Cell culture device and method

Abstract

The present disclosure provides a cell culture device comprising: a culture chamber for culturing suspension cells; a first flow path adjacent to the culture chamber, and having a first culture medium supply port and a first culture medium discharge port; a second flow path having a second culture medium supply port and a second culture medium discharge port; a semi-permeable membrane disposed in at least a portion between the first flow path and the second flow path; and a seeding port provided to at least a portion of the culture chamber.

Description

Cell culture apparatus and method

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

[0002] In the medical field, enzymes, hormones, antibodies, cytokines, and viruses (viral proteins) used in treatments and vaccines are industrially produced and utilized using animal cells. Furthermore, in the fields of regenerative medicine and cultured meat production, it is necessary to industrially cultivate cells on a large scale. Thus, in recent years, the technology for culturing animal cells has become increasingly important, and development in this area is thriving.

[0003] Traditionally, animal cells have been cultured statically in culture dishes such as petri dishes. However, as the culture period lengthened, problems arose such as the accumulation of waste products in the culture medium and the depletion of nutrients in the medium, leading to cell death. Furthermore, repeated cell division presented numerous challenges, including excessive cell density and depletion of oxygen necessary for respiration. Moreover, culturing large quantities of cells requires the exchange of large amounts of culture medium. Therefore, a structure that is not limited by the volume of the culture medium and a device that can frequently supply fresh culture medium were necessary to store and supply large quantities of medium.

[0004] For example, Patent Document 1 discloses a cell culture vessel comprising a culture chamber for culturing cells, a storage chamber for storing fluids, and a substance exchange membrane that separates the culture chamber and the storage chamber and selectively allows a predetermined substance to pass through without allowing cells to pass through.

[0005] Furthermore, Patent Document 2 discloses a cell culture apparatus having a cell culture vessel partitioned into two chambers, a cell culture chamber and a culture medium circulation chamber, with an oxygen supply and a carbon dioxide remover connected to the culture medium circulation chamber.

[0006] Furthermore, Patent Document 3 discloses a cell culture device comprising a culture chamber for containing cells, and a culture medium introduction channel and a culture medium discharge channel communicating with the culture chamber, wherein a porous filter is provided between the culture chamber and the culture medium discharge channel.

[0007] Furthermore, Patent Document 4 discloses a cell culture apparatus that uses a polyimide porous membrane as a cell culture substrate.

[0008] Japanese Patent Publication No. 2021-185877, Japanese Patent Publication No. Hei 5-38281, Japanese Patent Publication No. 2011-244713, International Publication No. 2016 / 121768

[0009] When using conventional cell culture equipment to increase the concentration of components produced by culturing cells, additional steps such as concentrating the culture supernatant were required. Furthermore, obtaining components produced by culturing large quantities of cells required large quantities of fresh culture medium, leading to increased culture medium costs.

[0010] In order to solve the above problems, the present inventors conducted diligent research and, as a result, devised a device having a culture chamber for cell culture, a first channel for flowing culture medium, a second channel for flowing culture medium, a semipermeable membrane between the first channel and the second channel, and a seeding port in the culture chamber, thereby completing the present invention. In other words, although not limited thereto, the present invention includes the following embodiments.

[0011] [1] A cell culture apparatus for culturing suspension cells, comprising: a culture chamber for culturing cells; a first channel having a first culture medium supply port and a first culture medium outlet, provided adjacent to a part or all of the outer surface of at least one side of the culture chamber; a second channel having a second culture medium supply port and a second culture medium outlet; a semipermeable membrane disposed in at least a part of the space between the first channel and the second channel; and a seeding port provided in at least a part of the culture chamber. [2] The cell culture apparatus according to item 1, wherein a porous membrane is provided between the first channel and the culture chamber. [3] The cell culture apparatus according to item 1 or 2, wherein a first oxygen permeable membrane is provided on a part of the outer surface of the first channel and / or at least one side of the culture chamber. [4] The cell culture apparatus according to any one of items 1 to 3, wherein a second oxygen permeable membrane is provided on a part or all of the outer surface of at least one side of the second channel. [5] A cell culture apparatus according to any one of items 1 to 4, wherein the thickness of the culture chamber is 0.05 to 5.0 mm. [6] The permeability of the porous membrane is 5 to 100 cm. ３ / (cm ２- A cell culture apparatus according to any one of items 2 to 5, wherein the fractional molecular weight of the semipermeable membrane is 100 kDa or less. [7] A cell culture apparatus according to any one of items 1 to 6, wherein the suspension cells are hybridomas.

[0012] [9] A method for producing cells or useful components produced from cells, comprising seeding cells in the culture chamber of a cell culture apparatus described in any one of items 1 to 8, flowing a first medium through the first channel at a first flow rate, and culturing the cells while flowing a second medium through the second channel at a second flow rate, wherein the flow rate of the second medium is faster than the flow rate of the first medium.

[10] The method according to item 9, wherein the cell culture apparatus further comprises a porous membrane between the first channel and the culture chamber.

[11] The porous membrane 1 cm ２ The method according to item 10, wherein the first culture medium is flowed at a rate of 10 mL or less per day while culturing.

[12] The method according to any one of items 9 to 11, comprising having a first culture medium recovery tank provided downstream of the first flow path, and recovering the first culture medium by concentrating the useful components produced from the cells.

[0013] According to the present invention, when increasing the concentration of components produced from cells, the step of concentrating the culture supernatant can be omitted. Furthermore, according to the present invention, the amount of expensive culture medium used can be reduced, contributing to a reduction in culture medium costs. In addition, according to the present invention, the amount of waste liquid can be reduced, contributing to a reduction in waste liquid disposal costs.

[0014] This is a schematic cross-sectional view of a cell culture apparatus of the present invention in one embodiment. This is a schematic cross-sectional view of a cell culture apparatus of the present invention in one embodiment. This is a schematic diagram (top view) of each component constituting the cell culture apparatus of the present invention in one embodiment. This is a schematic diagram (perspective view) showing each component constituting the cell culture apparatus of the present invention in one embodiment spread out vertically to explain it. This is a schematic diagram showing an example of how the cell culture apparatus of the present invention is used in one embodiment. This is a schematic cross-sectional view showing an example of how the cell culture apparatus used in the reference example is used. This is a graph showing the amount of antibody produced per day from cells when cultured using the cell culture apparatus of the example and the reference example.

[0015] An embodiment of the present invention will be described below with reference to the drawings, but the scope of the present invention is not limited to the embodiment described herein, and various modifications can be made without departing from the spirit of the invention. Furthermore, if multiple upper and lower limits are given for a particular parameter, any combination of these upper and lower limits can be used to create a suitable numerical range.

[0016] In this specification, terms such as "first," "second," "third," etc., are used to distinguish one element from other elements. For example, the first element may be referred to as the second element, and similarly the second element as the first element, and this will not depart from the scope of the present invention.

[0017] <Cell Culture Apparatus (First Embodiment (Cell Culture Apparatus 1) and Second Embodiment (Cell Culture Apparatus 1a))> Figures 1A and 1B show schematic diagrams representing cross-sections of the cell culture apparatus 1 and 1a of the present invention in one embodiment. Note that the cell culture apparatus 1 (Figure 1A) has the same configuration as the cell culture apparatus 1a (Figure 1B), except that it does not have a porous membrane 40 between the first culture chamber 10 and the flow channel 20, so it will be described in common here.

[0018] Figure 2 shows a schematic diagram (top view) of each component constituting the cell culture apparatus 1a of the present invention in one embodiment. Figure 3 is a schematic diagram (perspective view) showing the components shown in Figure 2 spread out vertically to explain their positional relationships.

[0019] Note that the sizes and positional relationships of the components shown in the schematic diagrams of cell culture apparatuses 1 and 1a are exaggerated for illustrative purposes; therefore, their height, width, and depth, or their positional relationships, can be adjusted as appropriate for the purpose.

[0020] In one embodiment, the cell culture apparatus 1 comprises a culture chamber 10 for culturing cells 90, a first channel 20 having a first culture medium supply port 21 and a first culture medium outlet 22 and provided adjacent to a part or all of the outer surface of at least one side of the culture chamber 10, a second channel 30 having a second culture medium supply port 31 and a second culture medium outlet 32, a semipermeable membrane 50 disposed in at least a part of the space between the first channel 20 and the second channel 30, and a seeding port 120 provided in at least a part of the culture chamber 10 (Figure 1A).

