Cell Culture Carriers

Abstract

To provide a cell culture carrier that can culture more cells.SOLUTION: The cell culture carrier according to the present invention capable of culturing a larger amount of cells is provided with a laminate made by laminating, on a fiber substrate layer (corresponding to a fiber aggregate according to the prior art), a main cell culture layer which has a higher air permeability than the fiber substrate layer, the air permeability of the main cell culture layer being higher than 204 cm3 / cm2 sec. In the present invention, when cells are seeded on the cell culture substrate, cell growth and proliferation are promoted not only on a main surface of the fiber substrate layer on the seeded side but also in the voids of the main cell culture layer, so that the seeded cells can easily proliferate in the thickness direction of the main cell culture layer. In this way, the cell culture carrier capable of culturing a larger amount of cells is provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a cell culture carrier. [Background technology]

[0002] In recent years, advances in bioscience and related technologies have led to the use of cultured cells in drug discovery research, regenerative medicine, and aesthetics such as cosmetic surgery. For example, stem cells (iPS cells, ES cells, hematopoietic stem cells, bone marrow stem cells, adipose stem cells, etc.) are cultured, and the cultured cells or their culture supernatants are transplanted, administered, or applied inside or outside the body according to the above-mentioned purposes to promote tissue regeneration or cell transplantation therapy aimed at fundamental healing is being carried out.

[0003] A method for culturing cells involves seeding cells prepared by extraction from a living body or the like onto a cell culture carrier, and culturing the cells in the desired manner (e.g., the desired number and shape of cells, the desired state of cell separation, the desired state of cell differentiation, etc.).

[0004] For the above-mentioned purpose, the applicant has proposed a cell culture carrier made of a fiber assembly (specific examples include JP 2015-159778 (Patent Document 1) and JP 2016-146785 (Patent Document 2)). Through the studies related to the above-mentioned Patent Documents 1 and 2, the applicant has found that the average fiber diameter of the fiber assembly is preferably 3 μm or less to facilitate cell culture. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication 2015-159778 [Patent Document 2] Patent Publication No. 2016-146785 Summary of the Invention [Problem to be solved by the invention]

[0006] The applicant of the present application has investigated cell culture carriers made of fiber assemblies according to the prior art as disclosed in Patent Documents 1 and 2, in order to provide a cell culture carrier capable of culturing a larger amount of cells.

[0007] As a result of the investigation, it was found that when using cell culture carriers composed only of fiber assemblies with a dense structure, such as an average fiber diameter of 3 μm or less, as in conventional technology, it was not easy to obtain large amounts of cells through culture.

[0008] The reason for this is that the fiber assembly has a dense structure, so the seeded cells have difficulty penetrating into the fiber assembly, and the cells tend to grow mainly in a planar manner on the main surface of the fiber assembly. In other words, the amount of cell growth is proportional to the size of the main surface of the fiber assembly, and there was a problem in that a cell culture carrier made of a huge fiber assembly had to be prepared in order to obtain a large amount of cells by culture. [Means for solving the problem]

[0009] The present invention relates to "A cell culture carrier that is a nonwoven fabric, The nonwoven fabric is It is an organic fiber nonwoven fabric. A fiber base material layer and a fiber base material layer having higher air permeability than the fiber base material layer. It is an organic fiber nonwoven fabric. A laminate with a main cell culture layer, The fibers constituting the fiber substrate layer and the main cell culture layer are the same, The thickness of the main cell culture layer is 0.23 to 0.34 mm; The air permeability of the main cell culture layer is 348~438cm 3 / cm 2 / sec, Cell culture carriers." It is. Effect of the Invention

[0010] As a result of further investigations, the applicant of the present application has found that a cell culture carrier comprising a laminate in which a main cell culture layer having a higher air permeability than a fiber base layer (corresponding to the fiber assembly according to the above-mentioned prior art) is laminated, and that the air permeability of the main cell culture layer can be increased to 204 cm 3 / cm 2 By increasing the strain rate to more than 100 s, it was found that a cell culture carrier capable of culturing a larger number of cells could be realized.

[0011] The applicant of the present application found that there is a correlation between the air permeability of each component of the cell culture carrier and the amount of cell proliferation in the component. Although the reason for this is not completely clear, it is thought that cells can easily enter the inside of a component with high air permeability such as the main cell culture layer and can easily proliferate in the thickness direction of the component, compared to a component with low air permeability such as the fiber substrate layer. Furthermore, the applicant has determined that the air permeability of the component with high air permeability is 204 cm 3 / cm2·sec, the amount of cell proliferation can be significantly increased. Although the reason for this is not completely clear, it is thought that the culture medium can circulate easily inside the component, efficiently supplying the culture medium to the cells and promoting their growth and proliferation. Therefore, by seeding cells onto the cell culture substrate of the present invention, the seeded cells can easily grow in the thickness direction of the main cell culture layer, not only on the main surface of the fiber substrate layer on which the cells are seeded, but also in the voids of the main cell culture layer, whereby cell growth and proliferation are promoted.

