be used for each culture cycle.

[0055] When the cell suspension is transferred from the cell bag 22 to the culture container 21 and the culture medium is transferred from the supply bag 23, cell culture is started in the culture container 21 (step S4). At this time, the cells in the cell suspension are attached to the bottom surface of the culture layer of the culture container 21. During the cell culture, the temperature, CO2 concentration, etc. in the culture part 12 are appropriately adjusted. The temperature is adjusted to, for example, 37°C. The CO2 concentration is adjusted to, for example, 5%. In addition, when cell culture is performed, each valve V1 to V5 is in a closed state.

[0056] (Calculation of cell number)

[0057] When a predetermined time has passed since the start of cell culture, the number of cells in the culture container 21 is calculated. The timing for calculating the number of cells in the culture container 21 is, for example, preset. Specifically, first, as shown in FIG6 , the control unit 13 opens the valve V3 and drives the third pump 28c. As a result, part of the culture medium in the culture container 21 is transported to the sensor 26 (as shown by the solid arrow in FIG6 ). At this time, the amount of culture medium transported from the culture container 21 to the sensor 26 is a part of the culture medium contained in the culture container 21, for example, 20 ml. In addition, at this time, the cells attached to the bottom surface of each culture layer of the culture container 21 will not peel off. The sensor 26 measures the lactic acid concentration in the culture medium transported from the culture container 21 as a first physical quantity (step S5). After the sensor 26 measures the first physical quantity, the control unit 13 closes the valve V3. The step of measuring the first physical quantity corresponds to the first measurement step of the present invention.

[0058] After that, the calculation unit 14 calculates the number of cells in the culture container 21 based on the first physical quantity measured by the sensor 26 and the error output value (step S6). Specifically, the calculation unit 14 calculates the number of cells in the culture container 21 based on the correction physical quantity calculated by subtracting the error output value from the value of the first physical quantity. For example, the number of cells corresponding to the correction physical quantity is predetermined for each cell type and each culture medium type. The step of calculating the number of cells in the culture container 21 by the calculation unit 14 is equivalent to the cell number calculation step of the present invention. Next, the control unit 13 determines whether the number of cells in the culture container 21 calculated by the calculation unit 14 reaches the preset target cell number (step S7).

[0059] When the number of cells in the culture container 21 reaches the target number of cells (S7: YES), the cells are recovered (step S8). The step of recovering cells will be described later. If the number of cells in the culture container 21 does not reach the target number of cells (S7: NO), the cells are continued to be cultured in the culture container 21 (step S9).

[0060] (Change medium)

[0061] After the cell culture is carried out for a predetermined time, the culture medium in the culture container 21 is replaced (step S10). The predetermined time is a time pre-set according to the type of cells, the type of culture medium, etc. In order to explain the replacement of the culture medium in detail, first, as shown in FIG7 , the control unit 13 opens the valve V4 and drives the second pump 28b. As a result, the culture medium in the culture container 21 is transported to the drainage bag 24 (as shown by the solid arrow in FIG7 ). At this time, almost all of the culture medium for culturing cells in the culture container 21 is transported to the drainage bag 24. Next, the control unit 13 closes the valve V4, opens the valve V2, and drives the first pump 28a. As a result, the culture medium contained in the supply bag 23 is transported to the culture container 21 (as shown by the solid arrow in FIG5 ). At this time, the required amount of culture medium contained in the supply bag 23 is transported to the culture container 21. The required amount is as described above. That is, the required amount varies depending on the type of the culture container 21, for example, one culture layer of the culture container 21 is 200 to 300 ml. As described above, the culture medium in the culture container 21 is replaced.

[0062] When the replacement of the culture medium in the culture container 21 is completed, the process returns to step S4 and the cell culture in the culture container 21 is restarted. Then, when a predetermined time has passed since the cell culture was restarted, the sensor 26 measures the lactic acid concentration in the culture medium in the culture container 21 as the first physical quantity (step S5). Then, the calculation unit 14 calculates the number of cells in the culture container 21 based on the first physical quantity measured again by the sensor 26 and the error output value measured by the sensor 26 as the output value of the lactic acid concentration in the culture medium initially supplied to the culture container 21 (step S6).