[0021] In another embodiment, the cell culture apparatus 1a, in addition to the configuration of the cell culture apparatus 1 described above, further includes a boundary made of a porous membrane 40 between the first channel 20 and the culture chamber 10 (Figure 1B). As a result, when the first culture medium 24 is continuously or intermittently flowed through the first channel 20, the components of the liquid 13 filling the culture chamber 10 and the components of the first culture medium 24 are exchanged via the porous membrane 40. Even if the porous membrane 40 is not present as in the cell culture apparatus 1, when the first culture medium 24 is continuously or intermittently flowed through the channel 20, the components of the liquid 13 filling the culture chamber 10 and the components of the first culture medium 24 are exchanged, however, it is preferable to flow the first culture medium 24 at a flow rate such that the cells 90 do not flow out of the culture medium outlet 22.

[0022] The culture chamber 10 only needs to have enough space to accommodate the liquid 13 and to allow for the cultivation of cells 90. For example, the thickness of the culture chamber 10 is 0.05 to 5.0 mm in the direction perpendicular to the surface on which the cell culture apparatus of the present invention is installed. If the culture chamber 10 has a thickness of this magnitude, the components of the liquid 13 filling the culture chamber 10 and the components of the first culture medium 24 are efficiently exchanged through the porous membrane 40, and the environment inside the culture chamber 10 can be maintained in a state suitable for culturing cells 90. Alternatively, for example, a smaller thickness of the culture chamber 10 (for example, about 0.05 to 2.00 mm) can improve the efficiency of component exchange from the first channel 20 and the gas exchange efficiency from the oxygen permeable membrane 60.

[0023] The second culture medium 34 flowing through the channel 30 is exchanged with components of the liquid 13 or the first culture medium 24 filling the culture chamber 10 via the semipermeable membrane 50, and the second culture medium 34 may be contained within the culture chamber 10.

[0024] In one embodiment, the culture chamber 10 may have a cell seeding port 12, and a cell seeding line 120 may be connected to the cell seeding port 12. A cell supply means 121 may be connected to the cell seeding line 120, and the cell supply means 121 may be, for example, a fluid supply device such as a syringe, a pipette, or a pump. The cell supply means 121 may be arbitrarily attached to and detached from the cell seeding line 120, for example, a stopcock may be provided at the end of the cell seeding line 120, and if the cell supply means 121 is not connected, the end of the cell seeding line 120 may be closed by the stopcock.

[0025] In another embodiment, the culture chamber 10 may be an open-system cell culture apparatus 1 or 1a, in which cells 90 are seeded in a sterile space and then the cell culture apparatus 1 is assembled as shown in Figure 3. However, it is preferable that the cell culture apparatus 1 or 1a is a closed-system cell culture apparatus 1 or 1a that is already formed in the shape shown in Figure 3 before use.

[0026] The first culture medium 24 can be appropriately selected depending on the type of cells 90 to be cultured, but a medium with buffering properties is preferred. The conditions for culturing the cells 90 can be appropriately determined depending on the type of cells 90, etc. Suitable culture methods for animal cells, plant cells, insect cells, and bacterial cells are known, and the first culture medium 24 can also be appropriately prepared depending on the type of cells 90.

[0027] Suspension cells are cells that may come into contact with the culture vessel during the culture process but are not anchorage-dependent and proliferate without extending pseudopods to adhere to the culture vessel. They may also be spheroids, which are self-organized cells formed by the aggregation of suspension cells. Suspension cells may be cells that do not originally possess adhesive properties (such as hematopoietic cells or leukemia cells), or cells that originally possessed adhesive properties but have lost or lost them due to the collection process, experimentation, or disease (such as cells isolated from animals or metastatic cancer cells).

[0028] In this specification, in addition to the suspension cells described above, cells that are originally adherent cells but have been artificially adapted to a suspension state (such as stem cells) and are in a state where they do not depend on adhesion for proliferation or metabolism may also be used. For example, cells such as CHO cells, HEK293 cells, and hybridoma cells are given as examples, but are not limited thereto. Preferred suspension cells used in the present invention are CHO cells, HEK293 cells, S f9 cells, mesenchymal stem cells, iPS cells, iPS-derived cells, hybridoma cells, and NS0 cells, more preferably CHO cells, HEK293 cells, mesenchymal stem cells, and hybridoma cells, and even more preferably CHO cells and hybridoma cells.

[0029] Cell 90 is not particularly limited as long as it is capable of expressing the desired substance. The substance may be naturally expressed within the cell, or it may be transformed to produce the substance by genetic engineering techniques. Preferably, the cell is transformed by genetic engineering techniques to express the substance. Suitable methods are known for the transformation of animal cells, plant cells, insect cells, and bacteria. (e.g., MOLECULAR CLONING: A Laboratory Manual (Fourth Edition), Michael R Green and Joseph Sambrook, 2012, (Cold Spring Harbor Laboratory Press), Mutation Research 760 (2014) 36-45, Reviews in Mutation Research)

[0030] The methods for culturing animal cells and the cell culture media are described, for example, in the Lonza Cell Culture Media Catalog. The first medium 24 and the second medium 34 may be, for example, Eagle Medium, Dulbecco's Modified Eagle Medium (DMEM), DMEM:F12 Medium, Glasgow Minimum Essential Medium, Grace Insect Medium, Hamm Medium, Iskov Modified Eagle Medium, RPMI-1640 Medium, L-15 Medium, McCoy 5A Medium, M199 Medium, etc., and these may be modified media depending on the cells 90 being cultured. These media may be those to which serum has been added, or they may be serum-free media.

[0031] Furthermore, the liquid 13 can be appropriately selected depending on the type of cells 90 to be cultured, but a buffering medium is preferred. For example, it may be PBS, Eagle medium, Dulbecco's modified Eagle medium (DMEM), DMEM:F12 medium, Glasgow minimal essential medium, Grace insect medium, Hamm medium, Iskov modified Eagle medium, RPMI-1640 medium, L-15 medium, McCoy 5A medium, M199 medium, etc., and these may be modified media depending on the cells 90 to be cultured. These media may have serum added to them, or they may be serum-free media. It is preferable that the first medium 24 and the liquid 13 are of the same type.

[0032] The inner shape of the culture chamber 10 is not particularly limited as long as it enables culturing of the cells 90 and has a space sufficient to fill the liquid 13. For example, it may be a prism (polygonal column) such as a triangular prism, a quadrangular prism, a pentagonal prism, or a hexagonal prism, or it may be a cylinder, without limitation. Considering the component exchange efficiency between the culture chamber 10 and the first flow path 20, it is preferable that the area in contact with the first flow path 20 is large.

[0033] The culture chamber 10 preferably has a culture surface on at least one side where the cells 90 can be cultured. At least one side of the culture chamber 10 may be coated with a substance that promotes adhesion (for example, collagen, gelatin, fibrin gel, etc.).

[0034] The first flow path 20 is fluidly connected to the first medium supply port 21 and the first medium discharge port 22. The first medium 24 is supplied from the first medium supply line 210 connected to the first medium supply port 21, and the first medium 24 is discharged to the first medium discharge line 220 connected to the first medium discharge port 22. A pump 80 is provided in the middle of the first medium supply line 210. The pump 80 may be, for example, a tube pump (peristaltic pump), a piezo pump, or a syringe pump, and any pump that can send out fluid can be used. The pump 80 may be connected to the first medium discharge line 220. For example, it may be a mechanism that causes the first medium 24 to flow by the first medium discharge line 220 becoming a negative pressure.

[0035] The first medium supply port 21 and the first medium discharge port 22 may be provided at the lower part or the side part of the first flow path 20 as shown in FIGS. 1A and 1B. The thickness of the first flow path 20 is not particularly limited, and for example, it may have a thickness of 0.05 to 5.0 mm in the direction perpendicular to the surface on which the cell culture device of the present invention is installed. The thickness of the first flow path 20 can be appropriately adjusted by changing the thickness of the first flow path forming layer 23 that forms the first flow path 20. When the first flow path 20 has the porous membrane 40 in the portion adjacent to the culture chamber 10, the components of the liquid 13 filling the culture chamber 10 and the components of the first medium 24 are efficiently exchanged through the porous membrane 40, and the environment in the culture chamber 10 plays a role in maintaining a state suitable for culturing the cells 90. Also, the useful components produced by the cells 90 move to the first medium 24 in the first flow path 20.

