Cell culture device and method

Abstract

A cell culture device according to the present disclosure comprises: a culture chamber for culturing suspension cells; a flow path having a culture medium supply port and a culture medium discharge port, and provided adjacent to part or all of the outer side of at least one surface of the culture chamber; an oxygen permeable membrane provided to at least part of the flow path and / or the culture chamber; and a cell seeding port provided to at least part 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, leading to cell death. Furthermore, repeated cell division led to numerous issues, such as excessive cell density and depletion of oxygen necessary for respiration. To solve these problems, devices have been developed that culture cells while perfusing the culture medium.

[0004] For example, Patent Document 1 discloses a culture apparatus for culturing cells under microgravity conditions such as outer space, and discloses that one end of the culture space is equipped with an oxygen-permeable membrane material.

[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. 2002-153256, Japanese Patent Publication No. Hei 5-38281, Japanese Patent Publication No. 2011-244713, International Publication No. 2016 / 121768

[0009] Conventional cell culture systems have problems supplying oxygen and nutrients when large numbers of cells are present, requiring frequent culture medium changes. This leads to instability in the culture system and the potential for cell death. There is a need for the development of a new cell culture system that provides easy oxygen supply to cells, minimizes damage to cells, and enables stable, long-term culture.

[0010] The inventors of the present invention have conducted diligent research to solve the above problems and have succeeded in completing a cell culture device that simplifies the configuration of the cell culture device itself and allows for long-term culture while maintaining cell activity, by configuring the device with a culture chamber for cell culture and a separate oxygen permeable membrane. In other words, although not limited thereto, the present invention includes the following embodiments.

[0011] [1] A cell culture apparatus comprising: a culture chamber for culturing suspension cells; a medium supply port and a medium discharge port; a flow channel provided adjacent to a part or all of the outer surface of at least one side of the culture chamber; an oxygen permeable membrane provided in the flow channel and / or at least a part of the culture chamber; and a cell seeding port provided in at least a part of the culture chamber. [2] The oxygen permeability of the oxygen permeable membrane is 10,000 to 10,000,000 cm². ３ / m ２ - A cell culture apparatus as described in item 1, wherein the temperature is 24 h·atm. [3] A cell culture apparatus as described in item 1 or 2, wherein the thickness of the culture chamber is 0.05 mm to 5.0 mm. [4] A cell culture apparatus as described in any one of items 1 to 3, wherein the flow channel has a porous membrane in a portion adjacent to the culture chamber, and when culture medium is continuously or intermittently flowed through the flow channel, the components of the liquid filling the culture chamber and the components of the culture medium are exchanged through the porous membrane. [5] A cell culture apparatus as described in any one of items 1 to 3, wherein the air permeability of the porous membrane is 0.1 to 100 cm. ３ / (cm ２- The cell culture apparatus described in item 4, wherein the oxygen permeable membrane is provided in both the channel and the culture chamber, according to any one of items 1 to 5. [7] The cell culture apparatus described in any one of items 1 to 6, wherein the suspended cells are hybridomas.

[0012] [8] A method for producing cells or useful components from cells, comprising seeding cells in the culture chamber of a cell culture apparatus described in any one of items 1 to 7, and culturing the cells while continuously or intermittently flowing the culture medium from the culture medium supply port to the culture medium outlet. [9] The method according to item 8, wherein the flow path has a porous membrane in a portion adjacent to the culture chamber.

[10] The porous membrane 1 cm ２ The method according to item 9, wherein the culture medium is flowed at a rate of 0.1 mL to 10 mL / day per cell.

[11] The method according to item 8 or 9, wherein the cell culture apparatus is rotated and then the cells are cultured further.

[12] The method according to any one of items 8 to 11, wherein the cells are cultured for 30 days or more to continuously produce useful components.

[0013] According to this disclosure, even when a large number of cells are aggregated, sufficient oxygen and nutrient supply can be provided, enabling a stable culture system. Furthermore, a new cell culture device and culture method using the same can be provided, which minimizes damage to cells and enables stable, long-term culture.

[0014] This is a schematic diagram showing a cross-section of the cell culture apparatus 1 of the present invention in one embodiment. This is a schematic diagram showing a cross-section of the cell culture apparatus 1a of the present invention in one embodiment. This is a schematic diagram (top view) showing each component constituting the cell culture apparatus 1a of the present invention in one embodiment. This is a schematic diagram (perspective view) showing each component constituting the cell culture apparatus 1a of the present invention spread out vertically to explain each component constituting the cell culture apparatus 1a of the present invention in one embodiment. This is a schematic diagram showing an example of how the cell culture apparatus 1 or 1a of the present invention is used in one embodiment. This is a diagram for explaining another way in which the cell culture apparatus 1a of the present invention is used. (A) Type A culture. (B) Type B culture. This is a graph (Examples 1 to 4) showing the antibody concentration obtained from cells when cultured using the cell culture apparatus (1, 1a) of the present invention. This is a schematic diagram showing a cross-section of the cell culture apparatus 1c without ventilation holes 700 used in Reference Example 5. This is a graph showing a comparison of the amount of antibody produced per unit of culture medium of hybridomas cultured using each cell culture apparatus used in Examples 1 to 4 and Reference Example 1.

