Cell culture system and method for proliferative testing of cells

Abstract

This invention provides a cell culture system that, when cells are cultured in a static state using a culture container made of a breathable component, can detect the proliferation of cells within the culture container at a desired moment during culture. The cell culture system of this invention uses a culture container (20) made of at least a portion of a breathable component to culture cells in a static state. The cell culture system includes: an oxygen concentration sensor (21) that measures the oxygen concentration near the culture surface within the culture container (20); and a proliferation detection unit (631) that detects cell proliferation based on the oxygen permeability of the breathable component, the measurement value of the oxygen concentration sensor, the oxygen concentration around the culture container (20), and the oxygen consumption per cell.

Description

Technical Field

[0001] This invention relates to a cell culture technique, and more particularly to a cell culture system for detecting cell proliferation when cells are cultured in a static state using a culture container made of a breathable component. Background Technology

[0002] In recent years, fields such as pharmaceutical production, gene therapy, regenerative medicine, and immunotherapy have required the efficient and large-scale culture of cells and tissues in artificial environments.

[0003] In this situation, a method is proposed to automatically culture large quantities of cells in a closed system using bag-shaped culture containers made of breathable components.

[0004] In large-scale cell culture, it is important to monitor cell proliferation and confirm that the culture has been performed appropriately. Additionally, to assess cell proliferation, it is best to determine, for example, the cell count in the culture vessel at the desired point in the culture process.

[0005] The following three methods can be cited as ways to confirm the number of cells in a culture vessel.

[0006] (1) Collect a portion of the culture medium in the culture container (sampling) and use a counting plate or similar device to count the number of cells.

[0007] (2) The number of cells is determined by image processing of photographs taken of cells in culture containers using a microscope and a camera.

[0008] (3) The number of cells is determined based on the reduction of dissolved oxygen in the culture vessel.

[0009] Existing technical documents

[0010] Patent documents

[0011] Patent Document 1: Japanese Patent Application Publication No. 63-15150

[0012] Patent Document 2: Japanese Patent Application Publication No. 6-121667 Summary of the Invention

[0013] The problem that the invention aims to solve

[0014] However, in method (1), a portion of the culture vessel needs to be opened in order to sample the culture medium, which increases the risk of contamination. In addition, when culturing adherent cells, there is the problem that it is not possible to peel off only a portion of the cells during the culture process and to count the cell count at the desired time.

[0015] Furthermore, in method (2), while measurements are possible when culturing suspension cells at low density, cell stacking occurs as cell density increases after the middle stage of culture, making measurement difficult. Additionally, the method requires a microscope, camera, and image processing equipment, which complicates the structure of the cell culture system.

[0016] Furthermore, in method (3), it is necessary to measure the reduction of dissolved oxygen in the culture container. Therefore, it is necessary to make it a sealed state so that gas does not move from inside the culture container to the outside or gas does not flow from the outside into the culture container. Alternatively, if there is space on the culture medium inside the culture container, it is necessary to block the dissolution of oxygen into the culture medium by replacing the space with nitrogen or the like, and measure the amount of dissolved oxygen consumed by the cells in the culture medium.

[0017] However, when using culture containers made of breathable components for cell culture, it is not possible to detect changes in the concentration of dissolved oxygen consumed by the cells in the culture medium alone. In addition, it is necessary to measure the volume of dissolved oxygen by stirring within the culture container, which makes static culture impossible.

[0018] Here, Patent Document 1 discloses a method for measuring the reduction of dissolved oxygen in a sample solution containing microorganisms while the sample solution is sealed in a container; however, this method requires the use of an airtight culture container.

[0019] Furthermore, Patent Document 2 discloses a cell culture apparatus that calculates the time variation of oxygen levels in relation to the number of cells in the culture container based on oxygen levels measured by an oxygen content measuring device that measures the amount of oxygen dissolved in the culture medium. However, this cell culture apparatus requires the use of a sealed container that prevents gas from flowing into the culture container from the outside, and in order to change from the normal culture state to a measurable state, it is necessary to replace the space above the culture medium in the culture container with nitrogen gas to block the dissolution of oxygen into the culture medium. After changing the culture conditions through this operation, the consumption of dissolved oxygen in the culture medium is measured.

[0020] The inventions described in these patent documents are not suitable for use in cases where cells are cultured in a static state using culture containers made of breathable components.

[0021] The present invention was made in view of the above circumstances, and its object is to provide a cell culture system that, when cells are cultured in a culture container made of a breathable component in a static state, can detect the proliferation of cells in the culture container at a desired time during culture, and provides a method for detecting cell proliferation.

[0022] Methods for solving problems

[0023] To achieve the above objectives, the cell culture system of the present invention is a cell culture system that uses a culture container made of at least a breathable component to culture cells in a static state, and is configured to include: an oxygen concentration sensor that measures the oxygen concentration near the culture surface inside the culture container; and a proliferation detection unit that detects the proliferation of the cells based on the oxygen permeability of the breathable component, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture container, and the oxygen consumption of each cell.

[0024] In addition, it is preferable to configure the cell culture system of the present invention as follows, wherein the proliferation detection unit calculates the number of cells per unit area as the proliferation of the cells using the following formula (1).

[0025] C=G×(M / 100-D / 100) / S···Equation (1)

[0026] C: Number of cells per unit area (cells / cm²) 2 );

[0027] G: Oxygen permeability of breathable components (mg / (cm³)) 2 ·hr·atm));

[0028] M: Oxygen concentration (%) around the culture vessel;

[0029] D: Oxygen concentration sensor reading (%);

[0030] S: Oxygen consumption per unit time of 1 cell (mg / (hr·cell)).

[0031] Furthermore, it is preferable to configure the cell culture system of the present invention in such a way that the proliferation detection unit uses the estimated value of the oxygen concentration in the culture container at the desired time as the measured value of the oxygen concentration sensor. The estimated value of the oxygen concentration is obtained based on the ratio of the slope of the peak value of the oxygen concentration increase when new culture medium is added to the culture container multiple times to the slope of the oxygen concentration change when the culture container is stable.

[0032] Furthermore, it is preferable to configure the cell culture system of the present invention such that the oxygen concentration around the culture container is the same as the oxygen concentration inside the incubator containing the culture container or the oxygen concentration in the atmosphere.

[0033] Furthermore, it is preferable to configure the cell culture system of the present invention such that the oxygen concentration sensor is a fluorescent type.

[0034] Furthermore, it is preferable to configure the cell culture system of the present invention such that the oxygen concentration sensor is fixed to the culture surface inside the culture container using an open-pore resin component.

[0035] Furthermore, the cell proliferation detection method of the present invention utilizes a culture container made of at least a portion of a breathable component to culture cells in a static state. The method employs the following steps: The oxygen concentration near the culture surface within the culture container is measured using an oxygen concentration sensor disposed within the culture container; the number of cells per unit area is calculated based on the oxygen permeability of the breathable component, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture container, and the oxygen consumption of each cell.

[0036] Invention Effects

[0037] According to the present invention, a cell culture system is provided that, when cells are cultured in a static state using a culture container made of a breathable component, the proliferation of cells within the culture container at a desired time during culture is detected, and a method for detecting cell proliferation is also provided. Attached Figure Description

[0038] Figure 1 This is a schematic diagram illustrating the configuration of a cell culture system according to an embodiment of the present invention.

[0039] Figure 2 This is an explanatory diagram showing the distribution of oxygen concentration within the culture vessel of a cell culture system according to an embodiment of the present invention.

[0040] Figure 3 This is an explanatory diagram showing the fixed arrangement of the oxygen concentration sensor inside the culture vessel of the cell culture system according to an embodiment of the present invention.

[0041] Figure 4 This is a graph showing the results of oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to a culture bag in cell culture in Experiment 1.

[0042] Figure 5 This is a graph showing the results of oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to a culture bag in cell culture in Experiment 2.

[0043] Figure 6 The graph (1) shows the results of the oxygen concentration measurement obtained by using a fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Experiment 3.

