Cell culture method and cell culture apparatus

Abstract

The objective is to provide a cell culture method and cell culture apparatus that can suppress foaming of the culture medium without the use of antifoaming agents, and that are useful as a technology for maintaining good culture conditions, such as supplying gases like oxygen and removing dissolved carbon dioxide. [Solution] A cell culture apparatus 1 is used, which comprises a culture vessel 10 in which culture medium A is sealed, a gas separation membrane element 20 placed in the culture vessel 10 immersed in culture medium A, and a stirring means 30. The culture medium A is stirred, and cells are cultured while the culture medium A is perfused to the outside of the multiple hollow fiber membranes 21 of the gas separation membrane element 20. The gas separation membrane element 20 is used to perform both (i) vacuuming from the gas outlet 23a to separate and remove dissolved carbon dioxide in the culture medium A through the multiple hollow fiber membranes 21, and (ii) supplying gas to the culture medium A through the multiple hollow fiber membranes 21 by allowing air to pass through the gas outlet 23a.

Description

Technical Field

[0001] The present invention relates to a cell culture method and a cell culture apparatus.

Background Art

[0002] In the initial production steps of cultured meat using animal cells in the production of biopharmaceuticals such as antibody production and the recently developed alternative meat production, a large number of cells are cultured. In cell culture, it is necessary to continuously supply oxygen throughout the culture period and remove carbon dioxide accumulated during culture. Patent Document 1 discloses supplying oxygen by sending air as bubbles using a sparger during culture, and removing dissolved carbon dioxide in the culture solution by passing a sweep gas through a carbon dioxide permeable membrane using gas exchange means provided with a carbon dioxide permeable membrane.

[0003] However, the method of sending bubbles into the culture solution has poor oxygen supply efficiency, there is a risk that the bubbles may damage the cells, and there is a problem that the culture solution foams. Patent Document 2 discloses a cell culture method of supplying oxygen to a culture solution using a hollow fiber membrane during culture. Further, Patent Document 3 discloses suppressing foaming by adding an antifoaming agent to the culture solution.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, the cell culture method described in Patent Document 2 does not take into account the removal of carbon dioxide accumulated during culture. Furthermore, since it is difficult to remove the defoaming agent from the culture medium, adding the defoaming agent to the culture medium as in Patent Document 3 results in the problem of the defoaming agent remaining in the final product.

[0006] The present invention aims to provide a cell culture method and a cell culture apparatus that are useful as a technology for maintaining good culture conditions, such as supplying gases like oxygen and removing dissolved carbon dioxide, while suppressing foaming of the culture medium without using an antifoaming agent. [Means for solving the problem]