[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 1 and 2 show schematic diagrams of cell culture apparatuses 1 and 1a in one embodiment. Note that cell culture apparatus 1 (Figure 1) has the same configuration as cell culture apparatus 1a (Figure 2), except that it does not have a porous membrane 40 between the culture chamber 10 and the flow channel 20, so it will be described in common here.

[0018] Figure 3 shows a schematic diagram (top view) of each component constituting the cell culture apparatus 1a of the present invention in one embodiment. Figure 4 is a schematic diagram (perspective view) showing the components shown in Figure 3 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 the cell culture apparatus 1 (and 1a and 1b) 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 present disclosure provides a cell culture apparatus 1 (Figure 1) comprising: a culture chamber 10 for culturing cells; a flow channel 20 having a culture medium supply port 21 and a 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; an oxygen permeable membrane (30, 31) provided in the flow channel 20 and / or at least a part of the culture chamber 10; and a cell seeding port 12 provided in at least a part of the culture chamber.

[0021] In another embodiment, the present disclosure provides a cell culture apparatus 1a (Figure 2) in which, in addition to the configuration of the cell culture apparatus 1 described above, the flow channel 20 has a porous membrane 40 in a portion adjacent to the culture chamber 10, and when the culture medium 23 is continuously or intermittently flowed through the flow channel 20, the components of the liquid 13 filling the culture chamber 10 and the components of the culture medium 23 are exchanged via the porous membrane 40.

[0022] Cell culture apparatuses 1 and 1a include a culture chamber 10 for culturing cells 60, a flow channel 20 having a culture medium supply port 21 and a 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, an oxygen permeable membrane (30, 31) provided in the flow channel 20 and / or at least a part of the culture chamber 10, and a cell seeding port 12 provided in at least a part of the culture chamber. In one embodiment, the oxygen permeable membrane (30, 31) may be provided in both the flow channel 20 and the culture chamber 10. The flow channel 20 may have a porous membrane 40 in the part adjacent to the culture chamber 10, and when culture medium 23 is continuously or intermittently flowed through the flow channel 20, the components of the liquid 13 filling the culture chamber 10 and the components of the culture medium 23 are exchanged via the porous membrane 40. Even in cases where the porous membrane 40 is not present, as in the cell culture apparatus 1, when the culture medium 23 is continuously or intermittently flowed through the flow path 20, the components of the liquid 13 filling the culture chamber 10 and the components of the culture medium 23 are exchanged. However, it is preferable to flow the culture medium 23 at a flow rate such that the cells 60 do not flow out from the culture medium outlet 22. Since the cell seeding port 12 is provided in the cell culture apparatus 1 and 1a, for example, when culturing over a long period, a cell suspension containing cells 60 can be added to the culture chamber 10 at any time.

[0023] The culture chamber 10 only needs to have enough space to accommodate the liquid 13 and to allow for the cultivation of cells 60. 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 culture medium 23 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 60. On the other hand, for example, if the thickness of the culture chamber 10 is smaller (for example, about 0.05 to 2.00 mm), the efficiency of component exchange from the flow path and the gas exchange efficiency from the oxygen permeable membrane 30 will be higher.

[0024] In one aspect, the culture chamber 10 may have a cell seeding port 12 communicating with the culture chamber 10, and further, 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 device capable of supplying a fluid such as a syringe, a pipette, or a pump. The cell supply means 121 may be optionally detachable from the first cell seeding line 120. For example, a valve is provided at the end of the cell seeding line 120, and when the cell supply means 121 is not connected, the end of the cell seeding line 120 may be closed by the valve.

[0025] In another aspect, the culture chamber 10 may be an open-system cell culture device 1 or 1a that assembles the cell culture device 1 or 1a as shown in FIG. 4 after seeding the cells 60 in a sterile space. However, it is preferably a closed-system cell culture device 1 or 1a that has already been formed in the shape of FIG. 4 before use.

[0026] The culture medium 23 may be appropriately selected according to the type of cells 60 to be cultured, but a medium having a buffering effect is preferred. The conditions for culturing the cells 60 can be appropriately determined according to the type of cells 60 and the like. Culture methods suitable for each of animal cells, plant cells, insect cells, and bacteria are known, and the culture medium 23 can also be appropriately prepared according to the type of cells 60.

[0027] Suspension cells refer to cells that can contact the culture vessel during the culture process, have no scaffold-dependence, and the cells themselves grow without extending pseudopods and adhering to the culture vessel. The suspension cells may be spheroids formed by aggregation and self-organization of suspension cells. The suspension cells may be cells that originally have no adhesiveness (such as blood cell line cells, leukemia cells, etc.), or cells that originally had adhesiveness but have weakened adhesiveness or lost their adhesiveness during the process of collection, experiment, or lesion (such as cells separated and collected from animals, cancer cells in a metastatic state, etc.).