[0044] Figure 7This is a graph (2) showing the results of oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to a culture bag in cell culture in Experiment 3.

[0045] Figure 8 This is a schematic diagram showing a portion of the composition of the culture bag in the cell culture system of this embodiment used in Experiment 4 and the method for detecting cell proliferation.

[0046] Figure 9 This is a graph showing the results of oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to the culture bag in cell culture in Experiment 4.

[0047] Figure 10 This is a graph showing the results of oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to the culture bag in cell culture in Experiment 5. Detailed Implementation

[0048] The following provides a detailed description of the embodiments of the cell culture system and the method for detecting cell proliferation of the present invention. However, the present invention is not limited to the specific content of the following embodiments and examples.

[0049] The cell culture system of this embodiment is characterized in that cells are cultured in a static state using a culture container made of at least a portion of a breathable component. The cell culture system includes: an oxygen concentration sensor that measures the oxygen concentration near the culture surface inside the culture container; and a proliferation detection unit that detects cell proliferation based on the oxygen permeability of the breathable component, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture container, and the oxygen consumption of each cell.

[0050] Specifically, the cell culture system of this embodiment can be configured as follows: Figure 1 The structure shown.

[0051] First, the general configuration of the cell culture system of this embodiment will be described.

[0052] In the cell culture system of this embodiment, the culture container 20 is disposed on the stage 11 housed within the incubator 10. The culture container 20 has two openings, one of which is connected via a pipe to a culture medium supply container 40 housed in the cold storage 20. The other opening of the culture container 20 is connected via a pipe to a waste liquid container 50 within the incubator 10.

[0053] A liquid delivery mechanism is provided on the pipes connecting the culture container 20 to the culture medium supply container 40 and on the pipes connecting the culture container 20 to the waste liquid container 50. As this liquid delivery mechanism, a pump capable of low-speed and high-precision liquid delivery, such as a peristaltic pump or a syringe pump, is preferably used.

[0054] Furthermore, the control device 60 controls the liquid delivery mechanism between the culture container 20 and the culture medium supply container 40 at a given time, delivering culture medium from the culture medium supply container 40 to the culture container 20. Additionally, the control device 60 controls the liquid delivery mechanism between the culture container 20 and the waste liquid container 50 at a given time, delivering culture medium from the culture container 20 to the waste liquid container 50.

[0055] By supplying oxygen and carbon dioxide gas to the incubator 10 from oxygen cylinder O (oxygen supply device) and carbon dioxide cylinder C (carbon dioxide supply device) respectively, the gas concentration inside the incubator 10 can be controlled, thereby adjusting the gas concentration around the culture container 20.

[0056] That is, the incubator 10 is connected to oxygen cylinder O and carbon dioxide cylinder C via valves, and the opening and closing of each valve is controlled by the control device 60 to control the gas supply from each cylinder to the incubator 10, thereby adjusting the oxygen concentration in the incubator 10.

[0057] For example, a CO2 incubator or the like can be used as the incubator 10. The platform 11 within the incubator 10 can be made of perforated metal or the like, and the culture container 20 can be placed on the platform 11 and stored in a closed manner. It should be noted that the platform 11 can also be omitted.

[0058] Since the cell culture system of this embodiment can be used to culture cells without using the incubator 10, the incubator 10 can also be omitted.

[0059] The culture container 20 of the cell culture system of this embodiment will now be described in detail.

[0060] The culture container 20 is a cell culture container with a breathable closed system, at least part of which is made of breathable components. For example, a culture container formed by heat-sealing the periphery of two rectangular breathable membranes can be used.

[0061] The culture container 20 can be shaped such that the shape near the container opening is inclined relative to the opening, thereby facilitating the flow of culture medium to the opening. Alternatively, it can be rectangular or other rectangular shapes. The number of openings in the culture container 20 is not limited to two; it can have one or more openings.

[0062] In the cell culture system of this embodiment, a fluorescent oxygen concentration sensor 21 is provided on the culture surface inside the culture container 20. Additionally, a fluorescent light-receiving and emitting part 22 capable of transmitting and receiving fluorescence from the fluorescent oxygen concentration sensor 21 is provided inside the incubator 10.

[0063] The fluorescent light-receiving and light-emitting part 22 is disposed, for example, on the lower side of the stage 11. When the stage 11 is transparent, it can pass through the stage 11 or through the through hole provided in the stage 11 to transmit and receive fluorescence for the fluorescent oxygen concentration sensor 21. It should be noted that the fluorescent light-receiving and light-emitting part 22 can also be disposed in contact with the lower side of the culture container 20.

[0064] By controlling the fluorescent light-receiving and light-emitting part 22 using the control device 60, the oxygen concentration inside the culture container 20 can be measured.

[0065] like Figure 2 As shown, the dissolved oxygen concentration in the culture medium within the culture vessel 20 varies greatly depending on the location. Specifically, in the culture medium near the cell-containing culture surface (the upper surface of the bottom permeable membrane 202), oxygen consumption due to cell activity is intense, resulting in rapid oxygen depletion and gradually decreasing to 0% as cell density increases. In contrast, while the concentration also depends on the liquid thickness within the culture vessel 20, for example, when the liquid thickness is 10 mm or more, in the culture medium near the top surface (the lower surface of the top permeable membrane 201) of the culture vessel 20 on the opposite side of the culture surface, the oxygen concentration is difficult to reduce relative to the initial state (e.g., approximately 21% in the atmosphere) when no cells are present nearby. In the culture medium near the center of the culture vessel 20, the oxygen concentration is at an intermediate level (e.g., 10%).

[0066] Therefore, in the cell culture system of this embodiment, by placing the fluorescent oxygen concentration sensor 21 on the culture surface inside the culture container 20, the oxygen concentration inside the culture container 20 based on the oxygen consumption caused by the cells can be properly measured.

[0067] Furthermore, as will be described later, in the cell culture system of this embodiment, by taking into account both the oxygen permeability of the culture container 20 and the oxygen concentration around the culture container 20, the oxygen consumption caused by the cells can be properly calculated.

[0068] Furthermore, according to the cell culture system of this embodiment, even when the oxygen concentration in the culture container 20 is consistently around 0% to 1% and it is difficult to measure the oxygen concentration using the fluorescent oxygen concentration sensor 21, the amount of oxygen consumed by the cells can be appropriately estimated.

[0069] Here, the mechanism of air permeability of the breathable membrane is explained.

[0070] Dissolved gases in the culture medium within the culture vessel dissolve at a concentration that balances with the partial pressure surrounding the vessel. The concentration of dissolved oxygen in the culture medium placed in an incubator, without control of gas concentration within the incubator, is approximately 21%, similar to atmospheric concentration. As cells consume dissolved oxygen within the culture vessel, the dissolved oxygen in the surrounding medium decreases. To maintain oxygen concentration balance with the area outside the culture vessel, oxygen permeates through the membrane into the vessel.

[0071] The permeability (velocity) of a membrane is determined by the partial pressure difference of the gas across the membrane, the affinity between the membrane and the gas, and the diffusivity of gas molecules moving within the membrane.

[0072] The following explains the principle of oxygen concentration measurement using a fluorescent oxygen concentration sensor.

[0073] When light hits the fluorescent element of a fluorescent oxygen concentration sensor, the fluorescent material absorbs the light and becomes excited, then returns to its ground state while emitting fluorescence. If oxygen is present near the fluorescent material, the excitation energy is lost, reducing the fluorescence intensity. Therefore, the more oxygen present, the lower the fluorescence intensity. In the fluorescent light-receiving and emitting section, oxygen dissolved in the culture medium contacts the fluorescent element, and the amount of oxygen is determined based on the change in luminescence intensity caused by the number of oxygen molecules in contact.

