Substrate for suspension culture of adhesive cells

Abstract

To provide a substrate for suspension culture of adhesive cells, which is used for suspension culture of adhesive cells capable of producing a substance selected from proteins, glycoproteins, viruses, and viral vectors, wherein the substrate exhibits superior culture performance and enables further increase in the production of the substance.SOLUTION: A substrate for suspension culture of adhesive cells comprises a laminate in which a porous layer and a nonwoven fabric layer having an apparent density of less than 0.440 g / cm3 are laminated.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a substrate for suspension culture of adherent cells, which is used for suspension culture of adherent cells capable of producing a substance selected from proteins, glycoproteins, viruses, and viral vectors. [Background technology]

[0002] To obtain large amounts of components (substances selected from proteins, glycoproteins, viruses, and viral vectors) produced by adherent cells, the cells are grown in a free-floating state in a medium filled in a container (suspension culture). Since the adherent cells release the above-mentioned components into the medium during the culture process, the above-mentioned components can be obtained in large amounts by recovering the medium.

[0003] To efficiently culture adherent cells and facilitate their removal from the medium, the adherent cells are cultured in suspension with the adherent cells attached to the substrate, while the adherent cells are immersed in a culture solution and cultured in suspension. Patent Document 1, for example, discloses a sheet for in vitro cell culture, comprising a fibrous matrix attached to a porous support sheet. Patent Document 1 also discloses that a plasma-treated polyester nonwoven fabric can be used as the matrix for adhering adherent cells, and that a polypropylene mesh can be used as the porous support sheet supporting the matrix. Patent Document 1 also discloses that the porosity of the porous support sheet should be in the range of 40 to 95%, with a preferred porosity being 60 to 80%, to allow cells and nutrients to enter the porous support sheet and for waste products to be removed from the sheet. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 8-29077 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors further investigated conventional substrates for suspension culture of adherent cells in order to provide a substrate for suspension culture of adherent cells with excellent culture performance. [Means for solving the problem]

[0006] As a result, it was found that by adjusting the apparent density of the nonwoven fabric layer laminated to a porous layer such as a mesh, it is possible to further increase the production amount of the above-mentioned components and provide a substrate for suspension culture of adherent cells with excellent culture performance. Specifically, when the apparent density of the nonwoven fabric layer is 0 g / cm 3 Greater than 0.440g / cm 3 It has been found that when the concentration is less than 1000 ppm, the amount of the above-mentioned components produced is further increased, making it possible to provide a substrate for suspension culture of adherent cells with excellent culture performance.

[0007] Furthermore, because the specific gravity of the constituent fibers of the nonwoven fabric layer is greater than 1, the substrate for suspension culture is prevented from floating on the surface of the culture medium in a culture medium composed primarily of water, which has a specific gravity of 1. Furthermore, even when a substrate that freely floats in a container is present on the surface of the culture medium, the nonwoven fabric layer side of the substrate for suspension culture tends to face the side opposite the liquid surface. As a result, adherent cells that adhere and are cultured primarily on the nonwoven fabric layer side can be kept in constant contact with the culture medium. As a result, a substrate for suspension culture of adherent cells with even better culture performance can be provided.

[0008] Furthermore, when the porous layer has triangular openings, the nonwoven fabric layer can be effectively supported with a smaller number of porous layers. Specifically, the nonwoven fabric layer can be supported more efficiently than a porous layer with square openings. As a result, the opening rate of the porous layer can be increased, allowing a larger amount of culture solution to pass through the nonwoven fabric layer, thereby providing a substrate for suspension culture of adherent cells with even better culture performance.