[0028] In this specification, in addition to the above floating cells, cells that were originally adherent cells but have been artificially adapted to a floating suspension state (such as stem cells), and are in a state where they do not depend on adhesion in terms of proliferation and metabolism, may also be used. For example, cells such as CHO cells, HEK293 cells, and hybridoma cells are exemplified, but are not limited thereto. As the floating cells used in the present invention, preferably, CHO cells, HEK293 cells, Sf9 cells, mesenchymal stem cells, iPS cells, iPS-derived cells, hybridoma cells, and NS0 cells are used, more preferably, CHO cells, HEK293 cells, mesenchymal stem cells, and hybridoma cells, and even more preferably, CHO cells and hybridoma cells.

[0029] The cells are not particularly limited as long as they can express the desired substance. The substance may be naturally expressed in the cells, or may be transformed by genetic engineering techniques to produce the substance. Preferably, the cells are transformed by genetic engineering techniques so as to express the substance. Suitable methods for the transformation of animal cells, plant cells, insect cells, and bacteria are known, respectively. (For example, refer to 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 cell culture methods and culture media for animal cells are described, for example, in the Lonza Cell Culture Media Catalog. The culture medium 23 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 60 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 60 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 60 to be cultured. These media may have serum added to them, or they may be serum-free media. It is preferable that the medium 23 and the liquid 13 are of the same type.

[0032] The shape of the inside of the culture chamber 10 is not particularly limited as long as it has enough space to culture cells 60 and fill the liquid 13. For example, it may be a prism (polygonal prism) such as a triangular prism, square prism, pentagonal prism, or hexagonal prism, or it may be a cylinder, and is not limited. Considering the efficiency of component exchange between the culture chamber 10 and the flow channel 20, it is preferable that the area in contact with the flow channel 20 is large.

[0033] The culture chamber 10 preferably has a culture surface on at least one side on which cells 60 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 flow path 20 is connected to a culture medium supply port 21 and a culture medium outlet 22. Culture medium 23 is supplied from a culture medium supply line 51 connected to the culture medium supply port 21 and discharged to a culture medium outlet line 52 connected to the culture medium outlet 22. A pump 50 is provided in the middle of the culture medium supply line 51. The pump 50 may be, for example, a tubular pump (peristallic pump), a piezo pump, or a syringe pump; any pump capable of dispensing fluid can be used. The pump 50 may also be connected to the culture medium outlet line 52, and for example, the culture medium 23 may be discharged by creating negative pressure in the culture medium outlet line 52.

[0035] The culture medium supply port 21 and culture medium outlet 22 may be located at the top or side of the channel 20, as shown in Figures 1 and 2. The thickness of the channel 20 is not particularly limited, but for example, it may be 0.5 to 5.0 mm in the direction perpendicular to the surface on which the cell culture apparatus of the present invention is installed. In one embodiment, the channel 20 has a 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 culture medium 23 are efficiently exchanged through the porous membrane 40, playing a role in maintaining the environment inside the culture chamber 10 in a state suitable for culturing cells 60. In addition, the porous membrane 40 prevents living cells from flowing out into the channel 20. In addition, dead cells can pass through the porous membrane 40 and flow out into the channel 20.

[0036] Living and dead cells can be defined, for example, using Countess® II FL (Thermo Fisher Scientific). Furthermore, cell viability can be calculated according to the following formula ("Formula 1").

[0037] The shape of the 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 culture medium 23 to flow from the culture medium supply port 21 to the culture medium outlet 22 at a constant flow velocity and / or pressure. For example, as shown in "(3)" of Figure 3, the flow path forming layer 24 that forms the flow path 20 may have an opening that is approximately square or approximately circular in the part that forms the flow path 20, and the part where the culture medium supply port 21 and the culture medium outlet 22, which are in point-symmetric positions, are connected may also be open. In addition, although not shown, the culture medium supply port 21 and the culture medium outlet 22 may have openings in line-symmetric positions.

[0038] Furthermore, the flow path 20 and the culture chamber 10 may be sandwiched between a first support 70 and a second support 71, as shown in Figures 1 and 2. The first support 70 and the second support 71 may be fixed together, for example, by male and female screws. The first support 70 and the second support 71 may be provided with ventilation holes 700 for exposing the first oxygen permeable membrane 30 and the second oxygen permeable membrane 31 to air. Multiple ventilation holes 700 may be provided, as shown in Figures 1 and 2, or only one may be provided per support 70.

[0039] The porous membrane 40 is a membrane (film) having numerous small voids inside or on its surface. 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 0.1 to 100 cm. ３ / (cm ２ It is preferable that the material is a filtration membrane, nonwoven fabric (for example, the nonwoven fabric described in Japanese Patent Application Publication No. 2019-126342), or polymer porous membrane. The degree of air permeability can be determined, for example, by measuring it using the JIS L1096 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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").

[0044]

[0045] (In the formula, Sa represents the average value of the pore area.)

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In this specification, the thickness of a polymer porous membrane can be measured using a contact-type thickness gauge.

[0052] 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.

[0053] 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").

[0054]

[0055] (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.)

[0056] 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.

[0057] 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.

[0058] <<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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Any diamine can be used in the production of the above-mentioned porous polyimide membrane. Specific examples of diamines include the following:

[0074] 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.

