JP7354543B2 - Nonwoven fabric-containing cell culture module - Google Patents

Description

The present invention relates to a cell culture module having a cell culture member made of a hydrophilized nonwoven fabric and a porous polymer membrane.

In recent years, proteins such as enzymes, hormones, antibodies, cytokines, and viruses (viral proteins) used in treatments and vaccines have been industrially produced using cultured cells. However, these protein production technologies have issues with efficiency, which has affected the timely and stable supply of biopharmaceuticals, which require a continuous and widespread supply. . Therefore, in order to establish efficient, stable, and rapid production methods, innovative technologies that increase protein production, such as technologies for culturing cells at high density and highly efficient continuous production methods, are required. was.

Anchorage-dependent adherent cells that adhere to culture substrates are sometimes used as cells that produce proteins. Since these cells proliferate in an anchorage-dependent manner, they must be cultured by adhering to the surface of a petri dish, plate, or chamber. Conventionally, in order to culture such adherent cells in large quantities, it was necessary to increase the surface area for adhesion. However, in order to increase the culture area, it is necessary to increase the space, which reduces the culture efficiency and causes the equipment to become complicated and bulky.

As a method for culturing adherent cells in large quantities while reducing the culture space, a culture method using microporous carriers, particularly microcarriers, has been developed (for example, Patent Document 1). A cell culture system using microcarriers needs to be sufficiently stirred and dispersed to prevent the microcarriers from aggregating with each other. Therefore, a volume that can sufficiently stir and diffuse the culture solution in which the microcarriers are dispersed is required, so there is an upper limit to the density of cells that can be cultured. In addition, in order to separate the microcarriers from the culture medium, it is necessary to use a filter that can separate fine particles, which causes a decrease in the productivity of proteins such as biopharmaceuticals and vaccines. Under these circumstances, an innovative cell culture methodology for culturing cells at high density has been desired.

Conventionally, in the culture of adherent cultured cells, a culture method using fiber aggregates made of polyester fibers for the purpose of high-density culture (Patent Document 2, scaffolds and templates for tissue regeneration and repair of skin, periodontal tissue, jawbone, etc.) A method using a nonwoven fabric sheet as a method (Patent Document 3), a method of improving the efficiency of both cell adhesion and proliferation by controlling both the orientation and average fiber diameter between fibers constituting a fiber bundle (Patent Document 3). Patent Document 4), and a method for proliferating osteoblasts that uses a nonwoven fabric containing a bone substitute material as a cell scaffolding material (Patent Document 5) is also known. BioNOC II™ carriers, which are macroporous carriers composed of polyester nonwoven fabrics, are commercially available. On the other hand, there is also a report that investigated the influence of the cross-sectional shape of non-woven fabric (polyvinylidene fluoride fiber) on the proliferation of mesenchymal stromal cells in three-dimensional culture for the purpose of cell transplantation using non-woven fabric (polyvinylidene fluoride fiber) as a scaffold (non-patent paper). Reference 1).

Polyimide porous membranes have been used for applications mainly related to batteries, such as filters, low dielectric constant films, and electrolyte membranes for fuel cells, even before the filing of this application. In particular, Patent Documents 6 to 8 have excellent permeability to substances such as gas, high porosity, excellent smoothness on both surfaces, relatively high strength, and high porosity in the film thickness direction. describes a polyimide porous membrane having a large number of macrovoids that has excellent resistance to compressive stress. All of these are polyimide porous membranes created using amic acid. A cell culture method has been reported that includes applying and culturing cells to a polyimide porous membrane (Patent Document 9).

International Publication No. 2003/054174 Japanese Patent Application Publication No. 8-33473 Japanese Patent Application Publication No. 2015-158026 International Publication No. 2016/068279 Japanese Patent Application Publication No. 2012-192105 International Publication No. 2010/038873 JP2011-219585A JP2011-219586A International Publication No. 2015/012415

Schellengber, A., et al., PLOS One, vol. 9, e94353 (2014)

An object of the present invention is to provide a cell culture module equipped with a cell culture member made of a nonwoven fabric or a hydrophilized nonwoven fabric and a porous polymer, and a method for culturing cells using the cell culture module.

The present inventors discovered that a cell culture member consisting of a nonwoven fabric and a porous polymer membrane having a predetermined structure is suitable for cell culture, and can promote production of useful proteins from cells by the cell culture, and the present invention completed. That is, although not limited, the present invention preferably includes the following aspects.

[1] A cell culture member made of a nonwoven fabric and a polymer porous membrane,
A cell culture module having two or more culture medium inlets and a casing in which the cell culture member is housed, wherein:
The nonwoven fabric is a nonwoven fabric composed of core-sheath composite fibers having a weight of 20% or more,
The basis weight of the nonwoven fabric is 1 to 50 g/ ^m2 ,
The core and sheath of the core-sheath type composite fiber are made of a polyolefin polymer, the melting point of the polymer in the sheath is lower than the melting point of the polymer in the core, and the sheath-core ratio is 90:10 to 10:90. and
The nonwoven fabric has pores; and the polymer porous membrane has a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. A porous polymer membrane with a three-layer structure,
The average pore diameter of the pores present in surface layer A is smaller than the average pore diameter of pores present in surface layer B,
The macrovoid layer has a partition wall coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B,
pores in surface layers A and B communicate with the macrovoids;
Here, within the casing (i) the two or more independent cell culture members are aggregated,
(ii) the cell culture member is folded;
(iii) the cell culture member is rolled and/or
(iv) the cell culture member is tied into a rope;
The above-mentioned cell culture module is housed.
[2] The cell culture module according to [1], wherein the nonwoven fabric is subjected to a hydrophilic treatment.
[3] The cell culture module according to [1] or [2], wherein the core-sheath type composite fiber has a fineness of 0.05 to 2.2 decitex (dTex).
[4] The cell culture module according to any one of [1] to [3], wherein the core-sheath type composite fibers form binding portions at contact intersections by thermal fusion.
[5] According to any one of [1] to [4], where the nonwoven fabric contains two or more types of core-sheath type conjugate fibers having different finenesses, sheath-core ratios, and/or fiber diameters. Cell culture module as described.
[6] The cell culture according to any one of [1] to [5], wherein the diameter of the medium outflow port is larger than the diameter of the cells and smaller than the diameter through which the nonwoven fabric and the porous polymer membrane flow out. module.
[7] The hydrophilization treatment is selected from the group consisting of fluorine gas treatment, normal pressure plasma treatment, vacuum plasma treatment, corona treatment, graft polymerization treatment of hydrophilic monomers, sulfonation treatment, and surfactant imparting treatment. The cell culture module according to any one of [1] to [6].
[8] The cell culture module according to any one of [1] to [7], wherein the nonwoven fabric has an average pore of 0.01 to 100 μm.
[9] The cell culture module according to any one of [1] to [8], wherein the nonwoven fabric has a total thickness of 5 to 500 μm.
[10] The cell culture module according to any one of [1] to [9], wherein the area ratio of the binding portion formed by the core-sheath composite fibers is 5 to 30%.
[11] The cell culture module according to any one of [1] to [10], wherein the polymer porous membrane has pores with an average pore diameter of 0.01 to 100 μm.
[12] The cell culture module according to any one of [1] to [11], wherein the surface layer A has an average pore diameter of 0.01 to 50 μm.
[13] The cell culture module according to any one of [1] to [12], wherein the surface layer B has an average pore diameter of 20 to 100 μm.
[14] The cell culture module according to any one of [1] to [13], wherein the polymer porous membrane has a total thickness of 5 to 500 μm.
[15] The cell culture module according to any one of [1] to [14], wherein the polymer porous membrane is a polyimide porous membrane.
[16] The cell culture module according to [15], wherein the porous polyimide membrane contains a polyimide obtained from a tetracarboxylic dianhydride and a diamine.
[17] The polyimide porous membrane is obtained by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine and a coloring precursor, and then heat-treating the composition at 250°C or higher. The cell culture module according to [15] or [16], which is a colored polyimide porous membrane.
[18] The cell culture module according to any one of [1] to [17], wherein the polymer porous membrane is a polyethersulfone (PES) porous membrane.
[19] The cell culture module according to any one of [1] to [18], wherein the casing has a mesh-like structure.
[20] The cell culture module according to any one of [1] to [19], wherein the casing is made of a non-flexible material.

According to the present invention, suspended cells can be efficiently adsorbed and stably cultured using a conventional suspension culture container. Further, according to the present invention, cells can be easily removed without using a conventional filter membrane. Furthermore, by using a modularized cell culture member housed in a casing, suspended cells are easily adsorbed, making it possible to simply and stably culture cells.