[0012] As described above, the present invention can provide a cell culture carrier capable of culturing a larger amount of cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the present invention, various configurations, such as the following configurations, can be appropriately selected. The various measurements described in the present invention were performed under atmospheric pressure unless otherwise specified. The measurements were performed under a temperature condition of 25°C. The results of the various measurements described in the present invention were obtained by measuring to a value one digit smaller than the desired value, and the value was rounded off to calculate the desired value, unless otherwise specified. As a specific example, when the desired value is to the first decimal place, the value was measured to the second decimal place, and the obtained value to the first decimal place was rounded off to calculate the value to the first decimal place, and this value was used as the desired value. The upper limit values ​​and lower limit values ​​exemplified in the present invention can be combined in any combination.

[0014] The fiber substrate layer in the present invention refers to a layer having air permeability composed of fibers, such as a layer derived from a fabric (a fiber web or nonwoven fabric, or a woven or knitted fabric, etc.). In particular, when the fiber substrate layer is a layer of fibers composed of randomly intertwined fibers, such as a layer derived from a fiber web or nonwoven fabric, the material constituting the fiber substrate layer can be efficiently utilized for cell culture, and a cell culture carrier capable of culturing a larger amount of cells can be provided, which is preferable.

[0015] The physical properties of the fiber substrate layer, such as the basis weight, thickness, or porosity, can be appropriately adjusted so that cells can be suitably cultured.

[0016] The weight of the fiber base layer is 0.01 to 100 g / m 2 and 0.05 to 70 g / m 2 and 0.1 to 40 g / m 2 In addition, "basis weight" refers to the area per square meter on the main surface of the object to be measured. 2 The thickness is the mass converted into a mass per unit area. The thickness is preferably 1000 μm or less, more preferably 750 μm or less, and even more preferably 500 μm or less. On the other hand, it is practical for the thickness to be 1 μm or more. Note that the "thickness" refers to the thickness measured in a direction perpendicular to the main surface of the target object when a load of 0.01 N is applied using a LITEMATIC (MITUTOYO, VL-50S) (contact area of ​​the terminal used for measurement: 28.77 mm 2) means the measured length.

[0017] The porosity may be 70 to 99%, 80 to 95%, or 85 to 90%. This "porosity" refers to a value obtained by the following formula. P = [1-M / (T × d)] × 100 Here, M is the basis weight of the object to be measured (unit: g / m 2 ), T is the thickness of the object to be measured (unit: μm), d is the average density of the various components that make up the object to be measured (unit: g / cm 3 ) respectively.

[0018] The average fiber diameter of the fibers constituting the fiber base layer can be 0.01 to 100 μm, 0.02 to 50 μm, or 0.05 to 30 μm. The term "average fiber diameter" refers to the arithmetic average value of the fiber diameters of 50 fibers measured based on a 5000x electron microscope photograph of a part of a measurement object containing the fibers. However, in the case of thick fibers whose fiber diameters cannot be observed at a magnification of 5000x, the term refers to the arithmetic average value of the fiber diameters of 50 fibers measured based on a 500x electron microscope photograph. In addition, when the cross-sectional shape of the fiber is non-circular, the diameter of a circle having the same area as the cross-sectional area is regarded as the fiber diameter.

[0019] The air permeability of the fiber base material layer can be appropriately adjusted as long as the configuration of the present invention is satisfied, but the lower limit is 0 cm 3 / cm 2 The upper limit of the aeration rate is lower than that of the main cell culture layer, and can be appropriately adjusted as long as the configuration of the present invention is satisfied.

[0020] The term "air permeability" as used herein refers to a value obtained by measurement based on JIS L1913-2010 [6.8.1 (Fragile type method)]. The air permeability of the fiber substrate layer and the main cell culture layer can be confirmed by measuring the air permeability of samples taken by the following method.

[0021] (How to check air permeability 1) (1) When each layer constituting the cell culture carrier can be easily peeled off from the cell culture carrier, the air permeability of each layer is measured. (2) Of the layers whose air permeability was measured, the air permeability was 204 cm 3 / cm 2 The layer higher than sec is assumed to be the main cell culture layer. (3) Among the layers whose air permeability was measured, the layer containing fibers adjacent to the layer assumed to be the primary cell culture layer and having a lower air permeability than the layer assumed to be the primary cell culture layer was determined as the fiber substrate layer. If a fiber substrate layer that satisfies the above conditions exists, the layer assumed to be the primary cell culture layer was determined as the primary cell culture layer.

[0022] (How to check air permeability 2) (1) If the individual layers constituting the cell culture carrier cannot be easily peeled off from the cell culture carrier, an electron microscope photograph (using a magnification that allows the entire thickness of the cell culture carrier to be observed) of a cross section of the cell culture carrier cut in the thickness direction should be taken. (2) Visually check the electron micrograph and measure the thickness of each layer constituting the cell culture carrier. The "thickness" here refers to the length of the shortest distance from the intersection (A) of one of the main surfaces of each layer to the intersection (B) of the other main surface of the line segment connecting the two main surfaces of the cell culture carrier in the electron micrograph at the shortest distance. (3) The cell culture carrier is cut in a direction parallel to one of the main surfaces of the cell culture carrier, one by one, for the thickness of each layer measured as described above, to obtain each layer. Then, the air permeability of each layer is measured. (4) Of the layers whose air permeability was measured, the air permeability was 204 cm 3 / cm 2 The layer higher than sec is assumed to be the main cell culture layer. (5) Among the layers whose air permeability was measured, the layer containing fibers adjacent to the layer assumed to be the primary cell culture layer and having a lower air permeability than the layer assumed to be the primary cell culture layer was determined as the fiber substrate layer. If a fiber substrate layer that satisfies the above conditions exists, the layer assumed to be the primary cell culture layer was determined as the primary cell culture layer.