[0007] The present invention has the following aspects. [1] A gas separation membrane element comprising a plurality of hollow fiber membranes and a housing into which at least one end of the plurality of hollow fiber membranes is inserted and fixed, wherein a gas outlet is formed in the housing, is immersed in a culture medium sealed in a culture vessel, The culture medium is stirred, and the cells are cultured while the culture medium is perfused to the outside of the plurality of hollow fiber membranes. A cell culture method comprising using the gas separation membrane element to perform both (i) vacuuming from the gas outlet and separating and removing dissolved carbon dioxide from the culture medium via the plurality of hollow fiber membranes, and (ii) supplying gas to the culture medium via the plurality of hollow fiber membranes by allowing air to pass through the gas outlet and [2] The cell culture method according to [1], wherein the gas outlet of the gas separation membrane element is connected to the outside of the culture vessel via a connection port provided in the culture vessel. [3] The cell culture method according to [1] or [2], wherein the separation and removal of dissolved carbon dioxide in (i) and the supply of gas in (ii) are switched in any one or more of the gas separation membrane elements. [4] At least one first gas separation membrane element whose gas outlet is connected to the outside of the culture vessel via one of the connection ports provided in the culture vessel, A second gas separation membrane element comprising at least one housing into which multiple hollow fiber membranes are inserted and fixed at both ends, each housing having a gas outlet, and the two gas outlets are connected to the outside of the culture vessel via two connection ports provided in the culture vessel, The cell culture method according to [1], wherein the first gas separation membrane element separates and removes the dissolved carbon dioxide in (i) and the second gas separation membrane element supplies the gas in (ii). [5] The cell culture method according to any one of [1] to [4], wherein the hollow fiber membrane is a composite hollow fiber membrane comprising a gas-permeable homogeneous layer and a porous support layer that supports the homogeneous layer. [6] The cell culture method according to [5], wherein the homogeneous layer has a flux ratio of oxygen to nitrogen of 2 or more, and a flux ratio of carbon dioxide to oxygen of 2 or more. [7] A cell culture apparatus comprising a culture vessel in which a culture medium is enclosed, at least one gas separation membrane element disposed within the culture vessel immersed in the culture medium, and a stirring means, The gas separation membrane element comprises a plurality of hollow fiber membranes and a housing into which at least one end of the plurality of hollow fiber membranes is inserted and fixed, and a gas outlet is formed in the housing. The stirring means is a means for stirring the culture solution so that the culture solution perfuses the outside of the plurality of hollow fiber membranes. A cell culture apparatus in which, with the gas outlet connected to at least one of a vacuum pump and a gas supply device outside the culture vessel, the gas separation membrane element performs both (i) vacuuming from the gas outlet and separating and removing dissolved carbon dioxide from the culture medium via the plurality of hollow fiber membranes, and (ii) supplying gas to the culture medium via the plurality of hollow fiber membranes by allowing air to pass through the gas outlet. [8] The cell culture apparatus according to [7], wherein the gas outlet of the gas separation membrane element is connected to the outside of the culture vessel via a connection port provided in the culture vessel. [9] The cell culture apparatus according to [7] or [8], wherein any one or more of the gas separation membrane elements are connected to both a vacuum pump and a gas supply device, and can be switched between the separation and removal of dissolved carbon dioxide in (i) and the supply of gas in (ii).

[10] At least one first gas separation membrane element for separating and removing the dissolved carbon dioxide in (i), wherein the gas outlet is connected to the outside of the culture vessel through one of the connection ports provided in the culture vessel, The cell culture apparatus according to [7], comprising at least one second gas separation membrane element for supplying the gas described in (ii), the second gas separation membrane element having two housings into which both ends of a plurality of hollow fiber membranes are respectively inserted and fixed, and a gas outlet is formed in each of the housings, the two gas outlets being connected to the outside of the culture vessel via each of two connection ports provided in the culture vessel.

[11] The cell culture apparatus according to any one of [7] to

[10] , wherein the hollow fiber membrane is a composite hollow fiber membrane comprising a gas-permeable homogeneous layer and a porous support layer that supports the homogeneous layer.

[12] The cell culture apparatus according to

[11] , wherein the homogeneous layer has at least one of the following conditions: the flux ratio of oxygen to nitrogen is 2.0 or more, and the flux ratio of carbon dioxide to oxygen is 2.0 or more. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a cell culture method and a cell culture apparatus that are useful as a technique for maintaining good culture conditions, such as supplying gases like oxygen and removing dissolved carbon dioxide, while suppressing foaming of the culture medium without using an antifoaming agent. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram showing a cell culture apparatus as an example of an embodiment. [Figure 2] This is a schematic diagram showing another example of a cell culture apparatus in the embodiment. [Figure 3]It is a schematic diagram showing a cell culture device of another example of the embodiment. [Figure 4] It is a schematic diagram showing a cell culture device of another example of the embodiment. [Figure 5] It is a schematic diagram showing a cell culture device of another example of the embodiment. [Figure 6] It is a schematic diagram showing a cell culture device of another example of the embodiment.

Mode for Carrying Out the Invention

[0010] Hereinafter, an example of the cell culture method and cell culture device of the present invention will be shown and described with reference to the drawings. In the following description, the dimensions and the like of the illustrated figures are examples, and the present invention is not necessarily limited thereto, and it can be appropriately changed and implemented without changing the gist thereof.

[0011] [[ID=二十]] [Cell Culture Device] Hereinafter, an example of the cell culture device of the present invention will be described based on FIG. 1. The cell culture device 1 of the present embodiment includes a culture container 10, a gas separation membrane element 20, and a stirring means 30. The cell culture device 1 may include a temperature control device, a pH adjuster supply device, a culture solution supply device, etc. that a normal cell culture device has.