[0075] These can be used individually or in mixtures of two or more. The diamines used can be appropriately selected according to the desired properties.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] (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.

[0080] 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.

[0081] 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.

[0082] <<Polyethersulfone (PES) porous membrane>>

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.).

[0087] 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.

[0088] 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.

[0089] 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.

[0090] The 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 the surface layer A and the surface layer B, wherein the macrovoid layer has partitions bonded to the surface layers A and B, and a plurality of macrovoids having an average pore diameter in the membrane plane direction of 10 μm to 500 μm surrounded by the partitions and the surface layers A and B. The partitions of the macrovoid layer have a thickness of 0.1 μm to 50 μm, the surface layers A and B each have a thickness of 0.1 μm to 50 μm, one of the surface layers A and B has a plurality of pores with an average pore diameter exceeding 5 μm and not exceeding 200 μm, and the other has a plurality of pores with an average pore diameter of 0.01 μm or more and less than 200 μm. The surface opening rate of one of the surface layers A and B is 15% or more, and the surface opening rate of the other surface layer is 10% or more. The pores of the surface layers A and B communicate with the macrovoids. The PES porous membrane has a total membrane thickness of 5 μm to 500 μm and a porosity of 50% to 95%.

[0091] <<Filter membrane>> The filter membrane as a porous membrane that can be used in the present invention should not affect cell culture and can prevent the outflow of the cultured cells. Preferably, the air permeability is 0.1 to 100 cm ３ / (cm ２ · s).

[0092] Examples of the filter membrane include a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, etc. Preferably, a microfiltration membrane is preferably used. As an example of the microfiltration membrane, a polytetrafluoroethylene (PTFE) membrane can be mentioned. For example, JMWP09025 of Millipore may be used.

[0093] <<Non-woven fabric>> The non-woven fabric as a porous membrane that can be used in the present invention should not affect cell culture and can prevent the outflow of the cultured cells. Preferably, the air permeability is 5 to 100 cm ３ / (cm ２ · s). For example, the non-woven fabric described in JP-A-2019-126342 may be used.

[0094] The nonwoven fabric to which the present invention can be applied is composed of one or more types of core-sheath type composite fibers relative to the weight of the nonwoven fabric. When the fibers are fused together to form a nonwoven fabric, it is preferable that the core-sheath type composite fibers make up 20% by weight or more of the nonwoven fabric. The content of the core-sheath type composite fibers may be 30% by weight or more, 40% by weight or more, or 50% by weight or more.

[0095] The core and sheath of the core-sheath composite fiber are preferably made of a polyolefin polymer. Examples of polyolefin polymers constituting the core and sheath include aliphatic α-monoolefins having 2 to 16 carbon atoms, such as homopolyolefins or copolymerized polyolefins of ethylene, propylene, 1-butene, 1-pentene, 3-methyl-1-butene, 1-hexene, 1-octene, 1-dodecene, and 1-octadecene. The aliphatic α-monoolefin may be copolymerized with other olefins and / or small amounts (up to about 10% by weight of the polymer) of other ethylene-based unsaturated monomers, such as butadiene, isoprene, pentadiene-1,3, styrene, and α-methylstyrene.

[0096] When combining the polymer constituting the core and the polymer constituting the sheath, it is preferable to combine them such that the melting point of the polymer in the sheath is lower than the melting point of the polymer in the core. For example, polymers with a melting point difference of 20 to 50°C are selected from the above-mentioned polymers. As will be described later, it is even more preferable to select a polyolefin polymer constituting the sheath whose melting point is, for example, 40°C or more lower than the melting point of the polyester polymer constituting the core. Core-sheath type composite fibers having the above properties are not limited to those having a combination in which the polymer in the sheath is polyethylene and the polymer in the core is polypropylene.

[0097] The core-sheath type composite fiber (nonwoven fabric) used in the present invention can be manufactured using a general method for manufacturing core-sheath type composite fiber (nonwoven fabric). Typically, such a manufacturing method may include, but is not limited to, the following steps: 1) Using a composite spinning apparatus equipped with two unscrew extruders and a composite spinning nozzle, spinning a high-melting-point component (e.g., polypropylene):low-melting-point component (e.g., polyethylene) in a desired mass ratio at a spinning temperature of 280°C to produce undrawn fibers; 2) Gathering the undrawn fibers and stretching them with a heatable stretching roller to produce drawn fibers, for example, with a stretching ratio of 4 times or more and a desired single-fiber fineness; 3) Producing short fibers of a desired fiber length (e.g., several mm) with a rotary cutter; and 4) Using the short fibers above, producing a nonwoven fabric using a general wet or dry process.

[0098] In the wet manufacturing method of step 4) described above, more specifically, short fibers are uniformly dispersed in water to which a viscosity modifier has been added to prepare a dispersion. Next, this dispersion is made into paper on a mesh to produce a wet web. The obtained wet web is then sandwiched between two rubber heating plates and dried by pressing it for 1 minute at a temperature of 140°C using a general-purpose heating and pressing machine. This melts the low-melting-point components of the short fibers, causing the short fibers to bond together and obtain a nonwoven fabric. In this invention, for example, but not limited to, the composite fiber "Airimo" manufactured by Ube Eximo Co., Ltd. can be used.