[0023] In addition, when the materials used to form the fiber substrate layer or the main cell culture layer of the cell culture support (e.g., a fabric used to form the fiber substrate layer or another fabric used to form the main cell culture layer) are known, the air permeability of each of the materials can be measured and the measured values ​​can be regarded as the air permeability of the fiber substrate layer or the main cell culture layer.

[0024] The primary cell culture layer in the present invention refers to a layer having air permeability, such as a layer derived from a material having air permeability and voids, such as a fabric (a fiber web or nonwoven fabric, or a woven or knitted fabric), a porous film, a foam, etc. In particular, when the primary cell culture layer is a fiber layer formed by randomly intertwining fibers derived from a fiber web or nonwoven fabric, the material constituting the primary cell culture layer can be efficiently utilized for cell culture, and a cell culture carrier capable of culturing a larger amount of cells can be provided, which is preferable.

[0025] The physical properties of the main cell culture layer, such as the basis weight, thickness, or porosity, can be appropriately adjusted so that cells can be cultured suitably. 2 and the thickness can be 0.05 to 100 g / m 2 and 0.1 to 40 g / m 2 The thickness can be 10 mm or less, 5 mm or less, or 1.5 mm or less. On the other hand, it is practical for the thickness to be 1 μm or more. Also, the porosity can be 70 to 99%, 80 to 95%, or 85 to 90%.

[0026] When the main cell culture layer is, for example, a fiber layer derived from a fabric (such as a fiber web or nonwoven fabric, or a woven or knitted fabric), the average fiber diameter of the fibers constituting the main cell culture layer can be 0.01 to 100 μm, 0.02 to 50 μm, or 0.05 to 30 μm.

[0027] The cell culture carrier according to the present invention is characterized in that the main cell culture layer has a higher air permeability than the fiber substrate layer. The applicant found that cells easily penetrate into the main cell culture layer, which is the component with higher air permeability, and that the internal space of the main cell culture layer is utilized to facilitate cell proliferation in the thickness direction. The applicant also found that the air permeability of the main cell culture layer is 204 cm 3 / cm 2 We found that when the incubation time was higher than 10 min, the amount of cell proliferation could be significantly increased.

[0028] Therefore, compared to performing cell culture by seeding cells on the surface of a cell culture carrier having only a fiber substrate layer, by performing cell culture by seeding cells on a cell culture carrier having a laminate formed by stacking a fiber substrate layer and a main cell culture layer that satisfies the configuration of the present invention, a larger number of cells can be cultured.

[0029] The air permeability of the main cell culture layer can be appropriately adjusted as long as the constitution of the present invention is satisfied. 3 / cm 2 sec., and 3 / cm 2 sec or more, 300 cm 3 / cm 2 sec or more, 330 cm 3 / cm 2 ·sec or more. Although the upper limit of the aeration rate can be appropriately selected, if the aeration rate of the main cell culture layer is excessively high, cells tend to fall off the cell culture carrier (more precisely, the main cell culture layer of the cell culture carrier), and the material constituting the main cell culture layer is difficult to efficiently use for cell culture, which may make it difficult to provide a cell culture carrier capable of culturing a larger amount of cells. Therefore, the aeration rate of the main cell culture layer should be set to 580 cm 3 / cm 2 sec or less, and preferably 560 cm 3 / cm 2 sec or less, and 550 cm 3 / cm 2 ·sec or less is preferable.

[0030] As described above, the main cell culture layer may be any layer having air permeability, and is not limited to being a fiber layer derived from, for example, fabric (fiber web or nonwoven fabric, or woven or knitted fabric, etc.). However, when the main cell culture layer is also a fiber layer, it is preferable because the constituent fibers of the main cell culture layer can be efficiently utilized for cell culture to provide a cell culture carrier capable of culturing a larger amount of cells.

[0031] The type of fiber constituting the fiber substrate layer, or the fiber substrate layer and the main cell culture layer, can be selected as appropriate, but may be a fiber layer containing fibers made of an organic resin, or a fiber layer containing fibers made of an inorganic component.

[0032] The type of organic resin can be appropriately selected, and examples of the organic resin include polyolefin resins (e.g., polyethylene, polypropylene, polymethylpentene, polyolefin resins having a structure in which a portion of a hydrocarbon is substituted with a nitrile group or a halogen such as fluorine or chlorine), styrene resins, polyvinyl alcohol resins, polyether resins (e.g., polyether ether ketone, polyacetal, modified polyphenylene ether, aromatic polyether ketone, etc.), polyester resins (e.g., polyethylene terephthalate, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene naphthalate, polybutylene naphthalate, polycarbonate, polyarylate, wholly aromatic polyester resins, etc.), polyimide resins, It can be constructed using known organic resins such as resin, polyamideimide resin, polyamide-based resin (e.g., aromatic polyamide resin, aromatic polyetheramide resin, nylon resin, etc.), resin having a nitrile group (e.g., polyacrylonitrile, etc.), urethane-based resin, epoxy-based resin, polysulfone-based resin (e.g., polysulfone, polyethersulfone, etc.), fluorine-based resin (e.g., polytetrafluoroethylene, polyvinylidene fluoride, etc.), cellulose-based resin, polybenzimidazole resin, acrylic-based resin (e.g., polyacrylonitrile-based resin copolymerized with acrylic acid ester or methacrylic acid ester, modacrylic-based resin copolymerized with acrylonitrile and vinyl chloride or vinylidene chloride, etc.).