[0009] Accordingly, the present invention relates to the following inventions: [1] A substrate for suspension culture of adherent cells, used for suspension culture of adherent cells capable of producing a substance selected from a protein, a glycoprotein, a virus, and a viral vector, comprising: Porous layer and apparent density of 0.440g / cm 3 and a nonwoven fabric layer of less than 1000 mm. Substrate for suspension culture of adherent cells. [2] The specific gravity of the constituent fibers of the nonwoven fabric layer is 1 g / cm 3 A substrate for the suspension culture of larger, adherent cells.[1] [3] The substrate for suspension culture of adherent cells according to [1] or [2], wherein the porous layer has triangular openings. [Effects of the Invention]

[0010] According to the present invention, the apparent density of the nonwoven fabric layer is 0 g / cm 3 Greater than 0.440g / cm 3 Since the amount of the suspension culture medium is less than 1000 mg / L, the amount of the above-mentioned components produced is further increased, and a substrate for suspension culture of adherent cells with excellent culture performance can be provided.

[0011] In a preferred embodiment of the present invention, when the specific gravity of the constituent fibers of the nonwoven fabric layer is greater than 1, adherent cells are primarily cultured on the nonwoven fabric layer side, and are brought into contact with the culture medium, thereby providing a substrate for suspension culture of adherent cells with excellent culture performance.

[0012] In another preferred embodiment of the present invention, when the porous layer has triangular openings, the nonwoven fabric layer can be effectively supported with a small number of porous layers, which increases the opening rate of the porous layer and allows a larger amount of culture solution to pass through the nonwoven fabric layer, thereby providing a substrate for suspension culture of adherent cells with even better culture performance. [Brief explanation of the drawings]

[0013] [Figure 1] (a)(b) Graphs showing the results of measuring the amount of viral vector contained in the recovered culture medium supernatant after 10 days of culture in experimental system A using the substrate for suspension culture of adherent cells prepared in Example 1. [Figure 2] (a)(b) Graphs showing the results of measuring the total number of adherent cells adhering to the culture substrate after two days of culture in experimental system A using the substrate for suspension culture of adherent cells prepared in Example 1. [Figure 3] 1 is a graph showing the results of measuring the number of viable adherent cells adhered to each culture substrate in a vial after culturing for one day using experimental system B using the substrate for suspension culture of adherent cells prepared in Example 2. [Figure 4] This graph shows the results of carrying out 4-day culture in experimental system C using the substrate for suspension culture of adherent cells prepared in Reference Example 1, and measuring the number of viable cells of adherent cells adhering to each culture substrate in a vial. DETAILED DESCRIPTION OF THE INVENTION

[0014] In the present invention, various configurations can be appropriately selected, for example, the following configurations. Unless otherwise specified, the various measurements described in this invention were performed under atmospheric pressure. Furthermore, measurements were performed at a temperature of 25°C. Unless otherwise specified, the various measurement results described in this invention were measured to a value one decimal place smaller than the desired value, and the value was calculated by rounding the value. Specifically, when the desired value is measured to one decimal place, the value was measured to two decimal places, and the obtained value was rounded to one decimal place to calculate the desired value. The upper and lower limits exemplified in this invention can be combined in any combination.

[0015] In the present invention, the porous layer forming the laminate is a sheet-like structure having a three-dimensional space (voids) through which the medium for adherent cells, products produced by adherent cells, and waste products secreted by adherent cells can pass. The porous layer is not particularly limited as long as it has sufficient strength to prevent deformation of the nonwoven fabric during culture (particularly during shaking culture). Examples of porous layers include fiber sheets, more specifically, woven fabrics, knitted fabrics, fiber webs, nonwoven fabrics, meshes, porous films, and foams. Meshes, especially those with a high opening ratio, can efficiently reinforce the nonwoven fabric layer. As a result, a substrate for suspension culture of adherent cells can be provided that is more liquid-permeable and therefore has even better culture performance.

[0016] When a fiber sheet is used as the porous layer, the fibers constituting the fiber sheet are not particularly limited as long as they can form a sheet-like structure that satisfies the above conditions, and for example, either organic fibers or inorganic fibers, or a combination of organic fibers and inorganic fibers, can be used.

