Cell aggregates and methods for producing cell aggregates

Abstract

To provide a cell aggregate capable of cryopreservation and a method for producing the cell aggregate.SOLUTION: Provided are: a cell aggregate comprising adherent cells and an extracellular matrix, the cell aggregate containing living cells even in a part located at a depth of 30 μm or more from the surface of the aggregate; and a method for producing a cell aggregate, the method comprising supporting adherent cells on a molded body formed by fibers containing an ethylene-vinylalcohol copolymer and having a non-woven fiber structure and culturing the cells under stirring.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cell aggregate comprising adherent cells and an extracellular matrix, and to a method for producing the cell aggregate. [Background technology]

[0002] In recent years, in cell transplantation therapy, there has been growing interest in transplanting cell aggregates (also called cell clusters or cell spheroids) created by three-dimensional culture, which better reflects the in vivo cell-cell interactions and cell-extracellular matrix interactions, rather than single cells cultured in a monolayer, in order to allow transplanted cells to fully exert their function.

[0003] Methods for producing cell aggregates have been reported for some time. Representative methods include the non-adhesive surface cell culture method, which involves culturing cells using a culture plate with a non-adhesive bottom to cause cells floating in the culture medium to adhere to each other; the hanging drop cell culture method, which involves dropping a cell suspension onto the inside of the lid of a culture plate and culturing the dome-shaped droplet, formed by surface tension, upside down so that the cells gather at the bottom of the droplet due to gravity and adhere to each other to form a cell aggregate; and the rotary cell culture method, which involves culturing cells in a cell culture chamber so that the cells come into contact with each other within the chamber and form a cell aggregate. Furthermore, cell aggregates of mesenchymal stem cells rich in collagen, produced in a growth medium supplemented with collagen-producing factors, and frozen grafts for mesenchymal stem cell transplantation, obtained by cryopreserving these cell aggregates with a cryopreservation agent, have been reported (Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 7105487 [Overview of the project] [Problems that the invention aims to solve]

[0005] In cell transplantation therapy using cell aggregates, it is necessary to prepare cell aggregates each time a transplant surgery is performed, but there is no guarantee that the number of cells and cell function of the cell aggregates can be made uniform. On the other hand, if cell aggregates that meet a certain quality standard are cryopreserved and thawed and transplanted at the time of transplant surgery, a stable transplant effect can be expected, but conventional techniques have made it difficult to cryopreserve cell aggregates because the cell viability after thawing is low. Therefore, the present invention aims to provide cryopreservable cell aggregates and a method for producing said cell aggregates. [Means for solving the problem]

[0006] As a result of diligent research, the inventors have surprisingly succeeded in obtaining cell aggregates by supporting adhesive cells on a molded body having a nonwoven fiber structure and containing ethylene-vinyl alcohol copolymer fibers, and culturing the cells under stirring. Even more surprisingly, it was found that the cells in the central part of the cell aggregates survived even after freeze-thawing, indicating a high cell viability rate, and that they could be cryopreserved. Thus, the present invention was completed.

[0007] In other words, the present invention provides the following: (1) A cell aggregate comprising adherent cells and extracellular matrix, wherein the aggregate also contains viable cells at depths of 30 μm or more from the surface. (2) The cell aggregate described in (1) above, wherein after the freeze-thaw of the aggregate, viable cells are also present in the area at a depth of 30 μm or more from the surface of the aggregate. (3) The cell aggregates described in (1) or (2) above, which are granular and have a diameter greater than 1.5 mm. (4) A cell aggregate according to any one of (1) to (3) above, wherein less than 50% of the extracellular matrix is ​​collagen. (5) A cell aggregate described in any of (1) to (4) above, which is in a frozen state. (6) A method for producing cell aggregates, comprising supporting adherent cells on a molded body having a nonwoven fiber structure and comprising fibers containing an ethylene-vinyl alcohol copolymer, and culturing the cells under stirring. (7) The manufacturing method according to (6), wherein the fiber containing the ethylene-vinyl alcohol copolymer is a moist heat adhesive fiber. (8) The manufacturing method according to (6) above, wherein the fiber containing the ethylene-vinyl alcohol copolymer is a composite fiber containing a non-moist heat-adhering fiber. (9) The manufacturing method according to (8) above, wherein the composite fiber is a core-sheath type composite fiber comprising a sheath made of an ethylene-vinyl alcohol copolymer and a core made of a polyester resin. (10) A method for producing a product according to any one of (6) to (9) above, comprising supporting adhesive cells on the molded body having an apparent density of 0.02 to 0.7 g / cm3 and culturing the cells under stirring at 30 to 140 rpm. [Effects of the Invention]

[0008] According to the present invention, cell aggregates can be obtained by a simple method of supporting adherent cells on a molded body having a nonwoven fiber structure and consisting of fibers containing an ethylene-vinyl alcohol copolymer, and culturing the cells under stirring. Compared to cell aggregates obtained by conventional techniques, the cell aggregates obtained by the present invention are larger in size, have improved cell viability during culture, and have improved cell viability after freeze-thaw cycles. The cell aggregates of the present invention are freezeable because the cells in the center of the aggregate survive even after freeze-thaw cycles. Thus, by using the cell aggregates of the present invention, it is not necessary to prepare cell aggregates for each treatment, and the lead time (the time from the decision to treat to the start of treatment) for cell transplantation therapy is shortened. [Brief explanation of the drawing]

[0009] [Figure 1] Microscopic image showing the results of a Live-Dead assay of cell aggregates. [Figure 2]Microscopic image showing the results of a Live-Dead assay on cell aggregates after freeze-thawing. [Figure 3] Microscopic image showing the result of picrosilius red staining of cell aggregates. [Modes for carrying out the invention]

[0010] 1.Cell aggregates In this specification, "cell aggregate" refers to a mass of cells formed by the aggregation or condensation of multiple cells. The cell aggregate of the present invention is an aggregate of adherent cells and contains extracellular matrix in addition to cells. The cell aggregate of the present invention is a cell aggregate produced outside the body (in vitro or ex vivo).

