Cell culture method and cell culture device

Abstract

The cell culture method includes the step of culturing cells (8) in a culture medium (10) that satisfies at least one of the following conditions (I) and (II): condition (I): the lactic acid concentration in the culture medium is less than 7.5 mM; condition (II): the ammonia concentration in the culture medium is less than 2.5 mM.

Description

Technical Field

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

[0002] In recent years, in the fields of pharmaceutical product manufacturing and regenerative medicine, it is necessary to artificially culture cells or microorganisms in large quantities with high efficiency. Cells that need to be cultured in large quantities include antibody-producing cells such as Chinese hamster ovary cells (CHO cells), pluripotent stem cells such as embryonic stem cells (ES cells) or artificial pluripotent stem cells (iPS cells), etc. As long as these cells can be cultured in large quantities and stably for a long time, biological substances such as monoclonal antibodies and differentiation-induced tissues derived from pluripotent stem cells can be produced efficiently.

[0003] As a method for industrially culturing cells in large quantities, suspension agitation culture using culture tanks such as spinner flasks is considered. On the other hand, in suspension agitation culture, there is a tendency for the equipment scale to become larger. Therefore, in order to seek to reduce costs, it is effective to increase the cell density during culture. However, it is known that when the cell density is continuously increased, the proliferation of cells is inhibited. The reason is that the concentration of waste (metabolites) in the culture solution (liquid culture medium) increases due to the high density of cells, thereby reducing the proliferation activity of the cells. As representative wastes that affect cells, lactic acid and ammonia are known.

[0004] Therefore, to ensure stable cell growth at high density, it is preferable to remove lactic acid and ammonia accumulated in the culture medium. In contrast, Patent Document 1, for example, discloses a cell culture apparatus that connects a cell culture tank to a composition adjustment tank using a liquid feed line. This liquid feed line is equipped with a culture medium composition adjustment membrane that allows components to pass through depending on their concentration. In this cell culture apparatus, waste products accumulated in the culture medium migrate toward the composition adjustment liquid, reducing their concentration in the culture medium. Simultaneously, nutrients whose concentrations decrease during culture flow from the composition adjustment liquid into the culture medium for replenishment. This maintains the environment in the culture medium at a state suitable for cell culture.

[0005] [Prior art literature]

[0006] [Patent Document]

[0007] Patent Document 1: International Publication No. 2015 / 122528 Summary of the Invention

[0008] [Problems to be Solved by the Invention]

[0009] The cell culture apparatus disclosed in Patent Document 1 utilizes the principle of dialysis to remove waste from the culture fluid. Therefore, in order to achieve sufficient waste removal, the volume of the composition adjustment liquid tank is set to more than 10 times the volume of the cell culture tank. In addition, the concentrations of lactic acid and ammonia in the culture fluid are not monitored. In other words, in terms of adjusting the lactic acid concentration and ammonia concentration, the timing and amount of the culture fluid transported from the cell culture tank to the composition adjustment liquid tank have not been fully discussed. Therefore, there is a problem that the required liquid volume is huge, which will cost money. In particular, when the culture fluid itself is used as the composition adjustment liquid, a large amount of expensive culture medium will be consumed, which will cost even more.

[0010] The present invention has been made in view of the above circumstances, and one of its objects is to provide a technology that can culture cells in large quantities while achieving cost reduction.

[0011] [Technical solutions for solving technical problems]

[0012] In order to solve the above-mentioned problems, one embodiment of the present invention is a cell culture method comprising the step of culturing cells in a culture medium that satisfies at least one of the following conditions (I) and (II).

[0013] Condition (I): The lactic acid concentration in the culture medium is less than 7.5 mM.

[0014] Condition (II): The ammonia concentration in the culture medium is less than 2.5 mM.

[0015] According to this scheme, it is possible to culture cells in large quantities while reducing costs.

[0016] In the above embodiment, the cells may be pluripotent stem cells. In addition, the lactic acid concentration in condition (I) may be 5 mM or less. In addition, the ammonia concentration in condition (II) may be 1 mM or less.

[0017] Another embodiment of the present invention provides a cell culture apparatus comprising: a culture container containing cells and a culture medium; and a regulator for regulating the culture medium so that the culture medium satisfies at least one of the following conditions (I) and (II).

[0018] Condition (I): The lactic acid concentration in the culture medium is less than 7.5 mM.

[0019] Condition (II): The ammonia concentration in the culture medium is less than 2.5 mM.

[0020] In the above embodiment, the adjustment unit may include: a storage container storing a supplemental solution; a supply pump for delivering the supplemental solution from the storage container to the culture container; a concentration sensor for detecting at least one of the lactic acid concentration and the ammonia concentration in the culture solution; and a control unit for driving the supply pump based on the detection result of the concentration sensor. Alternatively, the adjustment unit may include: a substance exchanger containing a substance exchange membrane and a composition adjustment solution, wherein the culture solution is supplied to perform substance exchange between the composition adjustment solution and the culture solution via the substance exchange membrane; a circulation pump for circulating the culture solution between the culture container and the substance exchanger; a concentration sensor for detecting at least one of the lactic acid concentration and the ammonia concentration in the culture solution; and a control unit for driving the circulation pump based on the detection result of the concentration sensor. Furthermore, the adjustment unit may include an adsorbent for adsorbing at least one of lactic acid and ammonia in the culture solution.