[0074] As the material for the culture container 20, resin membranes and the like can be appropriately used, such as polyolefin resins like polyethylene and polypropylene. Examples include copolymers of polyethylene and α-olefins, copolymers of ethylene and vinyl acetate, and ionomers using copolymers of ethylene and acrylic acid, methacrylic acid, and metal ions. Additionally, polyolefins, styrene-based elastomers, and polyester-based thermoplastic elastomers can also be used. Furthermore, soft vinyl chloride resins, polybutadiene resins, ethylene-vinyl acetate copolymers, chlorinated polyethylene resins, polyurethane-based thermoplastic elastomers, polyester-based thermoplastic elastomers, silicone-based thermoplastic elastomers, styrene-based elastomers such as SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butene-styrene), SEPS (styrene-ethylene-propylene-styrene), polyolefin resins, and fluorinated resins can also be used.

[0075] Furthermore, the breathable component used in at least a part of the culture container 20 is preferably a thermoplastic resin with particularly excellent breathability among the aforementioned materials, such as polyethylene such as LLDPE, polyolefin resins such as polypropylene, etc. Additionally, the breathable component is preferably made of a transparent material.

[0076] In addition, such as Figure 3As shown, in a variation of the cell culture system of this embodiment, it is preferable to fix the fluorescent oxygen concentration sensor 21a to the culture surface inside the culture container 20a using an open-pore resin component 23a (the upper surface of the bottom permeable membrane 202a in this example). In this case, by sandwiching the fluorescent oxygen concentration sensor 21a between the open-pore resin component 23a and the culture surface, the fusion region H of the open-pore resin component 23a can be fused to the culture surface and fixed.

[0077] For example, LLDPE and other polyethylene, polyolefin resins such as polypropylene, can be appropriately used as materials for open-cell resin components 23a. Examples include copolymers of polyethylene, ethylene and α-olefins, copolymers of ethylene and vinyl acetate, and ionomers using copolymers of ethylene and acrylic acid, methacrylic acid, and metal ions.

[0078] Since the open-pore resin component 23a is a component that fixes the fluorescent oxygen concentration sensor 21a to the culture surface while simultaneously bringing the fluorescent oxygen concentration sensor 21a into contact with the culture medium, a mesh material can be appropriately used. Alternatively, a porous material such as a sponge can also be used as the open-pore resin component 23a.

[0079] There are no particular limitations on the specific mesh material and porous material. However, it is preferable to use materials with a melting point close to and the same material as the culture container 20a. For example, polyethylene mesh materials (NBCMeshtec Co., Ltd., PE 200 mesh, etc.) and polyethylene porous materials (Teijin Co., Ltd., Miraim, large diameter ≤3μm) can be used appropriately.

[0080] By configuring the cell culture system of this embodiment to fix the fluorescent oxygen concentration sensor 21a with the open resin member 23a, it is possible to prevent the fluorescent oxygen concentration sensor 21a from detaching and moving when injecting culture medium into the culture container 20a or draining culture medium from the culture container 20a.

[0081] The cells cultured using culture container 20 are not particularly limited. They can be suspended cells such as lymphocytes and dendritic cells cultured in a culture medium, or adherent cells such as artificial pluripotent stem cells (iPS cells), neural stem cells, embryonic stem cells (ES cells), mesenchymal stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells, and lymphocytes from the activation process cultured on the culture surface inside the culture container.

[0082] In addition, the cells cultured using culture container 20 can also be cells that form aggregates such as spheres or organoids. The cell culture system and cell proliferation detection method of this embodiment can also be applied to the culture of aggregates.

[0083] The control device 60 of the cell culture system of this embodiment will now be described in detail.

[0084] Control device 60 (control unit) such as Figure 1 As shown, it includes an input / output unit 61, a fluorescent sensor input / output unit 62, a control unit 63, an operation unit 64, and a power supply unit 65.

[0085] The input / output unit 61 is connected to a pump disposed between the culture container 20 and the culture medium supply container 40, and a pump disposed between the culture container 20 and the waste liquid container 50. Based on input information from the control unit 63, the operation of these pumps is controlled. As a result, culture medium is supplied from the culture medium supply container 40 to the culture container 20, and culture medium is discharged from the culture container 20 to the waste liquid container 50.

[0086] Furthermore, the input / output unit 61 is connected to the gas sensor 12 of the incubator 10, the valve disposed between the oxygen cylinder O and the incubator 10, and the valve disposed between the carbon dioxide cylinder C and the incubator 10, and sends input information from the gas sensor 12 to the control unit 63. Additionally, the input / output unit 61 controls the operation of these valves based on the input information from the control unit 63. Thus, oxygen can be supplied from the oxygen cylinder O to the incubator 10, and carbon dioxide gas can be supplied from the carbon dioxide cylinder C to the incubator 10, enabling the gas concentration within the incubator 10 to be controlled at a desired level.

[0087] The fluorescence sensor input / output unit 62 is connected to the fluorescence receiving and emitting unit 22. Based on the input information from the control unit 63, it controls the fluorescence emission of the fluorescence receiving and emitting unit 22 and sends information based on the light received from the fluorescence oxygen concentration sensor 21 to the control unit 63. Thus, the oxygen concentration near the culture surface within the culture vessel 20 can be measured, and the control unit 63 can use this oxygen concentration for cell proliferation detection.

[0088] The control unit 63 consists of a PLC (programmable logic controller) and the like. The desired control content is pre-programmed and stored, and the operation of each part can be controlled based on the program.

[0089] That is, the control unit 63 sends information for controlling the pump and valve to the input / output unit 61 at a given time. Alternatively, the information for controlling the pump and valve can also be sent to the input / output unit 61 based on input information, time information, and other various information from the fluorescent sensor input / output unit 62.

[0090] In addition, the control unit 63 is equipped with a proliferation detection unit 631 for detecting the proliferation of cells in the culture container 20.

[0091] The proliferation detection unit 631 detects cell proliferation based on the oxygen permeability of the permeable component, the measurement value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption of each cell. Specifically, the proliferation detection unit 631 can calculate the number of cells per unit area as the cell proliferation rate.

[0092] The oxygen permeability of the breathable components and the oxygen consumption per cell are calculated in advance according to the intended use and stored in the proliferation detection unit 631, etc.

[0093] As described below, the oxygen consumption of one PBMC cell was calculated to be 1.0e-9 mg / (hr·cell). Additionally, the oxygen consumption of one Jurkat (lymphocyte lineage) cell was calculated to be 1.74e-9 mg / (hr·cell).

[0094] Determining oxygen permeability (mg / (cm³)) 2 Before calculating the oxygen permeability (·hr·atm), first determine the actual culture method. That is, after determining the container size, the amount of culture medium filled, the location of the fluorescent sensor, and whether a pressure clamp is used, calculate the oxygen permeability as shown below.

[0095] For example, a bottom area of ​​50 cm² can be obtained by laminating two sheets of polyethylene film with a thickness of 110 μm. 2 In Experiment 1 described later, the culture bag served as the culture container 20. A fluorescent oxygen concentration sensor 21 was fixed approximately in the center of the inner surface of the culture bag. The culture was carried out while the culture container 20 was clamped into a pressing fixture and pressure was applied. The bottom surface of the pressing fixture was a transparent resin plate, and a fluorescent light-receiving and light-emitting part 22 was fixed on the resin plate at the position corresponding to the fluorescent oxygen concentration sensor 21.

[0096] The culture medium was then heated to 37°C, the culture temperature, and injected into culture vessel 20 along with the cells. Two cell lines, PBMCs (Peripheral Blood Mononuclear Cells) and Jurkat (lymphocyte lineage), were used. The change in oxygen concentration was measured twice for each cell line, and the oxygen permeability was calculated as shown below, yielding its average value.

[0097] PBMCs were cultured using AlyS505N-7 (manufactured by Cell Science Research Institute Co., Ltd.) with 2% FBS added, and 35 ml of the medium was filled into a culture bag along with the cells. Alternatively, Jurkat medium was used, consisting of AlyS505N-0 (manufactured by Cell Science Research Institute Co., Ltd.) with 2% FBS added, and 35 ml of this medium was filled into a culture bag along with the cells.

[0098] The cell filling rate per unit area was set to 2.1e6 cells / cm². 2 After filling the culture bag with PBMCs, the cells settled and the oxygen concentration stabilized approximately 2 hours later. At this point, the oxygen concentration was 6%.