[0012] The culture container 10 is not particularly limited as long as it can enclose the culture solution A and cell culture can be performed. Specific examples of the culture container 10 include, for example, a metal culture tank such as stainless steel and a resin single-use bag. The shape and dimensions of the culture container 10 are not particularly limited and can be appropriately designed according to the application.

[0013] The gas separation membrane element 20 includes a sheet-like object 22 composed of a plurality of hollow fiber membranes 21, and a first housing 23 and a second housing 24 provided on both ends of the plurality of hollow fiber membranes 21, respectively. The gas separation membrane element 20 is positioned inside the culture vessel 10, immersed in the culture medium A.

[0014] The first housing 23 and the second housing 24 are substantially hollow members into which both ends of each hollow fiber membrane 21 of the sheet-like material 22 are inserted and fixed. Both ends of each hollow fiber membrane 21 of the sheet-like material 22 in the longitudinal direction are fixed by potting resin or the like while inserted into the first housing 23 and the second housing 24, respectively. In other words, the sheet-like material 22 is held between the first housing 23 and the second housing 24. In this example, at least the end face of each hollow fiber membrane 21 on the side inserted into the first housing 23 is open. That is, at least the inside of each hollow fiber membrane 21 is in communication with the inside of the first housing 23. The end face of each hollow fiber membrane 21 on the second housing 24 side may be open or sealed with potting resin. Alternatively, as shown in Figure 2, each hollow fiber membrane 21 may be folded back into a U-shape, and only one end opposite to the folded portion may be inserted and fixed into a single first housing 23.

[0015] The first housing 23 has a gas outlet 23a, to which a gas line 41 is connected. The gas line 41 is led to the outside through a connection port 11 provided in the culture vessel 10, and is connected to a gas supply device 51 and a vacuum pump 52 outside the culture vessel 10. Thus, the gas outlet 23a of the first housing 23 is connected to the outside of the culture vessel 10 through a single connection port 11 provided in the culture vessel 10. In the example shown in Figure 1, the gas line 42 is branched midway, allowing for arbitrary switching between a state where the gas outlet 23a is connected to the gas supply device 51 and a state where the gas outlet 23a is connected to the vacuum pump 52.

[0016] When the gas outlet 23a and the gas supply device 51 are connected, (ii) gas can be supplied to the culture medium A via the multiple hollow fiber membranes 21 by passing air from the gas outlet 23a into the gas separation membrane element 20. Specifically, the gas sent from the gas supply device 51 is supplied to the gas separation membrane element 20 from the gas outlet 23a via the gas line 41. The gas supplied to the gas separation membrane element 20 enters the interior of each hollow fiber membrane 21 from the interior of the first housing 23, and the gas that has permeated each hollow fiber membrane 21 is supplied to the culture medium A.

[0017] When the gas outlet 23a and the vacuum pump 52 are connected, (i) the gas separation membrane element 20 can be evacuated from the gas outlet 23a, and dissolved carbon dioxide in the culture medium A can be separated and removed through the multiple hollow fiber membranes 21. Specifically, by operating the vacuum pump 52, the gas separation membrane element 20 is evacuated from the gas outlet 23a. As a result, the inside of each hollow fiber membrane 21 becomes negative pressure, so that dissolved carbon dioxide in the culture medium A permeates into the inside of each hollow fiber membrane 21 as gas and is discharged outside the culture container 10 through the gas line 41 from the gas outlet 23a of the first housing 23.

[0018] Thus, the cell culture apparatus 1 can perform both (i) vacuuming from the gas inlet 23a and separating and removing dissolved carbon dioxide from the culture medium A via multiple hollow fiber membranes 21, and (ii) supplying gas to the culture medium A via multiple hollow fiber membranes 21 by allowing air to pass through the gas inlet 23a. By using the gas separation membrane element 20 for both the separation and removal of dissolved carbon dioxide in (i) and the gas supply in (ii), it is possible to maintain a good culture state while suppressing foaming of the culture medium without using an antifoaming agent.