[0021] Furthermore, any combination of the above-described constituent elements, or substitution of constituent elements or expressions of the present invention in methods, apparatuses, systems, etc., may also be effective as aspects of the present invention.

[0022] [Effects of the Invention]

[0023] According to the present invention, it is possible to culture cells in large quantities while reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the cell culture apparatus according to the first embodiment.

[0025] Figure 2 This is a schematic diagram of a cell culture apparatus according to a second embodiment.

[0026] Figure 3 This is a schematic diagram of a cell culture device according to a third embodiment.

[0027] Figure 4 This is a schematic diagram of a cell culture device according to a fourth embodiment.

[0028] Figure 5 This is a graph showing the relationship between lactate concentration and cell density.

[0029] Figure 6 are optical microscopy images of cells cultured in lactate-supplemented medium.

[0030] Figure 7 This is a graph showing the relationship between lactate concentration and gene expression level.

[0031] Figure 8 This is a graph showing the relationship between ammonia concentration and cell density.

[0032] Figure 9are light microscopy images of cells cultured in ammonia-supplemented medium.

[0033] Figure 10 This is a graph showing the relationship between ammonia concentration and gene expression level. DETAILED DESCRIPTION

[0034] Hereinafter, the present invention will be described based on preferred embodiments with reference to the accompanying drawings. The embodiments do not limit the invention, but are merely illustrative, and not all features and combinations described in the embodiments are the substantive contents of the invention. The same or equivalent components, members, and processes shown in the drawings are marked with the same figure numerals, and repeated descriptions are appropriately omitted. In addition, the scales or shapes of the various parts shown in the drawings are set for ease of description and will not be interpreted in a restrictive manner unless otherwise specified. In addition, terms such as "first" and "second" used in this specification or claims do not indicate any order or importance, but are only used to distinguish a certain configuration from other configurations. In addition, in the drawings, a part of the components that are not important in describing the embodiments will be omitted.

[0035] The present inventors have conducted intensive research on the effects of lactic acid and ammonia, representative waste products, on cells, and have identified the concentrations of lactic acid and ammonia that adversely affect cells. Furthermore, through repeated and detailed research, they discovered that, in the case of pluripotent stem cells, lactic acid and ammonia affect cell proliferation and undifferentiated states, respectively, at different concentrations.

[0036] (Implementation 1)

[0037] The cell culture method of the present embodiment includes the step of culturing cells in a culture medium that satisfies at least one of the following conditions (I) and (II).

[0038] Condition (I): Lactic acid concentration in the culture medium is less than 7.5 mM (7.5 × 10 －3 mol / L).

[0039] Condition (II): The ammonia concentration in the culture medium is less than 2.5 mM.

[0040] When lactic acid is present in the culture medium, a lactic acid concentration of less than 7.5 mM can suppress the inhibition of cell growth. Furthermore, the inhibition of cell growth due to a decrease in the culture medium pH caused by lactic acid can be suppressed. Furthermore, a lactic acid concentration of less than 10 mM can maintain cells in an undifferentiated state. Therefore, by culturing cells in a culture medium that meets condition (I), the effects of lactic acid on both cell growth and the undifferentiated state can be reduced.

[0041] When ammonia is present in the culture medium, inhibition of cell growth can be suppressed at an ammonia concentration of less than 10 mM. Furthermore, at an ammonia concentration of less than 2.5 mM, cells can be maintained in an undifferentiated state. Therefore, by culturing cells in a culture medium that satisfies condition (II), the effects of ammonia on both cell growth and the undifferentiated state can be reduced.

[0042] Preferably, the lactic acid concentration in condition (I) is 5 mM or less. This can further suppress the inhibition of cell proliferation by lactic acid. Furthermore, preferably, the ammonia concentration in condition (II) is 1 mM or less. This can further maintain the undifferentiated state of the cells.

[0043] Cells cultured in a culture medium include, for example, pluripotent stem cells such as human iPS cells, human ES cells, and human Muse cells; adult stem cells such as mesenchymal stem cells (MSC cells) and nephron precursor cells; tissue cells such as human proximal tubular epithelial cells, human distal tubular epithelial cells, and human collecting duct epithelial cells; antibody-producing cell lines such as human fetal kidney cells (HEK293 cells); and antibody-producing cell lines derived from animals other than humans, such as Chinese hamster ovary cells (CHO cells) and insect cells (SF9 cells). Preferably, the cells are pluripotent stem cells.

[0044] Cells are preferably cultured in suspension. As culture vessels for suspension culture, spinner flasks, large stainless steel culture tanks, cell culture bottles, cell culture bags, etc. can be used. The culture medium can be appropriately selected according to the cells to be cultured.

[0045] (Cell Culture Device)

[0046] Next, the cell culture apparatus according to this embodiment will be described. Figure 1 This is a schematic diagram of a cell culture apparatus according to Embodiment 1. Cell culture apparatus 1 includes a culture vessel 2 and a control unit 6 as its main components. Culture vessel 2 is a bioreactor that houses cells 8 and culture fluid 10. Control unit 6 adjusts culture fluid 10 so that it satisfies at least one of the aforementioned conditions (I) and (II). Control unit 6 in this embodiment includes a storage container 12, a supply pump 14, a concentration sensor 4, and a control unit 16.