[0033] The type of fiber made of an inorganic component can be appropriately selected, and examples of the fiber include lithium, beryllium, boron, carbon, sodium, magnesium, aluminum, silicon, phosphorus, sulfur, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, cadmium, indium, tin, antimony, tellurium, cesium, barium, lanthanum, hafnium, tantalum, tungsten, mercury, thallium, lead, bismuth, cerium, and praseodymium. Examples of the oxides of zinc, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, or lutetium include oxides of 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.

[0034] The above-mentioned fibers can be obtained by known methods such as melt spinning, dry spinning, wet spinning, direct spinning (melt blowing, spunbonding, electrostatic spinning, etc.), a method of extracting fibers having a small fiber diameter by removing one or more resin components from composite fibers, and a method of beating fibers to obtain split fibers.

[0035] The above-mentioned fibers may be composed of one type of resin or inorganic component, or may be composed of multiple types of resins or inorganic components. Fibers composed of multiple types of resins may be in the form of what is generally called composite fibers, such as core-sheath type, sea-island type, side-by-side type, orange type, bimetal type, etc.

[0036] The fibers constituting the fiber sheet may include irregular cross-section fibers other than the roughly circular and elliptical fibers. The irregular cross-section fibers may have a fiber cross section of a hollow shape, a polygonal shape such as a triangular shape, an alphabetical shape such as a Y-shape, an irregular shape, a multi-lobed shape, a symbolic shape such as an asterisk shape, or a shape in which a plurality of these shapes are combined.

[0037] The fabric can be prepared by, for example, a dry method in which the fibers are fed into a carding device or an air-laying device to entangle the fibers; a wet method in which the fibers are dispersed in a solvent and laid into a sheet to entangle the fibers; or a method in which the fibers are spun into yarn using a direct spinning method (such as a melt-blowing method, a spunbonding method, an electrostatic spinning method, or a method in which a spinning solution and a gas flow are discharged in parallel to spin the yarn (for example, the method disclosed in JP-A-2009-287138), etc.) and the yarn is collected.

[0038] The fibers constituting the prepared fiber web may be entangled and / or integrated to prepare a nonwoven fabric. Examples of methods for entangling and / or integrating the fibers constituting the fiber web include a method for entangling the fibers with needles or water flow, and a method for subjecting the fiber web to heat treatment, etc., and bonding and integrating the fibers constituting the fiber web with known binders or heat-fusible fibers. The heat treatment method can be appropriately selected, and examples of the heat treatment method include a method for heating or pressurizing with a roll (for example, heating and pressurizing can be performed using a heat roll), a method for heating the web by a heater such as an oven dryer, a far-infrared heater, a dry heat dryer, or a hot air dryer, and a method for irradiating infrared rays without pressure.

[0039] When the fabric is a woven fabric or knitted fabric, the woven fabric or knitted fabric can be prepared by weaving or knitting the fibers prepared as described above. In addition to the fiber web, a nonwoven fabric, woven fabric, knitted fabric, etc. may be subjected to the above-mentioned method of entangling and / or integrating the constituent fibers.

[0040] The fibers constituting each layer of the cell culture carrier of the present invention may contain additives, particles, etc. that have low cytotoxicity, cytotoxicity, reproductive toxicity, teratogenicity, etc., inside or on the surface of the fibers. For example, additives such as flame retardants, fragrances, pigments, antibacterial agents, antifungal materials, photocatalyst particles, emulsifiers, dispersants, surfactants, particles that foam when heated, inorganic particles such as hydroxyapatite, and antioxidants may be contained. In addition, in order to favorably adhere and grow cells, the fibers may contain extracellular matrices such as collagen and fibronectin, cell growth factors such as EGF and FGF, hormones, cytokines, etc., inside or on the surface of the fibers.

[0041] Specific methods for producing the fiber substrate layer, or the fiber substrate layer and the main cell culture layer, include, for example, a method disclosed in JP 2009-287138 A in which gas is discharged parallel to the spinning solution discharged from a liquid discharge section, and a shear force is applied to the spinning solution in a straight line to form fibers, electrostatic spinning, melt-blowing, spunbonding, flash spinning, etc.

[0042] As an example, it is preferable to adopt a main cell culture layer made of a material having calcium phosphate particles supported on the surface, since this provides a cell culture carrier capable of culturing a larger amount of cells. In particular, it is preferable to adopt a main cell culture layer made of fibers having a thermoplastic resin on the surface, and in which calcium phosphate particles are fixed to the fiber surface by thermal fusion of the thermoplastic resin, since this provides a cell culture carrier capable of culturing an even larger amount of cells.

[0043] Furthermore, it is possible to prepare a cell culture carrier having a thin fiber layer in which the constituent fibers are uniformly dispersed, thereby allowing the constituent fibers of the fiber base layer to be efficiently utilized for cell culture, thereby providing a cell culture carrier capable of culturing a larger number of cells, and therefore it is preferable to prepare the fiber base layer by a direct spinning method.