[0011] In this specification, "adherent cells" refers to cells that proliferate by adhering to a culture medium during cell culture. These cells may originate from any organism, such as animals (e.g., mammals, non-mammals), plants, insects, etc., and are not particularly limited. Examples of adherent cells, though not limited to these, include osteoblasts, chondrocytes, hematopoietic cells, epithelial cells (e.g., mammary epithelial cells), endothelial cells (e.g., vascular endothelial cells), epidermal cells, fibroblasts, mesenchymal-derived cells, cardiomyocytes, myobiocytes, smooth muscle cells, living organism-derived skeletal muscle cells, human tumor cells, fibroblasts, EB virus mutant cells, hepatocytes, renal cells, bone marrow cells, macrophages, hepatocytes, pancreatic β-cells, small intestinal cells, mammary gland cells, salivary gland cells, thyroid cells, skin cells, somatic stem cells (e.g., mesenchymal stem cells), etc.

[0012] As used in the specification of this application, the "extracellular matrix" refers to insoluble substances present outside cells in an organism and non-cellular components that fill the spaces between cells. The extracellular matrix is mainly composed of water, proteins, and polysaccharides, and is known to have different compositions and forms depending on the biological tissue. The extracellular matrix contained in the cell aggregates of the present invention may preferably be an extracellular matrix produced by the adherent cells themselves contained in the cell aggregates. The extracellular matrix contained in the cell aggregates of the present invention contains a relatively small amount of collagen. For example, less than about 50% of the extracellular matrix, preferably about 48% or less, more preferably about 46% or less is collagen.

[0013] The cell aggregates of the present invention have, for example, a granular shape. The size of the granular cell aggregates may be, for example, about 1.5 mm or more, preferably about 2 mm or more, more preferably about 5 mm or more as the diameter (when it is not a sphere, the longest diameter, hereinafter referred to as the "major axis"). The cell aggregates of the present invention may be, for example, granular aggregates having a major axis of about 1.5 mm to about 5 mm. The diameter of the cell aggregates can be measured by conventional methods, for example, by observing images under a microscope.

[0014] Furthermore, in the cell aggregates of the prior art, the cells in the central part of the aggregates die after freeze-thawing, whereas the cell aggregates of the present invention are characterized in that the cells in the central part of the aggregates survive even after freeze-thawing, and the viability of the cells after freeze-thawing is improved compared to the prior art. Also, in the cell aggregates of the prior art, for those with a large size exceeding about 1.5 mm in diameter, the cells in the central part die even without freeze-thawing. In contrast, the cell aggregates of the present invention are characterized in that the cells in the central part survive even for those with a large size, and the viability of the cells is improved compared to the prior art.

[0015] Here, the central part refers to, depending on the size of the cell aggregate, for example, the depth of about 30 μm or more, preferably about 40 μm or more from the surface of the cell aggregate (that is, the inside of the cell aggregate about 30 μm or more, preferably about 40 μm or more away from the surface of the cell aggregate). The distance from the surface to the depth of the cell aggregate can be measured, for example, by a microscopic image. Also, the survival of cells in the cell aggregate can be confirmed by microscopic observation. Thus, the cell aggregate of the present invention is a cell aggregate that can be frozen, that is, a cell aggregate that can be frozen and thawed.

[0016] Therefore, the cell aggregate of the present invention may be a cell aggregate in a frozen state. The cell aggregate is preferably frozen in a cryopreservation solution. As the above cryopreservation solution, for example, a cryopreservation solution containing 10% DMSO (Dimethyl sulfoxide), 20% FBS (fetal bovine serum), and 70% DMEM (Dulbecco's Modified Eagle Medium) and the like known in the art may be used, or a commercially available cryopreservation solution may also be used. Examples of commercially available cryopreservation solutions include, but are not limited to, CELLBANKER1 (manufactured by Xenogen Pharma Co., Ltd.). The freezing of the cell aggregate may be performed at a temperature at which freezing is possible, for example, at 0°C or lower, -70°C or lower, or -80°C or lower. The cell aggregate may further be stored in a frozen state. Although not limited, for example, it may be stored for 24 hours or more, or even for a long term of 1 month or more. Freezing and cryopreservation may be performed in a freezer or in liquid nitrogen. The freezing temperature and the cryopreservation temperature may be the same or different. The freezing temperature may be any temperature at which freezing is possible and is not particularly limited. The cryopreservation temperature may be any temperature at which the frozen state can be maintained and is not particularly limited. The freezing temperature, the cryopreservation temperature, and the storage period may be appropriately selected by those skilled in the art. For example, the cell aggregate may be placed in a freezer at about -70°C to -90°C for about 24 hours to 72 hours and then transferred to liquid nitrogen at about -196°C and stored for 24 hours or more.

[0017] The cell aggregates of the present invention can be used, for example, as cell transplants, as well as in cell-based tests such as screening for anticancer drugs. In particular, since the cell aggregates of the present invention are freeze-thawable, they can be prepared and stored in advance before use, making them highly convenient.

[0018] 2. Method for producing cell aggregates The cell aggregates of the present invention can be produced by a method (hereinafter referred to as "the production method of the present invention") which includes supporting adherent cells on a molded body having a nonwoven fiber structure and comprising fibers containing an ethylene-vinyl alcohol copolymer, and culturing the cells under stirring. According to the production method of the present invention, adherent cells proliferate in the gaps of the molded body, and the cells aggregate or clump together to form cell aggregates, which are then naturally separated from the molded body. Further stirring promotes proliferation and separation. That is, stirring promotes the supply of oxygen and nutrients necessary for cell survival into the nonwoven fiber structure, thereby promoting cell proliferation. Furthermore, the promotion of cell proliferation leads to the formation of cell aggregates by providing the number of cells necessary for the formation of cell aggregates within the nonwoven fiber structure. In addition, stirring also has the effect of physically separating the cell aggregates formed within the nonwoven fiber structure from the nonwoven fiber structure. Here, it is preferable that the fibers containing the ethylene-vinyl alcohol copolymer are moist heat adhesive fibers.