[0047] The storage container 12 stores a supplementing solution 18. The supplementing solution 18 may be the same as the culture solution 10 in the culture container 2, or may be a high-concentration culture solution obtained by concentrating the culture solution 10. Alternatively, the supplementing solution 18 may be a solution other than the culture medium, the components of which are adjusted according to the type of cells 8.

[0048] The storage container 12 and the culture container 2 are connected by a supply tube 20. A supply pump 14 is connected to the middle of the supply tube 20. As the supply pump 14, a well-known pump such as a peristaltic pump or a diaphragm pump can be used.

[0049] The concentration sensor 4 detects at least one of the lactic acid concentration and the ammonia concentration in the culture solution 10. As the concentration sensor 4, a well-known sensor such as a culture medium component analyzer can be used. In addition, the detection method of the lactic acid concentration and the ammonia concentration can adopt a well-known method such as a colorimetric method using a predetermined measuring reagent, an enzyme electrode method utilizing the substrate specificity of an enzyme, and high-speed liquid chromatography (HPLC). The concentration sensor 4 and the culture container 2 are connected by a sampling tube 5. The culture solution 10 in the culture container 2 is collected into the concentration sensor 4 via the sampling tube 5. A filter (not shown) is provided at the end of the culture container 2 side connected to the sampling tube 5. As a result, the situation where the cells 8 are sucked into the sampling tube 5 is suppressed. The concentration sensor 4 samples the culture solution 10 at predetermined intervals and performs component measurement. For example, a solenoid valve 5a is provided in the sampling tube 5. By controlling the opening and closing of the solenoid valve 5a using the control unit 16, the flow of the culture solution 10 into the concentration sensor 4 is switched and cut off. Thus, the measurement of the components of the culture solution 10 by the concentration sensor 4 is repeatedly performed at predetermined timings.

[0050] The control unit 16 drives the supply pump 14 based on the detection results of the concentration sensor 4. Specifically, when the concentration sensor 4 detects the lactic acid concentration, the supply pump 14 is controlled to satisfy condition (I). When the concentration sensor 4 detects the ammonia concentration, the supply pump 14 is controlled to satisfy condition (II). When the concentration sensor 4 detects both concentrations, the supply pump 14 is controlled to satisfy both conditions (I) and (II). Furthermore, even when the concentration sensor 4 detects both concentrations, the control unit 16 may control the supply pump 14 to satisfy only one of the conditions.

[0051] The control unit 16 is implemented as a hardware configuration using components or circuits such as a CPU or memory of a computer, or as a software configuration using a computer program, etc. Figure 1 In the embodiment, it is appropriately described as a functional block implemented by their cooperation. It should be understood by those skilled in the art that the functional block can be implemented in various ways by a combination of software and hardware.

[0052] When the supply pump 14 is driven by the control unit 16, the supplementary liquid 18 is transported from the storage container 12 to the culture container 2 via the supply pipe 20. As a result, the supplementary liquid 18 in the storage container 12 is filled into the culture container 2. Lactic acid and ammonia discharged from the cells 8 are accumulated in the culture solution 10 in the culture container 2. On the other hand, the supplementary liquid 18 is a solution with a lower lactic acid concentration and ammonia concentration than the culture solution 10. Preferably, the supplementary liquid 18 does not contain lactic acid and ammonia. Therefore, by adding the supplementary liquid 18 to the culture solution 10, the lactic acid concentration and ammonia concentration in the culture solution 10 are reduced. In addition, the culture medium components such as glucose and protein required for the culture of the cells 8 are supplemented to the culture solution 10.

[0053] The control unit 16 performs feedback control on the supply pump 14 based on the detection results of the concentration sensor 4 so that the culture solution 10 satisfies condition (I) and / or condition (II). Specifically, the control unit 16 repeatedly receives the detection results of the concentration sensor 4 at a predetermined timing. After adjusting the culture solution 10 so that it satisfies condition (I), the control unit 16 determines whether the lactic acid concentration in the culture solution 10 is less than 7.5 mM based on the detection results of the concentration sensor 4. When the lactic acid concentration is 7.5 mM or higher, the control unit 16 drives the supply pump 14. Furthermore, when the lactic acid concentration is less than 7.5 mM, the control unit 16 stops driving the supply pump 14.

[0054] Preferably, a concentration range is defined with a predetermined margin relative to the threshold value of 7.5 mM. The control unit 16 then drives the supply pump 14 when the concentration exceeds this range and stops it when the concentration falls below this range. More preferably, the concentration range is defined with an upper limit of 7.5 mM. That is, the control unit 16 stops driving the supply pump 14 when the lactate concentration falls below a predetermined value below 7.5 mM. Alternatively, the upper limit of the concentration range may be set to a value lower than 7.5 mM to more reliably prevent the lactate concentration in the culture solution 10 from exceeding 7.5 mM.

[0055] Furthermore, when the culture solution 10 is adjusted to satisfy condition (II), the control unit 16 determines whether the ammonia concentration in the culture solution 10 is less than 2.5 mM based on the detection result of the concentration sensor 4. If the ammonia concentration is 2.5 mM or higher, the control unit 16 drives the supply pump 14. If the ammonia concentration is less than 2.5 mM, the control unit 16 stops driving the supply pump 14.