[0099] In this case, the oxygen permeability G can be calculated as follows.

[0100] G = Cell filling volume per unit area × Oxygen consumption per cell / Change in oxygen concentration

[0101] =2.1e6×1.0e-9 / (21 / 100-6 / 100)

[0102] =1.40e-2(mg / (cm) 2 ·hr·atm))

[0103] The cell filling rate per unit area was set to 4.0e6 cells / cm². 2 After filling the culture bags with PBMCs, the oxygen concentration around the culture bags was controlled at 30%. After approximately 2 hours, the cells settled, and the oxygen concentration stabilized at 2%.

[0104] In this case, the oxygen permeability G can be calculated as follows.

[0105] G = Cell filling volume per unit area × Oxygen consumption per cell / Change in oxygen concentration

[0106] =4.0e6×1.0e-9 / (30 / 100-2 / 100)

[0107] =1.43e-2(mg / (cm) 2 ·hr·atm))

[0108] The cell filling rate per unit area was set to 1.1e6 cells / cm². 2 After Jurkat was filled into the culture bags, the cells settled and the oxygen concentration stabilized approximately 2 hours later. At this point, the oxygen concentration was 8%.

[0109] In this case, the oxygen permeability G can be calculated as follows.

[0110] G = Cell filling volume per unit area × Oxygen consumption per cell / Change in oxygen concentration

[0111] =1.1e6×1.74e-9 / (21 / 100-8 / 100)

[0112] =1.47e-2(mg / (em) 2 ·hr·atm))

[0113] The cell filling rate per unit area was set to 1.9e6 cells / cm². 2 After filling the culture bags with Jurkat, the oxygen concentration around the culture bags was controlled at 30%. After approximately 2 hours, the cells settled, and the oxygen concentration stabilized at 5%.

[0114] In this case, the oxygen permeability G can be calculated as follows.

[0115] G = Cell filling volume per unit area × Oxygen consumption per cell / Change in oxygen concentration

[0116] =1.9e6×1.74e-9 / (30 / 100-5 / 100)

[0117] =1.32e-2(mg / (cm) 2 ·hr·atm))

[0118] Based on the above results, the oxygen permeability G of the culture bag used under the above conditions is determined as follows.

[0119] G=(1.40e-2+1.43e-2+1.47e-2+1.32e-2) / 4

[0120] =1.4e-2(mg / (cm) 2 ·hr·atm))

[0121] The oxygen consumption per cell (mg / (hr·cell)) can be calculated as follows.

[0122] First, with the cell count known beforehand, inject the cells and culture medium into an airtight, sealed container. To minimize the introduction of air bubbles into the sealed container, use a syringe or similar tool to remove them. Calculate the dissolved oxygen level based on the volume of the sealed container.

[0123] Specifically, a 10 ml sealed container was filled with culture medium heated to 37°C. At 37°C (atmospheric oxygen concentration approximately 21%), 6.86e-2 mg of oxygen was dissolved in 10 ml of the culture medium (6.86e-2 mg / L @ 37°C (saturated dissolved oxygen)). The change in oxygen concentration in the sealed container during culture was measured, and the decrease in dissolved oxygen concentration per hour was calculated.

[0124] When PBMCs were injected into a 10 ml sealed container at a density of 2e6 cells / ml, 2e7 cells were suspended in the container. The oxygen concentration decreased by 0.1% per minute, or 6% per hour.

[0125] Since a 6% decrease relative to 21% saturated dissolved oxygen is equivalent to a decrease of 6 / 21 = 0.29, the oxygen consumption per unit time is as follows.

[0126] 6.86e-2mg×0.29=1.99e-2mg / hr

[0127] Therefore, the oxygen consumption S (mg / (hr·cell)) of each PBMC is calculated as follows.

[0128] S = 1.99e-2 / 2e7 = 1.0e-9 mg / (hr·unit)

[0129] Using the same method, the oxygen consumption S (mg / (hr·cell)) of one Jurkat cell was calculated as follows.

[0130] S = 1.74e-9 mg / (hr·unit)

[0131] The oxygen concentration sensor measures the oxygen concentration near the culture surface in the culture container 20, which is measured by the fluorescent oxygen concentration sensor 21. The value is input from the fluorescent light-receiving and emitting part 22 to the proliferation detection part 631 via the fluorescent sensor input / output part 62.

[0132] The oxygen concentration around the culture container is the same as the oxygen concentration inside the incubator 10 that houses the culture container 20. In the absence of controlled oxygen concentration inside the incubator 10, or when the incubator 10 is not in use, the oxygen concentration is the atmospheric oxygen concentration (approximately 21%). This concentration is preset in the control unit 63.

[0133] Here, under static culture conditions, the oxygen level through the culture container is in equilibrium with the oxygen consumed by the cells, so the following formula holds true.

[0134] G×(M / 100-D / 100)=C×S

[0135] G: Oxygen permeability of breathable components (mg / (cm³)) 2 ·hr·atm));

[0136] M: Oxygen concentration (%) around the culture vessel;

[0137] D: Oxygen concentration sensor reading (%);

[0138] C: Number of cells per unit area (cells / cm²) 2 );

[0139] S: Oxygen consumption per unit time of 1 cell (mg / (hr·cell)).

[0140] Therefore, in the cell culture system of this embodiment, the proliferation detection unit 631 can use the following formula (1) to calculate the number of cells per unit area as the cell proliferation.

[0141] C=G×(M / 100-D / 100) / S···Equation (1)

[0142] C: Number of cells per unit area (cells / cm²) 2 );

[0143] G: Oxygen permeability of breathable components (mg / (cm³)) 2 ·hr·atm));

[0144] M: Oxygen concentration (%) around the culture vessel;

[0145] D: Oxygen concentration sensor reading (%);

[0146] S: Oxygen consumption per unit time of 1 cell (mg / (hr·cell)).

[0147] The number of cells per unit area calculated using the cell culture system of this embodiment is not a strict measurement of the actual number of cells, but rather an estimated value of the number of cells based on the culture environment of the culture container 20.

[0148] When culturing cells in large quantities, it is important to confirm that the culture has been performed properly, so it is very helpful to estimate the number of cells in the culture vessel by estimation.

[0149] In addition, in the cell culture system of this embodiment, the proliferation detection unit 631 preferably uses the estimated value of the oxygen concentration in the culture container 20 at the desired time as the measured value D of the oxygen concentration sensor in the above formula (1). The estimated value is obtained based on the ratio of the slope of the peak value of the oxygen concentration increase when new culture medium is added to the culture container 20 multiple times to the slope of the oxygen concentration change when the culture container 20 is stable.

[0150] That is, as shown in Experiment 5 described later, when cell culture progresses and the cell density in culture container 20 increases, even if the oxygen concentration around culture container 20 is high, the oxygen concentration near the culture surface in culture container 20 is less than 1%, which exceeds the measurement limit of fluorescent oxygen concentration sensor 21 and cannot be accurately measured.

[0151] Even in this case, the cell culture system according to this embodiment can estimate the number of cells per unit area in the culture container 20 by means of the above-described method.

[0152] That is, by using the estimated value D' of the oxygen concentration in the culture bag at the desired moment as the measured value of the oxygen concentration sensor, the number of cells per unit area can be calculated based on the following formula (2), where the estimated value D' is based on the ratio of the slope of the peak change of oxygen concentration when new culture medium is added to the culture bag multiple times to the slope of the change of oxygen concentration when the culture bag is stable.

[0153] C=G×(M / 100-D' / 100) / S···Equation (2)

[0154] It should be noted that the peak oxygen concentration at which new culture medium is added is affected by the temperature of the added medium. Therefore, it is preferable to add the medium while keeping the temperature constant.

[0155] Furthermore, the method for calculating the estimated value D' is described in detail in Experiment 5.

[0156] The operation unit 64 includes a display unit such as a touch panel, which sends user input information to the control unit 63 to execute PLC settings, etc. Additionally, it displays input information from the control unit 63.