[0019] In this specification, "nonwoven fiber structure" refers to a board-like structure formed by bonding fibers together without weaving them.

[0020] In this specification, "moist heat adhesive fiber" means a fiber containing a moist heat adhesive resin that can exhibit adhesive function by high-temperature steam or hot water. In this specification, "moist heat adhesive resin" means a resin that softens by high-temperature steam or hot water and can exhibit adhesive function.

[0021] Examples of heat-adhering resins include thermoplastic resins, particularly hydrophilic resins and water-soluble resins, that soften in hot water (e.g., 80-120°C, especially around 95-100°C) and are self-adhering or can adhere to other fibers. Examples include cellulosic resins (C1-3 alkylcellulose ethers such as methylcellulose, hydroxyC1-3 alkylcellulose ethers such as hydroxymethylcellulose, carboxyC1-3 alkylcellulose ethers such as carboxymethylcellulose or their salts), polyalkylene glycol resins (polyC2-4 alkylene oxides such as polyethylene oxide and polypropylene oxide), polyvinyl resins [polyvinylpyrrolidone, polyvinyl ether, vinyl alcohol polymers (vinyl alcohol polymers containing α-C2-10 olefin units such as ethylene and propylene, especially ethylene-vinyl alcohol copolymers), polyvinyl acetate]. Examples include [such as acrylic resins], acrylic copolymers and their salts [polymers containing units composed of acrylic monomers such as (meth)acrylic acid and its salts, (meth)acrylamide, in particular (meth)acrylic copolymers containing (meth)acrylamide units], modified vinyl copolymers (polymers or salts of vinyl monomers such as isobutylene, styrene, ethylene, vinyl ether, etc., and unsaturated carboxylic acids such as maleic anhydride or their anhydrides), polymers with hydrophilic substituents (polyesters, polyamides, polystyrene or their salts with sulfo groups, carboxyl groups, hydroxyl groups, etc.), and aliphatic polyester resins [polylactic acid resins (e.g., polylactic acid)]. Furthermore, among polyolefin resins, polyester resins, polyamide resins, polyurethane resins, thermoplastic elastomers or rubbers (such as styrene elastomers), resins that can be softened with hot water or high-temperature steam to exhibit adhesive function can also be used as moist heat adhesive resins.

[0022] The moist heat adhesive resin may be used alone or in combination of two or more types.

[0023] As the moist heat adhesive resin, vinyl alcohol polymers such as ethylene-vinyl alcohol copolymers, polylactic acid resins such as polylactic acid, and (meth)acrylic copolymers containing (meth)acrylamide units are preferred, vinyl alcohol polymers containing α-C2-10 olefin units such as ethylene and propylene are more preferred, and ethylene-vinyl alcohol copolymers are even more preferred.

[0024] Ethylene-vinyl alcohol copolymers can be obtained by saponifying ethylene-vinyl ester copolymers.

[0025] Examples of vinyl ester monomers used in the production of the aforementioned ethylene-vinyl ester copolymer include vinyl formate, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl caprylate, vinyl laurate, vinyl palmitate, vinyl stearate, vinyl oleate, and vinyl benzoate. Among these, vinyl acetate is the most preferred from a cost perspective.

[0026] The ethylene unit content (copolymerization ratio) in the ethylene-vinyl alcohol copolymer is, for example, 10 to 60 mol%, preferably 20 to 55 mol%, and more preferably 30 to 50 mol%. Having ethylene units within this range provides the unique property of having moist heat adhesion but no hot water solubility. If the proportion of ethylene units is too low, the ethylene-vinyl alcohol copolymer easily swells or gels with low-temperature water vapor (water), and its form changes easily after just one wetting with water. On the other hand, if the proportion of ethylene units is too high, hygroscopicity decreases, making it difficult for fiber adhesion by moist heat to occur, thus making it difficult to manufacture molded articles with practical strength. When the proportion of ethylene units is particularly in the range of 30 to 50 mol%, the resin of the fibers softens, and adhesion points between fibers are easily formed, resulting in particularly excellent processability of the molded article into sheet or plate-like structures.

[0027] The degree of saponification of vinyl alcohol units in ethylene-vinyl alcohol copolymers is, for example, about 90 to 99.99 mol%, preferably 95 to 99.98 mol%, and more preferably about 96 to 99.97 mol%. If the degree of saponification is too low, the thermal stability decreases, and stability is reduced by thermal decomposition and gelation. On the other hand, if the degree of saponification is too high, it becomes difficult to manufacture the fibers themselves. Here, the degree of saponification of vinyl alcohol units refers to the extent to which the alkoxycarbonyl groups (also known as ester groups, RO-CO-) in the vinyl alcohol units are hydrolyzed and replaced by hydroxyl groups, and the degree of saponification is expressed as the percentage of hydroxyl groups to the total number of carboxyl groups and hydroxyl groups. The degree of saponification is measured by titration with sodium hydroxide.

[0028] The viscosity-average degree of polymerization (P) of the ethylene-vinyl alcohol copolymer can be selected as needed, but is preferably around 200 to 2500, more preferably 300 to 2000, and more preferably 400 to 1500. A degree of polymerization within this range provides an excellent balance between spinnability and adhesiveness of the resulting fibers. This viscosity-average degree of polymerization (P) is calculated from the viscosity (η) of the ethylene-vinyl alcohol copolymer using the following formula. logP = 1.613 log{η} × 10 4 ) / 7.15}

[0029] From a spinning perspective, the melt index (MI) of the ethylene-vinyl alcohol copolymer is important. MI indicates the degree of fluidity of the molten thermoplastic resin and is a typical index for quality control of thermoplastic resins. MI measurement is performed in accordance with JIS K7210. The MI of the ethylene-vinyl alcohol copolymer can be selected as needed. From the viewpoint of balancing the spinnability and adhesiveness of the resulting fibers, the MI of the ethylene-vinyl alcohol copolymer measured under conditions of a temperature of 190°C and a load of 2160g is preferably 1.0 to 10g / 10min, more preferably 1.3 to 7.0g / 10min, and even more preferably 1.5 to 6.6g / 10min.