[0056] Preferably, a concentration range is defined with a predetermined margin relative to the threshold value of 2.5 mM. Furthermore, the control unit 16 drives the supply pump 14 when the concentration exceeds this range, and stops the supply pump 14 when the concentration falls below this range. More preferably, the concentration range is defined with an upper limit of 2.5 mM. That is, the control unit 16 stops driving the supply pump 14 when the ammonia concentration falls below a predetermined value below 2.5 mM. Alternatively, the upper limit of the concentration range may be set to a value lower than 2.5 mM to more reliably prevent the ammonia concentration in the culture solution 10 from exceeding 2.5 mM.

[0057] When the culture solution 10 is adjusted to satisfy both conditions (I) and (II), the control unit 16 stops driving the supply pump 14 when both conditions are satisfied, and drives the supply pump 14 when neither one nor both conditions are satisfied.

[0058] The culture container 2 is connected to a waste liquid tank (not shown) via a discharge tube 22. A discharge pump 24 is connected midway through the discharge tube 22. A known pump, similar to the supply pump 14, can be used as the discharge pump 24. The controller 16 also controls the operation of the discharge pump 24. The controller 16 drives the discharge pump 24 in conjunction with the operation of the supply pump 14. The operation of the discharge pump 24 causes the culture fluid 10 in the culture container 2 to be delivered to the waste liquid tank via the discharge tube 22. This maintains a constant level of fluid in the culture container 2. A filter (not shown) is provided at the end of the culture container 2 connected to the discharge tube 22. This prevents cells 8 from being drawn into the discharge tube 22. The discharge tube 22 can also be used as a sampling tube 5. In this case, the concentration sensor 4 collects the culture fluid 10 via the discharge tube 22. Alternatively, the discharge pump 24 can be driven separately from the supply pump 14.

[0059] As described above, the cell culture method of the present embodiment includes the step of culturing cells in the culture solution 10 that satisfies at least one of the following conditions (I) and (II).

[0060] Condition (I): The lactic acid concentration in the culture solution 10 is less than 7.5 mM.

[0061] Condition (II): The ammonia concentration in the culture solution 10 is less than 2.5 mM.

[0062] Furthermore, the cell culture apparatus 1 of this embodiment includes a culture container 2 that accommodates cells 8 and a culture solution 10 , and a regulator 6 that regulates the culture solution 10 so as to satisfy at least one of the above-mentioned conditions (I) and (II).

[0063] By adjusting the culture medium 10 so that it satisfies at least one of conditions (I) and (II), the adverse effects of at least one of lactic acid and ammonia on cells can be reduced. This allows cells 8 to be cultured in large quantities at high density. Furthermore, by adjusting the culture medium 10 so that it satisfies both conditions (I) and (II), the adverse effects of both lactic acid and ammonia on cells can be reduced. This allows cells 8 to be cultured in large quantities at high density and with greater reliability.

[0064] Furthermore, in this embodiment, the concentrations of lactate and ammonia that affect cells 8 are determined, and the concentrations of lactate and ammonia are monitored to adjust the culture medium 10. This reduces the consumption of the composition adjustment solution and the culture medium, compared to conventional cell culture apparatuses that blindly dialyze the culture medium. This reduces costs. Therefore, according to this embodiment, it is possible to culture cells 8 in large quantities while maintaining low costs.

[0065] Furthermore, when cells 8 are pluripotent stem cells, in addition to culturing cells 8 at high density and in large quantities by reducing lactate and / or ammonia concentrations, it is also possible to maintain the undifferentiated state of cells 8, that is, the multipotency of cells 8. Consequently, it is possible to obtain a large number of cells 8 suitable for producing differentiation-induced tissues. This allows for the production of a homogeneous cell population and suppresses the reduction in differentiation-induced efficiency, thereby reducing the costs required for cell-based pharmaceutical production and regenerative medicine.

[0066] Furthermore, in the cell culture apparatus 1 of this embodiment, the adjustment unit 6 includes a storage container 12 that stores a supplementing liquid 18; a supply pump 14 that delivers the supplementing liquid 18 from the storage container 12 to the culture container 2; a concentration sensor 4 that detects at least one of the lactic acid concentration and the ammonia concentration in the culture solution 10; and a control unit 16 that drives the supply pump 14 based on the detection result of the concentration sensor 4. Adding the supplementing liquid 18 to the culture solution 10 reduces the lactic acid concentration and the ammonia concentration in the culture solution 10. Furthermore, the control unit 16 drives the discharge pump 24 in conjunction with the supply pump 14 to discharge the culture solution 10 from the culture container 2. In other words, in this embodiment, continuous culture, in which old culture medium is removed and fresh culture medium is added, suppresses increases in waste product concentration.

[0067] (Implementation Method 2)

[0068] Except for the difference in the structure of the cell culture apparatus, Embodiment 2 has the same configuration as Embodiment 1. Hereinafter, this embodiment will be described focusing on the configurations that differ from Embodiment 1, and the common configurations will be briefly described or omitted. Figure 2 Schematic diagram of the cell culture device according to embodiment 2.

[0069] The cell culture apparatus 1 of this embodiment mainly includes a culture container 2 and a control unit 6. The culture container 2 contains cells 8 and a culture medium 10. The control unit 6 controls the culture medium 10 so that it satisfies at least one of the above-mentioned conditions (I) and (II). The control unit 6 of this embodiment includes a storage container 12, a supply pump 14, a concentration sensor 4, and a control unit 16.