[0063] (Recovery of cells)

[0064] When the number of cells in the culture container 21 reaches the target number of cells (S7: YES), the cells are recovered (step S8). Specifically, first, the control unit 13 opens the valve V4 and drives the second pump 28b. As a result, all the culture medium in the culture container 21 is transported to the drainage bag 24 (as shown by the solid arrow in FIG7). Next, the control unit 13 closes the valve V4 and connects a stripping liquid bag (not shown) to the branch path 27b2 of the connection path 27 instead of the cell bag 22. The stripping liquid bag contains a stripping liquid for stripping cells attached to the bottom surface of each culture layer of the culture container 21 from the bottom surface. The cell bag 22 and the stripping liquid are replaced by the operator, for example. The replacement of the cell bag 22 and the stripping liquid is performed while maintaining a sterile state. Next, the control device 13 opens the valve V1 and drives the first pump 28a (the opening and closing states of each valve are shown in FIG3). As a result, all the stripping liquid contained in the stripping liquid bag (not shown) is transported to the culture container 21. Next, the control unit 13 closes the valve V1, opens the valve V2, and drives the first pump 28a (the opening and closing states of each valve are shown in FIG5 ). As a result, the culture medium contained in the supply bag 23 is transported to the culture container 21. In this state, the control unit 13 closes the valve V2, stops driving the first pump 28a, and stirs the culture medium in the culture container 21. Thus, a cell suspension in which cells are suspended in the culture medium is contained in the culture container 21. In this state, the controller 13 opens the valve V5 to drive the second pump 28b. Thus, the cell suspension in the culture container 21 is transported to the recovery bag 25. In this way, the recovery of cells is completed.

[0065] Thereby, the cell culture step in the cell culture apparatus 1 is completed.

[0066] (Effect)

[0067] As described above, the cell culture device 1 of this embodiment has a sensor 26 for measuring the lactic acid concentration in the culture medium. The sensor 26 measures the lactic acid concentration in the culture medium in the culture container 21 during cell culture as a first physical quantity. In addition, the sensor 26 measures the lactic acid concentration in the culture medium in the supply bag 23, that is, the output value of the lactic acid concentration in the culture medium supplied to the culture container 21 is used as an error output value. In addition, the cell culture device 1 has a calculation unit 14, and the calculation unit 14 calculates the number of cells in the culture container 21 based on the value of the correction physical quantity calculated by subtracting the error output value from the value of the first physical quantity. In addition, the cell number calculation method of this embodiment performs: a first measurement step of measuring the first physical quantity, a second measurement step of measuring the error output value, and a cell number calculation step of calculating the number of cells in the culture container 21 based on the correction physical quantity calculated by subtracting the error output value from the value of the first physical quantity.

[0068] According to this embodiment, the correction physical quantity is calculated by subtracting the error output value derived from the substance originally contained in the culture medium from the value of the first physical quantity in the culture medium in the culture container 21. As a result, the correction physical quantity related to the number of cells in the culture container 21, that is, the concentration of lactic acid, which is a cell metabolite not affected by the substance contained in the culture medium, can be obtained. Therefore, by deriving the number of cells based on the correction physical quantity, the number of cells in the culture container 21 can be calculated with high accuracy.

[0069] In addition, in the cell culture apparatus 1 of the present embodiment, one sensor 26 is used to measure both the first physical quantity and the error output value. Even if the same type of sensor is used, the measurement performance may be slightly different due to batch differences. Then, when different sensors are used to measure the lactic acid concentration in the culture medium in the culture container 21 and the lactic acid concentration in the culture medium in the supply bag 23, a measurement error may occur between the sensors. According to the present embodiment, since the lactic acid concentration in the culture medium in the culture container 21 and the lactic acid concentration in the culture medium in the supply bag 23 are measured using a single sensor 26, the measurement error between the sensors can be eliminated.

[0070] In addition, in this embodiment, the physical quantity of the cell metabolite that increases with cell proliferation is the concentration of lactic acid. Generally speaking, the concentration of a substance with a large total amount can be measured with higher accuracy than the concentration of a substance with a small total amount. Lactic acid is excreted in large quantities as a cell metabolite, and the concentration of lactic acid can be measured with high accuracy compared to the concentration of other substances.

[0071] (Variation)

[0072] A modified example obtained by modifying the above-described embodiment will be described below. However, members having the same configuration as those of the above-described embodiment are denoted by the same reference numerals, and description thereof is appropriately omitted.