FIG. 1 shows one embodiment of a cell culture module. A cell culture member is housed within the casing. FIG. 2 shows one embodiment of a cell culture module. A cell culture member is housed within the casing. FIG. 3 shows one embodiment of a cell culture module. (A) A cell culture member is housed in a mesh-like casing. (B) An embodiment of a casing consisting of a mesh-like net and a framework is shown. Figure 4 shows an embodiment of a device for use in conjunction with the application of cell culture modules in spinner flasks. (A) Rotating device. The device to which the cell culture module is applied is placed in a spinner flask, and the rotary device itself is rotated for use. (B) Fixed device. The device with the cell culture module applied is placed at the bottom of a spinner flask. Use the spinner flask by rotating the stirrer in the center of the device. FIG. 5 shows an embodiment in which a cell culture module is applied to a flexible bag-type culture container for culture. Figure 6 shows one embodiment of using a mesh module during shaking culture. FIG. 7 shows a model diagram of cell culture using a polyimide porous membrane. FIG. 8 shows one embodiment of a cell culture module. FIG. 9 shows one embodiment of a cell culture device. FIG. 10 shows one embodiment of a cell culture device. (A) A diagram showing the configuration of a cell culture device. (B) It is a figure which shows the cell culture part in which a cell culture device is mounted in (A). FIG. 11 shows one embodiment of a cell culture device. The basic configuration is the same as that in FIG. 10, but the culture medium discharge ports of each stage are shifted counterclockwise by 30 degrees, so that the culture medium can be easily discharged. FIG. 12 shows one embodiment of a cell culture device. FIG. 13 shows one embodiment of a cell culture device. (A) The configuration of the cell culture device is shown. (B) A diagram showing a cylindrical container (without spiral flow path). FIG. 14 shows one embodiment of a cell culture device. (A) It is a schematic diagram showing a cylindrical container (with a spiral flow path). (B) A diagram showing a cylindrical container (with a spiral flow path). Figure 15 shows one embodiment of cell culture. FIG. 16 is a diagram showing a WAVE type bioreactor to which the cell culture module of the present invention is applied in one embodiment. (A) A bag containing a cell culture module, and (B) a cell adsorption process to the cell culture module using WAVE25 are shown. FIG. 17 is a diagram showing a cell culture device in one embodiment of the present invention. (A) A cell culture module, (B) a cell culture section, and (C) a cell culture device in one embodiment. FIG. 18 is a diagram showing a cell culture device in one embodiment. FIG. 19 is a conceptual diagram showing the manner in which the medium is dropped from the dropletized medium supply means in the cell culture device of one embodiment. (A) Drop type, (B) Mesh type, and (C) Shower type. (D) It is a diagram showing a lid applied to a cell culture device of one embodiment, which is used for supplying drop-type and mesh-type droplets. A mesh bundle formed by winding stainless steel mesh is inserted into the medium supply port of the lid. FIG. 20 is a diagram showing a siphon cell culture device in one embodiment. FIG. 21 is a diagram showing the structure of cell culture modules by type. FIG. 22 is a diagram showing the structure of a bag-type bioreactor.

1. Cell culture module One aspect of the present invention is
A cell culture member made of a nonwoven fabric and a porous polymer membrane;
A cell culture module having two or more culture medium inlets and a casing in which the cell culture member is housed, wherein:
The nonwoven fabric is a nonwoven fabric composed of core-sheath composite fibers having a weight of 20% or more,
The basis weight of the nonwoven fabric is 1 to 50 g/ ^m2 ,
The core and sheath of the core-sheath type composite fiber are made of a polyolefin polymer, the melting point of the polymer in the sheath is lower than the melting point of the polymer in the core, and the sheath-core ratio is 90:10 to 10:90. and
The nonwoven fabric has pores; and the polymer porous membrane has a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. A porous polymer membrane with a three-layer structure,
The average pore diameter of the pores present in surface layer A is smaller than the average pore diameter of pores present in surface layer B,
The macrovoid layer has a partition wall coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B,
pores in surface layers A and B communicate with the macrovoids;
Here, within the casing (i) the two or more independent cell culture members are aggregated,
(ii) the cell culture member is folded;
(iii) the cell culture member is rolled and/or
(iv) the cell culture member is tied into a rope;
Regarding the cell culture module contained therein. The cell culture module is also referred to below as "cell culture module of the invention". In addition, in this specification, the description of "cell culture module" can be simply described as "module", and even if mutually changed, it means the same thing.

As used herein, the term "cell culture module" refers to a cell culture substrate applicable to cell culture containers, cell culture devices, and cell culture systems. Several embodiments of cell culture modules are shown in FIGS. 1-3, FIG. 8, and FIG. 17(A). The cell culture module of the invention can be used with embodiments such as FIGS. 4-6, 9-18, 20, and 22.

In the cell culture module of the present invention, since the nonwoven fabric and the porous polymer membrane are housed in the casing, the nonwoven fabric and the porous polymer membrane are prevented from being continuously deformed in the casing. This prevents stress from being applied to cells grown within the nonwoven fabric and/or polymer porous membrane, suppresses apoptosis, etc., and enables stable and large-scale cell culture.

The casing included in the cell culture module of the present invention has two or more culture medium inlets, so that the cell culture medium is supplied into the casing and discharged to the outside. The diameter of the medium inlet of the casing is preferably larger than the diameter of the cells so that the cells can flow into the casing. Further, it is preferable that the diameter of the medium outflow port is smaller than the diameter through which the nonwoven fabric and the polymer porous membrane flow out from the medium outflow port. The diameter smaller than the diameter through which the nonwoven fabric and polymer porous membrane flow can be appropriately selected depending on the shape and size of the nonwoven fabric and polymer porous membrane housed in the casing. For example, when the nonwoven fabric or porous polymer membrane is string-shaped, there is no particular limitation as long as it has an appropriate diameter that is smaller than the width of the short side of the nonwoven fabric or porous polymer membrane and does not cause the nonwoven fabric or porous polymer membrane to flow out. It is preferable that the number of medium inlets and inlets is as large as possible so that the cell culture medium can be easily supplied and/or discharged into and out of the casing. Preferably it is 5 or more, preferably 10 or more, preferably 20 or more, preferably 50 or more, preferably 100 or more. Part or all of the casing of the medium inlet may have a mesh-like structure. Moreover, the casing itself may be mesh-shaped. In the present invention, the mesh-shaped structure has, for example, a vertical, horizontal, and/or diagonal lattice-like structure, and each opening forms a medium inlet and outlet to the extent that fluid can pass through. However, it is not limited to this.

The casing included in the cell culture module of the present invention preferably has enough strength to not be deformed by movement of the medium under normal culture conditions, such as agitation culture or shaking culture conditions, and is made of a non-flexible material. Preferably. Furthermore, the casing is preferably made of a material that does not affect cell growth during cell culture. Examples of such materials include polyolefins (e.g., polyethylene, polypropylene, etc.), polymers such as nylon, polyester, polystyrene, polycarbonate, polymethyl methacrylate, and polyethylene terephthalate, stainless steel, and metals such as titanium. but not limited to. As used herein, "the casing does not deform" means that the casing does not deform under a load that is applied under a normal culture environment, but does not mean that it does not deform absolutely.

In the cell culture module of the present invention, (i) two or more independent cell culture members are aggregated in the casing,
(ii) the cell culture member is folded;
(iii) the cell culture member is wound into a roll, and/or
(iv) the cell culture member is tied into a rope;
It is accommodated.

In this specification, "two or more independent cell culture members are collectively housed within the casing" means that two or more independent polymer porous membranes are housed within a certain space surrounded by the casing. Refers to the state of being consolidated and contained. In the present invention, the two or more independent cell culture members are fixed at least one location on the cell culture member and at least one location within the casing by any method, and the cell culture members do not move within the casing. It may be fixed in a fixed state. Furthermore, the two or more independent cell culture members may be small pieces. The shape of the small piece may be any shape, such as a circle, an ellipse, a square, a triangle, a polygon, or a string, but is preferably substantially square. In the present invention, the size of the small piece can be any size, but when it is approximately square, the length can be any length, but for example, it is preferably 80 mm or less, preferably 50 mm or less, The length is more preferably 30 mm or less, even more preferably 20 mm or less, and may be 10 mm or less. In addition, if the small piece of the cell culture member is approximately square, the length of one side of the piece should be set along the inner wall of the casing or longer than the length of one side of the inner wall so that the cell culture member does not move within the casing. It may be formed short (for example, about 0.1 mm to 1 mm short). This prevents stress from being applied to cells grown within the cell culture member, suppresses apoptosis, etc., and allows stable and large amounts of cells to be cultured. In addition, in the case of a string-shaped cell culture member, as described below, (ii) the cell culture member is folded, (iii) the cell culture member is rolled up, and/or (iv) The cell culture member may be tied into a rope and housed in the casing. Moreover, in order to collectively accommodate two or more independent cell culture members in the casing, arbitrary numbers of nonwoven fabrics and polymer porous membranes may be laminated in an arbitrary order. In this case, a partition (insole) may be provided between the nonwoven fabric and the polymer porous membrane, between the nonwoven fabric and the nonwoven fabric, and between the polymer porous membrane and the polymer porous membrane. As an example of a cell culture member in which a nonwoven fabric and a porous polymer membrane are laminated, in one embodiment, a nonwoven fabric (for example, 3 sheets) and a polymer porous membrane (for example, 3 sheets) are alternately laminated, and a total of 6 sheets are stacked in one layer. As a unit, it is also possible to use a cell culture member in which three units are stacked with two partitions in between (see FIG. 21). In another embodiment, one unit is a structure in which a laminate of nonwoven fabrics (for example, 4 sheets) is sandwiched between upper and lower polymer porous membranes, and 3 units are laminated via two partitions. It can also be used as a cell culture member (see FIG. 21). In addition, by providing the partition, the culture medium can be efficiently supplied between the laminated nonwoven fabrics and polymer porous membranes. The partition has the function of forming an arbitrary space between the laminated nonwoven fabric and the polymer porous membrane, between the nonwoven fabric and the nonwoven fabric, and between the polymer porous membrane and the polymer porous membrane to efficiently supply the culture medium. For example, a planar structure having a mesh structure can be used, although there is no particular limitation. The material of the partition may be, for example, polystyrene, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, or stainless steel mesh, but is not limited thereto. In the case of having a partition having a mesh structure, it is sufficient to have a mesh opening that is large enough to supply a medium between the stacked cell culture members, and can be selected as appropriate.

As used herein, the term "folded cell culture member" means that it is folded within the casing and is moved within the casing by frictional force with each surface of the cell culture member and/or the surface within the casing. This is a cell culture member that has become empty. As used herein, "folded" may mean that the cell culture member has creases or may not have creases.

In the present specification, "a cell culture member rolled up in a roll" refers to a cell culture member rolled up in a roll shape, and the cell culture member is rolled up into a casing due to frictional force with each surface of the cell culture member and/or the surface inside the casing. refers to a cell culture member that has become immobile within the cell culture chamber. In addition, in the present invention, a cell culture member woven into a rope shape refers to, for example, a plurality of strip-shaped cell culture members woven into a rope shape by an arbitrary method so that the cell culture members do not move each other due to the frictional force between the cell culture members. Refers to the state of cell culture equipment. (i) A cell culture member in which two or more independent cell culture members are aggregated, (ii) a folded cell culture member, (iii) a cell culture member rolled up into a roll, and (iv) tied into a rope. A combination of cell culture members may be housed in the casing.