[0019] (ii) In the gas supply, oxygen is an example of the gas supplied to culture medium A. However, the gas supplied to culture medium A is not limited to oxygen; for example, if culture medium A is alkaline before culturing, carbon dioxide may be supplied to culture medium A in the early stages of culturing in order to adjust the pH of culture medium A.

[0020] It is preferable that the hollow fiber membranes 21 constituting the sheet-like material 22 are arranged separately from each other, so as to improve the efficiency of gas supply and the separation and removal of dissolved carbon dioxide. The hollow fiber membrane 21 is not particularly limited as long as it is impermeable to culture medium A, can supply the desired gas to culture medium A, and can remove dissolved carbon dioxide from culture medium A. In other words, the hollow fiber membrane 21 is not particularly limited as long as it is impermeable to culture medium A, and can permeate the gas supplied to culture medium A and carbon dioxide.

[0021] A composite hollow fiber membrane is preferred as the hollow fiber membrane 21 because it offers excellent strength and allows for more efficient degassing and aeration. The composite hollow fiber membrane is comprised of a gas-permeable homogeneous layer and a porous support layer that supports the homogeneous layer. The layer configuration of the composite hollow fiber membrane is not particularly limited; for example, it may be a two-layer configuration with a porous support layer inside the homogeneous layer, or a three-layer configuration with porous support layers on both the inside and outside of the homogeneous layer.

[0022] The material used to form the homogeneous layer is not particularly limited, and any known material can be used. Examples include silicone rubber resins, polyolefin resins, fluorine-containing resins, cellulose resins, polyphenylene oxide, poly-4-vinylpyridine, and urethane resins. These materials may be used individually or in combination of two or more.

[0023] The homogeneous layer preferably satisfies at least one of the following conditions: the flux ratio of oxygen to nitrogen (O2 / N2) is 2.0 or higher, and the flux ratio of carbon dioxide to oxygen (CO2 / O2) is 2.0 or higher; it is more preferable that both conditions are met. If the flux ratio (O2 / N2) is 2.0 or higher, the efficiency of oxygen supply to culture medium A will be high even when air is supplied from the gas supply device 51. If the flux ratio (CO2 / O2) is 2.0 or higher, it will be easier to separate and remove dissolved carbon dioxide from culture medium A while maintaining the culture state.

[0024] The flux ratio (O2 / N2) of the homogeneous layer is preferably 2.0 or higher, and more preferably 3.0 or higher. There is no particular upper limit to the flux ratio (O2 / N2), and it can be, for example, 5.0 or lower. The flux ratio (O2 / N2) of a homogeneous layer is a value calculated from the flow rates of each gas when pure gas is supplied to the membrane at a constant pressure.

[0025] The flux ratio (CO2 / O2) of the homogeneous layer is preferably 2.0 or higher, and more preferably 3.0 or higher. There is no particular upper limit to the flux ratio (CO2 / O2), and it can be, for example, 10 or less. The flux ratio (CO2 / O2) of the homogeneous layer is a value calculated from the flow rates of each gas when pure gas is supplied to the membrane at a constant pressure.

[0026] When using hollow fiber membranes 21 with high selective oxygen permeability, if air is continuously supplied to the culture medium A from the gas supply device 51, the nitrogen concentration inside each hollow fiber membrane 21 will become high, and the oxygen supply efficiency may gradually decrease. However, in the example shown in Figure 1, the gas separation membrane element 20 is connected to both the vacuum pump and the gas supply device, and it is possible to switch between (i) separation and removal of dissolved carbon dioxide and (ii) gas supply. Therefore, even if the nitrogen concentration inside the hollow fiber membrane 21 becomes high due to the gas supply in (ii), when it is switched to the separation and removal of dissolved carbon dioxide in (i), the gas with the high nitrogen concentration is discharged outside the culture vessel 10 through the gas line 41 from the gas outlet 23a. This makes it possible to suppress the decrease in oxygen supply efficiency due to the high nitrogen concentration inside the hollow fiber membrane 21.