[0099] The fibers constituting the nonwoven fabric are integrated by having heat-sealed portions (also referred to as "bonding portions" in this specification) to maintain their shape as a nonwoven fabric. The heat-sealed portions are formed by heating to a temperature above the melting point of the sheath component and below the melting point of the core component. In the heat-sealed portions, the sheath component melts or softens and contributes to adhesion, but the core component does not completely melt due to the heat, maintaining its fiber shape and contributing to improving the tear strength of the nonwoven fabric. Therefore, in order to melt or soften the sheath component in the heat-sealed portions while maintaining the fiber shape of the core component, a difference of, for example, 30°C or more in the melting points of the two can be established. This allows the core component to be unaffected by heat during the heat-sealing process, while the sheath component is reliably melted and fixed by fusion at the heat-sealed portions.

[0100] The "fineness" of the core-sheath composite fibers that constitute the nonwoven fabric is generally used to represent the thickness of the fiber and is expressed as the weight (g) per 10,000 meters of fiber (i.e., decitex (dTex)). The fineness of the core-sheath composite fibers (nonwoven fabric) used in the present invention is not limited to any fineness suitable for cell culture, but is preferably 0.05 to 2.2 decitex.

[0101] Furthermore, the core-sheath composite ratio (mass ratio) is preferably sheath portion / core portion = 20 / 80 to 50 / 50. By making the ratio of the core portion equal to or greater than that of the sheath portion, excellent mechanical properties are achieved and practical strength can be maintained. However, if the ratio of the core portion exceeds 80% by mass, the ratio of the sheath portion, which is the adhesive component, becomes smaller, so the adhesive strength at the heat-sealed portion tends to decrease. Therefore, the upper limit of the core portion ratio is preferably 80% by mass.

[0102] The fiber diameter of the core-sheath type composite fiber can be any diameter suitable for cell culture, and is determined by the fineness and specific gravity of the composite fiber, but is preferably 1 to 20 μm, more preferably 2 to 10 μm, and even more preferably 3 to 8 μm.

[0103] The basis weight of the nonwoven fabric is 1 to 50 g / m². ２ It is acceptable if the amount is such that, for example, 3 to 50 g / m2 is preferable.

[0104] The nonwoven fabric used in the present invention may be composed of two or more core-sheath composite fibers with different fineness, sheath-core ratio, and / or fiber diameters. For example, the different finenesses may consist of coarse fineness fibers with a fineness of 1 to 2.2 decitex and fine fineness fibers with a fineness of 0.05 to 0.5 decitex. In the present invention, the mass ratio of coarse fineness fibers to fine fineness fibers is preferably coarse fineness fibers / fine fineness fibers = 20 / 80 to 80 / 20. The coarse fineness fibers and fine fineness fibers may be mixed in the nonwoven fabric in a blended state, or they may exist in a laminated state where a web is formed by depositing coarse fineness fibers on one side and a web is formed by depositing fine fineness fibers on the other side.

[0105] As described above, the fibers constituting the nonwoven fabric are heat-bonded and fixed together by heat-sealed joints. In the heat-sealed joints, the sheath component of the core-sheath composite fiber melts and solidifies to form a heat-bonded joint that functions as an adhesive component. Alternatively, a number of scattered heat-sealed joints can be formed by heat embossing or ultrasonic welding. The shape of each heat-sealed joint can be any shape, such as circular, elliptical, rhombus, triangular, woven pattern, grid pattern, etc. The area of ​​each heat-sealed joint is 0.2 to 3 mm². ２ This degree is preferable. Furthermore, the area ratio of the heat-sealed portion to the area of ​​the nonwoven fabric is preferably 10% to 30%. If it is less than 10%, it is difficult to achieve both the excellent mechanical properties and rigidity that are the objectives of the present invention.

[0106] The nonwoven fabric used in the present invention may be a nonwoven fabric that has been treated to be hydrophilic. The hydrophilization of the nonwoven fabric is not limited to but can be performed by fluorine gas treatment, atmospheric pressure plasma treatment, vacuum plasma treatment, corona treatment, graft polymerization treatment of hydrophilic monomers, sulfonation treatment, or surfactant application treatment, with fluorine gas treatment and atmospheric pressure plasma treatment being preferred.

[0107] The total film thickness of the nonwoven fabric used in the present invention 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 25 to 75 μm.

[0108] The nonwoven fabric used in the present invention is preferably sterilized. The sterilization treatment is not particularly limited, but examples include gas sterilization, sterilization with disinfectants such as ethanol, and any other sterilization treatment such as electromagnetic wave sterilization such as ultraviolet light or gamma rays.

[0109] In one embodiment, the cell culture apparatus 1a may be provided as a device formed by stacking the components shown in Figure 3, namely (1) the second support 71, (2) the second oxygen permeable membrane 31, (3) the channel forming layer 24, (4) the porous membrane 40, (5) the culture chamber forming layer 11, (6) the first oxygen permeable membrane 30, and (7) the first support 70, in the order from the bottom layer (7) 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 3 in the reverse order from the bottom layer, from (1) to (7). 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 (7) is individually packaged.