As used herein, "a state in which the cell culture member does not move within the casing" refers to a state in which the cell culture member does not continuously change shape when the cell culture module is cultured in a cell culture medium. This refers to the state in which the product is housed inside the casing. In other words, the cell culture member itself is in a state where it is restrained by the fluid so as not to continuously make undulating movements. Since the cell culture member remains stationary within the casing, stress is not applied to the cells growing within the cell culture member, and cells are stably cultured without dying due to apoptosis, etc. It becomes possible.

As the cell culture module of the present invention, commercially available culture devices and systems for culturing cells can be used. For example, it is also applicable to a culture device in which the culture container is made of a flexible bag, and it is possible to use it while floating in the culture container. Further, the cell culture module of the present invention can be applied to and cultured in a stirring culture type container such as a spinner flask, for example. In addition, as the culture container, it is applicable to both open containers and closed containers. For example, petri dishes, flasks, plastic bags, test tubes, and large tanks for cell culture can be used as appropriate. Examples include cell culture dishes manufactured by BD Falcon, Nunc Cell Factory manufactured by Thermo Scientific, and the like.

2. Application of cell culture module to cell culture device In this specification, “cell culture device” is a term that is generally treated synonymously with cell culture system, bioreactor, or reactor, and is the same even when interchanged. has the meaning of The cell culture module of the present invention can be applied to the cell culture apparatus illustrated below. Moreover, it is also applicable to commercially available devices other than the devices exemplified below.

(1) Siphon-type culture device The cell culture module of the present invention can be applied to the siphon-type culture device shown in FIGS. 9 and 20. A siphon culture device includes a cell culture member (preferably a cell culture module containing the cell culture member), a cell culture section containing the cell culture member, and a liquid reservoir containing the cell culture section inside. a culture medium supply means disposed at the top of the liquid reservoir, an inverted U-shaped tube communicating with the bottom of the liquid reservoir, and a culture medium recovery device disposed at the bottom of the other end of the inverted U-shaped tube. and a medium discharge means disposed on the medium recovery means. Here, when the liquid level of the medium supplied from the medium supply means to the liquid reservoir reaches the top of the inverted U-shaped tube, the medium is intermittently discharged to the medium recovery means according to the siphon principle. This is a cell culture device characterized by:

(2) Cylindrical gas phase culture device The cell culture module of the present invention can be applied to the cylindrical gas phase culture device shown in FIGS. 10, 11, and 18. In one embodiment, a cylindrical gas phase culture apparatus includes a cell culture member (preferably a cell culture module including the cell culture member), a cell culture section in which the cell culture member is housed, and a cell culture section that accommodates the cell culture member. It includes a medium supply means arranged at the upper part and a medium recovery means arranged at the lower part of the cell culture section. Here, the cell culture unit is a cell culture device including a bottom portion having one or more medium outlet, and a side portion disposed substantially perpendicularly to the bottom portion. In one embodiment, a cylindrical gas phase culture device includes a cell culture member (preferably a cell culture module including the cell culture member), a cell culture section in which the cell culture member is housed, and a cell culture unit containing the cell culture member. It includes a medium supply means arranged at the upper part of the culture section, and a medium recovery means arranged at the lower part of the cell culture section. Here, the medium recovery means is a cell culture device that is a part of an outer cylinder that accommodates the cell culture section.

(3) Mist and shower type culture device The cell culture module of the present invention can be applied to the mist and shower type culture device shown in FIG. What is a mist and shower type culture device?
A cell culture member (preferably a cell culture module including the cell culture member), a cell culture member mounting part on which the cell culture member is placed, and a casing in which the cell culture member mounting part is housed. , a dropletized medium supply section disposed inside the casing, a medium supply line communicating with the dropletized medium supply section, a medium storage section communicating with the medium supply line, and a medium supply line connected to the medium supply line. A pump installed in a part,
In this cell culture device, the cell culture member mounting portion includes a plurality of slit-shaped or mesh-shaped medium outlet ports.

(4) Gas phase exposure type rotary culture device The cell culture module of the present invention can be applied to the gas phase exposure type rotary culture device shown in FIGS. 13, 14, and 17. What is a gas phase exposure type rotary culture device?
A cell culture member (preferably a cell culture module including the cell culture member), a cell culture section having the cell culture member, a shaft passing through the cell culture section, and a rotation motor for rotating the shaft. , a medium tank in which at least a portion of the cell culture section is immersed,
The cell culture device is characterized in that the cell culture section rotates around the axis, and cells supported on the cell culture member are cultured alternately in a gas phase and a liquid phase.

3. Nonwoven Fabric The nonwoven fabric used in the present invention is composed of one or more types of core-sheath composite fibers relative to the weight of the nonwoven fabric. When making a nonwoven fabric by mutually fusing them, it is preferable that the amount of core-sheath composite fibers is 20% by weight or more based on the weight of the nonwoven fabric. Note that the content of the core-sheath type composite fiber may be 30% by weight or more, 40% by weight or more, or 50% by weight or more.

The core and sheath portions of the core-sheath type composite fiber are preferably made of a polyolefin polymer. The polyolefin polymer constituting the core and sheath includes aliphatic α-monoolefins having 2 to 16 carbon atoms, such as ethylene, propylene, 1-butene, 1-pentene, and 3-methyl-1-butene. , 1-hexene, 1-octene, 1-dodecene, and 1-octadecene homopolyolefins or copolymerized polyolefins. Aliphatic α-monoolefins may contain other olefins and/or small amounts (up to about 10% by weight of the polymer weight) of other ethylenically unsaturated monomers, such as butadiene, isoprene, pentadiene-1,3, styrene, α- It may also be copolymerized with similar ethylenically unsaturated monomers such as methylstyrene.

The combination of the polymer constituting the core and the polymer constituting the sheath is preferably 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, from the above polymers, those having a melting point difference of 20 to 50°C are selected. As will be described later, it is more preferable to select a polyolefin polymer that makes up the sheath portion and has a melting point lower than the melting point of the polyester polymer that makes up the core portion by, for example, 40° C. or more. Core-sheath type composite fibers having the above-mentioned properties include, but are not limited to, those having a combination in which the sheath polymer is polyethylene and the core polymer is polypropylene.

The core-sheath type composite fiber used in the present invention, which has the fineness and core-sheath composite ratio as described below, can be manufactured using a general method for manufacturing core-sheath type composite fiber (nonwoven fabric). It is. Typically, such a manufacturing method may include, but is not limited to, the following steps.
1) Using a composite spinning device equipped with two single-screw extruders and a composite spinning nozzle, the mass ratio of high melting point component (e.g. polypropylene) to low melting point component (e.g. polyethylene) was adjusted to the desired ratio at a spinning temperature of 280°C. to produce an undrawn fiber;
2) Gather the undrawn fibers and stretch them with a heatable drawing roller to produce drawn fibers, for example, at a draw ratio of 4 times or more and a desired single fiber fineness;
3) Produce short fibers having a desired fiber length (for example, several mm) using a rotary cutter; and 4) Produce a nonwoven fabric using the short fibers using a general wet manufacturing method or dry manufacturing method.

More specifically, in the wet manufacturing method of step 4), short fibers are uniformly dispersed in water to which a viscosity modifier has been added to prepare a dispersion liquid, and then this dispersion liquid is placed on a mesh to form paper. After producing a wet web, the resulting wet web is sandwiched between two rubber heating plates, and dried by pressing for 1 minute at a temperature of 140°C using a general-purpose heating press machine. As a result, the low melting point components of the short fibers are melted and the short fibers are bonded together, making it possible to obtain a nonwoven fabric. In the present invention, for example, but not limited to, the composite fiber "Airimo" manufactured by Ube Eximo Co., Ltd. can be used.

The fibers constituting the nonwoven fabric are integrated and maintain their shape as a nonwoven fabric by having a heat-fused portion (also referred to as a “binding portion” in this specification). The heat-fused portion is formed by heating the sheath component to a temperature above the melting point and below the core component's melting point. In the heat-fused portion, the sheath component melts or softens and contributes to adhesion. The core component does not completely melt under the influence of heat, but maintains the fiber form, contributing to improving the tear strength of the nonwoven fabric. Therefore, in the heat-sealed part, the sheath component melts or softens, while the core component maintains its fiber form by providing a difference of, for example, 30°C or more in their melting points. During the attachment process, the core component is not affected by heat, the sheath component is reliably melted, and the core component can be thermally bonded and fixed by melting and fixing at the heat-sealing portion.

The "fineness" of the core-sheath composite fibers that make up nonwoven fabrics is generally used to express the thickness of the fibers, and is expressed as the weight (g) per 10,000 meters of fiber (i.e., decitex (dTex)). be done. The fineness of the core-sheath type composite fiber used in the present invention is not limited as long as it is suitable for cell culture, but is preferably 0.05 to 2.2 dtex.

Further, the core/sheath composite ratio (mass ratio) is preferably sheath/core = 20/80 to 50/50. By making the ratio of the core part equal to or higher than that of the sheath part, excellent mechanical properties and practical strength can be maintained. In addition, when the ratio of the core exceeds 80% by mass, the ratio of the sheath, which is an adhesive component, decreases, and the adhesive strength at the heat-sealed part tends to decrease. Therefore, the upper limit of the ratio of the core is 80% by mass is preferred.

The fiber diameter of the core-sheath type conjugate fiber may be any diameter suitable for cell culture, and is determined by the fineness and specific gravity of the conjugate fiber, but is preferably 1 to 20 μm, for example. More preferably 2 to 10 μm, still more preferably 3 to 8 μm.

The basis weight of the nonwoven fabric may be 1 to 50 g/m ² , and preferably 3 to 50 g/m ² .