[0036] In one aspect, the flow path 20 has the porous membrane 40 in the portion adjacent to the culture chamber 10, and the components of the liquid 13 filling the culture chamber 10 and the components of the medium 23 are efficiently exchanged through the porous membrane 40, and the environment in the culture chamber 10 plays a role in maintaining a state suitable for culturing the cells 60. Also, the porous membrane 40 prevents living cells from flowing out into the flow path 20. Also, the porous membrane 40 allows dead cells to pass through the porous membrane 40 and flow out into the flow path 20.

[0037] Living cells and dead cells can be defined, for example, using Countess (registered trademark) II FL (Thermo fisher scientific). Also, the survival rate of the cells can be determined according to the following formula ("Equation 1").

[0038] The shape of the first flow path 20 is not particularly limited as long as it fulfills the above-mentioned role, but it is preferable that it is a shape that allows the first culture medium 24 to flow from the first culture medium supply port 21 to the first culture medium outlet 22 at a constant flow velocity and / or pressure. For example, as shown in "(5)" of Figure 2, the first flow path forming layer 23 that forms the first flow path 20 may have a substantially square opening in the portion that forms the first flow path 20, with an opening therefor where the first culture medium supply port 21 and the first culture medium outlet 22, which are located in point-symmetric positions, are connected. Although not shown, the first culture medium supply port 21 and the first culture medium outlet 22 may also have openings in line-symmetric positions.

[0039] A semipermeable membrane 50 is provided in at least a portion of the space between the first channel 20 and the second channel 30. In this specification, a semipermeable membrane 50 is a membrane that allows only molecules or ions of a certain size or smaller to pass through. In a system of a solute that does not pass through the semipermeable membrane 50 and a solvent that does, when solutions of two concentrations are brought into contact across the semipermeable membrane 50, osmotic pressure is generated at the boundary, and only the solvent passes through. Although not limited to the following, for example, a copper ferrocyanide precipitate membrane, a collodion membrane, a bladder membrane, regenerated cellulose (cellophane), acetylcellulose, polyacrylonitrile, Teflon®, a polyester polymer alloy, or a porous polysulfone membrane can be used. Depending on the purpose, a semipermeable membrane 50 that allows or does not allow any fractional molecular weight to pass through can be used. For example, when the purpose is to concentrate antibodies produced from cells, such as IgG (about 150 kDa), it is preferable that the semipermeable membrane 50 has a fractional molecular weight of 100 kDa or less. It can be appropriately selected according to the molecular weight of the target protein component.

[0040] The second culture medium supply port 31 and the second culture medium outlet 32 ​​may be located at the top or side of the second channel 30, as shown in Figures 1A and 1B. The thickness of the second channel 30 is not particularly limited, but for example, it may be 0.05 to 5.0 mm in the direction perpendicular to the surface on which the cell culture apparatus of the present invention is installed. The thickness of the second channel 30 can be appropriately adjusted by changing the thickness of the second channel forming layer 33 that forms the second channel 30. The second channel 30 has a semipermeable membrane 50 in the portion adjacent to the first channel 20, and the components of the first culture medium 24 filling the first channel 20 and the components of the second culture medium 34 are selectively exchanged through the semipermeable membrane 50 according to the properties of the semipermeable membrane 50, maintaining the first culture medium 24 in a state suitable for culturing cells 90, and also playing a role in concentrating arbitrary components, such as antibodies and proteins, in the first culture medium 24.

[0041] The shape of the first flow path is not particularly limited as long as it fulfills the above-mentioned role, but it is preferable that it is a shape that allows the first culture medium 24 to flow from the first culture medium supply port 21 to the first culture medium outlet 22 at a constant flow velocity and / or pressure. For example, as shown in "(5)" of Figure 2, the first flow path forming layer 23 that forms the first flow path 20 may have a substantially square opening in the portion that forms the first flow path 20, with an opening therefor where the first culture medium supply port 21 and the first culture medium outlet 22, which are located in point-symmetric positions, are connected. Although not shown, the first culture medium supply port 21 and the first culture medium outlet 22 may also have openings at line-symmetric positions.

[0042] The shape of the second flow path is not particularly limited as long as it fulfills the above-mentioned role, but it is preferable that it is a shape that allows the second culture medium 34 to flow from the second culture medium supply port 31 to the second culture medium outlet 32 ​​at a constant flow velocity and / or pressure. For example, as shown in "(3)" of Figure 2, the second flow path forming layer 33 that forms the second flow path 30 may have a substantially square opening in the portion that forms the second flow path 30, with an opening therefor where the second culture medium supply port 31 and the second culture medium outlet 32, which are located in point-symmetric positions, connect. Although not shown, the second culture medium supply port 31 and the second culture medium outlet 32 ​​may also have openings in line-symmetric positions.

[0043] In one embodiment, the first oxygen permeable membrane 60 may be provided on at least one of the first channel 20 and the culture chamber 10. For example, as shown in Figures 1A and 1B, the first oxygen permeable membrane 60 may be provided on the lower surface of the culture chamber 10, or, for example, a portion of the first oxygen permeable membrane 60 may be provided on the side surface of the first channel 20, but it is preferable that the first oxygen permeable membrane 60 be provided on the lower surface of the culture chamber 10.

[0044] In one embodiment, a second oxygen permeable membrane 61 is provided in the second flow channel 30. For example, in the second flow channel 30, the second oxygen permeable membrane 61 is provided on the surface facing the surface on which the semipermeable membrane 50 is provided.

[0045] In other embodiments, even if the first oxygen permeable membrane 60 and / or the second oxygen permeable membrane 61 are not provided, the first culture medium supply line 210 and / or the second culture medium supply line 310 may have, for example, an oxygen supply means (e.g., an oxygen supply device using bubbling).

[0046] The first oxygen permeable membrane 60 and / or the second oxygen permeable membrane 61 are formed by membranes that allow oxygen molecules to pass through but do not allow larger molecules to pass through, including physiologically active substances secreted by cells 90, foreign substances from outside. For example, the first oxygen permeable membrane 60 and / or the second oxygen permeable membrane 61 have an oxygen permeability of 10,000 to 10,000,000 cm⁻¹. ３ / m ２- Preferably, the oxygen permeable membrane has an oxygen permeability of 24 h·atm. If the oxygen permeability is as described above and it can be used for cell culture, it can be applied to the present invention. The first oxygen permeable membrane 60 and / or the second oxygen permeable membrane 61 are not limited to the following, but for example, silicone, latex, polyvinyl acetate, polycarbonate, polyolefin such as polyethylene and polypropylene, or layers thereof can be used. In addition, a membrane with physically fine pores can be used as an oxygen permeable membrane. For example, the ePTFE (expanded polytetrafluoroethylene) membrane developed by W. L. Gore & Associates, Inc. and PTFE (polytetrafluoroethylene) nonwoven fabric have high hydrophobicity, so moisture does not easily penetrate them, and they function as the first oxygen permeable membrane 60 and / or the second oxygen permeable membrane 61 even when in contact with moisture.

[0047] The second flow path 30 is fluidly connected to the second culture medium supply port 31 and the second culture medium discharge port 32. The second culture medium 34 is supplied from the second culture medium supply line 310 connected to the second culture medium supply port 31, and discharged to the second culture medium discharge line 320 connected to the second culture medium discharge port 32. A pump 81 is provided in the middle of the second culture medium supply line 310. The pump 81 may be, for example, a tubular pump (peristallic pump) or a piezo pump; any pump capable of dispensing fluid can be used. The pump 81 may also be provided in the middle of the second culture medium discharge line 320, and for example, it may be a mechanism that causes the second culture medium 34 to flow when the first culture medium discharge line 220 becomes negatively pressurized.

[0048] Furthermore, the first channel 20, the second channel 30, and the culture chamber 10 may be sandwiched between a first support 70 and a second support 71, as shown in Figures 1A and 1B, and the first support 70 and the second support 71 may be fixed together by male and female screws (not shown). The first support 70 and the second support 71 may be provided with ventilation holes 700 for exposing the first oxygen permeable membrane 60 and / or the second oxygen permeable membrane 61 to air. Multiple ventilation holes 700 may be provided, as shown in Figure 2, or only one may be provided.