[0157] The power supply unit 65 (such as a stabilizing power supply) supplies power to various parts within the control device 60.

[0158] Furthermore, although not shown, the control device 60 may also include a relay unit and a wiring interruption unit (circuit breaker). Additionally, a microcomputer or computer may be used to implement part or all of the control unit 63, operation unit 64, etc., in the control device 60.

[0159] The cell proliferation detection method of this embodiment is characterized by detecting cell proliferation in a cell culture using a culture container made of at least a portion of a breathable component, where cells are cultured in a static state. In this detection method, an oxygen concentration sensor disposed on the culture surface inside the culture container is used to measure the oxygen concentration near the culture surface inside the culture container. The number of cells per unit area is calculated based on the oxygen permeability of the breathable component, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture container, and the oxygen consumption of each cell.

[0160] In the cell proliferation detection method of this embodiment, the number of cells per unit area can be calculated using the following formula (1).

[0161] C=G×(M / 100-D / 100) / S···Equation (1)

[0162] C: Number of cells per unit area (cells / cm²) 2 );

[0163] G: Oxygen permeability of breathable components (mg / (cm³)) 2 ·hr·atm));

[0164] M: Oxygen concentration (%) around the culture vessel;

[0165] D: Oxygen concentration sensor reading (%);

[0166] S: Oxygen consumption per unit time of 1 cell (mg / (hr·cell)).

[0167] In addition, in the cell proliferation detection method of this embodiment, it is also preferable to use the estimated value of oxygen concentration in the culture container 20 at the desired time as the measured value D of the oxygen concentration sensor in the above formula (1). The estimated value is obtained based on the ratio of the slope of the peak value of oxygen concentration increase when new culture medium is added to the culture container 20 multiple times to the slope of the oxygen concentration change when the culture container 20 is stable.

[0168] As shown above, according to the cell culture system and cell proliferation detection method of this embodiment, when cells are cultured in a static state using a culture container made of an air-permeable component, the cell proliferation within the culture container at a desired time during culture can be detected, and the number of cells per unit area can be calculated as cell proliferation. Therefore, according to this embodiment, it is possible to confirm whether the culture has been performed appropriately during large-scale cell culture.

[0169] Example

[0170] The following describes the experiments conducted to confirm the effectiveness of the cell culture system and the cell proliferation detection method according to the embodiments of the present invention.

[0171] In Experiment 1, Jurkat (lymphocyte lineage cells) were cultured and their proliferation was assessed. In this experiment, culture was performed continuously without interruption during the experiment, and the estimated cell density was compared with the calculated number of cells per unit area based on this embodiment.

[0172] Comparison of the measured cell density of PBMCs (Peripheral Blood Mononuclear Cells) cultured in Experiment 2 with the calculated number of cells per unit area based on this embodiment.

[0173] In Experiment 3, while culturing PBMCs, the culture was interrupted at a given time to count the actual number of cells, and the measured value of the obtained cell density was compared with the calculated value of the number of cells per unit area based on this embodiment.

[0174] In Experiment 4, iPS cells were cultured, and the actual number of cells was counted after the culture was completed. The measured value of the obtained cell density was compared with the calculated value of the number of cells per unit area based on this embodiment.

[0175] Experiment 5 is an experiment conducted during the duration of the culture in Experiment 3, including a period in which the cell density in the culture vessel increased and the measured oxygen concentration near the culture surface was less than 1%. After the culture was completed, the actual number of cells was counted, and the measured value of the obtained cell density was compared with the calculated value of the number of cells per unit area based on this embodiment.

[0176] [Experiment 1]

[0177] Prepare a culture chamber made of linear low-density polyethylene with a thickness of 110 μm, dimensions of 120 mm × 65 mm, and a bottom area of ​​48 cm². 2 The culture bag (manufactured by Toyo Seikan Group Holdings Co., Ltd.) is used as the culture bag. This culture bag is formed by heat-sealing the periphery of two flat membranes. The oxygen permeability of this culture bag is 1.4e-2 mg / (cm³). 2 ·hr·atm)).

[0178] A fluorescent oxygen concentration sensor (PSt3 sensor chip, PreSens) is fixed approximately in the center of the inside of the culture bag.

[0179] The culture bag is incubated under pressure while being clamped into a pressing fixture. The bottom surface of the pressing fixture is a transparent resin plate, and a fluorescent light-receiving and emitting part is fixed on the resin plate at the position corresponding to the fluorescent oxygen concentration sensor. The fluorescent light-receiving and emitting part is attached to the fluorescent sensor input / output section of the control device, and an OXY-4mini (TITECH Co., Ltd.) is used as the fluorescent sensor input / output section.

[0180] The same applies to the fluorescent oxygen concentration sensor, the fluorescent light-receiving and emitting part, and the fluorescent sensor input / output part used in the following tests.

[0181] As cells, Jurkat (a cell line derived from human leukemia T cells) was used at a concentration of 1.20e6 cells / cm². 2 Inoculation at cell density (number of cells per unit area).

[0182] Using a medium containing 2% FBS added to AlyS505N-0 (manufactured by Cell Science Research Institute Co., Ltd.), 35 ml of the medium was filled into the culture bag along with the cells.

[0183] A multi-gas incubator (PHC Corporation, model MCO-5M-PJ) capable of high-oxygen culture was used as the incubator, and an automatic liquid delivery mechanism was installed inside the incubator.

[0184] The culture medium supply bags, filled with the culture medium, are stored in a cold storage at 10°C and connected to the culture bags via tubing. Additionally, waste liquid bags are connected to the culture bags via tubing. The culture medium is delivered using a controlled pipeline pump. The culture medium is heated while passing through an incubator at 37°C, reaching approximately 30°C upon entering the culture bags.

[0185] The control unit of the control system is used to control the pipeline pumps and the oxygen concentration in the incubator. It should be noted that in each experiment, a computer is used to construct the proliferation detection unit within the control unit.

[0186] Subsequently, the culture medium is automatically partially replaced at designated times, thereby removing the deteriorated culture medium from the culture medium bag and filling it with new culture medium.

[0187] Specifically, the culture medium is replaced at a given moment at the start of the culture. At this time, 25 ml of the 35 ml medium is removed, and the delivery rate is set to 2 ml / min. When fresh medium is added by replacing the medium, the oxygen concentration in the culture bag temporarily increases, as shown in the graphs described later.

[0188] In addition, the oxygen concentration in the incubator is increased at specified times.

[0189] Specifically, 23 hours after the start of culture, the percentage changed from 21% to 25%. Additionally, 32 hours after the start of culture, the percentage changed from 25% to 30%. Furthermore, 50 hours after the start of culture, the percentage changed from 30% to 35%. Finally, 53 hours after the start of culture, the percentage changed from 35% to 40%.

[0190] The graph showing the oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to the culture bag in cell culture of Experiment 1 is presented in Figure 1. Figure 4 middle.

[0191] Based on the measurement results, the estimated values ​​of cell density were compared with the calculated values ​​of the number of cells per unit area based on this embodiment at 10, 40, and 60 hours after the start of culture.

[0192] Specifically, Jurkat cells proliferate at a rate of approximately 1.4 times per 24 hours, showing relatively stable growth. Therefore, based on the cell density at seeding (1.20e6 cells / cm³),... 2 The estimated cell densities at 24 and 48 hours after the start of culture were 1.68e6 cells / cm³. 2(=1.20e6×1.4), 2.35e6 pieces / cm 2 (=1.68e6×1.4), based on these values, the estimated values ​​of cell density after 10, 40, and 60 hours are calculated as follows.

[0193] The estimated cell density after 10 hours was 1.40e6 cells / cm³. 2 (=(1.20e6×1.4-1.20e6)×10 / 24+1.20e6);

[0194] The estimated cell density after 40 hours was 2.13e6 cells / cm³. 2 (=(1.68e6×1.4-1.68e6)×16 / 24+1.68e6);

[0195] The estimated cell density after 60 hours was 2.82e6 cells / cm³. 2 (=(2.35e6×1.4-2.35e6)×12 / 24+2.35e6).