[0017] Examples of components of organic fibers include polyolefin resins (such as polyethylene, polypropylene, and polyolefin resins in which part of the hydrocarbon is substituted with a cyano group or a halogen such as fluorine or chlorine), ethylene-vinyl acetate copolymer resins, polymethylpentene, styrene resins, polyvinyl alcohol resins, polyether resins (such as polyether ether ketone, polyacetal, modified polyphenylene ether, and aromatic polyether ketone), polyester resins (such as polyethylene terephthalate, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene naphthalate, polybutylene naphthalate, polycarbonate, polyarylate, and wholly aromatic polyester resins), polyimide resins, polyamideimide resins, polyamide resins (such as aromatic polyamide resins, aromatic polyetheramide resins, and nylon resins), and resins having a nitrile group (such as polyamide-based resins). Examples of the polymerizable polymer include polyacrylonitrile, polyacrylonitrile copolymers, etc.), urethane resins, epoxy resins, polysulfone resins (e.g., polysulfone, polyethersulfone, etc.), fluorine-based resins (e.g., polytetrafluoroethylene, polyvinylidene fluoride, etc.), cellulose resins, polybenzimidazole resins, acrylic resins (e.g., polyacrylonitrile resins copolymerized with acrylic esters or methacrylic esters, modacrylic resins copolymerized with acrylonitrile and vinyl chloride or vinylidene chloride, etc.), polylactic acid, poly(lactic acid-glycolic acid) (PLGA), polycaprolactone, polypropylene succinate, or proteins (e.g., gelatin, collagen), water-soluble polymers (e.g., polyvinyl alcohol, polyethylene glycol, poly(N-vinylpyrrolidone), Nafion), poly(methyl methacrylate) (PMMA)), and the like can be used alone or in combination.

[0018] Examples of constituent components of inorganic fibers include SiO2, Al2O3, B2O3, TiO2, ZrO2, CeO2, FeO, Fe3O4, Fe2O3, VO2, V2O5, SnO2, CdO, LiO2, WO3, Nb2O5, Ta2O5, In2O3, GeO2, PbTi4O9, LiNbO3, BaTiO3, PbZrO3, KTaO3, Li2B4O7, NiFe2O4, and SrTiO3, and the fibers may be composed of an oxide of one of these components or two or more oxides.

[0019] When the fiber sheet is made of two or more types of fibers with different compositions, it may be a fiber sheet made of a mixture of various fibers, a fiber sheet having fiber layers with different compositions, a fiber sheet made of a mixture of the above-mentioned organic fibers and inorganic fibers, or a fiber sheet having an organic fiber layer and an inorganic fiber layer.

[0020] The specific gravity of the fibers constituting the porous layer is preferably such that the substrate for suspension culture can be prevented from floating on the surface of the culture medium in a culture medium composed mainly of water, which has a specific gravity of 1. Furthermore, even when a freely floating substrate in a container is present on the surface of the culture liquid, it is preferable to adjust the specific gravity of the constituent fibers of the porous layer so that the nonwoven fabric layer side of the substrate for suspension culture is easily positioned opposite the liquid surface. In this case, the specific gravity of the constituent fibers of the porous layer is preferably lighter than the specific gravity of the constituent fibers of the nonwoven fabric layer, so that adherent cells that are cultured by adhering mainly to the nonwoven fabric layer side can be kept in constant contact with the culture liquid. As a specific example, when the constituent fibers of the nonwoven fabric layer have a specific gravity greater than 1 (a specific gravity greater than that of water), it is preferable that the constituent fibers of the porous layer have a specific gravity of 1 or less (a specific gravity less than that of water).

[0021] The shape of the openings in the porous layer is not particularly limited, and examples thereof include polygons (e.g., triangles, rectangles, hexagons, and other polygons), circles, and ellipses. A triangular shape is preferable because the nonwoven fabric layer can be effectively supported with a small amount of porous layer, thereby increasing the opening rate of the porous layer and allowing a larger amount of culture solution to pass through the nonwoven fabric layer.

[0022] The nonwoven fabric layer forming the laminate is a sheet-like structure having three-dimensional spaces (voids) through which the medium for adherent cells, products produced by adherent cells, and waste products secreted by adherent cells can pass, and has an apparent density of 0 g / cm. 3 Larger, 0.440g / cm 3 The above description regarding the constituent components of the constituent fibers when a fiber sheet is used for the porous layer can be applied as is to the constituent components of the fibers constituting the nonwoven fabric layer and the configuration of the nonwoven fabric layer, as long as the apparent density of the nonwoven fabric layer is within the above range.