[0030] The cross-sectional shape (cross-sectional shape perpendicular to the length direction of the fiber) of the fiber containing an ethylene-vinyl alcohol copolymer (hereinafter sometimes referred to as "the fiber") may be a general solid cross-sectional shape such as a round cross-section or an irregular cross-section [flattened, elliptical, polygonal, 3-14 lobed, T-shaped, H-shaped, V-shaped, dogbone-shaped (I-shaped), etc.], or it may be a hollow cross-section.

[0031] The fiber in question may be a composite fiber composed of multiple resins. The composite fiber only needs to have an ethylene-vinyl alcohol copolymer on at least a portion of its surface, but from the viewpoint of adhesion, it is preferable that the ethylene-vinyl alcohol copolymer occupies at least a portion of the surface continuously in the longitudinal direction.

[0032] Examples of cross-sectional structures of composite fibers in which the ethylene-byl alcohol copolymer occupies the surface include core-sheath type, sea-island type, side-by-side type, multilayer laminated type, radial laminated type, and random composite type. Of these cross-sectional structures, the core-sheath type structure (i.e., a core-sheath type structure in which the sheath portion is composed of the ethylene-byl alcohol copolymer), in which the ethylene-byl alcohol copolymer occupies the entire surface continuously in the longitudinal direction, is preferred due to its high adhesiveness.

[0033] In the case of composite fibers, two or more types of moist heat adhesive resins may be combined, or a moist heat adhesive resin may be combined with a non-moist heat adhesive resin. In this specification, "non-moist heat adhesive resin" refers to a resin that does not exhibit adhesive function in the presence of either high-temperature steam or hot water.

[0034] Examples of non-moist heat-adhesive resins include water-insoluble or hydrophobic resins, specifically polyolefin resins, (meth)acrylic resins, vinyl chloride resins, styrene resins, polyester resins, polyamide resins, polycarbonate resins, polyurethane resins, and thermoplastic elastomers. These non-moist heat-adhesive resins can be used individually or in combination of two or more. Among these non-moist heat-adhesive resins, those with a melting point higher than that of moist heat-adhesive resins (e.g., ethylene-vinyl alcohol copolymers) are preferred from the viewpoint of heat resistance and dimensional stability (e.g., polypropylene resins, polyester resins, polyamide resins), and polyester resins and polyamide resins are more preferred from the viewpoint of excellent balance between heat resistance and fiber formation properties.

[0035] As the polyester resin, aromatic polyester resins such as poly-C2-4 alkylene arylate resins [polyethylene terephthalate resins such as polyethylene terephthalate (PET), polytrimethylene terephthalate, polybutylene terephthalate, polyethylene naphthalate, etc.] are preferred, and polyethylene terephthalate resins are more preferred. In addition to ethylene terephthalate units, polyethylene terephthalate resins may also contain units composed of other dicarboxylic acids [e.g., isophthalic acid, naphthalene-2,6-dicarboxylic acid, phthalic acid, 4,4'-diphenylcarboxylic acid, bis(carboxyphenyl)ethane, 5-sodium sulfoisophthalic acid, etc.] or diols (e.g., diethylene glycol, 1,3-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, cyclohexane-1,4-dimethanol, polyethylene glycol, polytetramethylene glycol, etc.] in a proportion of about 20 mol% or less.

[0036] Preferred polyamide resins include aliphatic polyamides and their copolymers such as polyamide 6, polyamide 66, polyamide 610, polyamide 10, polyamide 12, and polyamide 6-12, as well as semi-aromatic polyamides synthesized from aromatic dicarboxylic acids and aliphatic diamines. These polyamide resins may also contain units other than amide units.

[0037] In the case of composite fibers composed of an ethylene-bill alcohol copolymer and a non-moist heat-adhesive resin (fiber-forming polymer), the ratio (mass ratio) of the two can be appropriately selected depending on the structure (e.g., core-sheath type structure). The mass ratio of ethylene-bill alcohol copolymer to non-moist heat-adhesive resin is, for example, 90 / 10 to 10 / 90, preferably 80 / 20 to 15 / 85, and more preferably 60 / 40 to 20 / 80. If the proportion of ethylene-bill alcohol copolymer is too high, it becomes difficult to ensure the strength of the fiber, and if the proportion of ethylene-bill alcohol copolymer is too low, it becomes difficult to maintain a continuous ethylene-bill alcohol copolymer in the longitudinal direction of the fiber surface, resulting in reduced adhesion. This tendency is also true when the ethylene-bill alcohol copolymer is coated onto the surface of a non-moist heat-adhesive fiber.

[0038] Examples of composite fibers include, preferably, composite fibers formed from an ethylene-vinyl alcohol copolymer and a polyester resin, and composite fibers formed from an ethylene-vinyl alcohol copolymer and a polyamide resin, and more preferably, composite fibers formed from an ethylene-vinyl alcohol copolymer and a polyester resin.

[0039] Examples of the core-sheath type composite fiber include a core-sheath type composite fiber formed from a sheath made of an ethylene-vinyl alcohol copolymer and a core made of a non-moist heat-adhesive resin. Preferably, these are a core-sheath type composite fiber formed from a sheath made of an ethylene-vinyl alcohol copolymer and a core made of a polyester resin, and a core-sheath type composite fiber formed from a sheath made of an ethylene-vinyl alcohol copolymer and a core made of a polyamide resin. More preferably, these are a core-sheath type composite fiber formed from a sheath made of an ethylene-vinyl alcohol copolymer and a core made of a polyester resin.

[0040] The average fineness of the fibers in question is, for example, 0.01 to 100 dtex, preferably 0.1 to 50 dtex, more preferably 0.5 to 30 dtex, and particularly preferably 1 to 10 dtex. When the average fineness is within this range, an excellent balance is achieved between the strength and adhesive properties of the fibers. The average fineness is measured according to the method of JIS L 1015.