[0196] Furthermore, after 10, 40, and 60 hours, the calculated values ​​of the number of cells per unit area based on the cell culture system of this embodiment and the cell proliferation detection method are shown below. First, the oxygen permeability of the air-permeable component (mg / (cm²)) 2 The oxygen consumption per hr (atm) is 1.4e-2. Additionally, the oxygen consumption per cell per unit time (mg / (hr-cell)) is 1.74e-9.

[0197] Additionally, after 10 hours, the oxygen concentration (%) around the culture vessel was 21%, and the oxygen concentration sensor reading (%) was 5%.

[0198] Therefore, the number of cells per unit area C (cells / cm²) after 10 hours 2 As shown below.

[0199] C=1.4e-2×(21 / 100-5 / 100) / 1.74e-9=1.29e6

[0200] In contrast, the estimated cell density after 10 hours, as shown above, was 1.40e6 cells / cm³. 2 .

[0201] Additionally, after 40 hours, the oxygen concentration (%) around the culture vessel was 30%, and the oxygen concentration sensor reading (%) was 3%.

[0202] Therefore, the number of cells per unit area C (cells / cm²) after 40 hours 2 As shown below.

[0203] C=1.4e-2×(30 / 100-3 / 100) / 1.74e-9=2.17e6

[0204] In contrast, the estimated cell density after 40 hours, as shown above, was 2.13e6 cells / cm². 2 .

[0205] Furthermore, after 60 hours, the oxygen concentration (%) around the culture vessel was 40%, and the oxygen concentration sensor reading (%) was 2%.

[0206] Therefore, the number of cells per unit area C (cells / cm²) after 60 hours 2 As shown below.

[0207] C=1.4e-2×(40 / 100-2 / 100) / 1.74e-9=3.06e6

[0208] In contrast, the estimated cell density after 60 hours, as shown above, was 2.82e6 cells / cm³. 2 .

[0209] As shown above, it can be seen that, according to the cell culture system and cell proliferation detection method of this embodiment, when cells are cultured in a static state using a culture container made of a breathable component, an estimated value of the number of cells per unit area at the desired time during culture can be calculated.

[0210] [Experiment 2]

[0211] Prepare a culture bag identical to that used in Experiment 1. The oxygen permeability of this culture bag is 1.4e⁻² mg / (em). 2 ·hr·atm)).

[0212] A fluorescent oxygen concentration sensor is fixed approximately in the center of the inside of the culture bag.

[0213] The culture bag is placed in a pressing clamp and pressure is applied during cultivation. The bottom surface of the pressing clamp is a transparent resin plate, and a fluorescent light-receiving and light-emitting part is fixed on the resin plate at the position corresponding to the fluorescent oxygen concentration sensor.

[0214] Peripheral blood mononuclear cells (PBMCs) were used as the cell line. Before seeding into culture bags, PBMCs were activated for 3 days in flasks coated with anti-CD3 antibody and immobilized, and then seeded at a concentration of 1.6e6 cells / cm³. 2 Cells were seeded at a specific density in culture bags.

[0215] Using a medium containing 2% FBS added to AlyS505N-7 (manufactured by Cell Science Research Institute Co., Ltd.), 35 ml of the medium was filled into the culture bag along with the cells.

[0216] Oxygen concentration was not measured 3 days after inoculation into the culture bags, but was measured in the culture bags over the next 3 days, approximately every 60 hours.

[0217] A multi-gas incubator (PHC Corporation, model MCO-5M-PJ) capable of high-oxygen culture was used as the incubator, and an automatic liquid delivery mechanism was installed inside the incubator.

[0218] The culture medium supply bags filled with the culture medium are stored in a cold storage at 10°C and connected to the culture bags via tubing. Separately, the waste liquid bags are connected to the culture bags via tubing. The culture medium is delivered using a controlled pipeline pump.

[0219] Subsequently, the culture medium is automatically partially replaced at designated times, thereby removing the deteriorated culture medium from the culture medium bag and filling it with new culture medium.

[0220] Specifically, the culture medium is replaced at a given moment at the start of the culture. At this time, 25 ml of the 35 ml medium is removed, and the delivery rate is set to 2 ml / min. When fresh medium is added by replacing the medium, the oxygen concentration in the culture bag temporarily increases, as shown in the graphs described later.

[0221] In addition, the oxygen concentration in incubator 10 was not increased in this experiment, and no culture was conducted under high oxygen conditions.

[0222] Subsequently, 61 hours after the start of the assay, the cells in the culture bag were stirred to ensure uniform suspension in the culture medium. Approximately 0.5 ml of culture medium was collected from the culture bag using a syringe, stained with trypan blue, and the cell density was measured using a hemocytometer. The result was a cell density of 2.34 e6 cells / cm³ 61 hours after the start of the assay. 2 .

[0223] The graph showing the oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to the culture bag in cell culture during Experiment 2 is presented in [the figure]. Figure 5 middle.

[0224] In addition, after 61 hours, the calculated value of the number of cells per unit area based on the cell culture system of this embodiment and the cell proliferation detection method is as follows.

[0225] First, the oxygen permeability of the breathable components (mg / (cm³)) 2The oxygen consumption per hr atm (mg / (hr·cell)) is 1.4e-2. Additionally, the oxygen consumption per cell per unit time (mg / (hr·cell)) is 1.00e-9.

[0226] In addition, the oxygen concentration (%) around the culture vessel was 21%, and the oxygen concentration sensor reading (%) was 1.2%.

[0227] Therefore, the number of cells per unit area, C (cells / cm²), is... 2 As shown below.

[0228] C=1.4e-2×(21 / 100-1.2 / 100) / 1.00e-9=2.77e6

[0229] In contrast, the measured cell density, as shown above, is 2.34e6 cells / cm². 2 .

[0230] That is, as can be seen from this experiment, based on the cell culture system and cell proliferation detection method of this embodiment, when cells are cultured in a static state using a culture container made of a breathable component, an estimated value of the number of cells per unit area can be calculated.

[0231] [Experiment 3]

[0232] Prepare a culture bag identical to that used in Experiment 1. The oxygen permeability of this culture bag is 1.4e⁻² mg / cm³. 2 ·hr·atm)).

[0233] A fluorescent oxygen concentration sensor is fixed approximately in the center of the inside of the culture bag.

[0234] The culture bag is placed in a pressing clamp and pressure is applied during cultivation. The bottom surface of the pressing clamp is a transparent resin plate, and a fluorescent light-receiving and light-emitting part is fixed on the resin plate at the position corresponding to the fluorescent oxygen concentration sensor.

[0235] Peripheral blood mononuclear cells (PBMCs) were used as the cell line. Before seeding into culture bags, PBMCs were activated for 3 days in flasks coated with anti-CD3 antibody and immobilized, and then seeded at a concentration of 1.06 e6 cells / cm³. 2 Cells were seeded into culture bags at the specified cell density.

[0236] Using a medium containing 2% FBS added to AlyS505N-7 (manufactured by Cell Science Research Institute Co., Ltd.), 35 ml of the medium was filled into the culture bag along with the cells.

[0237] Oxygen concentration was not measured 4 days after inoculation into the culture bags, but was measured in the culture bags over the next 3 days, approximately 65 hours later.

[0238] However, Experiment 3 differed from Experiment 2. To determine the cell count, the automatic culture delivery system was stopped, the culture bag was stirred to ensure the cells were uniformly suspended in the culture medium, and approximately 0.1 ml of culture medium was collected from the culture bag using a syringe. The medium was then stained with trypan blue and the cell density was measured using a hemocytometer. It should be noted that the number of cells collected was extremely small and had no impact on the overall cell density.

[0239] In addition, the use of the incubator and the replacement of the culture medium were carried out in the same manner as in Experiment 1.

[0240] The graph showing the oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to the culture bag in cell culture during Experiment 3 is presented in Figure 3. Figure 6 and Figure 7 middle. Figure 6 The chart shows the oxygen concentration measurements up to the first cell count. Figure 7 The chart shows the results of oxygen concentration measurements up to the second cell count.