[0049] In one embodiment, the porous membrane 40 is a membrane (film) having numerous small voids inside or on its surface, and the material forming this porous membrane 40 is not particularly limited and may be an inorganic substance, an organic substance, or a composite thereof. The porous membrane 40 has an air permeability of 5 to 100 cm. ３ / (cm ２ It is preferable that the material is of type s, for example, a nonwoven fabric or a polymer porous membrane. The degree of air permeability can be determined, for example, by measuring it using the JIS L 1096 air permeability method A (Fragile method). As one embodiment of the porous membrane 40 applicable to the present invention, a polymer porous membrane formed from a polymer will be described below as an example.

[0050] In this specification, a "polymer porous membrane" has a surface layer A (hereinafter sometimes referred to as "surface A" or "mesh surface") and a surface layer B (hereinafter sometimes referred to as "surface B" or "large hole surface"). The shape of the pore diameters of surface A and surface B may be the same or different. Furthermore, there is no particular limitation on the size of the pore diameters (average pore diameters) of surface A and surface B, but it is preferable that the average pore diameter of the pores on surface B is larger than the average pore diameter of the pores on surface A.

[0051] The average pore diameter of the pores present on surface A (hereinafter sometimes referred to as "average pore diameter") is not particularly limited, but for example, it is preferably 0.01 μm or more, more preferably 0.05 μm or more, even more preferably 0.1 μm or more, and particularly preferably 0.5 μm or more. Its upper limit is preferably less than 200 μm, more preferably 150 μm or less, even more preferably 100 μm or less, and particularly preferably 50 μm or less, 40 μm or less, 30 μm or less, 20 μm or less, or 15 μm or less.

[0052] The average pore diameter of the pores on surface B is preferably larger than the average pore diameter of the pores on surface layer A. For example, it is preferably greater than 5 μm, more preferably 20 μm or more, even more preferably 30 μm or more, and particularly preferably 50 μm or more and 60 μm or more. The upper limit is preferably 200 μm or less, and more preferably 100 μm or less.

[0053] The average pore diameter of the pores in the surface layer of a polymer porous membrane can be calculated by measuring the pore area of ​​200 or more open areas from scanning electron microscope images of the porous membrane surface, and then calculating the average diameter assuming the pore shape is perfectly circular using the following formula ("Formula 2").

[0054]

[0055] (In the formula, Sa represents the average value of the pore area.)

[0056] The thicknesses of surface layer A and surface layer B may be the same or different, and are not particularly limited. For example, their respective thicknesses are preferably 0.01 μm or more, with an upper limit of preferably 50 μm or less, and more preferably 20 μm or less.

[0057] The polymer porous membrane used in the present invention may have an intermediate layer between surface A and surface B. This intermediate layer may be a solid layer without pores, or a porous layer having pores. If it has pores, it may be the same as or different from the surface layer of the polymer porous membrane. Preferably, the intermediate layer is a porous layer, and that porous layer forms a macrovoid layer.

[0058] The explanation will be given using a macrovoid layer, in which the intermediate layer is a porous layer, as an example. In this specification, "macrovoid layer" means a film in which the average pore diameter in the film plane direction of the layer is 10 μm or more. The average pore diameter in the film plane direction of the macrovoids in the macrovoid layer is not particularly limited, but for example, the upper limit of the average pore diameter is preferably 500 μm or less, more preferably 100 μm or less, and particularly preferably 80 μm or less. Furthermore, the thickness of the partitions (frames that form the holes in the macrovoid layer) in the macrovoid layer is not particularly limited, but for example, it is preferably 0.01 μm or more, the upper limit is preferably 50 μm or less, and more preferably 20 μm or less.

[0059] In one embodiment, at least one partition wall in the macrovoid layer may have pores that connect adjacent macrovoids. The average diameter of these connecting pores is preferably 0.01 μm or more, with an upper limit of preferably 100 μm or less, and more preferably 50 μm or less. There may be one or more of these connecting pores. In another embodiment, the partition walls in the macrovoid layer do not have pores.

[0060] The total thickness of the polymer porous film (the sum of the thickness of the A-side and B-side, and the intermediate layer if present) is not particularly limited, but may be 5 μm or more, 10 μm or more, 20 μm or more, or 25 μm or more, and may be 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less, or 50 μm or less. Preferably, it is 5 to 500 μm, and more preferably 10 to 100 μm.

[0061] In this specification, the thickness of a polymer porous membrane can be measured using a contact-type thickness gauge.

[0062] In this specification, the porosity of the polymer porous membrane is not particularly limited, but for example, it is preferably 40% or more, more preferably 50% or more, and even more preferably 60% or more, with an upper limit of preferably less than 95%, more preferably 90% or less, and even more preferably 85% or less.

[0063] In this specification, the porosity of a polymer porous membrane can be determined by measuring the film thickness and mass of a polymer porous membrane cut to a predetermined size, and then calculating the basis mass according to the following formula ("Formula 3").

[0064]

[0065] (In the formula, S represents the area of ​​the polymer porous membrane, d represents the total film thickness, w represents the measured mass, and D represents the density of the polymer. If the polymer is polyimide, the density is 1.34 g / cm³.) ３ (Let's assume that.)

[0066] In this specification, the polymer porous membrane is preferably a three-layer polymer porous membrane having a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B. Here, the average pore diameter of the pores in the surface layer A is preferably 0.01 μm or more and 15 μm or less, and the average pore diameter of the pores in the surface layer B is preferably 20 μm or more and 100 μm or less. The macrovoid layer has partitions bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partitions and the surface layers A and B. The thickness of the partitions in the macrovoid layer and the surface layers A and B is preferably 0.01 μm or more and 20 μm or less, the pores in the surface layers A and B communicate with the macrovoids, and the total film thickness is preferably 5 μm or more and 500 μm or less. The porosity of this polymer porous membrane is preferably 40% or more and less than 95%. In one embodiment, at least one partition in the macrovoid layer has one or more pores with an average pore size of 0.01 μm or more and 100 μm or less, preferably 0.01 μm or more and 50 μm or less, that connect adjacent macrovoids. In another embodiment, the partition does not have such pores.

[0067] In this specification, the polymer porous membrane preferably has the structural characteristics described above, but particularly preferably a polyimide porous membrane formed from polyimide porous material, or a polyethersulfone porous membrane formed from polyethersulfone (PES) can be used. For example, it may be a polyimide porous membrane or polyethersulfone (PES) as described in International Publication No. 2016 / 121768.

[0068] <<Polyimide Porous Membrane>> In this specification, "polyimide" is a general term for polymers containing imide bonds in their repeating units, preferably those containing 50 mol% or more of imide bonds in the total repeating units, and usually refers to aromatic polyimides in which aromatic compounds are directly linked by imide bonds. Aromatic polyimides have a rigid and strong molecular structure because aromatic compounds have a conjugated structure via imide bonds, and they have very high levels of thermal, mechanical, and chemical properties because the imide bonds have strong intermolecular forces.

[0069] The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane that mainly contains polyimide obtained from tetracarboxylic dianhydride and diamine, and more preferably a polyimide porous membrane consisting of polyimide obtained from tetracarboxylic dianhydride and diamine. "Mainly contains" means that the polyimide porous membrane does not essentially contain any components other than polyimide obtained from tetracarboxylic dianhydride and diamine, or it may contain other components, but these are additional components that do not affect the properties of polyimide obtained from tetracarboxylic dianhydride and diamine.

[0070] Polyamic acids are obtained by polymerizing a tetracarboxylic acid component and a diamine component. Polyamic acids are precursors for forming polyimides, which can be cyclized by thermal or chemical imidation to form polyimides.

[0071] Polyamic acids can be used even if a portion of the amic acid is imidized, as long as this does not affect the present invention. In other words, polyamic acids may be partially thermally imidized or chemically imidized.

[0072] When thermally imidizing polyamic acid, additives such as imidation catalysts, organophosphorus-containing compounds, inorganic fine particles, organic fine particles, and other microparticles may be added to the solution in which the polyamic acid is dissolved (hereinafter also referred to as the "polyamic acid solution") as needed. Furthermore, when chemically imidizing polyamic acid, additives such as chemical imidating agents, dehydrating agents, inorganic fine particles, organic fine particles, and other microparticles may be added to the polyamic acid solution as needed.