[0044] The fiber base layer is preferably bonded with an adhesive after fibers are accumulated by the method described in JP 2004-238749 A. More specifically, it can be produced by the steps of (1) spinning fibers from a spinning solution by electrostatic spinning (spinning step), (2) irradiating the fibers with ions of the opposite polarity to the fibers, accumulating them, and forming a fiber web (accumulating step), and (3) bonding the fiber web (particularly bonding them with an adhesive) to form a fiber assembly (bonding step).

[0045] In particular, an inorganic fiber web can be produced by the method described in JP 2010-185164 A, that is, (1) a step of spinning inorganic fibers by electrostatic spinning using a spinning sol solution containing an oxide of a metal element (spinning step), and (2) a step of irradiating ions of the opposite polarity to the inorganic fibers, accumulating them, and forming an inorganic fiber web (accumulation step).

[0046] In addition, it is preferable to carry out a heat treatment process after the accumulation process (2) to bond the inorganic fibers together, since the fibers have excellent shape stability and are less likely to deform during cell culture. In particular, it is preferable to carry out a heat treatment at 200°C or higher, preferably 300°C or higher, to bond the inorganic fibers at their intersections, since this provides excellent shape stability.

[0047] In addition, after the accumulation step (2) (after the heat treatment, if any), it is preferable to include a step (3) of bonding the inorganic fiber web (bonding step) so that the inorganic fiber web has excellent shape stability and is less likely to deform during cell culture. Examples of the bonding step include adhesion with an adhesive and partial fusion.

[0048] When bonding with this adhesive, it is preferable to use a sol solution containing an oxide of a metal element (particularly, a metal alkoxide hydrolysis condensate is preferable) as the adhesive, since it has excellent shape stability and the inorganic fiber is not easily detached. In particular, it is preferable to use a sol solution containing an oxide of the same metal element as the metal oxide constituting the inorganic fiber, since it has excellent adhesive strength, excellent shape stability, and the inorganic fiber is not easily detached. The adhesive may bond only a part of the inorganic fiber, such as the surface of the inorganic fiber web, but it is preferable to bond the entire inorganic fiber web including the inside so that it has excellent shape stability and fiber shedding. In order to bond the entire inorganic fiber web including the inside, for example, the inorganic fiber web can be bonded by immersing the inorganic fiber web in a sol solution containing an oxide of a metal element. It is preferable to remove the excess adhesive after applying the adhesive by ventilation so that the bulk of the inorganic fiber web is not crushed when the entire inorganic fiber web is bonded with the adhesive. By removing the excess adhesive by ventilation, a coating is not formed between the inorganic fibers. Furthermore, the adhesive can be bonded by drying naturally at room temperature or by removing the solvent in the adhesive through heat treatment.

[0049] Furthermore, after the (3) bonding step, it is preferable to carry out a firing treatment to mineralize the inorganic fibers and / or the sol solution (adhesive) containing an oxide of a metal element, and to improve the strength and rigidity of the inorganic fibers, the adhesive strength between the inorganic fibers, and the dimensional stability of the fiber assembly. The firing temperature can be appropriately adjusted, and can be, for example, 300°C or 500°C.

[0050] The inorganic fiber web prepared as described above may be subjected to a pressure step such as a calendar treatment to adjust the thickness.

[0051] A cell culture carrier can be prepared by laminating a fabric capable of forming a fiber substrate layer and a material capable of forming a main cell culture layer (e.g., a fabric). The method of laminating the fabric and the material can be appropriately selected, but may be a method of simply overlapping them, a method of laminating them by entangling and integrating them through entangling treatment such as needle punching or water flow entangling, a method of laminating and integrating them by bonding them using a binder liquid, a method of laminating and integrating them by bonding them through a thermally bondable fabric, a method of melting a component of the fabric and / or the material (e.g., adhesive fiber, etc.) to bond and integrate the layers, or the like.

[0052] The cell culture carrier of the present invention is A cell culture carrier having a laminate of a fiber layer (fiber substrate layer) containing fibers made of an inorganic component and a fiber layer (main cell culture layer) containing fibers made of an organic resin, A cell culture carrier having a laminate of a fiber layer (fiber substrate layer) containing fibers made of an organic resin and a fiber layer (main cell culture layer) containing fibers made of another organic resin, A cell culture carrier having a laminate of a fiber layer (fiber substrate layer) containing fibers made of an inorganic component and a fiber layer (main cell culture layer) containing fibers made of another inorganic component, Examples include:

[0053] The material constituting the cell culture carrier or the cell culture carrier may be subjected to a hydrophilization treatment process, such as sulfonation treatment, fluorine gas treatment, graft polymerization treatment with a vinyl monomer, surfactant treatment, discharge treatment, or hydrophilic resin imparting treatment.

[0054] The physical properties of the cell culture carrier, such as the basis weight and thickness, can be appropriately adjusted so that cells can be cultured suitably. The basis weight is 0.02 to 400 g / m 2 and can be 0.05 to 170 g / m 2 and 0.1 to 80 g / m 2 The thickness can be 11 mm or less, can be 7 mm or less, or can be 3 mm or less. On the other hand, it is practical for the thickness to be 1 μm or more.