[0027] The material used to form the porous support layer is not particularly limited, and known materials can be used. Examples include silicone rubber resins, polyolefin resins, fluorine-containing resins, cellulose resins, polyphenylene oxide, poly-4-vinylpyridine, urethane resins, polystyrene, polyether ether ketones, and polyether ketones. These materials may be used individually or in combination of two or more.

[0028] The average film thickness of the hollow fiber membrane 21 is preferably 20 to 150 μm, and more preferably 30 to 70 μm. If the average film thickness of the hollow fiber membrane 21 is above the lower limit of the above range, the durability of the hollow fiber membrane 21 is excellent. If the average film thickness of the hollow fiber membrane 21 is below the upper limit of the above range, it is easier to maintain good performance in gas supply or carbon dioxide separation and removal. The average thickness of the hollow fiber membrane was calculated by measuring the thickness of the hollow fiber membrane at multiple locations (5 or more locations) in the circumferential direction and taking the average value.

[0029] The stirring means 30 is a means for stirring the culture medium A so that the culture medium A perfuses the outside of the multiple hollow fiber membranes 21 of the gas separation membrane element 20. The stirring means 30 in the example shown in Figure 1 comprises a stirring shaft 31, a plurality of stirring blades 32 provided on the portion of the stirring shaft 31 that is located inside the culture vessel 10, and a drive motor 33 located outside the culture vessel 10 that rotates the stirring shaft 31 around its axis. The stirring means 30 is not limited to the configuration shown in Figure 1, as long as it can sufficiently stir the culture medium A. For example, it may be a means that shakes the entire culture container 10 to stir the culture medium A.

[0030] The gas supply device 51 can be any device capable of supplying gases such as oxygen or air to the gas separation membrane element 20. Examples include the use of gas cylinders or a device that supplies air or a mixed gas while controlling the pressure.

[0031] [Cell culture method] Below, as an example of the cell culture method of the present invention, a cell culture method using the cell culture apparatus 1 illustrated in Figure 1 will be described. In this embodiment, the gas separation membrane element 20 is immersed in the culture medium A sealed inside the culture vessel 10, and the culture medium A is stirred by the stirring means 30, while the cells are cultured while the culture medium A is perfused around the outside of the multiple hollow fiber membranes 21.

[0032] The cells to be cultured are not particularly limited; any cells from which the desired substance, such as antibodies or enzymes, can be obtained are acceptable. Examples of cells that can be cultured include animal cells, plant cells, insect cells, bacteria, yeast, and fungi.

[0033] Culture medium A is not particularly limited as long as it is suitable for the cells being cultured. The temperature of culture medium A during cultivation can be set appropriately according to the cells being cultured, preferably 30-40°C, and more preferably 35-38°C.

[0034] It is preferable to set the pH of culture medium A to a range suitable for the cells being cultured. Culture medium A may contain a carbonate with pH buffering capacity. Examples of carbonates include sodium bicarbonate, sodium carbonate, potassium carbonate, potassium bicarbonate, and magnesium carbonate.

[0035] In the cell culture method using the cell culture apparatus 1, the gas outlet 23a and the gas supply device 51 are connected, and gas such as oxygen or air is supplied from the gas supply device 51, and (ii) the gas is passed from the gas outlet 23a of the first housing 23 to the gas separation membrane element 20, and the gas is supplied to the culture medium A via the multiple hollow fiber membranes 21. This ensures that sufficient oxygen is supplied throughout the culture period. In the initial stages of culture, carbon dioxide may be supplied to the alkaline culture medium for the purpose of pH adjustment.

[0036] The supply of oxygen to culture medium A may be carried out continuously or intermittently. When supplying oxygen or carbon dioxide to culture medium A, high-purity oxygen or carbon dioxide may be supplied from the gas supply device 51, or air may be supplied from the gas supply device 51 if a hollow fiber membrane with high selective permeability of oxygen or carbon dioxide is used.

[0037] Furthermore, for example, in the later stages of culture, by switching to a state where the gas outlet 23a and the vacuum pump 52 are connected and operating the vacuum pump 52, (i) vacuum is drawn from the gas outlet and dissolved carbon dioxide in the culture medium A is separated and removed via the multiple hollow fiber membranes 21. This makes it possible to suppress the deterioration of the culture condition due to an increase in the dissolved carbon dioxide concentration in the culture medium A.