The nonwoven fabric used in the present invention may be composed of two or more types of core-sheath type conjugate fibers having different finenesses, sheath-core ratios, and/or fiber diameters. Regarding different finenesses, for example, the fibers may be composed of thick fibers having a fineness of 1 to 2.2 dtex and fine fibers having a fineness of 0.05 to 0.5 dtex. Further, in the present invention, the mass ratio of thick fibers to fine fibers is preferably thick fibers/fine fibers = 20/80 to 80/20. The thick fibers and the fine fibers may be mixed in a nonwoven fabric, or the web may have thick fibers deposited on one surface and fine fibers deposited on the other surface. Both may exist depending on the laminated state of the web.

As mentioned above, the fibers constituting the nonwoven fabric are thermally bonded and fixed to each other by the heat-fused portion and are integrated. In the thermally bonded portion, the sheath component of the core-sheath composite fiber is melted and solidified to form a thermally bonded portion that functions as an adhesive component. Further, as a method for forming the heat-sealed portions, a large number of scattered thermocompression-bonded portions can be formed by heat embossing or ultrasonic welding. The shape of each thermocompression bonding part may be any shape such as a circle, an ellipse, a rhombus, a triangle, a weave shape, a square shape, and a lattice shape. In addition, in consideration of the flexibility of the nonwoven fabric and the formation of scaffolds for cells, it is preferable that the particles be present in a scattered pattern. The area of each thermocompression bonded portion is preferably about 0.2 to 3 mm ² . Further, the area ratio of the thermocompression bonded portion to the area of the nonwoven fabric is preferably 10% or more and 30% or less. If it is less than 10%, it is difficult to achieve both excellent mechanical properties and rigidity, which are the objectives of the present invention.

The nonwoven fabric used in the present invention may be a hydrophilized nonwoven fabric. The nonwoven fabric can be made hydrophilic by, but not limited to, fluorine gas treatment, normal pressure plasma treatment, vacuum plasma treatment, corona treatment, graft polymerization treatment of hydrophilic monomers, sulfonation treatment, or surfactant imparting treatment. fluorine gas treatment and atmospheric pressure plasma treatment are preferred.

The total 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 500 μm or less, 300 μm or less, 100 μm or less, 75 μm or less, or 50 μm or less. There may be. Preferably it is 5 to 500 μm, more preferably 25 to 75 μm.

The nonwoven fabric used in the present invention is preferably sterilized. Examples of the sterilization treatment include, but are not particularly limited to, gas sterilization, sterilization using a disinfectant such as ethanol, and arbitrary sterilization treatment such as electromagnetic wave sterilization such as ultraviolet rays and gamma rays.

4. Polymer Porous Membrane The average pore diameter of the pores present in the surface layer A (hereinafter also referred to as "A surface" or "mesh surface") in the polymer porous membrane used in the present invention is not particularly limited, but for example, , 0.01 μm to less than 200 μm, 0.01 to 150 μm, 0.01 to 100 μm, 0.01 to 50 μm, 0.01 μm to 40 μm, 0.01 μm to 30 μm, 0.01 μm to 25 μm, 0.01 μm to 20 μm, Or 0.01 μm to 15 μm, preferably 0.01 μm to 25 μm.

The average pore diameter of the pores present in the surface layer B (hereinafter also referred to as "B surface" or "large pore surface") in the polymer porous membrane used in the present invention is the average pore diameter of the pores present in the surface layer A. Although not particularly limited as long as it is larger than, for example, more than 5 μm to 200 μm, 20 μm to 100 μm, 25 μm to 100 μm, 30 μm to 100 μm, 35 μm to 100 μm, 40 μm to 100 μm, 50 μm to 100 μm, or 60 μm to 100 μm, preferably, It is 30 μm to 100 μm.

（２）上記式Ｉによって求められた全ての孔径を以下の式ＩＩに適用し、孔の形状が真円であるとした際の面積平均孔径ｄａを求める。

In this specification, the average pore size on the surface of the polymer porous membrane is the area average pore size. The area average pore diameter can be determined according to (1) and (2) below. Note that the average pore diameter of portions other than the surface of the porous polymer membrane can also be determined in the same manner.
(1) Measure the pore area S of 200 or more pores from a scanning electron micrograph of the surface of the porous membrane, and calculate the pore diameter d of each from Formula I assuming that the pore area is a perfect circle.

(2) Applying all the pore diameters determined by the above formula I to the following formula II, the area average pore diameter da is determined assuming that the shape of the pores is a perfect circle.

The thickness of the surface layers A and B is not particularly limited, but is, for example, 0.01 to 50 μm, preferably 0.01 to 20 μm.

The average pore diameter of the macrovoids in the macrovoid layer in the polymer porous membrane in the membrane plane direction is not particularly limited, but is, for example, 10 to 500 μm, preferably 10 to 100 μm, and more preferably 10 to 80 μm. Furthermore, the thickness of the partition walls in the macrovoid layer is not particularly limited, but is, for example, 0.01 to 50 μm, preferably 0.01 to 20 μm. In one embodiment, at least one partition wall in the macrovoid layer has one or more pores with an average diameter of 0.01 to 100 μm, preferably 0.01 to 50 μm, which connect adjacent macrovoids. It has holes. In another embodiment, the partition walls in the macrovoid layer have no pores.

The total film thickness of the surface of the polymer porous membrane 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 500 μm or less, 300 μm or less, 100 μm or less, or 75 μm or less. Or it may be 50 μm or less. Preferably it is 5 to 500 μm, more preferably 25 to 75 μm.

The thickness of the polymer porous membrane used in the present invention can be measured using a contact thickness meter.

The porosity of the polymer porous membrane used in the present invention is not particularly limited, but is, for example, 40% or more and less than 95%.

（式中、Ｓは多孔質フィルムの面積、ｄは総膜厚、ｗは測定した質量、Ｄはポリマーの密度をそれぞれ意味する。ポリマーがポリイミドである場合は、密度は１．３４ｇ／ｃｍ³とする。） The porosity of the polymer porous membrane used in the present invention can be determined by measuring the thickness and mass of a porous film cut into a predetermined size, and calculating from the basis weight according to the following formula III.

(In the formula, S means the area of the porous film, d means the total film thickness, w means the measured mass, and D means the density of the polymer. If the polymer is polyimide, the density is 1.34 g/cm ³ )

The polymer porous membrane used in the present invention preferably has three layers, including a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. A polymer porous membrane having a structure in which the average pore diameter of the pores present in the surface layer A is 0.01 μm to 25 μm, and the average pore diameter of the pores present in the surface layer B is 30 μm to 100 μm, The macrovoid layer has a partition wall coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, and the partition wall of the macrovoid layer and the surface layer The thickness of layers A and B is 0.01 to 20 μm, the pores in the surface layers A and B communicate with macro voids, the total film thickness is 5 to 500 μm, and the porosity is 40% or more. is a polymeric porous membrane that is less than 95%. In one embodiment, at least one partition wall in the macrovoid layer has one or more pores with an average pore diameter of 0.01 to 100 μm, preferably 0.01 to 50 μm, communicating adjacent macrovoids. has. In another embodiment, the septum has no such pores.

The polymer porous membrane used in the present invention is preferably sterilized. Examples of the sterilization treatment include, but are not particularly limited to, any sterilization treatment such as dry heat sterilization, steam sterilization, sterilization with a disinfectant such as ethanol, and electromagnetic wave sterilization such as ultraviolet rays and gamma rays.

The polymer porous membrane used in the present invention is not particularly limited as long as it has the above-mentioned structural characteristics, but is preferably a polyimide porous membrane or a polyethersulfone (PES) porous membrane.

<Polyimide porous membrane>
Polyimide is a general term for polymers containing imide bonds in repeating units, and usually refers to an aromatic polyimide in which aromatic compounds are directly connected via imide bonds. Aromatic polyimide has a conjugated structure between aromatics and aromatics through imide bonds, so it has a rigid and strong molecular structure, and because the imide bonds have strong intermolecular forces, it can withstand very high levels of heat. It has physical, mechanical, and chemical properties.

The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane containing (as a main component) polyimide obtained from tetracarboxylic dianhydride and diamine, more preferably tetracarboxylic dianhydride. This is a polyimide porous membrane made of polyimide obtained from polyimide and diamine. "Contained as a main component" means that components other than the polyimide obtained from tetracarboxylic dianhydride and diamine are essentially not included, or may be included, as constituent components of the polyimide porous membrane. , means that it is an additional component that does not affect the properties of the polyimide obtained from tetracarboxylic dianhydride and diamine.

In one embodiment, the polyimide porous membrane that can be used in the present invention is formed 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 at 250°C. Also included are colored polyimide porous membranes obtained by heat treatment as described above.

Polyamic acid is obtained by polymerizing a tetracarboxylic acid component and a diamine component. Polyamic acid is a polyimide precursor that can be ring-closed to form polyimide by thermal imidization or chemical imidization.

Even if a part of the amic acid is imidized, the polyamic acid can be used as long as it does not affect the present invention. That is, the polyamic acid may be partially thermally imidized or chemically imidized.

When thermally imidizing a polyamic acid, an imidization catalyst, an organic phosphorus-containing compound, fine particles such as inorganic fine particles, organic fine particles, etc. can be added to the polyamic acid solution as necessary. Further, when chemically imidizing polyamic acid, a chemical imidizing agent, a dehydrating agent, fine particles such as inorganic fine particles, organic fine particles, etc. can be added to the polyamic acid solution as necessary. It is preferable to conduct the mixing under conditions in which the coloring precursor does not precipitate even if the above-mentioned components are blended into the polyamic acid solution.

As used herein, the term "colored precursor" refers to a precursor that is partially or completely carbonized by heat treatment at 250° C. or higher to produce a colored product.