[0055] The outer shape of the cell culture carrier can be appropriately adjusted and is not particularly limited, but can be, for example, a two-dimensional sheet shape (e.g., a long shape that can be wound into a roll, a circular shape that can be laid on the bottom surface of a well plate), a three-dimensional corrugated shape, a pleated shape, a cylindrical shape, etc. The cell culture carrier can have a cutout part, a punched part, or a notched part. EXAMPLES

[0056] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

[0057] The cell proliferation rate was calculated as follows. (Method for measuring cell proliferation rate) The cell culture carrier was punched out into a circle with a diameter of 15.4 mm, sterilized with 70% ethanol in a clean bench (1 hour at room temperature), and washed with phosphate buffered saline.Then, the punched out cell culture carrier after washing was placed on the bottom of each well of a 24-well plate (NUNC, product number: 142475). Then, 0.5 ml of a suspension of NIH3T3 cells (mouse fibroblast-like cell line, RCB2767) (concentration: 1 million cells / ml) was seeded onto the main surface opposite the bottom of the wells of each 24-well plate in each cell culture support, to give a cell volume of 500,000 cells / well. Next, a complete medium containing the following supplementary factors added to DMEM medium (Dulbecco's Modified Eagle Medium, Fujifilm Wako Pure Chemical Industries, Ltd., 044-29765) was poured into each well so that the total medium volume was 1.0 ml. 10% FBS (Sigma), 100U / mL penicillin G 100 μg / mL streptomycin sulfate (Fujifilm Wako Pure Chemical Industries, Ltd., 168-23191) Thereafter, the cells were cultured in an incubator at 37°C and 5% CO for 14 days. During the culture period, the entire medium was replaced once every 1 to 3 days. The cell count was performed by WST-8 assay using Cell Counting Kit-8 (Dojindo Laboratories, CK04). First, a complete medium containing 10% FBS (Sigma), 100 U / mL penicillin G, and 100 μg / mL streptomycin sulfate (Fujifilm Wako Pure Chemical Industries, 168-23191) was mixed with Cell Counting Kit-8 at a mixing ratio of 9:1 and added to the medium at 1 ml / well. The reaction time was 30 minutes, and the reaction was carried out at 37°C, and 100 μl of the supernatant after the reaction was collected. The mixture was then evenly dispensed into each well of a 96-well plate prepared separately, and the absorbance at 450 nm in each well was measured to calculate the number of cells present in each well. The number of cells present in each well was then added together to obtain the total number of cells. The cell proliferation rate (%) was calculated by substituting the total cell number on day 1 of culture and the total cell number on day 14 of culture, calculated as described above, into the following formula. Cell proliferation rate (%) = 100 × (total cell number on day 14 of culture / total cell number on day 1 of culture)

[0058] (Reference example) A 24-well plate (NUNC, product number: 142475) was prepared as a culture vessel. Based on the method described above (Method for measuring cell proliferation rate), the NIH3T3 cell suspension was used to seed the cells directly onto the bottom of the wells of the well plate without using a cell culture carrier, thereby culturing the cells in the wells, and the proliferation rate of the cells (unit: %) was calculated.

[0059] (Method for preparing inorganic fiber nonwoven fabric) Tetraethoxysilane as a metal compound, ethanol as a solvent, water for hydrolysis, and 1N hydrochloric acid as a catalyst were mixed in a molar ratio of 1:5:2:0.003, and refluxed at a temperature of 78°C for 10 hours. The solvent was then removed using a rotary evaporator for concentration, and the mixture was then heated to a temperature of 60°C to form a sol solution with a viscosity of 2 poise. The obtained sol solution was used to spin gel-like silica continuous fibers by electrostatic spinning (spinning step), and the gel-like silica continuous fibers were irradiated with ions of the opposite polarity to the gel-like silica continuous fibers, and accumulated on a collecting member provided in a direction perpendicular to the discharge direction of the spinning solution, to form a gel-like silica continuous fiber web (accumulation step). That is, the gel-like silica continuous fiber web was formed under the same spinning conditions as in Example 8 of JP-A-2005-264374. The spinning conditions were as follows: (1) Spinning nozzle: a metal injection needle (tip cut) with an inner diameter of 0.4 mm. Voltage is applied by the first high-voltage power source. (2) Distance between the spinning nozzle and the counter electrode: 200 mm, (3) Counter electrode and ion generating electrode (serves as both electrodes): A 1 mm thick alumina film (dielectric substrate) is sprayed onto a stainless steel plate (induction electrode), and a 30 μm diameter tungsten wire (discharge electrode) is stretched on top of it at equal intervals of 10 mm (the tungsten wire surface faces the spinning nozzle and is grounded, and a 50 Hz AC high voltage is applied between the stainless steel plate and the tungsten wire from an AC high voltage power supply), and a voltage is applied from a second high voltage power supply. (4) First high voltage power source: -16kV, (5) Second high voltage power supply: ±5 kV (AC surface peak voltage: 5 kV, 50 Hz), (6) Air flow: 25 cm / sec in the direction of the spinning solution discharge (horizontal direction), 15 cm / sec in the direction perpendicular to the direction of the spinning solution discharge (vertical direction), (7) Atmosphere inside the spinning vessel: temperature 25°C, humidity 40% RH or less. Next, the gelled silica continuous fiber web was dried by heat treatment at a temperature of 800° C. for 3 hours to prepare a dried silica continuous fiber web in which the dried silica continuous fibers were bonded at their intersections. Next, tetraethoxysilane as a metal compound, ethanol as a solvent, water for hydrolysis, and nitric acid as a catalyst were mixed in a molar ratio of 1:7.2:7:0.0039, and the mixture was reacted at a temperature of 25°C and stirring conditions of 300 rpm for 15 hours. After that, the mixture was diluted with ethanol so that the solid concentration of silicon oxide became 0.25%, to prepare a silica sol solution as an adhesive. Next, the dried continuous silica fiber web was immersed in the silica sol solution (adhesive), and the excess silica sol solution was removed by suction. The web was then baked at a temperature of 500°C for 3 hours to obtain an inorganic fiber nonwoven fabric consisting of only sintered continuous silica fibers (average fiber diameter: 800 nm, basis weight: 8.0 g / m2) in which the sintered continuous silica fibers throughout the web, including the interior, were bonded together with silica without forming a coating. 2 , thickness: 0.35 mm) was prepared.