[0038] As described above, the present invention uses a gas separation membrane element for both (i) the separation and removal of dissolved carbon dioxide from culture medium A and (ii) the supply of gas to culture medium A. This allows for the separation and removal of dissolved carbon dioxide and gas supply while suppressing foaming of the culture medium, thereby maintaining a good culture state. In particular, by efficiently removing carbon dioxide accumulated in the culture medium using a hollow fiber membrane in the later stages of culture, damage to cells is reduced, and cells can be cultured with high efficiency. Furthermore, compared to blowing bubbles with a sparger, damage to cells can be further reduced. In addition, it becomes unnecessary to add substances that become impurities in the final product, such as antifoaming agents.

[0039] Furthermore, in a configuration like cell culture apparatus 1, where the gas outlet formed in the housing of the gas separation membrane element is connected to the outside of the culture vessel via a single connection port, the number of connection ports to be provided in the culture vessel can be reduced, thus making it easier to prevent the structure of the culture vessel from becoming too complex. In addition, by reducing the number of connection ports, it becomes easier to prevent contamination inside the culture vessel.

[0040] Furthermore, the present invention is not limited to the cell culture apparatus 1 described above, or the cell culture method using the cell culture apparatus 1. In the example shown in Figure 1, there is one gas separation membrane element placed in the culture vessel, but there may be multiple elements. The number of gas separation membrane elements should be appropriately determined depending on the capacity of the culture vessel and the dimensions of the gas separation membrane elements. If multiple gas separation membrane elements are arranged in the culture vessel, each gas separation membrane element can be used to simultaneously supply gas to culture medium A and separate and remove dissolved carbon dioxide from culture medium A.

[0041] For example, the cell culture apparatus 1A illustrated in Figure 3, and the cell culture method using the cell culture apparatus 1A may also be used. In Figure 3, the same reference numerals are used for parts that are the same as in Figure 1, and their explanations are omitted. Cell culture apparatus 1A is similar in configuration to cell culture apparatus 1, except that two gas separation membrane elements 20A and 20B are arranged inside the culture vessel 10.

[0042] The gas outlets 23a formed in the first housings 23 of each of the two gas separation membrane elements 20A and 20B are both connected to an external gas supply device 51 and a vacuum pump 52 via a single connection port 11 through a gas line 41. In this way, even when multiple gas separation membrane elements are arranged in a culture vessel, the number of connection ports on the culture vessel can be reduced by ensuring that the gas outlet of each gas separation membrane element leads to the outside of the culture vessel via a single connection port. As a result, the structure of the culture vessel can be simplified, and contamination of the inside of the culture vessel can be easily suppressed.

[0043] The cell culture apparatus 1B illustrated in Figure 4, and the cell culture method using the cell culture apparatus 1B, may also be used. In Figure 4, the same reference numerals are used for parts that are the same as in Figure 1, and their descriptions are omitted. Cell culture apparatus 1B is similar in configuration to cell culture apparatus 1, except for the configuration described below. In the cell culture apparatus 1B, two gas separation membrane elements 20A and 20B are arranged inside the culture vessel 10. The gas inlet 23a of gas separation membrane element 20A is connected to the gas supply device 51 by a gas line 41, and the gas inlet 23a of gas separation membrane element 20B is connected to the vacuum pump by a gas line 42. In addition, the gas inlet 23a of each of the two gas separation membrane elements 20A and 20B are connected to the outside via two connection ports 11a and 11b provided in the culture vessel 10.

[0044] In cell culture apparatus 1B, gas is supplied to culture medium A (ii) using gas separation membrane element 20A, and dissolved carbon dioxide is separated and removed from culture medium A (i) using gas separation membrane element 20B. Within the culture vessel, the number of gas separation membrane elements for supplying gas to culture medium A is not limited to one, but may be two or more. Similarly, within the culture vessel, the number of gas separation membrane elements for separating and removing dissolved carbon dioxide from culture medium A is not limited to one, but may be two or more.