[0241] In this experiment, the oxygen concentration in incubator 10 was increased at specified times.

[0242] Specifically, in Figure 6 In the chart, at 4 hours after the start of the measurement, the percentage changed from 21% to 30%. Additionally, at 23 hours after the start of the measurement, the percentage changed from 30% to 40%. Furthermore, in... Figure 7 In the chart, the percentage changed from 40% to 50% at 6 hours after the start of the measurement.

[0243] After that, Figure 6 In the chart, 42 hours after the start of the assay, the culture bag was stirred to ensure uniform cell suspension in the culture medium. Approximately 0.1 ml of culture medium was collected from the culture bag, stained with trypan blue, and the cell density was measured using a hemocytometer. The result was a cell density of 4.04 e6 cells / cm³ 42 hours after the start of the assay. 2 .

[0244] After that, Figure 7 In the chart, 22.5 hours after the start of the assay, the culture bag was stirred to ensure uniform cell suspension in the culture medium. Approximately 0.1 ml of culture medium was collected from the culture bag, stained with trypan blue, and the cell density was measured using a hemocytometer. The result was a cell density of 5.2 e6 cells / cm² 22.5 hours after the start of the assay. 2 .

[0245] In addition, the calculated value of the number of cells per unit area based on the cell culture system and cell proliferation detection method of this embodiment is shown below.

[0246] First, the oxygen permeability of the breathable components (mg / (cm³)) 2 The oxygen consumption per hr atm (mg / (hr·cell)) is 1.4e-2. Additionally, the oxygen consumption per cell per unit time (mg / (hr·cell)) is 1.00e-9.

[0247] In addition, Figure 6 Forty-two hours after the start of the measurements, the oxygen concentration (%) around the culture vessel was 40%, and the oxygen concentration sensor reading (%) was 7%.

[0248] Therefore, the number of cells per unit area, C (cells / cm²), is... 2 As shown below.

[0249] C=1.4e-2×(40 / 100-7 / 100) / 1.00e-9=4.62e6

[0250] In contrast, the measured cell density, as shown above, is 4.04e6 cells / cm². 2 .

[0251] In addition, Figure 7 The oxygen concentration (%) around the culture vessel was 50% 22.5 hours after the start of the measurement in the graph, and the oxygen concentration sensor reading (%) was 12%.

[0252] Therefore, the number of cells per unit area, C (cells / cm²), is... 2 As shown below.

[0253] C=1.4e-2×(50 / 100-12 / 100) / 1.00e-9=5.32e6

[0254] In contrast, the measured cell density, as shown above, is 5.2e6 cells / cm². 2 .

[0255] That is, as can be seen from this experiment, based on the cell culture system and cell proliferation detection method of this embodiment, when cells are cultured in a static state using a culture container made of a breathable component, an estimated value of the number of cells per unit area can be calculated.

[0256] [Experiment 4]

[0257] Prepare a culture chamber made of linear low-density polyethylene with a thickness of 110 μm, dimensions of 120 mm × 65 mm, and a bottom area of ​​48 cm². 2The culture bags (manufactured by Toyo Seikan Group Holdings Co., Ltd.) are used as culture bags.

[0258] The culture bag, as Figure 8 As shown, the inner side has multiple protrusions, and the outer surface has multiple small protrusions. Specifically, it is formed by heat-sealing the periphery of two membranes. The membrane is made by processing multiple approximately triangular prisms with a spacing of 0.11 mm, a height of 0.19 mm, and an angle of 75° arranged side by side in a mountain-like pattern without gaps. The thinnest part of the membrane (thin-walled portion) has a thickness of 25 μm. The oxygen permeability of this culture bag is 8.0e-2 mg / (cm³). 2 (·hr·atm)). It should be noted that the oxygen permeability of this culture bag is calculated in the same way as the method described above using the culture bag used in Experiment 1 as an example.

[0259] A fluorescent oxygen concentration sensor is fixed approximately in the center of the inside of the culture bag.

[0260] Furthermore, the culture was conducted without using pressing clamps in this experiment. A fluorescent light-receiving and emitting part was positioned on the underside of the culture bag, corresponding to the position of the fluorescent oxygen concentration sensor.

[0261] As cells, iPS cells (strain 1231A3, Kyoto University iPS Cell Research Institute) were used at a rate of 1.7e4 cells / cm². 2 Cell density seeding was performed. In this experiment, both the upper and lower surfaces of the culture bag were used as culture surfaces, and the seeding cell density was the amount on both surfaces.

[0262] StemFit® (AK-02N, Ajinomoto Healthy Supply Co., Ltd.) was used as the culture medium, and 35 ml of the culture medium was filled into the culture bag along with the cells.

[0263] A multi-gas incubator (PHC Corporation, model MCO-5M-PJ) capable of high-oxygen culture was used as the incubator. It should be noted that in this experiment, an automatic culture medium delivery mechanism was not used; instead, the culture medium was changed manually.

[0264] The culture medium supply bags, filled with the culture medium, are stored in a cold storage at 10°C and connected to the culture bags via tubing. Additionally, waste liquid bags are connected to the culture bags via tubing. The culture medium is delivered using a controlled pipeline pump. The culture medium is heated while passing through an incubator at 37°C, reaching approximately 30°C upon entering the culture bags.

[0265] Subsequently, the entire culture medium is automatically replaced at designated times, thereby removing the deteriorated culture medium from the culture medium bag and filling it with new culture medium.

[0266] Specifically, the culture medium is replaced at a given moment at the start of the culture. At this time, 35 ml of the 35 ml medium is dispensed at a rate of 2 ml / min. When fresh medium is added by replacing the medium, the oxygen concentration in the culture bag temporarily increases, as shown in the graphs described later.

[0267] In addition, the oxygen concentration in incubator 10 was not increased in this experiment, and no culture was conducted under high oxygen conditions.

[0268] Subsequently, 63 hours after the start of the assay, the culture medium was removed from the culture bags, and cells were detached from the bags using cell detachment solution (ThermoFisher Scientific, Inc., TrypLE). The culture bags were then refilled with culture medium to suspend the detached cells. All the culture medium was then recovered from the bags using a syringe, and the cells were stained with trypan blue and their density was measured using a hemocytometer. The result was a cell density of 3.20 e6 cells / cm³. 2 .

[0269] The graph showing the oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to the culture bag in cell culture during Experiment 4 is presented in [the figure]. Figure 9 middle.

[0270] In addition, the calculated value of the number of cells per unit area based on the cell culture system and cell proliferation detection method of this embodiment is shown below.

[0271] First, the oxygen permeability of the breathable components (mg / (cm³)) 2 The oxygen consumption per hr atm (mg / (hr·cell)) is 8.0e-2. Additionally, the oxygen consumption per cell per unit time (mg / (hr·cell)) is 2.34e-9.

[0272] In addition, the oxygen concentration (%) around the culture vessel was 21%, and the oxygen concentration sensor reading (%) was 12%.

[0273] Therefore, the number of cells per unit area, C (cells / cm²), is... 2 As shown below.

[0274] C=8.0e-2×(21 / 100-12 / 100) / 2.34e-9=3.08e6

[0275] In contrast, the measured cell density, as shown above, is 3.20e6 cells / cm². 2 .

[0276] That is, as can be seen from this experiment, based on the cell culture system and cell proliferation detection method of this embodiment, when cells are cultured in a static state using a culture container made of a breathable component, an estimated value of the number of cells per unit area can be calculated.

[0277] [Experiment 5]

[0278] This experiment was conducted consecutively with Experiment 3. The oxygen concentration in the culture bag was measured over a period of approximately 60 hours over 3 days following the oxygen concentration measurement in Experiment 3.

[0279] The graph showing the oxygen concentration measurements obtained using a fluorescent oxygen sensor fixed to the culture bag in cell culture of Experiment 5 is presented in [the figure]. Figure 10 middle.