[0023] The apparent density of the nonwoven fabric layer is 0 g / cm 3 Larger, 0.440g / cm 3 less than 0.111 g / cm 3 ~0.400g / cm 3 and more preferably 0.136 g / cm 3 ~0.341g / cm 3 is.

[0024] The apparent density of the nonwoven fabric layer in this specification is not limited to the following, but can be determined, for example, by measuring the basis weight and thickness of the nonwoven fabric and dividing the basis weight by the thickness, as shown in the examples described later. In addition, the apparent density of the nonwoven fabric layer can be determined by removing unnecessary layers such as the porous layer from the substrate for suspension culture to obtain a nonwoven fabric constituting the nonwoven fabric layer, and measuring the basis weight and thickness of the obtained nonwoven fabric. That is, the apparent density of the nonwoven fabric can be calculated using the measurement results, and the calculated apparent density can be used as the apparent density of the nonwoven fabric layer. The "weight per unit area" is the mass per unit area of ​​the main surface of the object to be measured, obtained by the method specified in JIS L1085:1998, 6.2 "Mass per unit area". 2The "thickness" was determined by subjecting the object to be measured to a compression elasticity tester (Mitutoyo Corporation, Litematic VL-50S-B, using a probe with a circular shape of 28.77 mm in diameter that comes into contact with the object to be measured, and applying a force of 0.115 N between both main surfaces of the object to be measured by the probe during measurement). The porosity of the nonwoven fabric can be adjusted to provide a substrate for suspension culture of adherent cells with excellent culture performance. Specifically, the lower limit can be higher than 64%, 70% or more, or higher than 80%. Meanwhile, the upper limit can be lower than 95%, 94% or less, or 90% or less.

[0025] The porous layer and the nonwoven fabric layer can be laminated by a known method to obtain a laminate. For example, the laminate may be a laminate structure in which the porous layer and the nonwoven fabric layer are simply superposed on each other, or a laminate in which the layers are bonded together using a binder, a hot melt web, or fiber bonding, or by subjecting the layer to a bonding treatment such as heat sealing or ultrasonic welding.

[0026] Substances that can be produced by culturing adherent cells using the substrate for suspension culture of the present invention include proteins, glycoproteins, viruses, and viral vectors, but they may be natural substances that can be originally produced by adherent cells based on their genomic information, or exogenous substances that can be produced based on genetic information introduced into adherent cells by genetic recombination.

[0027] Adherent cells to which the substrate for suspension culture of the present invention can be applied are adhesive cells capable of producing the above-mentioned substances, and are not particularly limited as long as their culture systems have been established or can be established in the future. Examples include HEK293 cells, 293T cells, Vero cells, CHO cells, hybridomas consisting of B cells and myeloma cells, Cos cells, and MDCK cells. [Example]

[0028] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0029] Example 1: Experimental System A (Method for preparing a substrate for suspension culture of adherent cells) Hydroentangled polyethylene terephthalate nonwoven fabric (basis weight: 106 g / m) that has been given hydrophilicity through plasma treatment. 2 , average fiber diameter: 11.5 μm, specific gravity of constituent fibers: 1.4 g / cm 3 ) were prepared. Next, the prepared nonwoven fabrics were placed in a heat press (heating temperature: 130°C, treatment time: 1 minute) to adjust the thickness of the nonwoven fabric. The pressing pressure applied to each nonwoven fabric was individually changed. In this way, 10 types of nonwoven fabrics with different apparent densities were prepared, as shown in Table 1.