[0045] The cell culture apparatus 2 and the cell culture method using the cell culture apparatus 2 are also shown in Figure 5. In Figure 5, the same reference numerals are used for parts that are the same as in Figure 1, and their descriptions are omitted. Cell culture apparatus 2 is similar in appearance to cell culture apparatus 1, except for the configuration described below. In the cell culture apparatus 2, a first gas separation membrane element 20C and a second gas separation membrane element 20D are arranged inside the culture vessel 10.

[0046] The first gas separation membrane element 20C is a gas separation membrane element for separating and removing dissolved carbon dioxide from the culture medium A. The gas outlet 23a of the first gas separation membrane element 20C is connected to the outside of the culture vessel 10 via a single connection port 11b provided in the culture vessel 10. The first gas separation membrane element 20C can be the same as the gas separation membrane element 20.

[0047] The second gas separation membrane element 20D is a gas separation membrane element for supplying gas to culture medium A. In the second gas separation membrane element 20D, the end face of each hollow fiber membrane 21 that is inserted into the second housing 24 is also open, and a gas outlet 24a is formed in the second housing 24. The gas outlet 24a of the second gas separation membrane element 20D is connected to the gas supply device 51 by a gas line 41a. The gas outlet 23a of the second gas separation membrane element 20D is connected to the outside of the culture vessel 10 by a gas line 41b. In this way, the two gas outlets 23a and 24a of the second gas separation membrane element 20D are connected to the outside of the culture vessel 10 via two connection ports 11a and 11c.

[0048] In the cell culture apparatus 2, the second gas separation membrane element 20D is used to supply gas to the culture medium A in (ii), and the first gas separation membrane element 20C is used to separate and remove dissolved carbon dioxide from the culture medium A in (i). Gas is supplied to the culture medium A by using a hollow fiber membrane 21 with high selective oxygen permeability as the second gas separation membrane element 20D, for example, by supplying air from the gas supply device 51 through the gas line 41a. As a result, air entering the second housing 24 from the gas outlet 24a is distributed to each hollow fiber membrane 21, and oxygen in the air permeates each hollow fiber membrane 21 and is supplied to the culture medium A. The gas, whose nitrogen concentration has increased after oxygen supply, then flows to the first housing 23 and is discharged to the outside of the culture vessel 10 through the gas outlet 23a and the gas line 41b. In this way, the cell culture device 2 can supply oxygen to the culture medium A while discharging the gas, whose nitrogen concentration has increased after oxygen supply, to the outside of the second gas separation membrane element 20D. Therefore, it is possible to suppress the decrease in oxygen supply efficiency due to a high nitrogen concentration inside the hollow fiber membrane 21.

[0049] Within the culture vessel, the second gas separation membrane element for supplying gas to culture medium A is not limited to one, but may be two or more. Similarly, within the culture vessel, the first gas separation membrane element for separating and removing dissolved carbon dioxide from culture medium A is not limited to one, but may be two or more. Furthermore, if the amount of culture medium is large and the length of the element is long, for example, as shown in Figure 6, the first gas separation membrane element 20C may be configured to efficiently separate the gas by simultaneously reducing the pressure from both the first housing 23 and the second housing 24 using the vacuum pump 52.

[0050] Furthermore, without departing from the spirit of the present invention, the components in the above embodiments may be replaced with well-known components as appropriate, and the above-described modifications may be combined as appropriate. [Explanation of symbols]

[0051] 1,1A~1C,2...Cell culture apparatus, 10...Culture vessel, 11a~11c...Connection port, 20...Gas separation membrane element, 21...Hollow fiber membrane, 22...Sheet-like material, 30...Agitation means, 41...Gas line, 51...Gas supply device, 52...Vacuum pump, A...Culture medium.