[0070] The storage container 12 stores a supplementary liquid 18. The storage container 12 and the culture container 2 are connected by a supply pipe 20. A supply pump 14 is connected midway in the supply pipe 20. The concentration sensor 4 detects at least one of the lactic acid concentration and the ammonia concentration in the culture solution 10. The control unit 16 drives the supply pump 14 based on the detection result of the concentration sensor 4. When the supply pump 14 is driven by the control unit 16, the supplementary liquid 18 is transported from the storage container 12 to the culture container 2 via the supply pipe 20. As a result, the supplementary liquid 18 in the storage container 12 is filled into the culture container 2. The driving timing of the supply pump 14 controlled by the control unit 16 is the same as that in embodiment 1.

[0071] Unlike embodiment 1, the cell culture device 1 of this embodiment does not have a discharge pipe 22 and a discharge pump 24. Therefore, the amount of liquid in the culture container 2 will gradually increase. That is, in this embodiment, the increase in waste concentration is suppressed by continuously supplying the supplementary liquid 18 to the culture container 2 through a fed-batch culture. In this configuration, both the number of cells and the amount of culture liquid in the culture container 2 will increase as the culture period passes. Therefore, the cell density in the culture liquid 10 can be maintained constant. In addition, the size (capacity) of the culture container 2 is set in consideration of the culture period of the cells 8, the amount of culture liquid 10 contained in the culture container 2 at the start of the culture, and the amount of supplementary liquid 18 added. According to this embodiment, the same effect as embodiment 1 can be achieved.

[0072] (Implementation 3)

[0073] Except for the difference in the structure of the cell culture apparatus, Embodiment 3 has the same configuration as Embodiment 1. Hereinafter, this embodiment will be described focusing on the configurations that differ from Embodiment 1, and the common configurations will be briefly described or omitted. Figure 3 Schematic diagram of a cell culture device according to embodiment 3.

[0074] The cell culture apparatus 1 of this embodiment mainly includes a culture container 2 and a conditioning unit 6. The culture container 2 contains cells 8 and a culture medium 10. The conditioning unit 6 adjusts the culture medium 10 so that it satisfies at least one of the above-mentioned conditions (I) and (II). The conditioning unit 6 of this embodiment includes a substance exchanger 26, a circulation pump 28, a concentration sensor 4, and a control unit 16.

[0075] The material exchanger 26 houses a material exchange membrane 30 and a composition-adjusted liquid 32. Furthermore, by supplying the culture fluid 10 from the culture container 2, the material exchanger 26 performs material exchange between the composition-adjusted liquid 32 and the culture fluid 10 via the material exchange membrane 30. A known material exchange membrane such as a hollow fiber can be used as the material exchange membrane 30. The composition-adjusted liquid 32, like the supplemental liquid 18, can be the same as the culture fluid 10 in the culture container 2 or a highly concentrated culture fluid obtained by concentrating the culture fluid 10. Furthermore, a solution other than the culture medium can be used, the composition of which is adjusted according to the type of cells 8.

[0076] The culture container 2 and the substance exchanger 26 are connected by a circulation path 34. The circulation path 34 includes an outgoing path portion 34a, one end of which is connected to the culture container 2 and the other end is connected to the substance exchanger 26; and a return path portion 34b, one end of which is connected to the substance exchanger 26 and the other end is connected to the culture container 2. The ends of the outgoing path portion 34a and the return path portion 34b on the substance exchanger 26 side are connected by a substance exchange membrane 30. The substance exchange membrane 30 is immersed in the composition adjustment liquid 32. Therefore, a flow path for the culture solution 10 is formed between the culture container 2 and the substance exchanger 26 by the outgoing path portion 34a, the substance exchange membrane 30, and the return path portion 34b.

[0077] The circulation pump 28 circulates the culture solution 10 between the culture container 2 and the mass exchanger 26. Specifically, the circulation pump 28 includes a first pump 28a and a second pump 28b. The first pump 28a is connected midway along the outgoing path 34a and transports the culture solution 10 from the culture container 2 to the mass exchange membrane 30 of the mass exchanger 26. The second pump 28b is connected midway along the return path 34b and transports the culture solution 10 from the mass exchange membrane 30 to the culture container 2.

[0078] The concentration sensor 4 detects at least one of the lactic acid concentration and the ammonia concentration in the culture solution 10. The control unit 16 drives the circulation pump 28 based on the detection result of the concentration sensor 4. When the circulation pump 28 is driven by the control unit 16, the culture solution 10 circulates between the culture container 2 and the substance exchanger 26 via the circulation path 34. During this process, substances are exchanged between the culture solution 10 and the composition-adjusted liquid 32 via the substance exchange membrane 30. As a result, lactic acid and ammonia in the culture solution 10 move to the composition-adjusted liquid 32, and medium components such as glucose and protein in the composition-adjusted liquid 32 move to the culture solution 10. The driving timing of the circulation pump 28 controlled by the control unit 16 is the same as the driving timing of the supply pump 14 in the first embodiment.