[0041] The average fiber length of the fibers in question is, for example, 10 to 100 mm, preferably 20 to 80 mm, more preferably 25 to 75 mm, and particularly preferably about 35 to 55 mm. When the average fiber length is within this range, the fibers intertwine sufficiently, improving the mechanical strength of the molded product. The average fiber length is measured according to the method of JIS L 1015.

[0042] The crimp rate of the fiber in question is, for example, 1 to 50%, preferably 3 to 40%, more preferably 5 to 30%, and particularly preferably 10 to 20%. The crimp count is, for example, 1 to 100 crimps / inch, preferably 5 to 50 crimps / inch, and more preferably 10 to 30 crimps / inch. The crimp rate and crimp count are measured according to the method of JIS L 1015.

[0043] The above-mentioned "molded article comprising fibers containing an ethylene-bil alcohol copolymer and having a nonwoven fiber structure" may further contain non-moist heat-adhering fibers. In this specification, "non-moist heat-adhering fibers" means fibers that cannot exhibit adhesive function by either high-temperature steam or hot water.

[0044] Examples of non-moist heat-adhering fibers include polyester fibers (aromatic polyester fibers such as polyethylene terephthalate fiber, polytrimethylene terephthalate fiber, polybutylene terephthalate fiber, polyethylene naphthalate fiber, etc.), polyamide fibers (aliphatic polyamide fibers such as polyamide 6, polyamide 66, polyamide 11, polyamide 12, polyamide 610, polyamide 612, etc., semi-aromatic polyamide fibers, aromatic polyamide fibers such as polyphenylene isophthalamide, polyhexamethylene terephthalamide, poly-p-phenylene terephthalamide, etc.), and polyolefin fibers (polyethylene, polypropylene, etc.). Examples include C2-4 olefin fibers, acrylic fibers (such as acrylonitrile-vinyl chloride copolymers and other acrylonitrile-containing fibers), polyvinyl fibers (such as polyvinyl acetal fibers), polyvinyl chloride fibers (such as polyvinyl chloride, vinyl chloride-vinyl acetate copolymers, and vinyl chloride-acrylonitrile copolymer fibers), polyvinylidene chloride fibers (such as vinylidene chloride-vinyl chloride copolymer fibers and vinylidene chloride-vinyl acetate copolymer fibers), poly(p-phenylene)benzobisoxazole fibers, polyphenylene sulfide fibers, and cellulose fibers (e.g., rayon fibers, acetate fibers). These non-moist heat-adhering fibers can be used individually or in combination of two or more types.

[0045] The average fineness and average fiber length of non-moist heat-adhering fibers are similar to those of moist heat-adhering fibers.

[0046] The ratio of the subject fiber to the non-moist heat-adhesive fiber (mass ratio of the subject fiber to the non-moist heat-adhesive fiber) in the above-mentioned molded article is, for example, 20 / 80 to 100 / 0, preferably 30 / 70 to 100 / 0. When manufacturing a rigid molded article, a higher proportion of the subject fiber is preferable, and the mass ratio of the subject fiber to the non-moist heat-adhesive fiber is more preferably 80 / 20 to 100 / 0, even more preferably 90 / 10 to 100 / 0, and particularly preferably around 95 / 5 to 100 / 0. When the proportion of the subject fiber is within this range, a molded article can be obtained that ensures high surface hardness and bending behavior. When manufacturing a molded article utilizing the properties of the non-moist heat-adhesive fiber, the mass ratio of the subject fiber to the non-moist heat-adhesive fiber is more preferably 20 / 80 to 99 / 1, even more preferably 30 / 70 to 90 / 10, and particularly preferably around 40 / 60 to 80 / 20.

[0047] The fiber adhesion rate of the above molded article is preferably 10-85%, more preferably 10-70%, even more preferably 10-60%, and particularly preferably about 10-35%. In this specification, "fiber adhesion rate" refers to the ratio of the number of fibers that are adhered in pairs to the total number of fibers in the cross-section of the molded article having a nonwoven fiber structure (i.e., fiber adhesion rate = 100 × number of fibers that are adhered in pairs to the cross-section of the molded article / total number of fibers in the cross-section of the molded article). Therefore, a low fiber adhesion rate means that the proportion of fibers that adhere to each other (the proportion of fibers that are bundled and adhered) is small.

[0048] The fibers constituting the nonwoven fiber structure of the molded body described above are bonded at their respective contact points. However, in order to generate a large bending stress with as few contact points as possible, it is preferable that these bonding points are uniformly distributed along the thickness direction, from the surface of the molded body to the interior (center) and then to the back surface. If the bonding points are concentrated on the surface or interior, it becomes difficult to secure sufficient bending stress, and the shape stability in areas with few bonding points decreases.

[0049] Because the above-mentioned molded body is formed from the fibers in question, it exhibits excellent shape stability and ease of handling in cell culture. The molded body can consistently maintain the voids necessary for cell adhesion and proliferation throughout the culture period, thus demonstrating a high ability to preserve cell morphology in cell culture. Therefore, using the above-mentioned molded body as a culture carrier enables the immobilization of cells, particularly adherent cells, and three-dimensional culture of adherent cells.

[0050] Furthermore, the above-mentioned molded body has permeability to medium components (nutrient supply) and oxygen for cell maintenance in cell culture, as well as to waste removal, for example, 0.02 to 0.7 g / cm³. 3 Preferably 0.025 to 0.3 g / cm³ 3 More preferably 0.03 to 0.15 g / cm³ 3 More preferably 0.075 to 0.125 g / cm³ 3 It has an apparent density of [value]. By adjusting the apparent density to the above range, it becomes possible to continue cell proliferation for a long period of time. Here, "apparent density" is the value obtained by dividing the mass of a molded body having a nonwoven fiber structure containing voids by the volume of the molded body. Note that this apparent density can also be determined by measuring the basis weight (i.e., mass per unit area) and thickness of the molded body and dividing the basis weight by the thickness.