Claims

1. A gas separation membrane element comprising multiple hollow fiber membranes and a housing into which at least one end of the multiple hollow fiber membranes is inserted and fixed, and having a gas outlet formed in the housing, is immersed in a culture medium sealed in a culture vessel, The culture medium is stirred, and the cells are cultured while the culture medium is perfused to the outside of the plurality of hollow fiber membranes. A cell culture method comprising using the gas separation membrane element to perform both (i) vacuuming from the gas outlet and removing dissolved carbon dioxide from the culture medium via the plurality of hollow fiber membranes, and (ii) supplying gas to the culture medium via the plurality of hollow fiber membranes by allowing air to pass through the gas outlet and removing air.

2. The cell culture method according to claim 1, wherein the gas outlet of the gas separation membrane element is connected to the outside of the culture vessel through a single connection port provided in the culture vessel.

3. The cell culture method according to claim 1 or 2, wherein in any one or more of the gas separation membrane elements, the separation and removal of dissolved carbon dioxide in (i) and the supply of gas in (ii) are switched.

4. The gas outlet of the culture vessel is connected to the outside of the culture vessel via one of the connection ports provided in the culture vessel, and the gas separation membrane element comprises at least one first gas separation membrane element, A second gas separation membrane element comprising at least one housing into which multiple hollow fiber membranes are inserted and fixed at both ends, each housing having a gas outlet, and the two gas outlets are connected to the outside of the culture vessel via two connection ports provided in the culture vessel, The cell culture method according to claim 1, wherein the first gas separation membrane element separates and removes the dissolved carbon dioxide in (i), and the second gas separation membrane element supplies the gas in (ii).

5. The cell culture method according to any one of claims 1 to 4, wherein the hollow fiber membrane is a composite hollow fiber membrane comprising a homogeneous layer having gas permeability and a porous support layer supporting the homogeneous layer.

6. The cell culture method according to claim 5, wherein the homogeneous layer satisfies at least one of the following conditions: the flux ratio of oxygen to nitrogen is 2 or more, and the flux ratio of carbon dioxide to oxygen is 2 or more.

7. A cell culture apparatus comprising a culture vessel in which a culture medium is sealed, at least one gas separation membrane element positioned immersed in the culture medium within the culture vessel, and a stirring means, The gas separation membrane element comprises a plurality of hollow fiber membranes and a housing into which at least one end of the plurality of hollow fiber membranes is inserted and fixed, and a gas outlet is formed in the housing. The stirring means is a means for stirring the culture solution so that the culture solution perfuses the outside of the plurality of hollow fiber membranes. A cell culture apparatus in which, with the gas outlet connected to at least one of a vacuum pump and a gas supply device outside the culture vessel, the gas separation membrane element performs both (i) vacuuming from the gas outlet and separating and removing dissolved carbon dioxide from the culture medium via the plurality of hollow fiber membranes, and (ii) supplying gas to the culture medium via the plurality of hollow fiber membranes by allowing air to pass through the gas outlet.

8. The cell culture apparatus according to claim 7, wherein the gas outlet of the gas separation membrane element is connected to the outside of the culture vessel via a single connection port provided in the culture vessel.

9. The cell culture apparatus according to claim 7 or 8, wherein any one or more of the gas separation membrane elements are connected to both a vacuum pump and a gas supply device, and can be switched between the separation and removal of dissolved carbon dioxide in (i) and the gas supply in (ii).

10. The gas outlet is connected to the outside of the culture vessel via one of the connection ports provided in the culture vessel, and the system includes at least one first gas separation membrane element for separating and removing the dissolved carbon dioxide in (i), The cell culture apparatus according to claim 7, comprising at least one second gas separation membrane element for supplying the gas described in (ii), the second gas separation membrane element comprising two housings into which both ends of a plurality of hollow fiber membranes are respectively inserted and fixed, and a gas outlet is formed in each of the housings, the two gas outlets being connected to the outside of the culture vessel via two connection ports provided in the culture vessel.

11. The cell culture apparatus according to any one of claims 7 to 10, wherein the hollow fiber membrane is a composite hollow fiber membrane comprising a gas-permeable homogeneous layer and a porous support layer that supports the homogeneous layer.

12. The cell culture apparatus according to claim 11, wherein the homogeneous layer satisfies at least one of the following conditions: the flux ratio of oxygen to nitrogen is 2.0 or more, and the flux ratio of carbon dioxide to oxygen is 2.0 or more.

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