[0073] In one embodiment, the polyimide porous membrane that can be used in the present invention also includes a colored polyimide porous membrane obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component and a coloring precursor, and then heat-treating it at 250°C or higher.

[0074] In this specification, "coloring precursor" means a precursor that is partially or completely carbonized by heat treatment at 250°C or higher to produce a colored product.

[0075] The coloring precursors that can be used in the production of the above-mentioned polyimide porous membrane are preferably those that can be uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution and then thermally decomposed and carbonized to produce a colored product by heat treatment at 250°C or higher, preferably 260°C or higher, more preferably 280°C or higher, more preferably 300°C or higher, preferably in the presence of oxygen such as air, and more preferably those that produce a black colored product, and more preferably carbon-based coloring precursors.

[0076] When heated, the colored precursors appear to be carbonides, but structurally they contain heteroatoms other than carbon, and include layered structures, aromatic cross-linked structures, and disordered structures containing tetrahedral carbon.

[0077] The carbon-based coloring precursor is not particularly limited and includes, for example, tar or pitch such as petroleum tar, petroleum pitch, coal tar, and coal pitch, coke, polymers obtained from monomers containing acrylonitrile, and ferrocene compounds (ferrocene and ferrocene derivatives). Among these, polymers and / or ferrocene compounds obtained from monomers containing acrylonitrile are preferred, and polyacrylonitrile is preferred as the polymer obtained from monomers containing acrylonitrile.

[0078] When using a coloring precursor, the above-mentioned additives may be used. In this case, it is preferable to carry out the process under conditions in which the coloring precursor does not precipitate even when the additives are added to the polyamic acid solution.

[0079] Furthermore, in another embodiment, the polyimide porous membrane that can be used in the present invention also includes a polyimide porous membrane obtained by molding a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component, and then heat-treating it, without using the above-mentioned coloring precursor.

[0080] Polyimide porous membranes may be produced, with or without the use of a coloring precursor, by, for example, casting a polyamic acid solution consisting of 3 to 60% by mass of polyamic acid having an intrinsic viscosity number of 1.0 to 3.0 and 40 to 97% by mass of an organic polar solvent in a film form, immersing or contacting it with a solidification solvent in which water is an essential component to produce a porous polyamic acid membrane, and then heat-treating the porous polyamic acid membrane to imide it. In this method, the solidification solvent in which water is an essential component may be water, or a mixture of 5% by mass or more and less than 100% by mass of water and more than 0% by mass and 95% by mass or less of an organic polar solvent. Furthermore, after the above imide treatment, at least one side of the obtained porous polyimide membrane may be subjected to plasma treatment.

[0081] In the production of the above-mentioned porous polyimide membrane, any tetracarboxylic dianhydride can be used and can be appropriately selected according to the desired properties. Specific examples of tetracarboxylic dianhydrides include pyromellitic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA), 2,3,3',4'-biphenyltetracarboxylic dianhydride (a-BPDA), oxydiphthalic dianhydride, diphenylsulfone-3,4,3',4'-tetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl) sulfide dianhydride, 2,2-bis(3,4-dicarboxyphenyl)-1,1,1,3,3,3-hexafluoropropane dianhydride, 2,3,3',4'-benzophenonetetracarboxylic dianhydride, 3,3',4,4'-benzophenonetetracarboxylic dianhydride, bis(3,4-dicarboxyphenyl)methane dianhydride, and 2,2-bis(3,4-dicarboxyphenyl)propane dianhydride. Examples include anhydrides, p-phenylenebis(trimellitic acid monoester anhydride), p-biphenylenebis(trimellitic acid monoester anhydride), m-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic acid dianhydride, 1,3-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)benzene dianhydride, 1,4-bis(3,4-dicarboxyphenoxy)biphenyl dianhydride, 2,2-bis[(3,4-dicarboxyphenoxy)phenyl]propane dianhydride, 2,3,6,7-naphthalenetetracarboxylic acid dianhydride, 1,4,5,8-naphthalenetetracarboxylic acid dianhydride, and 4,4'-(2,2-hexafluoroisopropylidene)diphthalic acid dianhydride. Furthermore, it is also preferable to use aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfonetetracarboxylic acid. These can be used individually or in combination of two or more.

[0082] Among these, at least one aromatic tetracarboxylic dianhydride selected from the group consisting of biphenyltetracarboxylic dianhydrides and pyromellitic dianhydrides is particularly preferred. As the biphenyltetracarboxylic dianhydride, one containing 3,3',4,4'-biphenyltetracarboxylic dianhydride can be suitably used.

[0083] Any diamine can be used in the production of the above-mentioned porous polyimide membrane. Specific examples of diamines include the following:

[0084] 1) Benzene diamines with one benzene ring, such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, and 2,6-diaminotoluene; 2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenylmethane, 3,3'-dimethyl-4,4'-diaminobiphenyl, 2,2'-dimethyl-4,4'-diaminobiphenyl, 2,2'-bis(trifluoromethyl)-4,4'-diaminobiphenyl, 3,3'-dimethyl-4,4'-diaminodiphenylmethane, 3,3'-dicarboxy-4,4'-diaminodiphenylmethane, 3 ,3',5,5'-tetramethyl-4,4'-diaminodiphenylmethane, bis(4-aminophenyl) sulfide, 4,4'-diaminobenzanilide, 3,3'-dichlorobenzidine, 3,3'-dimethylbenzidine, 2,2'-dimethylbenzidine, 3,3'-dimethoxybenzidine, 2,2'-dimethoxybenzidine, 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, 3,3'-diaminodiphenyl Sulfide, 3,4'-diaminodiphenyl sulfide, 4,4'-diaminodiphenyl sulfide, 3,3'-diaminodiphenyl sulfone, 3,4'-diaminodiphenyl sulfone, 4,4'-diaminodiphenyl sulfone, 3,3'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4,4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-di Diamines with two benzene rings, such as aminodiphenylmethane, 2,2-bis(3-aminophenyl)propane, 2,2-bis(4-aminophenyl)propane, 2,2-bis(3-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 2,2-bis(4-aminophenyl)-1,1,1,3,3,3-hexafluoropropane, 3,3'-diaminodiphenyl sulfoxide, 3,4'-diaminodiphenyl sulfoxide, and 4,4'-diaminodiphenyl sulfoxide;3) 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, 1,4-bis(3-aminophenoxy)benzene, 1,4-bis(4-aminophenoxy)benzene, 1,3-bis(3-aminophenoxy)-4-trifluoromethylbenzene, 3,3'-diamino-4-(4-phenyl)phenoxybenzophenone, 3,3'-diamino-4,4'-di(4-phenylphenoxy)benzophenone, 1,3 Benzene-nuclear diamines such as bis(3-aminophenylsulfide)benzene, 1,3-bis(4-aminophenylsulfide)benzene, 1,4-bis(4-aminophenylsulfide)benzene, 1,3-bis(3-aminophenylsulfone)benzene, 1,3-bis(4-aminophenylsulfone)benzene, 1,4-bis(4-aminophenylsulfone)benzene, 1,3-bis[2-(4-aminophenyl)isopropyl]benzene, 1,4-bis[2-(3-aminophenyl)isopropyl]benzene, and 1,4-bis[2-(4-aminophenyl)isopropyl]benzene;4) 3,3'-bis(3-aminophenoxy)biphenyl, 3,3'-bis(4-aminophenoxy)biphenyl, 4,4'-bis(3-aminophenoxy)biphenyl, 4,4'-bis(4-aminophenoxy)biphenyl, bis[3-(3-aminophenoxy)phenyl] ether, bis[3-(4-aminophenoxy)phenyl] ether, bis[4-(3-aminophenoxy)phenyl] ether, bis[4-(4-aminophenoxy)phenyl] ether, bis[3-(3-aminophenoxy)phenyl] ketone, bis[3-(4-A Minophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(4-aminophenoxy)phenyl]ketone, bis[3-(3-aminophenoxy)phenyl]sulfide, bis[3-(4-aminophenoxy)phenyl]sulfide, bis[4-(3-aminophenoxy)phenyl]sulfide, bis[4-(4-aminophenoxy)phenyl]sulfide, bis[3-(3-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]sulfone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone, bis[4-(3-aminophenoxy)phenyl]ketone [nophenoxy)phenyl]sulfone, bis[4-(4-aminophenoxy)phenyl]sulfone, bis[3-(3-aminophenoxy)phenyl]methane, bis[3-(4-aminophenoxy)phenyl]methane, bis[4-(3-aminophenoxy)phenyl]methane, bis[4-(4-aminophenoxy)phenyl]methane, 2,2-bis[3-(3-aminophenoxy)phenyl]propane, 2,2-bis[3-(4-aminophenoxy)phenyl]propane, 2,2- Diamines with four benzene rings, such as bis[4-(4-aminophenoxy)phenyl]propane, 2,2-bis[3-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[3-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, 2,2-bis[4-(3-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane, and 2,2-bis[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane.