The colored precursor that can be used in the production of the polyimide porous membrane is uniformly dissolved or dispersed in a polyamic acid solution or a polyimide solution, and is preferably heated to a temperature of 250°C or higher, preferably 260°C or higher, more preferably 280°C or higher, and more preferably 280°C or higher. is thermally decomposed and carbonized by heat treatment at 300°C or higher, preferably at 250°C or higher, preferably at 260°C or higher, more preferably at 280°C or higher, even more preferably at 300°C or higher. Those that produce colored products are preferred, those that produce black colored products are more preferred, and carbon-based colored precursors are more preferred.

When the colored precursor is heated, it appears to be a carbonized product at first glance, but it has a structure that contains elements other than carbon, and has a layered structure, an aromatic cross-linked structure, and a disordered structure containing tetrahedral carbon. include.

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

In another embodiment, the porous polyimide membrane that can be used in the present invention is obtained by forming a polyamic acid solution obtained from a tetracarboxylic acid component and a diamine component without using the above-mentioned colored precursor, and Also included are polyimide porous membranes obtained by heat treatment.

A polyimide porous membrane produced without using a colored precursor is, for example, made from 3 to 60% by mass of a polyamic acid having an intrinsic viscosity of 1.0 to 3.0 and 40 to 97% by mass of an organic polar solvent. A porous film of polyamic acid is produced by casting a polyamic acid solution in the form of a film, immersing it in or contacting it with a coagulation solvent containing water as an essential component, and then heat-treating the porous film of polyamic acid to form an imide. It may also be manufactured by In this method, the coagulation solvent containing water as an essential component is 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. It's okay. Further, after the imidization, at least one side of the obtained porous polyimide film may be subjected to plasma treatment.

Any tetracarboxylic dianhydride can be used as the tetracarboxylic dianhydride that can be used in the production of the polyimide porous membrane, and can be appropriately selected depending on desired characteristics. Specific examples of tetracarboxylic dianhydride include pyromellitic dianhydride, 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA), 2,3,3',4' -Biphenyltetracarboxylic dianhydride such as 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, 2 , 2-bis(3,4-dicarboxyphenyl)propane dianhydride, p-phenylenebis(trimellitic acid monoester acid anhydride), p-biphenylenebis(trimellitic acid monoester acid anhydride), m- Terphenyl-3,4,3',4'-tetracarboxylic dianhydride, p-terphenyl-3,4,3',4'-tetracarboxylic 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 dianhydride, 1,4,5,8-naphthalenetetracarboxylic dianhydride Examples include anhydride, 4,4'-(2,2-hexafluoroisopropylidene) diphthalic dianhydride, and the like. It is also preferable to use aromatic tetracarboxylic acids such as 2,3,3',4'-diphenylsulfonetetracarboxylic acid. These can be used alone or in combination of two or more.

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

Any diamine can be used as the diamine that can be used in the production of the polyimide porous membrane. Specific examples of diamines include the following.
1) Benzenediamine with one benzene nucleus such as 1,4-diaminobenzene (paraphenylenediamine), 1,3-diaminobenzene, 2,4-diaminotoluene, 2,6-diaminotoluene;
2) Diaminodiphenyl ethers such as 4,4'-diaminodiphenyl ether and 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'-diaminodiphenylsulfide, 3,3'-diaminodiphenylsulfone, 3,4'-diaminodiphenylsulfone, 4,4'-diaminodiphenylsulfone, 3,3'-diaminobenzophenone, 3,3'-diamino-4,4'-dichlorobenzophenone, 3,3'-diamino-4, 4'-dimethoxybenzophenone, 3,3'-diaminodiphenylmethane, 3,4'-diaminodiphenylmethane, 4,4'-diaminodiphenylmethane, 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, Diamines with two benzene nuclei such as 1,3,3,3-hexafluoropropane, 3,3'-diaminodiphenylsulfoxide, 3,4'-diaminodiphenylsulfoxide, 4,4'-diaminodiphenylsulfoxide;
3) 1,3-bis(3-aminophenyl)benzene, 1,3-bis(4-aminophenyl)benzene, 1,4-bis(3-aminophenyl)benzene, 1,4-bis(4-amino phenyl)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-bis(3-aminophenyl sulfide)benzene, 1,3-bis(4-aminophenyl sulfide)benzene, 1,4-bis(4-aminophenyl sulfide)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] Diamines with three benzene nuclei such as benzene, 1,4-bis[2-(3-aminophenyl)isopropyl]benzene, 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-aminophenoxy)phenyl]ketone, bis[4-(3-amino) phenoxy)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[3-(4-aminophenoxy) phenyl]sulfone, bis[4-(3-aminophenoxy)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-amino phenoxy)phenyl]propane, 2,2-bis[3-(4-aminophenoxy)phenyl]propane, 2,2-bis[4-(3-aminophenoxy)phenyl]propane, 2,2-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, 2,2-bis[4-(4-aminophenoxy)phenyl]-1,1,1,3,3,3-hexafluoropropane and other diamines with four benzene nuclei.

These can be used alone or in combination of two or more. The diamine to be used can be appropriately selected depending on desired characteristics and the like.

Among these, aromatic diamine compounds are preferred, including 3,3'-diaminodiphenyl ether, 3,4'-diaminodiphenyl ether, 4,4'-diaminodiphenyl ether, paraphenylenediamine, and 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-amino Phenoxy)benzene and 1,4-bis(3-aminophenoxy)benzene can be preferably used. Particularly preferred is at least one diamine selected from the group consisting of benzenediamine, diaminodiphenyl ether, and bis(aminophenoxy)phenyl.

From the viewpoint of heat resistance and dimensional stability at high temperatures, the polyimide porous membrane that can be used in the present invention has a glass transition temperature of 240°C or higher, or a tetracarboxylic membrane that does not have a clear transition point at 300°C or higher. It is preferably formed from a polyimide obtained by combining an acid dianhydride and a diamine.

The polyimide porous membrane that can be used in the present invention is preferably a polyimide porous membrane made of the following aromatic polyimide from the viewpoint of heat resistance and dimensional stability at high temperatures.
(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 aromatic diamine units;
(ii) an aromatic polyimide consisting of 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) at least one tetracarboxylic acid unit selected from the group consisting of biphenyltetracarboxylic acid units and pyromellitic acid units; and at least one tetracarboxylic acid unit selected from the group consisting of benzenediamine units, diaminodiphenyl ether units, and bis(aminophenoxy)phenyl units. An aromatic polyimide consisting of a type of aromatic diamine unit.

The polyimide porous membrane used in the present invention preferably has three layers, including a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. A polyimide porous membrane having a structure, wherein the average pore diameter of the pores present in the surface layer A is 0.01 μm to 25 μm, and the average pore diameter of the pores present in the surface layer B is 30 μm to 100 μm, The macrovoid layer has a partition wall coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B, and the partition wall of the macrovoid layer and the surface layer The thickness of layers A and B is 0.01 to 20 μm, the pores in the surface layers A and B communicate with macro voids, the total film thickness is 5 to 500 μm, and the porosity is 40% or more. The polyimide porous membrane is less than 95%. Here, at least one partition wall in the macrovoid layer has one or more pores with an average pore diameter of 0.01 to 100 μm, preferably 0.01 to 50 μm, which connect adjacent macrovoids.

For example, polyimide porous membranes described in WO 2010/038873, JP 2011-219585, or JP 2011-219586 can also be used in the present invention.

<Polyether sulfone (PES) porous membrane>
PES porous membranes that can be used in the present invention include, and typically consist essentially of, polyethersulfone. The polyether sulfone may be synthesized by a method known to those skilled in the art, for example, a method in which a dihydric phenol, an alkali metal compound, and a dihalogenodiphenyl compound are subjected to a polycondensation reaction in an organic polar solvent, a method in which a dihydric phenol is It can be produced by a method in which an alkali metal di-salt is synthesized in advance and subjected to a polycondensation reaction with a dihalogenodiphenyl compound in an organic polar solvent.

Examples of the alkali metal compound include alkali metal carbonates, alkali metal hydroxides, alkali metal hydrides, alkali metal alkoxides, and the like. Particularly preferred are sodium carbonate and potassium carbonate.

Examples of dihydric phenol compounds include hydroquinone, catechol, resorcinol, 4,4'-biphenol, bis(hydroxyphenyl) alkanes (for example, 2,2-bis(hydroxyphenyl)propane, and 2,2-bis(hydroxyphenyl) methane), dihydroxydiphenyl sulfones, dihydroxydiphenyl ethers, or at least one hydrogen of their benzene ring is 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 an ethoxy group. Substituted items are listed. As the dihydric phenol compound, two or more of the above compounds can be used as a mixture.

The polyether sulfone may be a commercially available product. Examples of commercially available products include Sumika Excel 7600P and Sumika Excel 5900P (manufactured by Sumitomo Chemical Co., Ltd.).

The logarithmic viscosity of the polyethersulfone is preferably 0.5 or more, more preferably 0.55 or more, from the viewpoint of forming macrovoids in the porous polyethersulfone membrane. From the viewpoint of ease, it is preferably 1.0 or less, more preferably 0.9 or less, even more preferably 0.8 or less, particularly preferably 0.75 or less.

In addition, from the viewpoint of heat resistance and dimensional stability at high temperatures, the PES porous membrane or the polyether sulfone as its raw material must have a glass transition temperature of 200°C or higher, or a clear glass transition temperature. Preferably, it is not observed.

The method for producing the PES porous membrane that can be used in the present invention is not particularly limited, but for example,
A polyether sulfone solution containing 0.3% to 60% by mass of polyethersulfone with a logarithmic viscosity of 0.5 to 1.0 and 40% to 99.7% by mass of an organic polar solvent is cast into a film. and a step of producing a coagulated membrane having pores by immersing or bringing it into contact with a coagulating solvent containing a poor solvent or non-solvent of polyether sulfone as an essential component, and a coagulating membrane having pores obtained in the above step. The heat treatment includes a step of coarsening the pores to obtain a PES porous membrane, and the heat treatment heats the solidified membrane having the pores to a temperature equal to or higher than the glass transition temperature of the polyether sulfone, or to 240°C or higher. The method may include raising the temperature.