[0051] The above-mentioned molded body is substantially composed of the fibers in question without resin impregnation. Furthermore, its nonwoven fiber structure is formed by the adhesion of the fibers in question. The above-mentioned molded body has a sufficiently large cell adhesion surface and excellent shape stability, for example, when performing three-dimensional culture of adherent cells. Therefore, by using the above-mentioned molded body as a cell culture carrier in a bioreactor or the like, efficient cell proliferation becomes possible.

[0052] The shape of the molded body described above is not particularly limited, but may be spherical, plate-shaped, rod-shaped, square-shaped, elliptical, disc-shaped, cylindrical, or columnar. Among these shapes, spherical and disc-shaped are preferred. When the molded body is used as a cell culture carrier in a bioreactor, a spherical shape is preferred. In particular, when used in a fluid-type bioreactor, the fluidity of a spherical carrier is superior to that of other shapes. When used in a fixed-bed bioreactor, a spherical carrier is easy to pack, and because the contact points between carriers are small, aggregation is less likely to occur, making it easy to handle.

[0053] If the molded body is disc-shaped, its size is not particularly limited and can be appropriately selected from the viewpoint of ease of handling, for example, by referring to the size of commonly available culture plates. On the other hand, if the molded body is spherical, its average particle size is preferably 1 mm or more, more preferably 2 mm or more, and even more preferably 3 mm or more, from the viewpoint of facilitating the separation and recovery of the carrier, and preferably 20 mm or less, more preferably 10 mm or less, and even more preferably 6 mm or less, from the viewpoint of increasing the cell culture surface area per unit volume. The average particle size is obtained by measuring the maximum diameter of 10 randomly selected cell culture carriers using calipers and averaging them.

[0054] The above-described molded body has a nonwoven fiber structure in which the fibers are appropriately bonded, and therefore possesses high bending stress even though it is lightweight and low density. This molded body has breathability and heat insulation properties, as well as high hardness, and also excellent folding resistance and toughness. In other words, this molded body is formed into a plate shape, and even when a load is applied to the surface, local deformation is unlikely to occur. By bending and deforming in response to the applied stress, it absorbs that stress, resulting in high impact resistance, and it does not easily break or fracture even when subjected to a strong impact. Furthermore, this molded body can be composed substantially only of fibers, and there is no need to add chemical binders or special agents, so it does not use components that generate harmful components (such as volatile organic compounds such as formaldehyde), and is inert to cells.

[0055] Components other than the constituent fibers adhering to the molded body may be removed by washing or other means. Examples of such components include oils. Oils are used in general methods for manufacturing nonwoven fiber structures to suppress friction between fibers during the process of intertwining the fibers. The removal method is not particularly limited, but for example, washing may be done using water or organic solvents, acid / alkali components, or liquid components containing surfactants. The washing method is also not particularly limited, but the molded body may be washed by immersing it in the liquid component. When washing continuously, the molded body may be placed on a mesh conveyor and transported while the liquid component is passed through the inside of the molded body for washing. This washing may be done multiple times. Washing may be done in stages with different liquid components. Finally, the molded body may be washed once or more with water to prevent any washing components from remaining inside. After washing, the liquid components may be removed by suction or compression, and any remaining liquid inside the molded body may be evaporated and dried with hot air or the like.

[0056] When the above molded body is used for long-term cell culture, differences in cell density occur between the center and the outer edge of the molded body as cell proliferation continues over a long period. To avoid such uneven cell proliferation, one or more through-holes may be provided in the molded body. By providing through-holes, nutrient supply to the inside (center) of the molded body and waste removal are efficiently performed, resulting in uniform cell proliferation in the molded body. Cell proliferation can also be observed within the through-holes. The size and number of through-holes provided in the molded body can be appropriately determined depending on the size of the molded body and are not particularly limited. Depending on the size of the molded body, for example, one to four, preferably one or two, through-holes of about 0.5 mm to 2 mm, preferably about 0.7 μm to 1.5 mm, may be provided.

[0057] The above-mentioned molded article is manufactured by forming a nonwoven fiber structure using the above-mentioned fibers. Any method known in the art may be used for such manufacturing. For example, if the fibers are moist heat-adhering fibers, they can be manufactured by the following method. First, the fibers containing the fibers are webbed. Conventional methods can be used to form the web, such as direct methods like the spunbond method and melt-blown method, or dry methods such as the carding method using staple fibers or the air-laying method. Of these methods, the carding method using staple fibers is particularly commonly used. Examples of webs obtained using staple fibers include random webs, semi-random webs, parallel webs, and cross-wrap webs. Of these webs, semi-random webs and parallel webs are preferred when the proportion of bundled fused fibers is increased. Next, the obtained fiber web is sent to the next process by a mesh conveyor and then exposed to a stream of superheated steam or high-temperature steam (high-pressure steam) to obtain a molded article having a nonwoven fiber structure. In other words, as the fiber web transported by the mesh conveyor passes through the high-temperature steam stream ejected from the nozzle of the steam injection device, the fibers are bonded together three-dimensionally by the sprayed high-temperature steam.

[0058] The belt conveyor used is not particularly limited as long as it can compress the fiber web used for processing to the desired density while treating it with high-temperature steam, but an endless mesh conveyor is preferably used. Alternatively, a single general-purpose mesh conveyor may be used, or two mesh conveyors may be combined and the web transported between them as needed. This method of transport suppresses deformation of the web's shape due to external forces such as water, high-temperature steam, and vibrations of the mesh conveyor during processing. Furthermore, the density and thickness of the molded product with a nonwoven fiber structure after processing can be controlled by adjusting the spacing of the belt.