[0085] These can be used individually or in mixtures of two or more. The diamines used can be appropriately selected according to the desired properties.

[0086] Among these, aromatic diamine compounds are preferred, and 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether and paraphenylenediamine, 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-aminophenyl)benzene, 1,3-bis(4-aminophenoxy)benzene, and 1,4-bis(3-aminophenoxy)benzene can be suitably used. In particular, at least one diamine selected from the group consisting of benzenediamine, diaminodiphenyl ether and bis(aminophenoxy)phenyl is preferred.

[0087] The porous polyimide membrane that can be used in the present invention is preferably formed from a polyimide obtained by combining a tetracarboxylic dianhydride and a diamine, which has a glass transition temperature of 240°C or higher, or 300°C or higher and no clear transition point, from the viewpoint of heat resistance and dimensional stability at high temperatures.

[0088] The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane made of the following aromatic polyimides, from the viewpoint of heat resistance and dimensional stability at high temperatures.

[0089] (i) an aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and an aromatic diamine unit; (ii) an aromatic polyimide comprising a tetracarboxylic acid unit and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units, and bis(aminophenoxy)phenyl units, and / or (iii) an aromatic polyimide comprising at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units, and at least one aromatic diamine unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units, and bis(aminophenoxy)phenyl units.

[0090] The polyimide porous membrane that can be used in the present invention is preferably a three-layer polyimide porous membrane having a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layers A and B, wherein the average pore diameter of the pores in the surface layer A is preferably 0.01 μm or more and 15 μm or less, and the average pore diameter of the pores in the surface layer B is preferably 20 μm or more and 100 μm or less. The macrovoid layer preferably has a partition wall bonded to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, wherein the thickness of the partition wall of the macrovoid layer and the surface layers A and B is preferably 0.01 μm or more and 20 μm or less. The pores in the surface layers A and B communicate with the macrovoids, and the total film thickness is preferably 5 μm or more and 500 μm or less. Furthermore, the porosity of the polyimide porous membrane is preferably 40% or more and less than 95%. Here, at least one partition wall in the macrovoid layer may have one or more pores with an average pore size of 0.01 μm or more and 100 μm or less, preferably 0.01 μm or more and 50 μm or less, that connect adjacent macrovoids.

[0091] For example, the polyimide porous membranes described in International Publication No. 2010 / 038873, Japanese Patent Publication No. 2011-219585, or Japanese Patent Publication No. 2011-219586 can also be used in the present invention.

[0092] <<Polyethersulfone (PES) porous membrane>>

[0093] The PES porous membrane that can be used in the present invention contains a polyethersulfone and is typically substantially composed of a polyethersulfone. The polyethersulfone may be synthesized by methods known to those skilled in the art, for example, by polycondensation of a divalent phenol, an alkali metal compound and a dihalogenodiphenyl compound in an organic polar solvent, or by pre-synthesizing an alkali metal disal of a divalent phenol and polycondensation of it with a dihalogenodiphenyl compound in an organic polar solvent.

[0094] Examples of alkali metal compounds include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides, and alkali metal alkoxides. Sodium carbonate and potassium carbonate are particularly preferred.

[0095] Examples of divalent phenol compounds include hydroquinone, catechol, resorcinol, 4,4'-biphenol, bis(hydroxyphenyl)alkanes (e.g., 2,2-bis(hydroxyphenyl)propane and 2,2-bis(hydroxyphenyl)methane), dihydroxydiphenyl sulfones, dihydroxydiphenyl ethers, or compounds in which at least one hydrogen atom of the benzene ring is substituted with a lower alkyl group such as a methyl group, ethyl group, or propyl group, or a lower alkoxy group such as a methoxy group or ethoxy group. Two or more of the above compounds can be used as divalent phenol compounds.

[0096] Polyethersulfone may be a commercially available product. Examples of commercially available products include Sumika Excel 7600P and Sumika Excel 5900P (both manufactured by Sumitomo Chemical Co., Ltd.).

[0097] The logarithmic viscosity of the polyethersulfone is preferably 0.5 or higher, more preferably 0.55 or higher, from the viewpoint of good formation of macrovoids in the PES porous membrane, and preferably 1.0 or lower, more preferably 0.9 or lower, even more preferably 0.8 or lower, and particularly preferably 0.75 or lower, from the viewpoint of ease of manufacturing the PES porous membrane.

[0098] Furthermore, from the viewpoint of heat resistance and dimensional stability at high temperatures, it is preferable that the PES porous membrane, or the polyethersulfone used as its raw material, has a glass transition temperature of 200°C or higher, or that no clear glass transition temperature is observed.

[0099] The method for producing a PES porous membrane that can be used in the present invention is not particularly limited, but for example, it may include the steps of: casting a polyethersulfone solution containing 0.3% to 60% by mass of polyethersulfone having a logarithmic viscosity of 0.5 to 1.0 and 40% to 99.7% by mass of an organic polar solvent into a film shape, immersing or contacting it with a solidification solvent having polyethersulfone as a poor solvent or non-solvent as an essential component to produce a solidified film having pores; and heat-treating the solidified film having pores obtained in the above step to coarseen the pores to obtain a PES porous membrane, wherein the heat treatment includes raising the temperature of the solidified film having pores to above the glass transition temperature of the polyethersulfone, or to 240°C or higher.

[0100] A PES porous membrane that can be used in the present invention is preferably a PES porous membrane having a surface layer A, a surface layer B, and a macrovoid layer sandwiched between surface layer A and surface layer B, wherein the macrovoid layer has partitions bonded to surface layers A and B, and a plurality of macrovoids surrounded by the partitions and surface layers A and B, the average pore diameter in the membrane planar direction being 10 μm to 500 μm, the partitions of the macrovoid layer having a thickness of 0.1 μm to 50 μm, surface layers A and B each having a thickness of 0.1 μm to 50 μm, one of surface layers A and B having a plurality of pores with an average pore diameter of more than 5 μm and 200 μm or less, and the other having a plurality of pores with an average pore diameter of 0.01 μm or more and less than 200 μm, one of surface layer A and surface layer B having a surface opening ratio of 15% or more, and the other surface layer having a surface opening ratio of 10% or more. The pores of surface layer A and surface layer B communicate with the macrovoids, and the PES porous membrane has a total thickness of 5 μm to 500 μm and a porosity of 50% to 95%.

[0101] In one embodiment, the cell culture apparatus 1a may be provided as a device formed by stacking the components shown in Figure 2, namely (1) the second support 71, (2) the second oxygen permeable membrane 61, (3) the second channel forming layer 33, (4) the semipermeable membrane 50, (5) the first channel forming layer 23, (6) the porous membrane 40, (7) the culture chamber forming layer 11, (8) the first oxygen permeable membrane 60, and (9) the first support 70, in the order from the bottom layer (9) to (1), as shown in Figure 3. In another embodiment, the cell culture apparatus 1a may be provided as a device formed by stacking the components shown in Figure 2 in the reverse order from the bottom layer, from (1) to (9), as shown in Figure 3. In this case, the culture chamber 10 is provided as the upper layer. Alternatively, the device may be provided in the form of a kit in which each component (1) to (9) is individually packaged.

[0102] (1) The material of the member forming the second support 71 and (9) the first support 70 is not limited, but examples include polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, acrylic resin (e.g., polymethacrylate, polyacrylic acid, sodium polyacrylate, polyacrylonitrile, polyacrylamide, etc.), polysulfone, cellulose, cellulose derivatives, polysilicone, polymethylpentene, metals, etc.