[0073] (First Modification)

[0074] In the above embodiment, the sensor 26 measures the output value of the lactic acid concentration in the culture medium as the error output value, and the culture medium is initially supplied to the culture container 21. And the error output value is no longer remeasured when the culture medium is replaced. However, the sensor 26 can remeasure the output value of the lactic acid concentration in the culture medium resupplied to the culture container 21 as the error output value when the culture medium is replaced. Hereinafter, it will be described with reference to Figure 10. In Figure 10, the same symbols will be used for steps S1 to S10 common to the above embodiment. As shown in Figure 10, when the culture medium is replaced, the error output value is first remeasured before replacing the culture medium (step S10) (step S20). Specifically, the control unit 13 opens valves V2 and V3 and drives the third pump 28c. As a result, a portion of the culture medium in the supply bag 23 is transported to the sensor 26 (as shown by the solid arrow in Figure 4). At this time, the amount of culture medium transported from the supply bag 23 to the sensor 26 is a portion of the culture medium contained in the supply bag 23, for example, 20ml. The sensor 26 measures the output value of the lactic acid concentration in the culture medium delivered from the supply bag 23 as the error output value. That is, when the culture medium is replaced, the sensor 26 measures the output value of the lactic acid concentration in the culture medium resupplied to the culture container 21 as the error output value. After the sensor 26 measures the error output value, the control unit 13 closes the valve V3. After the error output value is remeasured, the culture medium in the culture container 21 is replaced (step S10). The step of replacing the culture medium is the same as that of the above embodiment.

[0075] That is, in the first variant, the culture medium in the culture container 21 is replaced during the culture process, and the sensor 26 measures the output value of the lactic acid concentration of the culture medium resupplied to the culture container 21 as the error output value when the culture medium is replaced. In addition, in the cell number calculation method of this embodiment, in the second measurement step, the output value of the lactic acid concentration of the culture medium resupplied to the culture container 21 when the culture medium is replaced is measured as the error output value.

[0076] The sensor 26 for measuring the lactic acid concentration may deteriorate with repeated measurements, resulting in a decrease in measurement capability. As a result, the measured value of the lactic acid concentration may deviate according to the measurement timing of the sensor 26. If there is a large deviation between the timing of measuring the first physical quantity and the timing of measuring the error output value of the sensor 26, the first physical quantity and the error output value will be measured by the sensor 26 with different measurement capabilities. Therefore, the corrected physical quantity calculated based on the first physical quantity and the error output value measured by the sensor 26 with the measurement capability deviation may deviate from the actual value. According to the present embodiment, the sensor 26 re-measures the output value of the lactic acid concentration in the culture medium re-supplied to the culture container 21 as the error output value at the timing of replacing the culture medium. Therefore, it is possible to suppress the large deviation between the timing of measuring the first physical quantity of the lactic acid concentration in the culture medium in the culture container 21 in the culture and the timing of measuring the error output value. As a result, it is possible to suppress the deviation of the corrected physical quantity from the actual value caused by the deviation of the measurement accuracy of the sensor 26 due to deterioration over time. By deriving the number of cells based on the corrected physical quantity obtained in this way, the number of cells in the culture container 21 can be calculated more accurately.

[0077] In addition, when culturing cells, multiple culture cycles can be performed. That is, after a culture cycle from the start of cell culture to the recovery of cells is completed, the next culture cycle can be performed using the subcultured cells. In this case, the culture medium used in one culture cycle may be different from the batch of the culture medium used in the next culture cycle. In other words, the batch of the culture medium supplied from the supply bag 23 to the culture container 21 in one culture cycle may be different from the batch of the culture medium supplied from the supply bag 23 to the culture container 21 in the next culture cycle. Here, even if the culture medium from different batches belongs to the same type, its composition may be slightly different. In this way, for example, the error output value in the culture medium supplied to the culture container 21 in the first culture cycle and the error output value in the culture medium supplied to the culture container 21 in the next culture cycle will vary depending on the culture medium batch. Assume that if the deviation of the error output value caused by the batch difference between the culture media is not taken into account, the value of the corrected physical quantity calculated by subtracting the error output value from the value of the first physical quantity may deviate from the actual value. In this regard, in the first variant, the sensor 26 re-measures the output value of the lactic acid concentration in the culture medium re-supplied to the culture container 21 as the error output value when the culture medium is replaced. Therefore, even if culture medium from different batches is used when multiple culture cycles are performed, the accurate value of the corrected physical quantity can be obtained in consideration of the variation in the error output value between the culture medium from different batches. By deriving the number of cells based on the corrected physical quantity obtained in this way, the number of cells in the culture container 21 can be calculated more accurately.