[0030] [Table 1]

[0031] In addition, a mesh made of a mixed resin of polyethylene and ethylene vinyl acetate copolymer resin (weight: 14 g / m 2 , Thickness: 0.11 mm, Has triangular openings, Specific gravity of constituent fibers: 1.0 g / cm 3 Next, the nonwoven fabrics and meshes prepared above were stacked to form a laminate, which was then placed in a heat press (heating temperature: 100°C, treatment time: 10 seconds, press pressure: 0.5 MPa) to bond the nonwoven fabrics and meshes together. Then, square pieces (6 mm per side) weighing 0.5 g were cut out of each laminate. The cut pieces weighing 0.5 g were used as substrates for suspension culture of adherent cells (hereinafter referred to as substrates). The basis weight, thickness, apparent density, and porosity of the nonwoven fabrics remained unchanged before and after the interlayer bonding.

[0032] (Method for culturing adherent cells and method for confirming the amount of viral vector produced by adherent cells) A 125 mL culture flask was filled with 29 mL of medium for adherent cells (DMEM medium adjusted to a final concentration of 10% fetal bovine serum, 100 units / mL penicillin G, and 100 μg / mL streptomycin sulfate), and a substrate comprising any of nonwoven fabrics 1 to 10 was placed inside. Furthermore, 1 mL of a cell suspension containing 293HEK-EB cells, which were the cells to be cultured, was added to the medium. The seeding amount of adherent cells was 2 × 10 per mL of the medium in the culture flask. 7 The concentration was adjusted to cells / mL. The culture flask was shaken for 3 days using a rotary shaker installed in an incubator (shaking conditions: temperature 37°C, rotation speed 80 rpm). Then, to transfect the cultured adherent cells, A plasmid containing the ZsGreen1 gene, A plasmid containing genes encoding Rep proteins (proteins necessary for viral replication) and Cap proteins (proteins that make up the capsid), a plasmid containing genes encoding adenovirus-derived E1a protein, E1b protein, E2 protein, E4 protein, etc. The above three types of plasmids were prepared, and the medium was replaced with transfection medium (DMEM medium adjusted to a final concentration of 100 units / mL penicillin G and 100 μg / mL streptomycin sulfate), and transfection was performed using the transfection reagent PEI-MAX.

[0033] After 10 days of shaking (after culturing the adherent cells for 10 days), the culture supernatant was collected and the amount of viral genome contained in the collected culture supernatant was determined by quantitative PCR. The amount of the viral genome thus determined was considered to be the amount of viral vector, and the amount of viral vector produced by the adherent cells and contained in the culture supernatant was determined. The results of a comparison of the amount of viral vectors produced by adherent cells when a substrate comprising any one of nonwoven fabrics 1 to 8 was used are shown in FIG. 1(a). Figure 1(b) shows the results of a comparison of the amount of viral vectors produced by adherent cells when a substrate comprising nonwoven fabric 1, nonwoven fabrics 9-10, or nonwoven fabric 7 was used. In Figure 1(b), the amount of viral vectors produced by adherent cells when a substrate comprising nonwoven fabric 9-10 or nonwoven fabric 7 was used is expressed as a percentage, with the amount of viral vectors produced by adherent cells when a substrate comprising nonwoven fabric 1 was used being taken as 100%. Note that only numbers are shown on the X-axis in Figure 1, and the nonwoven fabrics are not shown. For example, 1 on the X-axis in Figure 1 indicates the results when a substrate comprising nonwoven fabric 1 was used.

[0034] (Method for culturing adherent cells) The adherent cells were cultured by shaking the culture flask for 2 days in the same manner as described above (method for culturing adherent cells and method for confirming the amount of viral vector produced by adherent cells).

[0035] After shaking for 2 days as described above (after culturing the adherent cells for 2 days), the substrate was removed from the medium, and the adherent cells adhered to the removed substrate were treated with a cell treatment reagent (Reagent A100) from Chemometec to extract nuclei from the adherent cells. The number of nuclei obtained by measurement or counting was then measured using a cell counter NucleoCounter (Chemometec) or a nuclei counting chamber, and the total number of adherent cells adhered to the substrate was calculated. The results of a comparison of the total number of adherent cells adhering to the substrates comprising any one of the nonwoven fabrics 1 to 8 are shown in FIG. 2(a). Figure 2(b) shows the results of a comparison of the total cell counts of adherent cells adhered to a substrate comprising nonwoven fabric 1 and a substrate comprising any of nonwoven fabrics 9 and 10. In Figure 2(b), the total cell counts of adherent cells adhered to a substrate comprising either nonwoven fabrics 9 to 10 or nonwoven fabric 7 are expressed as a percentage, with the total cell count of adherent cells adhered to a substrate comprising nonwoven fabric 1 being set at 100%.