[0103] (3) The material of the member forming the second channel forming layer 33, (5) the first channel forming layer 23, and (7) the culture chamber forming layer 11 is not limited, but examples include polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, acrylic resin (e.g., polymethacrylate, polyacrylic acid, sodium polyacrylate, polyacrylonitrile, polyacrylamide, etc.), polysulfone, cellulose, cellulose derivatives, polysilicone, polymethylpentene, and metals. A sealing member may be provided between the members to prevent fluid leakage, but for example, if the material of the member forming the first channel forming layer 23 and (7) the culture chamber forming layer 11 is flexible and has a sealing function, it is preferable that liquids such as culture media do not leak, and polysilicone is more preferable.

[0104] In one aspect, in order to increase the number of cells 90, the cell culture device 1 or 1a may increase the area or volume of the culture chamber 10, or the area or volume of the culture chamber may be increased by connecting a plurality of cell culture devices 1 or 1a in parallel. In the latter case, for example, the first medium supply line 210 connected to one first medium supply tank 212 may branch midway and be connected in parallel to the first medium supply port 21. Also, the first medium discharge line 220 connected in parallel to the first medium discharge port 22 may merge midway and be connected to one first medium recovery tank 222. Further, the second medium supply line 310 connected to one second medium supply tank 312 may branch midway and be connected in parallel to the second medium supply port 31. Also, the second medium discharge line 320 connected in parallel to the second medium discharge port 32 may merge midway and be connected to one second medium recovery tank 322.

[0105] Method for producing cells or useful components from cells

[0106] In one aspect, the present invention is a method for producing cells or useful components from cells, comprising seeding cells 90 in the culture chamber 10 of the cell culture device 1 or 1a described above, flowing a first medium 24 at a first flow rate through the first flow path 20, and culturing while flowing a second medium 34 at a second flow rate through the second flow path 30, wherein the flow rate of the second medium 34 is faster than the flow rate of the first medium 24. By perfusion with the flow rate of the second medium 34 being faster than the flow rate of the first medium 24, useful components produced from the cells 90 are accumulated in the first medium 24, enhancing the concentration effect.

[0107] In one aspect, the first flow rate is per 1 cm of the area of the porous membrane 40 ２The culture is carried out while flowing the first culture medium at a rate of 10 mL or less per day. The first flow rate is preferably 10 mL or less per day (10 mL or less / day), and may be, for example, 9 mL or less / day, 8 mL or less / day, 7 mL or less / day, 5 mL or less / day, 3 mL or less / day, 1 mL or less / day, or even less. For example, it may be 0.5 mL or less / day. Since useful components are supplied and waste components are removed by the second flow path, the first flow rate may be 0 mL / day. The first and second flow rates can be adjusted by pumps (80, 81).

[0108] In one embodiment, the second flow velocity is not particularly limited, as it only needs to be a faster perfusion velocity than the first flow velocity. For example, it may be 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 10 times, or more than the first flow velocity.

[0109] Furthermore, in one embodiment, the present invention may include a first culture medium recovery tank 222 provided downstream of the first flow path 20, and may include concentrating the useful components produced from the cells 90 to recover the first culture medium 24.

[0110] The present invention provides a method for culturing cells 90 in the culture chamber 10 of a cell culture device 1 or 1a, while continuously or intermittently flowing culture medium from the culture medium supply port to the culture medium outlet. By using the cell culture device 1 or 1a described above, even when a large number of cells are gathered, sufficient oxygen and nutrient supply can be provided during cell culture, making it possible to provide a stable culture system. Furthermore, damage to the cells is minimized, and stable long-term culture is possible.

[0111] In one embodiment, the method of the present invention provides a method for culturing cells 90 for 30 days or more using a cell culture apparatus 1 or 1a, and for continuously producing useful components from the cells. That is, in the present invention, without performing subculturing operations as in the conventional method, it is possible to culture cells for a long period of time of 30 days or more, 60 days or more, 120 days or more, 200 days or more, or 300 days or more, and to continuously produce useful components from the cells. Furthermore, in one embodiment, the method of the present invention allows cells to be cultured for a period longer than that which can be achieved with conventional planar culture, for example, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, or 4.5 times or more than the planar culture period, and to continuously produce useful components from the cells.

[0112] In one embodiment, the amount of useful components obtained in the present invention can be determined according to the following formula ("Formula 4").

[0113] In this specification, "useful ingredients" refer to substances that can be produced by cells and can be used in the medical, food, and cosmetic fields, as well as for therapeutic and edible purposes. These substances can be produced naturally within cells or by genetic engineering technology and are selected from the group consisting of, for example, proteins (including polypeptides), glycoproteins, and viruses. Examples of proteins include physiologically active proteins such as erythropoietin, insulin, and albumin; cytokines such as tumor necrosis factor α, interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), and interferon; enzymes such as thrombin and trypsin; and monoclonal antibodies including antibody drugs.

[0114] Examples of glycoproteins include collagen, fibronectin, and hyaluronic acid.

[0115] Examples of viruses include influenza viruses, adenoviruses, lentiviruses, herpesviruses, and viral vectors based on these viruses.

[0116] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and change the present invention based on the description herein, and such modifications fall within the technical scope of the present invention.

[0117] The polymer porous membrane used in the following examples was a polyimide porous membrane, which was prepared by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic acid component, 3,3',4,4'-biphenyltetracarboxylic acid dianhydride (s-BPDA), and a diamine component, 4,4'-diaminodiphenyl ether (ODA), and a coloring precursor, polyacrylamide, and then heat-treating it at 250°C or higher. The obtained polyimide porous membrane was a three-layer polyimide porous membrane having surface layers A and B having multiple pores, and a macrovoid layer sandwiched between surface layers A and B. The average pore diameter of the pores in surface layer A was 18 μm, the average pore diameter of the pores in surface layer B was 31 μm, the film thickness was 25 μm, and the porosity was 75%.

[0118] Example 1: Hybridoma cell culture using a cell culture apparatus equipped with a semipermeable membrane 1. Cells and cell culture apparatus components

[0119] <Cells> [Hybridoma Cells] ・Hybridoma Mycl 9E10 (ECACC, Cat. No. 85102202)

[0120] <Culture medium and antimicrobial agent> ・Hybridoma-SFM (Thermo Fisher Scientific, Cat. No. 12045084) ・Gentamicin 10 mg / mL (Thermo Fisher Scientific, Cat. No. 15710064)

[0121] <Components> - Oxygen permeable membrane (Asahi Rubber Co., Ltd., Cat. No. ARFS-5030C, oxygen permeability 1,064,831cc / m ２• day atm (23℃) • Tubing (Saint-Gobain, Cat. No. SCFJ00003) • Tubing pump (Tokyo Rikakikai Co., Ltd., Cat. No. MP-3000) • Polyimide porous membrane (air permeability 50.6 cm) ３ / (cm ２ • s)) • Silicone sheet (culture chamber cambium, first channel forming layer, second channel forming layer) • Acrylic plate (first support, second support) • Semipermeable membrane (REPLIGEN, molecular weight cutoff 10,000, Cat. No. 132120)

[0122] The cell culture apparatus used in this Example 1 (corresponding to cell culture apparatus 1a in Figure 1B) is designed with the following dimensions. Unless otherwise specified, the cell culture apparatus C1 used in the reference example is of the same design.

[0123] - Culture chamber (corresponding to culture chamber 10): 30 mm long, 30 mm wide, 1.5 mm thick - Cell seeding port (corresponding to cell seeding port 120): 4 mm in diameter - Flow channels (corresponding to the first flow channel 20 and the second flow channel 30): 30 mm long, 30 mm wide, 1.5 mm thick - Culture medium supply port (corresponding to the first culture medium supply port 210 and the second culture medium supply port 310), culture medium outlet (corresponding to the first culture medium outlet 220 and the second culture medium outlet 320): 6.2 mm in diameter - Supports (corresponding to the first support 70 and the second support 71): As ventilation holes (700), 4 mm in diameter holes are formed in a 6 x 6 arrangement, and the distance between the centers of adjacent ventilation holes is 1.2 mm.