The PES porous membrane that can be used in the present invention preferably has a surface layer A, a surface layer B, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. And,
The macrovoid layer includes a partition wall coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B and having an average pore diameter of 10 μm to 500 μm in the film plane direction. have,
The partition wall of the macrovoid layer has 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,
Among the surface layers A and B, one has a plurality of pores with an average pore diameter of more than 5 μm and less than 200 μm, and the other has a plurality of pores with an average pore diameter of 0.01 μm and more and less than 200 μm,
One of the surface layer A and the surface layer B has a surface aperture ratio of 15% or more, and the other surface layer has a surface aperture ratio of 10% or more,
The pores of the surface layer A and the surface layer B communicate with the macro void,
The PES porous membrane has a total thickness of 5 μm to 500 μm and a porosity of 50% to 95%.
It is a PES porous membrane.

5. A cell culture method using a cell culture module One aspect of the present invention is
(1) applying a first medium containing suspended cells to a cell culture module;
(2) maintaining the temperature at which cell culture is possible and adsorbing the cells to the cell culture module; and
(3) culturing the cell culture module to which the cells have been adsorbed in a second medium in a culture container;
including;
Here, the cell culture module
A cell culture member including a hydrophilized nonwoven fabric and a porous polymer membrane;
A cell culture module having two or more culture medium inlets and a casing in which the cell culture member is housed, wherein:
The nonwoven fabric is a nonwoven fabric composed of core-sheath composite fibers having a weight of 20% or more,
The basis weight of the nonwoven fabric is 1 to 50 g/ ^m2 ,
The core and sheath of the core-sheath type composite fiber are made of a polyolefin polymer, the melting point of the polymer in the sheath is lower than the melting point of the polymer in the core, and the sheath-core ratio is 90:10 to 10:90. and
The nonwoven fabric has pores; and the polymer porous membrane has a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. A porous polymer membrane with a three-layer structure,
The average pore diameter of the pores present in surface layer A is smaller than the average pore diameter of pores present in surface layer B,
The macrovoid layer has a partition wall coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B,
pores in surface layers A and B communicate with the macrovoids;
Here, within the casing (i) the two or more independent cell culture members are aggregated,
(ii) the cell culture member is folded;
(iii) the cell culture member is rolled and/or
(iv) the cell culture member is tied into a rope;
It is housed,
Here, the present invention relates to a cell culture method in which the second medium does not contain a surfactant. The cell culture method of the present invention is also referred to below as the "cell culture method of the present invention."

The types of cells that can be used in the present invention are, for example, selected from the group consisting of animal cells, insect cells, plant cells, yeast, and bacteria. Animal cells are broadly classified into cells derived from animals belonging to the phylum vertebrates and cells derived from invertebrates (animals other than the phylum vertebrates). In this specification, the origin of animal cells is not particularly limited. Preferably, it refers to cells derived from animals belonging to the phylum Vertebrata. The phylum Vertebrate includes the supragnathina and the gnathostome, and the phylum gnathostome includes the classes Mammalia, Aves, Amphibia, Reptilia, and the like. Preferably, cells are derived from animals belonging to the class Mammalia, commonly referred to as mammals. Mammals are not particularly limited, but preferably include mice, rats, humans, monkeys, pigs, dogs, sheep, goats, and the like.

The types of animal cells that can be used in the present invention are not limited, but are preferably selected from the group consisting of pluripotent stem cells, tissue stem cells, somatic cells, and germ cells.

As used herein, "pluripotent stem cells" is intended to collectively refer to stem cells that have the ability to differentiate into cells of any tissue (pluripotency). Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), germline stem cells (GS cells), etc. . Preferably they are ES cells or iPS cells. iPS cells are particularly preferred because they pose no ethical problems. Any known pluripotent stem cells can be used, and for example, pluripotent stem cells described in International Publication No. 2009/123349 (PCT/JP2009/057041) can be used.

The term "tissue stem cell" refers to a stem cell whose differentiateable cell lineage is limited to a specific tissue, but which has the ability to differentiate into various cell types (pluripotency). For example, hematopoietic stem cells in the bone marrow become the source of blood cells, and neural stem cells differentiate into nerve cells. There are many other types of cells, including liver stem cells, which form the liver, and skin stem cells, which form skin tissue. Preferably, the tissue stem cells are selected from mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, skin stem cells, or hematopoietic stem cells.

"Somatic cells" refer to cells other than reproductive cells that constitute a multicellular organism. In sexual reproduction, it is not passed on to the next generation. Preferably, the somatic cells are hepatocytes, pancreatic cells, muscle cells, osteocytes, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, renal cells, lung cells, or , lymphocytes, red blood cells, white blood cells, monocytes, macrophages or megakaryocytes.

"Germ cells" refer to cells that play a role in transmitting genetic information to the next generation during reproduction. For example, it includes gametes for sexual reproduction, ie, eggs, egg cells, sperm, sperm cells, and spores for asexual reproduction.

The cells may be selected from the group consisting of sarcoma cells, established cell lines and transformed cells. "Sarcoma" is a cancer that occurs in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood, and includes soft tissue sarcoma, malignant bone tumors, and the like. Sarcoma cells are cells derived from sarcoma. The term "established cell line" refers to cultured cells that have been maintained outside the body for a long period of time and have certain stable properties, making semi-permanent subculture possible. PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human fetal kidney), HL-60 cells (derived from human white blood cells), HeLa cells (derived from human cervical cancer), Vero cells (derived from African green monkey kidney epithelial cells), MDCK cells (derived from dog kidney tubular epithelial cells), HepG2 cells (human liver cancer-derived cell line), BHK cells (neonatal hamster kidney cells), NIH3T3 cells (derived from mouse fetal fibroblast cells) There are cell lines derived from various tissues of various species including humans. A "transformed cell" refers to a cell whose genetic properties have been changed by introducing a nucleic acid (such as DNA) from outside the cell.

As used herein, "adherent cells" are generally cells that need to attach themselves to a suitable surface for proliferation, and are also referred to as adherent cells or anchorage-dependent cells. In some embodiments of the invention, the cells used are adherent cells. The cells used in the present invention are adherent cells, and more preferably cells that can be cultured even when suspended in a medium. Adherent cells that can be cultured in suspension can be obtained by acclimating adherent cells to a state suitable for suspension culture using a known method, and can be obtained, for example, from CHO cells, HEK293 cells, Vero cells, NIH3T3 cells. and cell lines derived from these cells. Even if the cells are not listed here, the cells used in the present invention are not particularly limited as long as they are adherent cells and can be cultured in suspension through acclimatization.

A model diagram of cell culture using the polymer porous membrane constituting the cell culture member is shown in FIG. FIG. 7 is a diagram to aid understanding, and each element is not to scale. In the cell culture method of the present invention, by applying cells to a polymer porous membrane and culturing them, a large amount of cells grow on the internal multifaceted connected pores and surface of the polymer porous membrane. It becomes possible to easily culture a large amount of cells. Furthermore, the cell culture method of the present invention allows a large amount of cells to be cultured while significantly reducing the amount of medium used for cell culture compared to conventional methods. For example, large amounts of cells can be cultured for long periods of time even when a portion or the entire porous polymer membrane is not in contact with the liquid phase of the cell culture medium. Furthermore, it is also possible to significantly reduce the total volume of the cell culture medium contained in the cell culture container with respect to the total volume of the polymer porous membrane including the cell living area.

In this specification, the volume that a porous polymer membrane that does not contain cells occupies in space, including the volume of its internal gaps, is referred to as the "apparent volume of the porous polymer membrane" (see FIG. 7). Then, cells are applied to the polymer porous membrane, and in a state where the cells are supported on the surface and inside of the polymer porous membrane, the polymer porous membrane, the cells, and the medium infiltrated inside the polymer porous membrane form a space as a whole. The volume occupied therein is referred to as the "polymer porous membrane volume containing the cell living area" (see FIG. 7). In the case of a porous polymer membrane with a thickness of 25 μm, the volume of the porous polymer membrane including the cell survival area is approximately 50% larger at most than the apparent volume of the porous polymer membrane. A plurality of polymeric porous membranes can be housed and cultured in one cell culture container, in which case the polymeric porous membrane includes a cell living area for each of the plurality of polymeric porous membranes carrying cells. The total volume may be simply described as "the total volume of the polymer porous membrane including the cell living area."

By using the method of the present invention, cells can be maintained effectively for a long period of time even under conditions where the total volume of the cell culture medium contained in the cell culture container is 10,000 times or less than the total volume of the cell culture member including the cell survival zone. It becomes possible to culture Furthermore, cells can be cultured satisfactorily over a long period of time even under conditions where the total volume of the cell culture medium contained in the cell culture container is 1000 times or less than the total volume of the cell culture member including the cell survival zone. Furthermore, cells can be cultured satisfactorily over a long period of time even under conditions where the total volume of the cell culture medium contained in the cell culture container is 100 times or less than the total volume of the cell culture member including the cell survival zone. Even under conditions where the total volume of the cell culture medium contained in the cell culture container is 10 times or less than the total volume of the cell culture member including the cell survival zone, cells can be cultured satisfactorily over a long period of time.

That is, according to the present invention, the space (container) in which cells are cultured can be made extremely small compared to the conventional cell culture apparatus that performs two-dimensional culture. Furthermore, when it is desired to increase the number of cells to be cultured, the volume for cell culture can be flexibly increased by a simple operation such as increasing the number of cell culture members to be stacked. With a cell culture device equipped with the cell culture member used in the present invention, it is possible to separate the space (container) for culturing cells and the space (container) for storing cell culture medium, and the number of cells to be cultured is It becomes possible to prepare the required amount of cell culture medium depending on the situation. The space (container) for storing the cell culture medium is not particularly limited, and may be made larger or smaller depending on the purpose, or may be a replaceable container.