[0059] When two mesh conveyors are combined, the steam injection device for supplying steam to the web is installed inside one of the mesh conveyors and supplies steam to the web through the mesh structure of the conveyor. A suction box may be installed on the opposite mesh conveyor. The suction box can suck up and discharge excess steam that has passed through the web. In order to steam treat both the front and back sides of the web at once, another suction box may be installed downstream of the mesh conveyor on the side where the steam injection device is installed, and the steam injection device may be installed inside the mesh conveyor on the opposite side where this suction box is installed. If there is no downstream steam injection device or suction box, the front and back sides of the fiber web can be steam treated by reversing the front and back sides of the treated fiber web and passing it through the treatment device again.

[0060] The shape of the mesh conveyor is not particularly limited, as long as it does not hinder the transport of the web or the high-temperature steam treatment. However, when high-temperature steam treatment is performed, the surface shape of the belt may be transferred to the surface of the fiber web depending on the conditions, so it is preferable to select an appropriate shape depending on the application. In particular, if a molded product with a flat surface is desired, a fine mesh net should be used. Note that the upper limit is around 90 mesh, and nets with finer mesh than this have low permeability and make it difficult for water vapor to pass through. From the viewpoint of heat resistance to steam treatment, the material of the mesh conveyor is preferably metal, heat-resistant polyester resin, polyphenylene sulfide resin, polyarylate resin (all aromatic polyester resin), aromatic polyamide resin, or other heat-resistant resins.

[0061] The high-temperature steam sprayed from the steam injection device is an airflow, and unlike water entanglement or needle punching, it penetrates the web without significantly moving the fibers within the web being treated. This penetration of the steam flow into the web, combined with the moist heat effect, efficiently covers the surface of each fiber within the web in a moist heat state, enabling uniform thermal bonding. Furthermore, because this process is carried out in a very short time under high-speed airflow, while sufficient heat conduction of the steam to the fiber surface occurs, the process ends before sufficient heat conduction to the inside of the fibers. Therefore, deformation such as crushing of the entire fiber web or loss of thickness due to the pressure and heat of the high-temperature steam is unlikely to occur. As a result, moist heat bonding is completed without significant deformation of the fiber web, and the degree of adhesion in the surface and thickness directions is generally uniform.

[0062] Furthermore, when obtaining molded articles with high surface hardness and bending strength, it is important to expose the web to high-temperature steam while it is compressed to the desired apparent density between mesh conveyors or rollers during the steam treatment process. In particular, when aiming to obtain relatively high-density molded articles, it is necessary to compress the fiber web with sufficient pressure during the high-temperature steam treatment. Moreover, it is possible to adjust the desired thickness and density by ensuring an appropriate clearance between the mesh conveyors or rollers. In the case of mesh conveyors, since it is difficult to compress the web all at once, it is preferable to set the tension of the mesh conveyors as high as possible and gradually narrow the clearance from upstream of the steam treatment point. Furthermore, by adjusting the steam pressure and processing speed, it is possible to process the web into a molded article with the desired bending hardness, surface hardness, lightness, and breathability.

[0063] In this case, if you want to increase the hardness, you can make the back of the mesh conveyor on the opposite side of the nozzle from the web out of the way by using a stainless steel plate or something similar to create a structure that prevents water vapor from passing through. In this case, the water vapor that has passed through the web being processed will be reflected here, and the fibers will be strongly bonded due to the heat retention effect of the water vapor. Conversely, if only light bonding is required, a suction box may be placed to discharge excess water vapor to the outside.

[0064] A nozzle for injecting high-temperature steam can be constructed using a plate or die with predetermined orifices arranged continuously in the width direction, and arranged so that the orifices are aligned in the width direction of the supplied web. One or more rows of orifices are sufficient, and multiple rows may be arranged in parallel. Alternatively, multiple nozzle dies, each having one row of orifices, may be installed in parallel.

[0065] When using a nozzle with an orifice in a plate, the plate thickness may be, for example, about 0.5 to 1 mm. There are no particular restrictions on the diameter and pitch of the orifice as long as the desired fiber fixation conditions are met, but the orifice diameter is usually about 0.05 to 2 mm, preferably 0.1 to 1 mm, and more preferably about 0.2 to 0.5 mm. The orifice pitch is usually about 0.5 to 3 mm, preferably 1 to 2.5 mm, and more preferably about 1 to 1.5 mm. If the orifice diameter is too small, it can lead to equipment problems such as low processing accuracy of the nozzle and difficulty in processing, as well as operational problems such as increased clogging. Conversely, if the orifice diameter is too large, the steam injection force will decrease. On the other hand, if the pitch is too small, the nozzle holes will become too dense, reducing the strength of the nozzle itself. On the other hand, if the pitch is too large, there may be cases where high-temperature steam does not sufficiently hit the entire surface of the web, resulting in uneven adhesion between fibers in the web and a decrease in the strength of the molded product.

[0066] Regarding high-temperature steam, there are no particular limitations as long as the desired fiber fixation is achieved, and the pressure should be set according to the material and form of the fiber used. However, the pressure is, for example, 0.05 to 2 MPa, preferably 0.05 to 1.5 MPa, and more preferably 0.1 to 1 MPa. If the steam pressure is too high, the fibers forming the web may move, causing disorder in the weave, or the fibers may melt too much, making it impossible to partially maintain their shape. On the other hand, if the pressure is too low, it may not be possible to supply the web with the heat necessary for fiber fusion, or the steam may not be able to penetrate the web, resulting in uneven fiber fusion in the thickness direction, and it may be difficult to control the uniform ejection of steam from the nozzle.

[0067] The temperature of the high-temperature steam is, for example, 70 to 150°C, preferably 80 to 120°C, and more preferably 90 to 110°C. The processing speed of the web with high-temperature steam is, for example, 200 m / min or less, preferably 0.1 to 100 m / min, and more preferably 1 to 50 m / min.

[0068] If necessary, a predetermined uneven shape can be applied to the mesh conveyor, and the resulting board product can also have this uneven shape applied by transferring it to the conveyor. Alternatively, it can be laminated with other materials to form a laminate, or processed into a desired shape (such as cylindrical, rectangular prism, spherical, or ellipsoidal) by molding.