[0079] As a result, the lactic acid concentration and ammonia concentration in the culture solution 10 are reduced. In addition, the culture medium components such as glucose and protein required for the culture of the cells 8 are supplemented to the culture solution 10. In addition, a filter (not shown) is provided at the end of one side of the culture container 2 connected to the outgoing portion 34a. This prevents the cells 8 from being sucked into the discharge pipe 22. In addition, an adsorbent for waste may be added to the material exchanger 26. This can reduce the concentration of waste dissolved in the component preparation solution 32, thereby reducing the amount of the component adjustment solution 32 required for material exchange. As the adsorbent, a known adsorbent such as silica, activated carbon, etc. that can adsorb at least one of lactic acid and ammonia can be used.

[0080] That is, in this embodiment, the culture solution 10 is caused to flow from the culture container 2 to the substance exchange membrane 30 in the substance exchanger 26 to perform substance exchange processing, and the waste is discharged into the component adjustment liquid 32 in the substance exchanger 26, thereby suppressing the increase in the waste concentration in the culture solution 10. According to this embodiment, the same effects as those of the first embodiment can be achieved.

[0081] (Implementation 4)

[0082] Except for the difference in the structure of the cell culture apparatus, Embodiment 4 has the same configuration as Embodiment 1. Hereinafter, this embodiment will be described focusing on the configurations that differ from Embodiment 1, and the common configurations will be briefly described or omitted. Figure 4 Schematic diagram of a cell culture device according to embodiment 4.

[0083] The cell culture apparatus 1 of this embodiment includes a culture container 2 and a conditioning unit 6. The culture container 2 contains cells 8 and a culture medium 10. The conditioning unit 6 conditions the culture medium 10 so that it satisfies at least one of the above-mentioned conditions (I) and (II). The conditioning unit 6 of this embodiment includes an adsorbent 36.

[0084] As the adsorbent 36, known adsorbents such as silica and activated carbon can be used. The adsorbent 36 can be a single type of adsorbent that adsorbs both lactic acid and ammonia, or a mixture of a first adsorbent that adsorbs lactic acid and a second adsorbent that adsorbs ammonia. By bringing the adsorbent 36 into contact with the culture medium 10, the culture medium 10 is adjusted so that it satisfies at least one of the above-mentioned conditions (I) and (II). The amount of adsorbent 36 required to satisfy condition (I) and / or condition (II) can be appropriately determined by a person skilled in the art. In addition, the adsorbent 36 is preferably an adsorbent with low toxicity to the cells 8.

[0085] The adsorbent 36 is, for example, in a granular form and is housed in the culture container 2. The adsorbent 36 is dispersed, precipitated, or suspended in the culture solution 10 within the culture container 2. In this case, to prevent phagocytosis by the cells 8, the adsorbent 36 is preferably larger than a predetermined size, for example, a size of 10 μm or larger. Since the culture solution 10 is in contact with the adsorbent 36, the lactic acid and / or ammonia in the culture solution 10 are adsorbed by the adsorbent 36. As a result, the lactic acid concentration and / or ammonia concentration in the culture solution 10 are reduced, thereby satisfying condition (I) and / or condition (II). Therefore, according to this embodiment, the same effect as embodiment 1 can be achieved.

[0086] The adjustment unit 6 may also have a structure in which an adsorption column (not shown) connected to the culture container 2 is filled with the adsorbent 36. In this case, the culture solution 10 in the culture container 2 is transferred to the adsorption column and contacts the adsorbent 36. Alternatively, the adjustment unit 6 may have a structure in which the adsorbent 36 is supported on the inner wall surface of the culture container 2. Examples of methods for supporting the adsorbent 36 on the inner wall surface of the culture container 2 include a method of bonding the adsorbent 36 to the inner wall surface of the culture container 2 or, if the culture container 2 is made of resin, a method of pre-molding the culture container 2 with a resin mixed with the adsorbent 36.

[0087] Alternatively, the adjustment unit 6 may have a structure in which the interior of the culture container 2 is divided into an upper and lower stage by a diaphragm such as a porous membrane, with the adsorbent 36 housed in the lower stage. In this structure, the cells 8 are housed in the upper stage. Furthermore, the culture fluid 10 can pass through the diaphragm and move between the upper and lower stages. On the other hand, the cells 8 and the adsorbent 36 cannot pass through the diaphragm. Since the culture fluid 10 contacts the adsorbent 36 housed in the lower stage, lactic acid and / or ammonia in the culture fluid 10 can be adsorbed by the adsorbent 36.

[0088] In addition, it is preferred that the adsorbent 36 is covered with a resin such as polyvinyl alcohol, collagen, alginic acid, gelatin, or a gel derived from a biological source. In this way, it is possible to suppress the outflow of microparticles that may affect the cells 8, etc. from the adsorbent 36 into the culture medium. Alternatively, the adsorbent 36 is formed by mixing a ceramic adhesive, a resin adhesive, and a gel derived from a biological source with an adsorption component. In this way, the outflow of microparticles can also be suppressed. Examples of ceramic adhesives include alumina adhesives and colloidal silica. Examples of resin adhesives include polyvinyl alcohol and carboxymethyl cellulose. Examples of gels derived from biological sources include collagen, alginic acid, gelatin, etc.

[0089] The above describes the embodiments of the present invention in detail. The aforementioned embodiments do not merely represent specific examples when implementing the present invention. The contents of the embodiments do not limit the technical scope of the present invention, and various design changes such as changes, additions, and deletions of constituent elements can be made without departing from the scope of the idea of ​​the invention specified in the claims. The new embodiment with design changes has the effects of the combined embodiment and the deformation. In the aforementioned embodiments, the contents that can make such design changes are emphasized by adding descriptions such as "in this embodiment" and "in this embodiment", but even in the contents without such descriptions, design changes are allowed. Any combination of the above constituent elements is also valid as a solution of the present invention.