[0060] Comparative Example 1 The inorganic fiber nonwoven fabric prepared as described above was used as a cell culture carrier. Then, cells were cultured using the cell culture carrier according to the method described in (Method for measuring cell proliferation rate) above, and the proliferation rate (unit: %) of the cells was calculated.

[0061] (Method for preparing organic fiber nonwoven fabrics 1 to 5) A fiber web (basis weight: 30 g / m) was obtained by blending 75% by mass of sheath-core composite fiber (core: polypropylene (melting point: 165°C), sheath: polyethylene (melting point: 130°C), fiber cross-sectional shape: circular, fineness: 6.6 dtex, fiber length: 64 mm) with 25% by mass of sheath-core composite fiber (core: polypropylene (melting point: 165°C), sheath: polyethylene (melting point: 130°C), fiber cross-sectional shape: circular, fineness: 1.7 dtex, fiber length: 38 mm) and feeding the blend to a carding machine. 2 ) was prepared. Approximately spherical sintered hydroxyapatite particles (manufactured by Sofsera, product name: micro-SHAp, product number: IHMO-100P000, particle size: 4.0 μm, aspect ratio: 1) were heated to 220° C. and sprayed onto both main surfaces of the fiber web together with an airflow heated to 158° C. At this time, the airflow containing the sintered hydroxyapatite particles was adjusted so that it passed over the main surface of the fiber web at a tempo of 5 m / min. After cooling to room temperature, the sintered hydroxyapatite particles that were not fixed to the surface of the fiber web were removed with air, resulting in an organic fiber nonwoven fabric (basis weight: 33.1 g / m) in which the sintered hydroxyapatite particles were fixed to the fiber surface by thermal fusion of the thermoplastic resin that constitutes the fiber surface. 2 ) was prepared. The prepared organic fiber nonwoven fabric was combined with a spacer to obtain a desired thickness and fed into a heat press (Ide Manufacturing Co., Ltd., IMC-11DE) to reduce the thickness, producing organic fiber nonwoven fabrics 1 to 5 with different thicknesses (each with a basis weight of 33.1 g / m 2 ) was prepared.

[0062] (Comparative Example 2, Examples 1 to 4) The inorganic fiber nonwoven fabric and the organic fiber nonwoven fabric prepared as described above were overlapped, and then the resulting laminate was combined with a spacer to obtain a desired thickness, and laminated and integrated using a heat press. The various laminates (laminated bodies of a fiber substrate layer and a main cell culture layer) thus prepared were used as cell culture carriers. Then, based on the method described in (Method for measuring cell proliferation rate) above, cells were cultured using the cell culture carriers, and the proliferation rate (%) of the cells was calculated.

[0063] The cell proliferation rates (%) obtained in the above manner in Reference Example, Comparative Examples 1 and 2, and Examples 1 to 4 are summarized in Table 1. In addition, the various components of the cell culture carriers according to Comparative Examples 1 and 2 and Examples 1 to 4 are also summarized in Table 1. Components that are not present are marked with "-" in the table.

[0064] [Table 1]

[0065] A comparison between Comparative Example 1 and Comparative Example 2 revealed that a cell culture carrier having a laminate of a fiber substrate layer and a main cell culture layer having a higher air permeability than the fiber substrate layer does not necessarily improve the cell proliferation rate or enable a larger number of cells to be cultured compared to a cell culture carrier having only a fiber substrate layer.

[0066] On the other hand, the results of comparing Comparative Example 2 with Examples 1 to 4 show that the cell culture carrier is provided with a laminate of a fiber base layer and a main cell culture layer having a higher air permeability than the fiber base layer, and the main cell culture layer has an air permeability of 204 cm 3 / cm 2 It was found that when the incubation time was higher than 10 min, the cell proliferation rate improved and a larger amount of cells could be cultured.

[0067] In addition, when the state inside the well plate in which cells were cultured in Example 4 was observed using a phase contrast microscope, it was found that there were significantly more cells in the wells that had fallen off from the cell culture carriers than in the Reference Example, Comparative Examples, and other Examples. The reason for this was thought to be that cells tend to fall off from the main cell culture layer, which has an excessively high degree of air permeability. Therefore, it was found that in a cell culture carrier having such a main cell culture layer, the fibers that make up the main cell culture layer are difficult to efficiently use for cell culture. On the other hand, the number of cells that had fallen off from the cell culture carriers present in the wells in Examples 1 to 3 was significantly smaller than in Example 4. From this, in order to provide a cell culture carrier that can efficiently culture cells and can culture a large amount of cells, the air permeability of the main cell culture layer should be 580 cm 3 / cm 2 sec or less (more preferably, 541 cm 3 / cm 2 It was found that a temperature of 100° C. or less is preferable.