Mass culture of cells in the cell culture method of the present invention means, for example, a cell culture medium in which all the cells are contained in the cell culture container after culturing using a cell culture member. 1.0 x 10 ⁵ or more, 1.0 x 10 6 or more, 2.0 x 10 ⁶ or more, 5.0 x 10 ^{6 or} more per milliliter of medium, 1.0×10 ⁷ or more, 2.0×10 ⁷ or more, 5.0×10 ⁷ or more, 1.0×10 8 or more, 2.0×10 ^{8 or} more, 5.0×10 This refers to culturing until the number of cells reaches ⁸ or more, 1.0 x 10 ⁹ or more, 2.0 x 10 ⁹ or more, or 5.0 x 10 ⁹ or more.

Note that various known methods can be used to measure the number of cells during or after culture. For example, a method for measuring the number of cells contained in a cell culture container after culturing using a cell culture member assuming that all cells are uniformly dispersed in the cell culture medium contained in the cell culture container is as follows: Any known method can be used as appropriate. For example, a cell counting method using CCK8 (see next text) can be suitably used. Specifically, Cell Counting Kit 8; a solution reagent manufactured by Dojindo Chemical Research Institute (hereinafter referred to as "CCK8") was used to count the number of cells in normal culture without using cell culture materials, and the absorbance and actual Find the correlation coefficient with the number of cells. Thereafter, the cells were applied and the cultured cell culture member was transferred to a medium containing CCK8, stored in an incubator for 1 to 3 hours, and the supernatant was extracted and the absorbance was measured at a wavelength of 480 nm. Calculate the number of cells from the correlation coefficient.

From another perspective, mass culture of cells means, for example, that the number of cells contained per square centimeter of the nonwoven fabric and/or polymer porous membrane is 1.0 × 10 ⁵ or more after culturing using a cell culture member. , 2.0×10 ⁵ or more, 1.0×10 ⁶ or more, 2.0×10 ⁶ or more, 5.0×10 ⁶ or more, 1.0×10 ⁷ or more, 2.0× It refers to culturing until the number of cells reaches 10 ⁷ or more, 5.0 x 10 ⁷ or more, 1.0 x 10 ⁸ or more, 2.0 x 10 ⁸ or more, or 5.0 x 10 ⁸ or more. The number of cells contained per square centimeter of the nonwoven fabric and/or polymer porous membrane can be appropriately measured using a known method such as a cell counter.

As used herein, "suspended cells" refer to, for example, cells obtained by forcibly suspending adherent cells in a medium using a proteolytic enzyme such as trypsin, or cells obtained through the above-mentioned conditioning process. Contains cells that can be cultured in suspension in a culture medium.

As used herein, "medium" refers to a cell culture medium for culturing cells, particularly animal cells. Medium is used synonymously with cell culture solution. Therefore, the medium used in the present invention refers to a liquid medium. The type of medium can be a commonly used medium, and is appropriately determined depending on the type of cells to be cultured.

In the method for culturing cells of the present invention, the first medium used in step (1) is not particularly limited as long as it is a medium that can culture cells, but for example, when culturing CHO cells, the first medium is manufactured by JX Energy Co., Ltd. BalanCD™ CHO GROWTH A can be used.

In the cell culture method of the present invention, the temperature at which cells can be cultured in step (2) may be any temperature at which cells can be adsorbed to the cell culture module, and is 10°C to 45°C, preferably 15°C to The temperature is 42°C, more preferably 20°C to 40°C, even more preferably 25°C to 39°C. Furthermore, in the cell culture method of the present invention, the time for adsorbing cells in step (2) is, for example, 5 minutes to 24 hours, preferably 10 minutes to 12 hours, and more preferably 15 minutes to 500 minutes. .

In the cell culture method of the present invention, the step (2) may include adsorbing the cells to the cell culture member of the cell culture module while shaking and/or stirring, or allowing the cells to adhere to the cell culture member of the cell culture module while standing still. Cells may be adsorbed to the cell culture member. Although the shaking method is not particularly limited, for example, a culture vessel containing the cell culture module of the present invention and cells may be placed on a commercially available shaking device and shaken. Shaking may be performed continuously or intermittently, for example, shaking and standing may be repeated alternately, and may be adjusted as appropriate. The stirring method is not particularly limited, but for example, the cell culture module of the present invention and cells may be placed in a commercially available spinner flask and stirred by rotating a stirrer. Stirring may be performed continuously or intermittently, for example, stirring and standing still may be repeated alternately, and may be adjusted as appropriate.

In the cell culture method of the present invention, the second medium used in step (3) is selected from a medium used for culturing adherent cells, such as D-MEM, E-MEM, IMDM, Ham's F -12 or the like can be used, but is not limited thereto. The second medium is preferably a medium that does not contain a component that inhibits adhesion of cells to the substrate, such as a surfactant. The second medium used is appropriately selected depending on the type of cells. In step (3), culturing in the second medium promotes the adhesion of the cells adsorbed to the cell culture member in step (2) into the cell culture member. Thereby, cells can be stably cultured without falling off the cell culture member. Further, the cell culture method of the present invention uses a cell culture module equipped with the above-described cell culture member. In the cell culture module used in the cell culture method of the present invention, the cell culture member is housed in the casing, thereby preventing the membranous cell culture member from being continuously deformed within the casing. There is. This prevents stress from being applied to cells grown within the cell culture member, suppresses apoptosis, etc., and allows stable and large amounts of cells to be cultured.

In the cell culture method of the present invention, commercially available culture devices and systems for culturing cells can be used in step (3). For example, the culture container may be a flexible bag-type culture container, or it is also possible to culture in a stirred culture container, such as a spinner flask. In addition, as the culture container, it is applicable to both open containers and closed containers. For example, petri dishes, flasks, plastic bags, test tubes, and large tanks for cell culture can be used as appropriate. Examples include cell culture dishes manufactured by BD Falcon, Nunc Cell Factory manufactured by Thermo Scientific, and the like. In addition, in the above cell culture method, by using a cell culture module, even adhesive cells that are not naturally suitable for suspension culture can be adsorbed to the cell culture module. This makes it possible to culture the cells while floating in the cell culture medium. As a device for floating culture, for example, a spinner flask or rotary culture manufactured by Corning Corporation can be used. Moreover, the step (3) may be carried out using the cell culture device described herein.

In the cell culture method of the present invention, step (3) can also be carried out using a continuous circulation type device that continuously adds and collects a medium to a culture container containing the cell culture module. be.

In the cell culture method of the present invention, the step (3) includes continuously or intermittently supplying the cell culture medium into the cell culture vessel from a cell culture medium supply means installed outside the culture vessel containing the cell culture module. It may also be a system that is supplied to At that time, a system may be used in which the cell culture medium circulates between the cell culture medium supply means and the cell culture container.

6. Method for removing suspended cells One aspect of the present invention is
(1) applying a cell culture member to a first medium containing suspended cells;
(2) maintaining the temperature at which cell culture is possible and adsorbing the cells to the cell culture member;
The present invention relates to a method for removing suspended cells, including: The cell removal method of the present invention is also referred to below as the "cell removal method of the present invention."

As the cell culture member used in the cell removal method of the present invention, the above-mentioned cell culture member can be used. The cell culture member may be the above-described cell culture module, or may be a cell culture member not housed in a casing.

In the method for culturing cells of the present invention, the first medium used in step (1) is not particularly limited as long as it is a medium that can culture cells, but for example, when culturing CHO cells, the first medium is manufactured by JX Energy Co., Ltd. BalanCD™ CHO GROWTH A can be used.

In the cell removal method of the present invention, the temperature at which cells can be cultured in step (2) may be any temperature at which cells can be adsorbed to the cell culture module, and is 10°C to 45°C, preferably 15°C to The temperature is 42°C, more preferably 20°C to 40°C, even more preferably 25°C to 39°C. Furthermore, in the cell culture method of the present invention, the time for adsorbing cells in step (2) is, for example, 5 minutes to 24 hours, preferably 10 minutes to 12 hours, and more preferably 15 minutes to 500 minutes. .

In the cell removal method of the present invention, the step (2) may include adsorbing cells to the nonwoven fabric and polymer porous membrane of the cell culture module while shaking and/or stirring, or allowing the cells to adsorb to the nonwoven fabric and polymer porous membrane of the cell culture module. Cells may be adsorbed to the nonwoven fabric and polymer porous membrane of the cell culture module. Although the shaking method is not particularly limited, for example, a culture vessel containing the cell culture module of the present invention and cells may be placed on a commercially available shaking device and shaken. Shaking may be performed continuously or intermittently, for example, shaking and standing may be repeated alternately, and may be adjusted as appropriate. The stirring method is not particularly limited, but for example, the cell culture module of the present invention and cells may be placed in a commercially available spinner flask and stirred by rotating a stirrer. Stirring may be performed continuously or intermittently, for example, stirring and standing still may be repeated alternately, and may be adjusted as appropriate.

Conventionally, in order to remove cells from a culture medium containing cells using the cell removal method of the present invention, operations such as centrifugation treatment and filter treatment were required. According to the method of the present invention, cells can be removed from the culture medium without using a filter or the like.

Hereinafter, the present invention will be described in more detail based on Examples. Note that the present invention is not limited to these examples. Those skilled in the art can easily make modifications and changes to the present invention based on the description in this specification, and these are included within the technical scope of the present invention.

The polyimide porous membrane used in the following examples consists of 3,3',4,4'-biphenyltetracarboxylic dianhydride (s-BPDA) as a tetracarboxylic acid component and 4,4 as a diamine component. A polyamic acid solution composition containing a polyamic acid solution obtained from '-diaminodiphenyl ether (ODA) and polyacrylamide as a coloring precursor is molded, and then heat treated at 250 ° C. or higher. It was prepared. The obtained porous polyimide membrane has a three-layer structure including a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. It is a membrane, and the average pore diameter of the pores present in the surface layer A is 19 μm, the average pore diameter of the pores present in the surface layer B is 42 μm, the thickness is 25 μm, and the porosity is 74 μm. %Met.
In addition, the nonwoven fabrics used in the examples were prepared using raw cotton manufactured by Ube Eximo Co., Ltd. with the configurations shown in Table 1 below, and were further subjected to treatments.