[0090] [Example]

[0091] Hereinafter, examples of the present invention will be described. However, the examples are merely illustrations for better explaining the present invention and do not limit the present invention in any way.

[0092] [Determining the lactate concentration that affects cell proliferation]

[0093] 1×10 3 Individual pluripotent stem cells were seeded in 24-well plates (Corning cellBIND surface: Corning) and cultured with pluripotent stem cell culture medium (StemFit: Ajinomoto Co., Ltd.). Culture matrix (iMatrix-511: Nippi) was added to the culture medium for 24 hours at a concentration of 0.5 μg / ml, and a ROCK inhibitor (Y-27632: Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the culture medium at a concentration of 10 μM. Furthermore, lactic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the culture medium after 24 hours of culture at various lactic acid concentrations. The lactic acid concentrations were 0 mM, 2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM, and 15.0 mM.

[0094] Adding lactic acid lowers the pH of the culture medium. Therefore, we prepared experimental groups with the culture medium pH corrected to 7.2 and uncorrected groups. In the uncorrected groups, the pH was 6.9 in a culture medium with a lactic acid concentration of 5.0 mM, 6.8 in a culture medium with a lactic acid concentration of 7.5 mM, 6.7 in a culture medium with a lactic acid concentration of 10 mM, and 6.5 in a culture medium with a lactic acid concentration of 15 mM.

[0095] The culture medium was changed every day for 4 days (therefore, the total culture period was 5 days). After 5 days of culture, the number of cells in each well was measured using a TC20 automatic cell counter (Bio-Rad) and the cell density was calculated. The results were recorded in Figure 5 Shown in. Figure 5 This is a graph showing the relationship between lactate concentration and cell density.

[0096] like Figure 5 As shown, regardless of whether pH correction was performed, a significant decrease in cell density was observed at lactate concentrations of 7.5 mM or higher. Furthermore, in the experimental group with pH correction, cell density at a lactate concentration of 15 mM was reduced by more than half compared to a lactate concentration of 0 mM. On the other hand, in the experimental group without pH correction, cell density was significantly reduced, falling below the cell density at lactate concentrations of 10 mM or higher and at a lactate concentration of 15 mM with pH correction. This confirms that lactate itself inhibits cell growth, and also inhibits cell growth by causing a decrease in pH.

[0097] The above results confirm that maintaining the lactate concentration below 7.5 mM is effective in suppressing cell growth inhibition. Furthermore, it has been confirmed that maintaining the lactate concentration below 5 mM can more reliably suppress cell growth inhibition caused by lactate.

[0098] [Determining the lactate concentration that affects the undifferentiated state of cells]

[0099] 1×10 3Pluripotent stem cells were seeded in 24-well plates (Corning cellBIND surface: Corning) and cultured with pluripotent stem cell culture medium (StemFit: Ajinomoto Co., Ltd.). Furthermore, culture matrix (iMatrix-511: Nippi Co., Ltd.) was added to the culture medium for 24 hours to a concentration of 0.5 μg / ml, and a ROCK inhibitor (Y-27632: Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a concentration of 10 μM. Furthermore, lactic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) at various lactic acid concentrations was added to the culture medium after 24 hours of culture. The lactic acid concentrations were set to 0 mM, 1.0 mM, 2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM, and 15.0 mM.

[0100] The culture medium was replaced every day for 4 days (thus, the total culture period was 5 days). The condition of each cell after 5 days of culture was observed under an optical microscope. Figure 6 This is an optical microscope image of cells cultured in a lactate-supplemented medium. Figure 6 As shown in the results, when the lactic acid concentration was 10 mM or higher, the cell colonies were significantly reduced in size, indicating that cell proliferation was inhibited.

[0101] Furthermore, mRNA was extracted and purified from each cell after 6 days of culture using the RNeasy Mini Kit (QIAGEN). Subsequently, cDNA was synthesized from the purified mRNA using the QuantiTect Reverse Transcription Kit (QIAGEN). Using this cDNA as a template, the expression levels of the Oct4, Nanog, T, Pax6, and Fox2 genes were measured by real-time PCR using the Thermal Cycler Dice Real Time System I (TAKARA BIO).

[0102] Oct4 and Nanog are undifferentiated markers, indicating that pluripotent stem cell properties have been maintained. T, Pax6, and Fox2 are differentiation markers, indicating that pluripotent stem cells have differentiated, rather than maintaining their pluripotent stem cell properties. Specifically, expression of the T gene indicates that cells have differentiated into mesoderm, expression of Pax6 indicates that cells have differentiated into ectoderm, and expression of Fox2 indicates that cells have differentiated into endoderm.

[0103] For each gene expression level at each lactic acid concentration, the ratio to the expression level at 0 mM lactic acid concentration was calculated. Figure 7 Shown in. Figure 7 This is a graph showing the relationship between lactate concentration and gene expression level. Figure 7As shown, at a lactate concentration of 10 mM, the expression level of the mesoderm differentiation marker T gene increased by more than 10 times compared to a lactate concentration of 0 mM. This confirms that maintaining the lactate concentration below 10 mM is effective in maintaining the undifferentiated state of cells.