[0110] (1) The materials of the members forming the second support 71 and (7) the first support 70 are 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.

[0111] (3) The material of the members forming the channel forming layer 24 and (5) 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 members forming the channel forming layer 24 and (5) 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.

[0112] In one embodiment, in order to increase the number of cells 60, the cell culture apparatus 1 and / or cell culture apparatus 1a may increase the area or volume of the culture chamber 10, or the area or volume of the culture chamber 10 may be increased by connecting multiple cell culture apparatuses 1 and / or cell culture apparatuses 1a in parallel. In the latter case, for example, a culture medium supply line 51 connected to one culture medium supply tank 510 may branch off midway and be connected in parallel to a culture medium supply port 21. Also, a culture medium discharge line 52 connected in parallel to a culture medium discharge port 22 may merge midway and be connected to one culture medium recovery tank 520.

[0113] The first oxygen permeable membrane 30 and the second oxygen permeable membrane 31 are formed of membranes that allow oxygen molecules to pass through but do not allow larger molecules to pass through, including physiologically active substances secreted by cells 60, foreign substances from outside. For example, the first oxygen permeable membrane 30 and the second oxygen permeable membrane 31 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 30 and the second oxygen permeable membrane 31 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 30 and the second oxygen permeable membrane 31 even when in contact with moisture.

[0114] <Method for producing useful components from cells or cells>

[0115] In one embodiment, the present invention provides a method for producing cells or useful components from cells, comprising seeding cells in the culture chamber of a cell culture apparatus and culturing them while continuously or intermittently flowing a culture medium from a culture medium supply port to a culture medium outlet. By using the above-described cell culture apparatus 1, even when a large number of cells are gathered, sufficient oxygen and nutrient supply can be provided, making it possible to provide a stable culture system. Furthermore, damage to cells is minimized, and stable long-term culture is possible.

[0116] In one embodiment, the cell culture apparatus 1 and / or cell culture apparatus 1a described above have a porous membrane (40) 1 cm ２ The present invention provides a method of culturing while flowing medium 23 at a rate of 0.1 to 10 mL / day. The flow rate of medium 23 can be appropriately adjusted by adjusting the operating speed of the pump 50.

[0117] In one embodiment, the present invention provides a method for further culturing cells after rotating the cell culture apparatus 1a relative to the planes of the first and second oxygen permeable membranes (30, 31) (for example, by about 180°, but not limited to that) (Figure 6). The porous membrane 40 prevents living cells from flowing out into the channel 20. After culturing the cell culture apparatus 1a for any period of time, it may be rotated by about 180° relative to the planes of the oxygen permeable membranes (30, 31). For example, it may be rotated by 180° relative to the planes of the oxygen permeable membranes (30, 31) from type A (Figure 6(A)) to type B (Figure 6(B)) in Figure 3, or vice versa. This promotes cell proliferation and / or the production of useful components from the cells.

[0118] In one embodiment, the method of the present invention provides a method for culturing cells 60 for 30 days or more using cell culture apparatus 1 and / or cell culture apparatus 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.

[0119] In one embodiment, the amount of useful components obtained in the present invention can be determined according to the following formula ("Formula 4").

[0120] 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.

[0121] Examples of glycoproteins include collagen, fibronectin, and hyaluronic acid.

[0122] Examples of viruses include influenza viruses, adenoviruses, lentiviruses, herpesviruses, and viral vectors based on these viruses.

[0123] 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.

[0124] 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%.

[0125] Example 1: Hybridoma cell culture using a cell culture device equipped with a polyimide porous membrane

[0126] 1. Cells and cell culture device components

[0127] <Cells> [Hybridoma Cells] ・Hybridoma Mycl 9E10 (ECACC, Cat. No. 85102202)

[0128] <Culture medium and antimicrobial agent> ・Hybridoma-SFM (Thermo Fisher Scientific, Cat. No. 12045084) ・Gentamicin 10 mg / mL (Thermo Fisher Scientific, Cat. No. 15710064)

[0129] <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 (used for creating channel cambium and culture chamber cambium)

[0130] The cell culture apparatus used in Example 1 (corresponding to cell culture apparatus 1a in Figure 2) is designed with the following dimensions. Unless otherwise specified, the cell culture apparatuses used in Examples 2-4 and Reference Example 1 were of the same design.

[0131] - 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 12): 4 mm in diameter - Channel (corresponding to channel 20): 30 mm long, 30 mm wide, 1.5 mm thick - Culture medium supply port (corresponding to culture medium supply port 21), culture medium outlet (corresponding to culture medium outlet 22): 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.

[0132] 2. Measurement method for cell culture device components

[0133] <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)". <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).

[0134] 3. Experimental Procedure

[0135] <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.

[0136] <Preparation of culture medium> Serum-free medium was prepared by adding 500 μL of Gentamicin (10 mg / mL) to 500 mL of Hybridoma-SFM (hereinafter referred to as "Culture Medium A").