[0068] (Preparation method of organic fiber nonwoven fabrics 6 to 9) A fiber web (basis weight: 30 g / m2) was prepared by blending 75% by mass of sheath-core composite fibers (core: polypropylene (melting point: 165°C), sheath: polyethylene (melting point: 130°C), fiber cross-sectional shape: circular, fineness: 6.6 dtex, fiber length: 64 mm) and 25% by mass of sheath-core composite fibers (core: polypropylene (melting point: 165°C), sheath: polyethylene (melting point: 130°C), fiber cross-sectional shape: circular, fineness: 1.7 dtex, fiber length: 38 mm) and passing the blend through a carding machine. Approximately spherical sintered hydroxyapatite particles (manufactured by Sofsera, product name: micro-SHAp, product number: IHMO-100P000, particle size: 4.0 μm, aspect ratio: 1) were heated to 220° C. and sprayed onto both main surfaces of the fiber web together with an airflow heated to 158° C. At this time, the airflow containing the sintered hydroxyapatite particles was adjusted so that it passed over the main surface of the fiber web at a tempo of 5 m / min. After cooling to room temperature, the sintered hydroxyapatite particles that were not fixed to the surface of the fiber web were removed with air, resulting in an organic fiber nonwoven fabric (basis weight: 33.1 g / m) in which the sintered hydroxyapatite particles were fixed to the fiber surface by thermal fusion of the thermoplastic resin that constitutes the fiber surface. 2 ) was prepared. In addition, the prepared organic fiber nonwoven fabric was combined with a spacer to achieve the desired thickness and fed into a heat press machine (Ide Manufacturing Co., Ltd., IMC-11DE) to crush the thickness, and various organic fiber nonwoven fabrics 6 to 9 with different thicknesses (each with a basis weight of 33.1 g / m 2 ) was prepared.

[0069] Comparative Example 3 The organic fiber nonwoven fabric 6 prepared as described above was used as a cell culture carrier. Then, based on the method described in (Method for measuring cell proliferation rate) above, cells were cultured using the cell culture carrier, and the proliferation rate (%) of the cells was calculated.

[0070] (Examples 5 to 7) The organic fiber nonwoven fabric 6 prepared as described above was overlapped with another organic fiber nonwoven fabric (any of organic fiber nonwoven fabrics 7 to 9), and then the two were combined with a spacer to achieve a desired thickness and laminated together using a heat press. The various laminates (laminated bodies of a fiber substrate layer and a main cell culture layer) thus prepared were used as cell culture carriers. Then, based on the method described in (Method for measuring cell proliferation rate) above, cells were cultured using the cell culture carriers, and the proliferation rate (%) of the cells was calculated.

[0071] The cell proliferation rates (%) obtained in the above manner in Reference Example, Comparative Example 3, and Examples 5 to 7 are summarized in Table 2. In addition, the various components of the cell culture carriers according to Comparative Example 3 and Examples 5 to 7 are also summarized in Table 2. Components that were not included are marked with "-" in the table.

[0072] [Table 2]

[0073] From the results of comparing Comparative Example 3 with Examples 5 to 7, it was found that the cell culture carrier had a laminate of a fiber base layer and a main cell culture layer having a higher air permeability than the fiber base layer, and the main cell culture layer had an air permeability of 204 cm 3 / cm 2 It was found that when the incubation time was higher than 10 min, the cell proliferation rate improved and a larger amount of cells could be cultured.

[0074] In addition, when the state inside the well plate in which the cells were cultured in Examples 5 to 7 was observed by a scanning microscope, the number of cells that had fallen off from the cell culture carriers present in the wells in Examples 5 to 7 was significantly smaller (significantly smaller than that in Example 4). Therefore, it was found that the cell culture carriers in Examples 5 to 7 were also cell culture carriers capable of culturing cells efficiently and culturing large amounts of cells.

[0075] From the above, it was found that the present invention can provide a cell culture carrier capable of culturing a larger amount of cells. [Industrial Applicability]

[0076] The cell culture carrier of the present invention makes it possible to culture a variety of cells, including established cell lines such as NIH3T3 cells, MG63 cells, and HepG2 cells, primary cultured cells, stem cells (iPS cells, ES cells, hematopoietic stem cells, bone marrow stem cells, adipose stem cells, etc.), and patient-derived cells, and allows for efficient culture of larger quantities of cells.

Claims

[Claim 1] A cell culture carrier which is a nonwoven fabric, The nonwoven fabric is a laminate of a fiber substrate layer which is an organic fiber nonwoven fabric and a main cell culture layer which is an organic fiber nonwoven fabric having a higher air permeability than the fiber substrate layer, The fibers constituting the fiber substrate layer and the main cell culture layer are the same, The thickness of the main cell culture layer is 0.23 to 0.34 mm; The main cell culture layer has an air permeability of 348 to 438 cm 3 / cm 2 / sec; Cell culture carrier.

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