[0280] Fifty-six hours after the start of the assay, the culture bag was stirred to ensure uniform cell suspension in the culture medium. Approximately 0.1 ml of culture medium was collected from the bag, stained with trypan blue, and the cell density was measured using a hemocytometer. The result was a cell density of 1.04 e7 cells / cm³ 56 hours after the start of the assay. 2 .

[0281] In this experiment, such as Figure 10 As shown, the cell density inside the culture bag became very high 25 hours after the start of the assay. Particularly at 56 hours after the start of the assay, the oxygen concentration inside the culture bag was less than 1%.

[0282] In this case, in the cell culture system and cell proliferation detection method of this embodiment, the number of cells per unit area is calculated using the following method.

[0283] First, during the culture time before the cell density in the culture bag becomes very high, the slope PT of the change in the peak oxygen concentration when new culture medium is added to the culture bag multiple times, and the slope QT of the change in oxygen concentration when the culture bag stabilizes are calculated, and their ratio (QT / PT) is calculated.

[0284] Specifically, in Figure 10 In the above, the slope PT of the change in the rising peak value of P1(5.5, 36.3) and P2(20.1, 34.1) is -15% (=(34.1-36.3) / (20.1-5.5)×100), and the slope QT of the change in the rising peak value of Q1(7.7, 9.3) and Q2(22.9, 2.6) is -44% (=(2.6-9.3) / (22.9-7.7)), and their ratio (QT / PT) is 2.9.

[0285] Then, the peak shortly before Q4 (56, less than 1) 56 hours after the start of the measurement was P4 (55.1, 22.3), and the peak shortly before Q3 (31.1, 2.3) when the oxygen concentration could be measured was P3 (29.5, 32.7).

[0286] Regarding the oxygen concentration at Q4, the change in oxygen concentration from P3 to P4 can be estimated based on the above ratio (QT / PT) compared to Q3.

[0287] Therefore, the estimated oxygen concentration D' can be calculated as follows.

[0288] D' = Oxygen concentration of Q3 - (Change in oxygen concentration from P3 to P4) × QT / PT

[0289] That is, by using the estimated value D' of the oxygen concentration in the culture bag at the desired moment as the measured value of the oxygen concentration sensor, the number of cells per unit area can be calculated based on the following formula (2), where the estimated value D' is based on the ratio of the slope of the peak change of oxygen concentration when new culture medium is added to the culture bag multiple times to the slope of the change of oxygen concentration when the culture bag is stable.

[0290] C=G×(M / 100-D' / 100) / S···Equation (2)

[0291] Therefore, based on the cell culture system of this embodiment and the cell proliferation detection method, the number of cells per unit area is calculated as follows.

[0292] Oxygen permeability of breathable components (mg / (cm³)) 2 The oxygen consumption per hr atm (mg / (hr·cell)) is 1.4e-2. Additionally, the oxygen consumption per cell per unit time (mg / (hr·cell)) is 1.00e-9.

[0293] D' = 2.3 - (22.3 - 32.7) × 2.9

[0294] = -27.9

[0295] C = 1.4e-2 × (50 / 100 + 27.9 / 100) / 1.00e-9 = 1.09e7 pieces / cm 2

[0296] In contrast, the measured cell density, as shown above, is 1.04e7 cells / cm³. 2 .

[0297] That is, as can be seen from this experiment, based on the cell culture system and cell proliferation detection method of this embodiment, when cells are cultured in a static state using a culture container made of a breathable component, an estimated value of the number of cells per unit area can be calculated.

[0298] This invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of this invention. For example, the culture container is not limited to the illustrated culture container; for example, a culture container with one opening, a culture container with three openings, a culture container with multiple holes on the culture surface, or other culture containers that can be modified appropriately according to the purpose of the culture can be used.

[0299] Industrial availability

[0300] This invention can be appropriately used in situations such as high-density, large-scale cell culture using cell culture bags.

[0301] All the documents described in this specification and the contents of the Japanese application specification that form the basis of the Paris priority claim are incorporated herein by reference.

[0302] Explanation of reference numerals in the attached figures

[0303] 10 Incubator, 11 Stage, 12 Gas Sensor, 20, 20a Culture Containers, 201, 201a Top Permeable Membrane, 202, 202a Bottom Permeable Membrane, 21, 21a Fluorescent Oxygen Concentration Sensor, 22, 22a Fluorescent Light Receiving and Emitting Part, 23a Open-Pore Resin Component, 30 Cold Storage, 40 Culture Medium Supply Container, 50 Waste Liquid Container, 60 Control Device, 61 Input / Output Unit, 62 Fluorescent Sensor Input / Output Unit, 63 Control Unit, 631 Proliferation Detection Unit, 64 Operation Unit, 65 Power Supply Unit, H Welding Area, i Cells, L Culture Medium, O Oxygen Cylinder, C Carbon Dioxide Cylinder.

Claims

1. A cell culture system, characterized in that, The cell culture system utilizes a culture vessel, at least part of which is made of breathable components, to culture cells in a static state. The cell culture system has the following features: An oxygen concentration sensor measures the oxygen concentration on the culture surface within the culture vessel; and The proliferation detection unit detects cell proliferation based on the oxygen permeability of the permeable component, the measurement value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption of each cell. The proliferation detection unit detects the proliferation of the cells cultured in a static state. The oxygen concentration around the culture container is the same as the oxygen concentration inside the incubator containing the culture container or the oxygen concentration in the atmosphere. The proliferation detection unit calculates the number of cells per unit area as the cell proliferation using the following formula (1): C = G × (M / 100 - D / 100) / S ··· Equation (1) C: Number of cells per unit area, expressed as cells / cm² 2 , G: Oxygen permeability of breathable components, expressed in mg / (cm³). 2 ·hr·atm), M: Oxygen concentration around the culture vessel, expressed as a percentage. D: The measured value of the oxygen concentration sensor, expressed in %. S: Oxygen consumption per unit time of one cell, expressed in mg / (hr·cell).

2. The cell culture system according to claim 1, characterized in that, The proliferation detection unit uses the estimated oxygen concentration in the culture vessel at the desired time as the measured value of the oxygen concentration sensor. The estimated oxygen concentration is based on the ratio of the slope of the peak increase in oxygen concentration when new culture medium is added to the culture vessel multiple times to the slope of the change in oxygen concentration when the culture vessel is stable.

3. The cell culture system according to claim 1 or 2, characterized in that, The oxygen concentration sensor is fluorescent.

4. The cell culture system according to claim 1 or 2, characterized in that, The oxygen concentration sensor is fixed to the culture surface inside the culture container using an open-pore resin component.

5. The cell culture system according to claim 4, characterized in that, The culture container and the open-pore resin component comprise any one of polyethylene, polypropylene, copolymers of ethylene and α-olefins, copolymers of ethylene and vinyl acetate, and ionomers obtained by using copolymers of ethylene and acrylic acid and / or methacrylic acid with metal ions.

6. A method for detecting cell proliferation in cell culture, characterized in that, This is a method for detecting cell proliferation in cell culture using a culture vessel with at least a portion made of breathable components, under static conditions. In the detection method, an oxygen concentration sensor disposed on the culture surface inside the culture container is used to measure the oxygen concentration on the culture surface inside the culture container. The number of cells per unit area is calculated based on the oxygen permeability of the permeable component, the measurement value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption of each cell. This allows for the detection of cell proliferation during static culture. The oxygen concentration around the culture container is the same as the oxygen concentration inside the incubator containing the culture container or the oxygen concentration in the atmosphere. The proliferation detection unit uses the following formula (1) to calculate the number of cells per unit area as the cell proliferation rate: C = G × (M / 100 - D / 100) / S ··· Equation (1) C: Number of cells per unit area, expressed as cells / cm² 2 , G: Oxygen permeability of breathable components, expressed in mg / (cm³). 2 ·hr·atm), M: Oxygen concentration around the culture vessel, expressed as a percentage. D: The measured value of the oxygen concentration sensor, expressed in %. S: Oxygen consumption per unit time of one cell, expressed in mg / (hr·cell).

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