[0036] (Consideration) 0.440g / cm 3 The substrates with nonwoven fabric layers having an apparent density of 0.440 g / cm or more had a significantly reduced amount of viral vectors compared to substrates with nonwoven fabric layers having a lower apparent density. 3 It has been found that a substrate having a nonwoven layer with an apparent density of less than 1000 sq. mm provides a large amount of components produced by adherent cells. The total number of adherent cells cultured on the substrate used in the above measurement was confirmed to be 0.440 g / cm 3 It has also been found that a large number of adherent cells can be cultured by using a substrate with a nonwoven fabric layer having an apparent density of less than 100 μm. The ability to culture a large number of adherent cells advantageously correlates with the yield of a large amount of components produced by the adherent cells.

[0037] Patent Document 1 discloses that the porosity (which is considered to correspond to the void ratio in the present invention) of the porous support sheet (which corresponds to the nonwoven fabric layer in the present invention) should be in the range of 40 to 95%, and a preferred porosity is 60 to 80%. However, contrary to this disclosure, the results of Example 1 showed that substrates for suspension culture of adherent cells comprising nonwoven fabrics with porosities of 82% (nonwoven fabric 5) to 90% (nonwoven fabric 1) were able to obtain larger amounts of components produced by adherent cells (specifically, viral vectors) and to culture larger amounts of adherent cells, compared to substrates for suspension culture of adherent cells comprising a nonwoven fabric with a porosity of 69% (nonwoven fabric 7), which would be preferable. Therefore, in order to realize a substrate for suspension culture of adherent cells that can obtain even larger amounts of components produced by adherent cells, it was clear that adjusting the porosity of the nonwoven fabric layer that constitutes the substrate was insufficient, and that the apparent density of the nonwoven fabric layer that constitutes the substrate had to be adjusted.

[0038] Example 2: Experimental System B (Method for preparing a substrate for suspension culture of adherent cells) In addition to the substrates comprising any of the nonwoven fabrics 1 to 8 used in Experimental System A, two new substrates comprising either of two new nonwoven fabrics, A1 or A2, prepared by changing the pressing pressure were prepared. These 10 substrates are summarized in Table 2.

[0039] [Table 2]

[0040] (Method for culturing adherent cells) Eighteen square pieces (6 mm per side) were cut out from each laminate, and the cut pieces were used as substrates for suspension culture of adherent cells (hereinafter referred to as substrates). Eighteen substrates were placed in a 37 mL vial together with 10 mL of pure water, and the vial was sterilized by autoclaving. The pure water was removed from the autoclaved vial, and the substrates remaining in the vial were washed with PBS(-). 4.0 mL of medium for adherent cells (EMEM medium adjusted to a final concentration of 10% fetal bovine serum, 100 units / mL penicillin G, and 100 μg / mL streptomycin sulfate) was added to the vial, followed by the addition of 1.0 mL of a cell suspension of adherent HEK293 cells. This resulted in a seeding density of 2.25 × 10 cells per vial. 6 It became cells. The vials were shaken for 1 day using a rotary shaker placed in an incubator (shaking conditions: temperature 37°C, rotation speed 100 rpm) to culture the adherent cells. After 1 day of shaking (after 1 day of culturing the adherent cells), the number of viable cells adhering to each substrate in the vial was determined using a BCA assay kit. The results are shown in Figure 3.