[0137] <Culturing Hybridoma Cells Using a Cell Culture Apparatus> The cell culture apparatus is used in combination with a pump that continuously delivers culture medium. Specifically, the pump is placed between the culture medium supply tank and the culture medium supply port, and the flow rate of the culture medium can be controlled by changing the pump flow rate setting.

[0138] After filling the cell culture device with culture medium A, add the hybridoma cells acclimatized as described above (2.7 × 10⁶). ６ Cells was suspended in 1 mL of medium A and seeded through the cell seeding port. 37°C, 5% CO2 ２ The samples were left undisturbed in an incubator for two days.

[0139] After standing, rotate the tube pump at 0.04 rpm (1 cm) ２ The cell culture apparatus was started at a flow rate of 0.7 mL / day, and culture medium A was continuously supplied for 77 days. In addition, on the 77th day, the cell culture apparatus was inverted (see Figure 6), and the cells were cultured for a total of 170 days from seeding. During this time, the culture medium was collected from the medium recovery tank 520 every 3 to 7 days, and the cells and culture supernatant were separated by centrifugation at 300 G for 5 minutes.

[0140] The IgG concentration in the culture supernatant was measured using a CedexBio culture medium component analyzer (Roche Diagnostics). The results are shown in Table 1.

[0141]

[0142] From the fourth day after cell seeding, the IgG concentration gradually increased, and a stable IgG level of 60-80 mg / L was obtained, with a temporary high concentration of 120 mg / L being achieved.

[0143] Example 2: Hybridoma cell culture using a cell culture device equipped with a filtration membrane

[0144] <Experimental Procedure> The porous membrane (40) of the cell culture apparatus (1a) prepared in Example 1 was used as a filtration membrane (Millipore, JMWP09025, air permeability 0.5 cm) ３ / (cm ２ - To a value less than s, insert the pump into a syringe pump (AS ONE Corporation, IC-3001, 1 cm) ２ The same cell culture apparatus as in Example 1 was used, except that the flow rate per unit was changed to 0.7 mL / day. Hybridoma cells (2.7 × 10⁶ cells) that had been acclimatized under the same culture conditions as in Example 1 were introduced into the apparatus. ６Cells were seeded. On day 36, the cell culture apparatus was inverted, and the cells were cultured for a total of 64 days from seeding. Every 7 days, the culture medium was collected from the culture medium collection tank 520 and separated into cells and culture supernatant by centrifugation at 300G for 5 minutes.

[0145] The number of cells was measured using an automated cell counter, Countess® II FL (Thermo Fisher Scientific). The results are shown in Table 2.

[0146]

[0147] As shown in Table 2, the amount of live cells flowing into the culture medium recovery tank 520 did not change before and after the inversion of the cell culture apparatus, confirming that almost no live cells were flowing out.

[0148] The IgG concentration in the culture supernatant was measured using the Cedex® Bio culture medium component analyzer (Roche Diagnostics). The results are shown in Table 3.

[0149]

[0150] IgG levels were high, around 100 mg / L, within 19 days of cell seeding. However, after the cell culture system was reversed on day 36, the concentration gradually decreased and eventually fell below the lower limit of detection.

[0151] Example 3: Hybridoma cell culture using a cell culture device with nonwoven fabric.

[0152] <Experimental Procedure> The porous membrane (40) of the cell culture apparatus (1a) shown in Figure 2 is made of nonwoven fabric (PP (polypropylene):PE (polyethylene) = 50:50 composition polyolefin nonwoven fabric, basis weight 20 g / m²). ２ Hydrophilic treatment with fluorine gas, air permeability 33.6 cm ３ / (cm ２ ・s)) to a syringe pump (AS ONE Corporation, IC-3001, 1cm ２ The same cell culture apparatus as in Example 1 was used, except that the flow rate per unit was changed to 0.8 mL / day. Hybridoma cells (2.7 × 10⁶ cells) that had been acclimatized under the same culture conditions as in Example 1 were introduced into the apparatus. ６Cells were seeded. On day 36, the cell culture apparatus was inverted, and the cells were cultured for a total of 64 days from seeding. Every 7 days, the culture medium was collected from the culture medium collection tank 520 and separated into cells and culture supernatant by centrifugation at 300G for 5 minutes.

[0153] The number of cells was measured using an automated cell counter, Countess® II FL (Thermo Fisher Scientific). The results are shown in Table 4.

[0154]

[0155] As shown in Table 4, very few living cells leaked out after the cell culture apparatus was inverted, but it was confirmed that a large number of dead cells leaked out after the inversion.

[0156] The IgG concentration in the culture supernatant was measured using a CedexBio® (Roche Diagnostics) culture medium component analyzer. The results are shown in Table 5.

[0157]

[0158] IgG levels were high, around 100 mg / L, for up to 19 days after cell seeding. After the cell culture system was reversed, the IgG concentration gradually decreased along with cell outflow, but then increased to 60 mg / L.