[0124] 2. Measurement method for cell culture device components

[0125] <Oxygen Permeability> 1) Equipment used: Gasperm-100 pressurized gas permeability meter manufactured by JASCO Corporation 2) Oxygen gas purity used: 99.5% or higher 3) Measurement conditions: Set temperature: 23°C, Set oxygen gas pressure: 1.0 atm 4) Method of calculating oxygen permeability: The value obtained by the measuring instrument at 23°C is converted to the amount of oxygen permeation (in CCs) per square meter of oxygen permeable cross-sectional area, per 24 hours of permeation time, and per atm. The result is defined as "cc / m²". ２ The temperature was expressed in units of "day atm (23°C)".

[0126] <Air permeability> 1) Equipment used: KES-F8 air permeability tester manufactured by Kato Tech Co., Ltd. 2) Measurement conditions: Cross-sectional area of ​​ventilation holes: 0.2π cm ２ Pressure difference 0.125 kPa 3) Method for calculating air permeability Based on JIS L1096 air permeability method A (Fragile method), the air permeability at a pressure difference of 0.125 kPa is converted from the air permeability resistance value obtained from the measuring instrument to a flow rate per unit area and per unit time, and this is used as the air permeability in "cm ３ / (cm ２ The units were expressed as "s" (s).

[0127] 3. Experimental Procedure

[0128] <Hybridoma Cell Acclimatization> Mycl 9E10, an immunoglobulin G (IgG)-producing hybridoma cell, was suspended in RPMI1640 medium supplemented with 10% FBS and gradually acclimatized to serum-free medium (Hybridoma-SFM) over 7 days by repeatedly subculturing the cells with dilution.

[0129] <Preparation of culture medium> Serum-free medium was prepared by adding 500 μL of Gentamicin (10 mg / mL) to 500 mL of Hybridoma-SFM. (Culture medium 1 (corresponding to "First Culture Medium 24" in Figure 1))

[0130] <Hybridoma Cell Culture Using a Cell Culture Apparatus> The cell culture apparatus was constructed by combining the apparatus with a tube pump that continuously delivers culture medium. Specifically, the tube pump was placed between the culture chamber and the culture medium supply port, and the flow rate of the culture medium was controlled by changing the pump rotation speed.

[0131] After filling channel 1 (a channel containing antibody-containing culture medium where the antibodies are concentrated: corresponding to "first channel 20" in Figure 1) and channel 2 (a channel without antibody-containing culture medium through which waste culture medium passes on the semipermeable membrane side: corresponding to "second channel 30" in Figure 1) with culture medium 1, the hybridoma cells acclimatized as described above (2.7 × 10⁶) were added. ６ Cells were suspended in 1 mL of medium 1 and seeded through the cell seeding port. 37°C, 5% CO2 ２ The samples were left undisturbed in an incubator for two days.

[0132] After standing, the tube pump connected to channel 1 was operated intermittently for 15 minutes per day at a rotation speed of 0.96 rpm. The tube pump connected to channel 2 was operated continuously at a rotation speed of 0.04 rpm, and the culture was carried out for 97 days. During this time, at intervals of 3 to 10 days, the culture medium collected from culture medium collection tanks 222 and 322 was centrifuged at 300 G for 5 minutes to separate the cells from the supernatant.

[0133] The number of cells was measured using an automated cell counter, Countess® II FL (Thermo Fisher Scientific). The results are shown in Tables 1 and 2.

[0134]

[0135]

[0136] As shown in Tables 1 and 2, it was found that the viability of living cells in the culture medium recovery tank 222 and culture medium recovery tank 322 was low. This indicates that the living cells in the culture chamber 10 did not flow out into the culture medium recovery tank 222 and culture medium recovery tank 322, but remained in the culture chamber.

[0137] The antibody concentration in the culture supernatant was measured using a Cedex® Bio culture medium component analyzer (Roche Diagnostics). The results are shown in Tables 3 and 4.

[0138]

[0139]

[0140] Reference Example 1 As Reference Example 1, the cell culture apparatus C1 shown in Figure 5 was used. The cell culture apparatus C1 in Figure 5 does not have the second channel and semipermeable membrane components corresponding to the cell culture apparatus 1a in Example 1. Hybridoma cells acclimatized in Example 1: 2.7 × 10 ６ Cells were suspended in 1 mL of medium 1 and seeded through the cell seeding port. 37°C, 5% CO2 ２ The samples were left undisturbed in an incubator for two days.

[0141] After standing, the tube pump connected to the flow path is rotated at 0.04 rpm (1 cm). ２The system was started at a flow rate of 0.7 mL / day, and the same culture medium 1 as in Example 1 was continuously supplied and incubated for 170 days.

[0142] The results of Example 1 and Reference Example 1 are shown in Figure 6.

[0143] 1, 1a Cell culture apparatus 10 Culture chamber 11 Culture chamber cambium 12 Cell seeding port 120 Cell seeding line 121 Cell supply means 13 Liquid 20 First channel 21 First culture medium supply port 210 First culture medium supply line 211 First supplied culture medium 212 First culture medium supply tank 22 First culture medium outlet 220 First culture medium discharge line 221 First discharged culture medium 222 First culture medium recovery tank 23 First channel cambium 24 First culture medium 30 Second channel 31 Second culture medium supply port 310 Second culture medium supply line 311 Second supplied culture medium 312 Second culture medium supply tank 32 Second culture medium outlet 320 Second culture medium discharge line 321 Second discharged culture medium 322 Second culture medium recovery tank 33 Second channel cambium 34 Second culture medium 40 Porous membrane 50 Semipermeable membrane 60 First oxygen permeable membrane 61 Second oxygen permeable membrane 70 First support 700 Ventilation holes 710 Ventilation holes 71 Second support 80, 81 Pump 90 Cells

[0144] C1 Cell culture apparatus C10 Culture chamber C11 Culture chamber cambium C12 Cell seeding port C120 Cell seeding line C20 Channel C21 Culture medium supply port C22 Culture medium outlet C23 Culture medium C24 Channel cambium C30, C31 Oxygen permeable membrane C40 Porous membrane C50 Pump C51 Culture medium supply line C52 Culture medium outlet line C60 Cells C70, C71 Support C700 Ventilation holes

Claims

1. A cell culture apparatus for culturing suspension cells, comprising: a culture chamber for culturing cells; a first channel having a first culture medium supply port and a first culture medium outlet, provided adjacent to a part or all of the outer surface of at least one side of the culture chamber; a second channel having a second culture medium supply port and a second culture medium outlet; a semipermeable membrane disposed between at least a part of the first channel and the second channel; and a seeding port provided in at least a part of the culture chamber.

2. The cell culture apparatus according to claim 1, wherein a porous membrane is provided between the first channel and the culture chamber.

3. The cell culture apparatus according to claim 1, wherein a first oxygen permeable membrane is provided on a part of the outside of at least one surface of the first channel and / or the culture chamber.

4. The cell culture apparatus according to claim 1, wherein a second oxygen permeable membrane is provided on part or all of the outer surface of at least one surface of the second flow channel.

5. The cell culture apparatus according to claim 1, wherein the thickness of the culture chamber is 0.05 to 5.0 mm.

6. The permeability of the porous membrane is 5 to 100 cm. ３ / (cm ２ The cell culture apparatus according to claim 2, wherein s) 7. The cell culture apparatus according to claim 1, wherein the fractional molecular weight of the semipermeable membrane is 100 kDa or less.

8. The cell culture apparatus according to claim 1, wherein the suspended cells are hybridomas.

9. A method for producing cells or useful components produced from cells, comprising seeding cells in the culture chamber of a cell culture apparatus described in claim 1, flowing a first culture medium through a first channel at a first flow rate, and culturing the cells while flowing a second culture medium through a second channel at a second flow rate, wherein the flow rate of the second culture medium is faster than the flow rate of the first culture medium.

10. The method according to claim 9, wherein the cell culture apparatus further comprises a porous membrane between the first channel and the culture chamber.

11. The porous membrane 1 cm ２ The method according to claim 10, wherein the culture is performed while flowing the first culture medium at a rate of 10 mL or less per day.

12. The method according to claim 9 or 10, comprising a first culture medium recovery tank provided downstream of the first flow path, and recovering the first culture medium by concentrating the useful components produced from the cells.

Images