[0041] (Consideration) 0.440g / cm 3 By using a substrate with a nonwoven fabric layer having an apparent density of less than 100 μm, it was possible to culture a large number of adherent cells. The ability to culture a large number of adherent cells leads to the production of a large amount of components produced by the adherent cells. In order to obtain a large amount of components produced by the adherent cells as in Experimental System A, we attempted to culture the adherent cells for a period longer than 4 days using the same substrate as in Example 2. No areas where the cultured adherent cells had detached were observed on the substrate.

[0042] <<Reference Example 1: Experimental System C>> (Method for preparing a substrate for suspension culture of adherent cells) Hydroentangled polyethylene terephthalate nonwoven fabric (basis weight: 56 g / m) that has been given hydrophilicity through plasma treatment. 2 , average fiber diameter: 12.1 μm, specific gravity of constituent fibers: 1.4 g / cm 3 ) were prepared. Next, the prepared nonwoven fabrics were placed in a heat press (heating temperature: 130°C, treatment time: 1 minute) to adjust the thickness of the nonwoven fabric. The pressing pressure applied to each nonwoven fabric was individually changed. In this way, nine types of nonwoven fabrics with different apparent densities were prepared, as shown in Table 3.

[0043] [Table 3]

[0044] Twelve square pieces (6 mm on a side) were cut from each of nine types of nonwoven fabric with different apparent densities. These pieces were used as substrates for suspension culture of adherent cells (hereafter referred to as substrates).

[0045] (Method for culturing adherent cells) Twelve substrates were placed in a 37 mL vial together with 10 mL of pure water, and the vial was sterilized by autoclaving. The pure water was removed from the autoclaved vial, and the substrates remaining in the vial were washed with PBS(-). 1.5 mL of medium for adherent cells (DMEM medium adjusted to a final concentration of 10% fetal bovine serum, 100 units / mL penicillin G, and 100 μg / mL streptomycin sulfate) was added to the vial, followed by the addition of 1.0 mL of a cell suspension of adherent NIH3T3 cells. This resulted in a seeding density of 1.5 x 10 cells per vial. 6 It became cells. The vials were shaken for 4 days using a rotary shaker (shaking board: temperature 37°C, rotation speed 100 rpm) installed in an incubator, and the adherent cells were cultured. After 4 days of shaking (after 4 days of culturing the adherent cells), the number of viable cells in the vials was determined using the WST-8 assay kit. The results are shown in Figure 4.

[0046] (Consideration) 0.440g / cm 3 Even when a nonwoven fabric having an apparent density of less than 100 μm was used as a substrate (a nonwoven fabric alone without being supported by a mesh), a large number of adherent cells could be cultured.

[0047] Example 2: Experimental System C In order to obtain a large amount of components produced by the adherent cells, as in Experimental System A, we attempted to culture the adherent cells for a period longer than 4 days using the same substrate as in Reference Example 1. However, areas on the substrate where the cultured adherent cells had detached were observed. Note that the physiological activity of the adherent cells detached from the substrate is reduced or lost, making it difficult to obtain the components produced by the adherent cells. The reason for this was thought to be that the nonwoven fabric used as the substrate was not supported by a mesh and was therefore easily deformed by shaking, which resulted in the adherent cells being detached from the nonwoven fabric during culture. Therefore, it was found that a substrate for suspension culture of adherent cells, which is used to obtain large amounts of components produced by adherent cells, must have a structure in which a porous layer and a nonwoven fabric layer are laminated. [Industrial Applicability]

[0048] The present invention can be used to culture in suspension adherent cells capable of producing a substance selected from proteins, glycoproteins, viruses, and viral vectors.

Claims

1. A substrate for suspension culture of adherent cells, used for suspension culture of adherent cells capable of producing a substance selected from a protein, a glycoprotein, a virus, and a viral vector, comprising: The porous layer has an apparent density of 0.440 g / cm ３ and a nonwoven fabric layer of less than 1000 mm. Substrate for suspension culture of adherent cells.

2. The specific gravity of the constituent fibers of the nonwoven fabric layer is 1 g / cm ３ The substrate for suspension culture of adherent cells according to claim 1, which is larger than the substrate.

3. The substrate for suspension culture of adherent cells according to claim 1 or 2, wherein the porous layer has triangular openings.

Images