[0078] (Other variations)

[0079] In the above embodiment, one sensor 26 measures both the first physical quantity and the error output value. However, a sensor for measuring the first physical quantity and a sensor for measuring the error output value may be provided separately.

[0080] In the above embodiment, the sensor 26 measures the lactate concentration as a physical quantity of the cell metabolite. However, the physical quantity measured by the sensor 26 is not limited to the lactate concentration. For example, when culturing cells in a differentiated state, in addition to lactate, the physical quantity measured by the sensor 26 may also include the concentrations of glutamic acid and kynurenine. Furthermore, when culturing cells in an undifferentiated state, in addition to lactate, the physical quantity measured by the sensor 26 may also include the concentrations of glutamic acid and 2-aminoadipic acid.

[0081] In the above embodiment, when the sensor 26 measures the lactic acid concentration in the culture medium supplied to the culture container 21 as an error output value, it measures the lactic acid concentration in the culture medium in the supply bag 23. However, the sensor 26 does not have to measure the lactic acid concentration in the culture medium in the supply bag 23. For example, the sensor 26 may be a culture medium of the same type as the culture medium in the supply bag 23, and measures the lactic acid concentration in a culture medium different from the culture medium contained in the supply bag 23. Specifically, the sensor 26 may measure the lactic acid concentration in the culture medium in a supply bag (not shown) other than the supply bag 23 connected to the connection path 27 (branch path 27b6). At this time, the culture medium in the supply bag (not shown) whose lactic acid concentration is measured by the sensor 26 is then connected to the connection path 27 at a predetermined timing and supplied to the culture container 21. In addition, another supply bag (not shown) can be loaded and unloaded relative to the connection path 27 while maintaining a sterile state.

[0082] In the above embodiment, the cell culture device 1 includes a calculation unit 14 that calculates the number of cells in the culture container 21 based on the corrected physical quantity. However, the calculation unit 14 can be built into an external PC. In this case, data of the first physical quantity and the error output value measured by the sensor 26 will be transmitted to the external PC. In addition, in the cell number calculation method, the operator can calculate the number of cells in the culture container 21 corresponding to the corrected physical quantity based on the corrected physical quantity calculated in the cell culture device 1. Alternatively, the operator calculates the corrected physical quantity based on the first physical quantity measured by the sensor 26 and the error output value, and further, the number of cells in the culture container 21 can be calculated based on the calculated corrected physical quantity.

[0083] Explanation of symbols

[0084] 1: Cell culture device,

[0085] 13: Control Department,

[0086] 14: Calculation unit (cell number calculation device),

[0087] 21: culture container,

[0088] 23: Supply bag,

[0089] 26: Sensor,

[0090] 27: Connection path.

Claims

1. A cell culture device, comprising: A culture container for containing cells and culture medium and culturing cells; a sensor that measures a first physical quantity and an error output value of a physical quantity of a metabolite of the cell that increases as the cell proliferates, wherein the first physical quantity is the physical quantity in the culture medium in the culture container during the culture of the cell, and the error output value is an output value of the physical quantity in the culture medium supplied to the culture container; as well as The cell number calculation unit calculates the number of cells in the culture container based on the correction physical quantity calculated by subtracting the error output value from the value of the first physical quantity.

2. The cell culture device according to claim 1, wherein: One of the sensors measures both the first physical quantity and the error output value.

3. The cell culture device according to claim 1 or 2, wherein: When the culture medium in the culture container is replaced during the culture process and the sensor measures the replacement of the culture medium, an output value of the physical quantity of the culture medium resupplied to the culture container is used as the error output value.

4. The cell culture device according to any one of claims 1 to 3, wherein: The first physical quantity is lactic acid concentration.

5. A method for calculating the number of cells, for calculating the number of cells in a culture container for accommodating cells and culture medium and culturing cells, the method for calculating the number of cells comprising the following steps: A first measuring step, measuring a first physical quantity of a metabolite of the cell that increases with proliferation of the cell, the first physical quantity being the physical quantity in the culture medium in the culture container during the culture of the cell; a second measuring step of measuring an error output value, the error output value being an output value of the physical quantity in the culture medium supplied to the culture container; as well as The cell number calculation step calculates the number of cells in the culture container based on the correction physical quantity calculated by subtracting the error output value from the value of the first physical quantity.

6. The method for counting the number of cells according to claim 5, wherein: The culture medium in the culture container is replaced during the culture process, and in the second measurement step, the output value of the physical quantity of the culture medium re-supplied to the culture container when the culture medium is replaced is measured as the error output value.

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