[0159] Example 4: Hybridoma cell culture using a cell culture apparatus without a porous membrane

[0160] <Experimental Procedure> Unlike cell culture apparatus 1 in Figure 1, a porous membrane is not used, and a syringe pump (AS ONE Corporation, IC-3001, 1 cm) is used. ２ The same cell culture apparatus as in Example 1 was used, except that the flow rate per unit was changed to 0.8 mL / day. Hybridoma cells (2.7 × 10⁶ cells) that had been acclimatized under the same culture conditions as in Example 1 were introduced into the apparatus. ６ Cells were seeded. On day 36, the cell culture apparatus was inverted, and the cells were cultured for a total of 64 days from seeding. Every 7 days, the culture medium was collected from the culture medium collection tank 520 and separated into cells and culture supernatant by centrifugation at 300G for 5 minutes.

[0161] The number of cells was measured using an automated cell counter, Countess® II FL (Thermo Fisher Scientific). The results are shown in Table 6.

[0162]

[0163] As shown in Table 6, before reversing the cell culture device, almost no cells leaked out, but after reversing the cell culture device, it was confirmed that a large number of dead cells leaked out.

[0164] The IgG concentration in the culture supernatant was measured using the Cedex® Bio culture medium component analyzer (Roche Diagnostics). The results are shown in Table 7.

[0165]

[0166] IgG levels were high, around 120 mg / L, for up to 19 days after cell seeding. After the cell culture system was reversed, the IgG concentration gradually decreased along with cell outflow, but then increased to 95 mg / L.

[0167] Reference Example 1: Hybridoma cell culture using a continuous culture device without ventilation holes

[0168] <Experimental Procedure> Using the cell culture apparatus (1c) (Figure 8), hybridoma cells (2.7 × 10) were further acclimatized under the same culture conditions as in Example 1. ６ Cells were sown. Note that 22 days after sowing, the acclimatized hybridoma cells were 2.7 × 10⁶. ６ Cells were added to the cell culture device and seeded. The cell culture device was not inverted, and the cells were cultured for 50 days from seeding. The culture supernatant was collected from the culture medium collection tank 520 every 3 to 7 days, and the culture medium was separated into cells and culture supernatant by centrifugation at 300G for 5 minutes.

[0169] The number of cells was measured using an automated cell counter, Countess® II FL (Thermo Fisher Scientific). The results are shown in Table 8.

[0170]

[0171] As shown in Table 8, very few live or dead cells leaked out of the cell culture apparatus.

[0172] The IgG concentration in the culture supernatant was measured using a Cedex® Bio culture medium component analyzer (Roche Diagnostics). The results are shown in Table 9.

[0173]

[0174] IgG levels were not produced immediately after the start of cell culture, and this did not improve even after additional cells were seeded on day 22.

[0175] Figure 9 shows the IgG concentration in the supernatant obtained from the cultures of Examples 1-4 and Reference Example 1. The amount of IgG produced per unit of culture medium was calculated by dividing the cumulative antibody amount by the total amount of culture medium recovered. When a culture apparatus equipped with an oxygen permeable membrane was used, a higher concentration of IgG was produced compared to when no such membrane was used.

[0176] 1, 1a, 1c 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 Flow channel 21 Medium supply port 22 Medium discharge port 23 Medium 24 Flow channel cambium 30 First oxygen permeable membrane 31 Second oxygen permeable membrane 40 Porous membrane 50 Pump 51 Medium supply line 510 Medium supply tank 511 Supply medium 52 Medium discharge line 520 Medium recovery tank 521 Discharged medium 60 Cells 70 First support 700 Ventilation holes 71 Second support

Claims

1. A cell culture apparatus for culturing suspension cells, comprising: a culture chamber for culturing cells; a channel having a culture medium supply port and a culture medium discharge port, and provided adjacent to a part or all of the outer surface of at least one side of the culture chamber; an oxygen permeable membrane provided in the channel and / or at least a part of the culture chamber; and a cell seeding port provided in at least a part of the culture chamber.

2. The oxygen permeability of the oxygen permeable membrane is 10,000 to 10,000,000 cm². ３ / m ２ The cell culture apparatus according to claim 1, wherein the temperature is 24 h·atm.

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

4. The cell culture apparatus according to claim 1, wherein the channel has a porous membrane in a portion adjacent to the culture chamber, and when culture medium is continuously or intermittently flowed through the channel, the components of the liquid filling the culture chamber and the components of the culture medium are exchanged through the porous membrane.

5. The permeability of the porous membrane is 0.1 to 100 cm. ３ / (cm ２ The cell culture apparatus according to claim 4, wherein s) 6. The cell culture apparatus according to claim 1, wherein the oxygen permeable membrane is provided in both the channel and the culture chamber.

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

8. A method for producing cells or useful components from cells, comprising seeding cells in the culture chamber of the cell culture apparatus described in claim 1, and culturing the cells while continuously or intermittently flowing a culture medium from the culture medium supply port to the culture medium outlet.

9. The method according to claim 8, wherein the channel has a porous membrane in a portion adjacent to the culture chamber.

10. The porous membrane 1 cm ２ The method according to claim 9, wherein the culture is performed while flowing the culture medium at a rate of 0.1 mL to 10 mL per day.

11. The method according to claim 9, wherein the cell culture apparatus is rotated and then cultured further.

12. The method according to claim 8, wherein the cells are cultured for 30 days or more to continuously produce useful components.

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