Example 1: Experimental results of a Petri dish-type rotating bioreactor using a cell culture module Anti-human IL-8 antibody-producing CHO-DP12 cells (ATCC CRL-12445) were conditioned and suspended in a medium (BalanCD (trademark) CHO Growth). A) was used for suspension culture until the number of viable cells per ml reached 3.20 x 10 ⁶ cells/ml (total number of cells 3.40 x 10 ⁶ cells/ml, viable cell rate 96%). continued. A total of 6 types of culture modules (2 types) were prepared on 3 types of nonwoven fabrics as shown in Figure 21, and after washing with diluted Milton, ultrapure water, and water containing 70% ethanol, they were sterilized. Dry and ready. Table 2 shows the configuration of each experiment. The size of the nonwoven fabric and polyimide porous membrane used in each module was 1.0 x 1.0 cm.

Place 4 of each of the 6 sterilized modules shown in Table 2 in a cell culture dish with a diameter of 10 cm, and add 8 ml of CHO cell monolayer culture medium KBM270 manufactured by Kohjin Bio Co., Ltd. and Balan CD, a suspension cell medium. (Trademark) A mixed solution of 3 ml of CHO Growth A was added and immersed for 5 minutes at a shaking speed of 35 rpm using a Kennis program shaker in a CO ₂ incubator. After stopping shaking, add CHO DP-12 suspension cell culture solution (total number of cells: 3.40×10 ⁶ cells/ml, number of living cells: 3.20×10 ⁶ cells/ml, number of dead cells: 1.30×10 ⁵ cells/ml). ml, viable cell rate 96%) was added, and after gently stirring by hand, the cells were adsorbed in a CO ₂ incubator for about 19 hours at a shaking speed of 35 rpm using a Kennis program shaker. The medium in the dish was discarded, the number of cells in the recovered medium was measured, and the cell adsorption rate was calculated. In all experiments, no living cells were observed and the adsorption rate was 100%.

12 ml of CHO cell monolayer culture medium KBM270 manufactured by Kohjin Bio Co., Ltd. was added to the empty petri dish, and culture was started by operating a program shaker at a shaking speed of 35 rpm. The medium was replaced every day (the old medium was discarded after sampling, and 12 ml of new medium was added), and the daily glucose consumption, lactic acid production, lactate dehydrogenase amount, and antibody production in the medium were measured using Roche Diagnostics. Measurement was performed using Cedex Bio manufactured by Stix. It has been found that depending on the type of module and the type of nonwoven fabric, glucose is consumed over time and antibodies and lactic acid are continuously produced. Table 3 shows the amount of antibodies produced. It has been found that in a preferred combination, continuous antibody production can be achieved.

Example 2: Small bag bioreactor experimental results using cell culture module In the same manner as in Example 1, anti-human IL-8 antibody-producing CHO-DP12 cells (ATCC CRL-12445) were conditioned and suspended in a culture medium ( BalanCD (trademark) CHO Growth A) was used for suspension culture, and the number of viable cells per ml was 3.20 x 10 ⁶ cells/ml, (total number of cells 3.40 x 10 ⁶ cells/ml, viable cell rate 96 %). A total of 5 types of modules (excluding A-2) of the module configurations shown in Table 2 were prepared and diluted with culture modules (2 types) in the combinations shown in Figure 21 on 3 types of nonwoven fabrics. After washing with Milton, ultrapure water, and water containing 70% ethanol, the sample was sterilized and dried to complete preparation. The size of the nonwoven fabric and polyimide porous membrane used in each module was 1.0 x 1.0 cm.

A Nipro gas-permeable culture bag (A350-NL) is sterilized along the dotted line shown in Figure 22 to create a tube-shaped bag, four sterilized modules are added thereto, and the bag is sterilized using a sealer. A bag-shaped bioreactor was created by fusing it to Add a mixture of 8 ml of CHO cell monolayer culture medium KBM270 manufactured by Kohjin Bio Co., Ltd. and 3 ml of suspended cell medium BalanCD (trademark) CHO Growth A into the bag, and soak for 5 minutes using a rocking mixer in a CO ₂ incubator. I let it happen. After stopping the mixer, add CHO DP-12 suspension cell culture solution (total number of cells: 3.40 x 10 ⁶ cells/ml, number of living cells: 3.20 x 10 ⁶ cells/ml, number of dead cells: 1.30 x 10 ⁵ cells). /ml, viable cell rate 96%) was added, and after gently stirring by hand, the cells were adsorbed in a CO ₂ incubator for about 19 hours using a rocking mixer. After the adsorption step was completed, the cell fluid in the bag was collected, the number of cells in the medium was measured, and the cell adsorption rate was calculated. Table 4 shows the cell adsorption rate calculated from the number of recovered cells.

12 ml of CHO cell monolayer culture medium KBM270 manufactured by Kohjin Bio Co., Ltd. was added to the empty bag, and culture was started by shaking the bag with a rocking mixer. The medium was replaced every day (the old medium was discarded after sampling, and 12 ml of new medium was added), and the daily glucose consumption, lactic acid production, lactate dehydrogenase amount, and antibody production in the medium were measured using Roche Diagnostics. Measurement was performed using Cedex Bio manufactured by Stix. It has been found that depending on the type of module and the type of nonwoven fabric, glucose is consumed over time and antibodies and lactic acid are continuously produced. Table 5 shows the amount of antibodies produced. It has been found that in a preferred combination, continuous antibody production can be achieved.

Claims (19)

A cell culture member made of a nonwoven fabric and a porous polymer membrane;
A cell culture module having two or more culture medium inlets and a casing in which the cell culture member is housed, wherein:
The nonwoven fabric is a nonwoven fabric composed of 20 % by weight or more of core-sheath composite fibers,
The basis weight of the nonwoven fabric is 1 to 50 g/ ^m2 ,
The core and sheath of the core-sheath type composite fiber are made of a polyolefin polymer, the melting point of the polymer in the sheath is lower than the melting point of the polymer in the core, and the sheath-core ratio is 90:10 to 10:90. and
The nonwoven fabric has pores; and the polymer porous membrane has a surface layer A and a surface layer B having a plurality of pores, and a macrovoid layer sandwiched between the surface layer A and the surface layer B. A porous polymer membrane with a three-layer structure,
The average pore diameter of the pores present in surface layer A is smaller than the average pore diameter of pores present in surface layer B,
The macrovoid layer has a partition wall coupled to the surface layers A and B, and a plurality of macrovoids surrounded by the partition wall and the surface layers A and B,
pores in surface layers A and B communicate with the macrovoids;
Here, within the casing (i) the two or more independent cell culture members are aggregated,
(ii) the cell culture member is folded;
(iii) the cell culture member is rolled and/or
(iv) the cell culture member is tied into a rope;
accommodated ,
The nonwoven fabric comprises two or more types of core-sheath composite fibers having different finenesses and/or sheath-core ratios.
Contains synthetic fibers,
The cell culture module described above, wherein the nonwoven fabric and the polymer porous membrane are alternately laminated, or the nonwoven fabric is sandwiched between the polymer porous membrane. The cell culture module according to claim 1, wherein the nonwoven fabric has been subjected to a hydrophilic treatment. The cell culture module according to claim 1 or 2, wherein the core-sheath type composite fiber has a fineness of 0.05 to 2.2 decitex (dTex). The cell culture module according to any one of claims 1 to 3, wherein the core-sheath type composite fibers form binding portions at contact intersections by thermal fusion. The cell culture module according to any one of claims 1 to 4 , wherein the diameter of the medium outflow port is larger than the diameter of the cells and smaller than the diameter through which the nonwoven fabric and the polymer porous membrane flow out. A claim in which the hydrophilic treatment is selected from the group consisting of fluorine gas treatment, normal pressure plasma treatment, vacuum plasma treatment, corona treatment, graft polymerization treatment of hydrophilic monomers, sulfonation treatment, and surfactant imparting treatment. The cell culture module according to any one of items 1 to 5 . The cell culture module according to any one of claims 1 to 6 , wherein the nonwoven fabric has an average pore diameter of 0.01 to 100 μm. The cell culture module according to any one of claims 1 to 7 , wherein the total thickness of the nonwoven fabric is 5 to 500 μm. The cell culture module according to any one of claims 1 to 8, wherein the area ratio of the binding portion formed by the core-sheath composite fibers is 5 to 30%. The cell culture module according to any one of claims 1 to 9 , wherein the polymer porous membrane has pores with an average pore diameter of 0.01 to 100 μm. The cell culture module according to any one of claims 1 to 10 , wherein the surface layer A has an average pore diameter of 0.01 to 50 μm. The cell culture module according to any one of claims 1 to 11 , wherein the surface layer B has an average pore diameter of 20 to 100 μm. The cell culture module according to any one of claims 1 to 12 , wherein the total membrane thickness of the polymer porous membrane is 5 to 500 μm. The cell culture module according to any one of claims 1 to 13 , wherein the polymer porous membrane is a polyimide porous membrane. The cell culture module according to claim 14 , wherein the porous polyimide membrane is a porous polyimide membrane containing polyimide obtained from a tetracarboxylic dianhydride and a diamine. The polyimide porous membrane is colored by molding a polyamic acid solution composition containing a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine and a coloring precursor, and then heat-treating the composition at 250°C or higher. The cell culture module according to claim 14 or 15 , which is a polyimide porous membrane. Cell culture module according to any one of claims 1 to 16 , wherein the polymeric porous membrane is a polyethersulfone (PES) porous membrane. The cell culture module according to any one of claims 1 to 17 , wherein the casing has a mesh-like structure. The cell culture module according to any one of claims 1 to 18 , wherein the casing is made of a non-flexible material.

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