[0104] [Determining the ammonia concentration that affects cell proliferation]

[0105] 1×10 3 Individual pluripotent stem cells were seeded in 24-well plates (Corning cellBIND surface: Corning) and cultured with pluripotent stem cell culture medium (StemFit: Ajinomoto Co., Ltd.). Furthermore, culture matrix (iMatrix-511: Nippi) was added to the culture medium for 24 hours at a concentration of 0.5 μg / ml, and a ROCK inhibitor (Y-27632: Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the culture medium at a concentration of 10 μM. Furthermore, ammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) with various ammonia (ammonium ion) concentrations was added to the culture medium after 24 hours of culture. The ammonia concentrations were 0 mM, 7.5 mM, 10.0 mM, 15.0 mM, and 25.0 mM.

[0106] The culture medium was changed every day for 4 days (therefore, the total culture period was 5 days). After 5 days of culture, the number of cells in each well was measured using a TC20 automatic cell counter (Bio-Rad) and the cell density was calculated. The results were recorded in Figure 8 Shown in. Figure 8 This is a graph showing the relationship between ammonia concentration and cell density.

[0107] like Figure 8 As shown in Figure 2, a significant decrease in cell density was observed when the ammonia concentration was 10.0 mM or higher. This confirms that maintaining the ammonia concentration below 10 mM is effective in suppressing cell growth inhibition. Furthermore, it was confirmed that maintaining the ammonia concentration below 7.5 mM can more reliably suppress cell growth inhibition caused by ammonia.

[0108] [Determining the ammonia concentration that affects the undifferentiated state of cells]

[0109] 1×10 3Pluripotent stem cells were seeded in 24-well plates (Corning cellBIND surface: Corning) and cultured with pluripotent stem cell culture medium (StemFit: Ajinomoto Co., Ltd.). Furthermore, culture matrix (iMatrix-511: Nippi) was added to the culture medium for 24 hours at a concentration of 0.5 μg / ml, and a ROCK inhibitor (Y-27632: Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the culture medium at a concentration of 10 μM. Furthermore, ammonium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) with various ammonia concentrations was added to the culture medium after 24 hours of culture. The ammonia concentrations were set to 0 mM, 0.1 mM, 1.0 mM, 2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM, and 20.0 mM.

[0110] The culture medium was replaced every day for 4 days (thus, the total culture period was 5 days). The condition of each cell after 5 days of culture was observed under an optical microscope. Figure 9 This is an optical microscope image of cells cultured in ammonia-supplemented medium. Figure 9 As shown in Figure 3, when the ammonia concentration is above 2.5 mM, morphological changes of cells can be observed.

[0111] Furthermore, mRNA was extracted and purified from each cell after 6 days of culture using the RNeasy Mini Kit (QIAGEN). Subsequently, cDNA was synthesized from the purified mRNA using the QuantiTect Reverse Transcription Kit (QIAGEN). Using this cDNA as a template, the expression levels of the Oct4, Nanog, T, Pax6, and Fox2 genes were measured by real-time PCR using the Thermal Cycler Dice Real Time System I (TAKARA BIO).

[0112] The expression level of each gene at each ammonia concentration was calculated relative to the expression level at an ammonia concentration of 0 mM. Figure 10 Shown in. Figure 10 This is a graph showing the relationship between ammonia concentration and gene expression level. Figure 10 As shown, at an ammonia concentration of 2.5 mM, the expression levels of the T gene, a marker for mesodermal differentiation, and the Pax6 gene, a marker for ectoderm differentiation, increased nearly 10-fold compared to an ammonia concentration of 0 mM. This confirms that maintaining an ammonia concentration below 2.5 mM is effective in maintaining the undifferentiated state of cells. Furthermore, it was confirmed that maintaining an ammonia concentration below 1 mM more reliably inhibited the promotion of cell differentiation by ammonia.

[0113] The above results confirm that in order to proliferate pluripotent stem cells in an undifferentiated state, the lactic acid concentration in the culture medium must be maintained at less than 7.5 mM and the ammonia concentration must be maintained at less than 2.5 mM.

[0114] [Industrial Applicability]

[0115] The present invention can be utilized in a cell culture method and a cell culture apparatus.

[0116] [Explanation of Reference Numerals]

[0117] 1 cell culture device, 2 culture container, 4 concentration sensor, 6 adjustment unit, 8 cells, 10 culture solution, 12 storage container, 14 supply pump, 16 control unit, 18 supplementary solution, 26 material exchanger, 28 circulation pump, 30 material exchange membrane, 32 component adjustment solution, 36 adsorbent.

Claims

1. A cell culture method, characterized in that: Include A step of culturing undifferentiated cells in a culture medium that satisfies both of the following conditions (I) and (II), by performing both treatments of removing lactic acid from the culture medium when the lactic acid concentration exceeds an upper limit value determined based on 7.5 mM, and removing ammonia from the culture medium when the ammonia concentration exceeds an upper limit value determined based on 1 mM. Condition (I): The lactate concentration in the culture medium is maintained at less than 7.5 mM during the culture period; Condition (II): The ammonia concentration in the culture medium is maintained below 1 mM during the culture period; By satisfying both of the above conditions (I) and (II), the above cells are maintained in an undifferentiated state; The cells are at least one of pluripotent stem cells and adult stem cells.

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