[0069] After partially bonding the fibers of the fiber web with moist heat in this manner, moisture may remain in the resulting molded body, so the web may be dried as needed. Regarding drying, it is necessary that the surface of the molded body that has come into contact with the drying heating element does not lose its fiber shape due to melting of the fibers after drying, and conventional methods can be used as long as the fiber shape is maintained. For example, large drying equipment such as cylinder dryers or tenters used for drying nonwoven fabrics may be used, but since the amount of residual moisture is often small and can be dried with relatively mild drying methods, non-contact methods such as far-infrared irradiation, microwave irradiation, electron beam irradiation, or methods using hot air are preferred.

[0070] Furthermore, as described above, the molded body is obtained by adhering the fibers of the present case with high-temperature steam, but it may also be adhered by other conventional methods, such as heat-pressure fusion bonding (such as heat embossing), mechanical compression (such as needle punching), etc. In addition, other conventional methods may be used for adhering the obtained molded bodies to each other.

[0071] When the fibers of the present case are wet-heat adhesive fibers, they can also be adhered by immersing the fiber web in hot water. However, in the method using immersion in hot water, it is difficult to control the fiber adhesion rate, and it is also difficult to obtain a molded body with high uniformity of the fiber adhesion rate. The reasons are presumed to be that the wet-heat adhesiveness varies depending on the position due to the influence of the air inevitably contained in the fiber web, the influence on the structure due to the extrusion of this air outside the fiber web, the deformation of the fine structure inside the fiber by the take-up roller when taking out the wet-heat adhered fiber web from the hot water, and the difference in the deformation of the fine structure in the vertical direction due to the weight of the hot water contained in the taken-out fiber web.

[0072] The molded body obtained as described above can be made into a desired shape by further processing it by, for example, known methods (such as pressing, cutting).

[0073] In the production method of the present invention, adherent cells are cultured using the above molded body as a cell culture carrier. That is, adherent cells are supported on the above molded body and cultured. The support of cells only requires that the cells are finally supported on the molded body, and any method can be used. Since the cells are adherent cells, the cells can easily adhere to the molded body by bringing the cells into contact with the molded body and are supported on or in the molded body. For example, adherent cells can be supported on the above molded body by methods such as dropping a cell suspension onto the above molded body, immersing the above molded body in a cell suspension, etc. The cell suspension is, for example, in a liquid such as water, a culture medium, etc., preferably 10 3 ~10 8 cells / ml, more preferably 10 3 ~10 6The suspension may have a concentration of approximately cells / ml. The adhesive cells are preferably supported in the gaps between the fibers of the molded body.

[0074] The adherent cells then supported on the molded body are cultured under agitation. For culture, the culture medium and culture temperature, etc., should be selected to be suitable for the cells to be cultured (i.e., the adherent cells supported on the molded body). Examples of culture conditions include those described in "Cultured Cell Handbook: All About the Basics and Analytical Methods of Cell Culture," edited by Toshio Kuroki and Nam-Ho Heo, Yodosha, published July 2004.

[0075] Preferably, a liquid culture medium is used, and any conventional culture medium suitable for the adherent cells to be cultured may be used. For example, when culturing animal cells, a culture medium containing various components such as various essential amino acids, various vitamins, and sugars such as glucose (e.g., Dulbecco's modified Eagle medium) may be used. The amount of these components in the culture medium should be appropriate for cell culture. Furthermore, the culture medium may also be, for example, fetal bovine serum medium, serum-free medium, or human serum medium.

[0076] Stirring may be performed, for example, using a stirrer or shaker. The stirring speed should be, for example, about 30 rpm to 140 rpm, preferably about 50 rpm to 90 rpm. Stirring should be performed for at least a portion of the culture period, preferably throughout the entire culture period.

[0077] In the manufacturing method of the present invention, stirring promotes the aggregation of adhesive cells in the molded body, resulting in the efficient formation of cell aggregates. Furthermore, stirring separates the cell aggregates formed in the molded body from the molded body and releases them into the culture medium. The cell aggregates thus produced can be easily recovered from the culture medium by methods such as filtration.

[0078] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to these examples. [Examples]

[0079] Example 1: Production of cell aggregates by the manufacturing method of the present invention 1 (1) Fabrication of molded body 0.1 g / cm³ 3 A disc-shaped molded body with an apparent density of 2 mm thickness and 6 mm diameter was fabricated. Two through holes of approximately 1 mm in diameter were provided in the molded body. As the fiber used, a core-sheath type composite staple fiber (Sophista, manufactured by Kuraray Co., Ltd., average fineness 1.7 dtex, average fiber length 51 mm, mass ratio of ethylene-vinyl alcohol copolymer / non-moist heat adhesive resin = 50 / 50, crimp count 22.9 pieces / inch, crimp rate 12.2%) was used, with a basis weight of approximately 100 g / m², obtained by carding. 2 A card web was created, and seven of these webs were stacked together to achieve a total weight of 700 g / m². 2 This was used as the card web. This card web was then transferred to a mesh conveyor equipped with a 50-mesh, 500mm wide stainless steel endless net.

[0080] This mesh conveyor consists of a pair of conveyors, a lower conveyor and an upper conveyor. Steam injection nozzles are installed on the underside of the belts of both conveyors, allowing high-temperature steam to be injected onto the web as it passes through the belt. Furthermore, metal rolls for adjusting the web thickness (hereinafter sometimes abbreviated as "web thickness adjustment rolls") are installed upstream of these nozzles. The lower conveyor has a flat top surface (i.e., the surface through which the web passes), while the upper conveyor has a curved bottom surface that follows the web thickness adjustment rolls. The web thickness adjustment rolls on the upper conveyor are paired with those on the lower conveyor. The upper conveyor is also movable vertically, allowing the distance between the web thickness adjustment rolls on the upper and lower conveyors to be adjusted to a predetermined interval. Furthermore, the upstream side of the upper conveyor is inclined at a 30-degree angle to the downstream section (relative to the lower surface of the downstream side of the upper conveyor) with the web thickness adjustment roll as the pivot point, and the downstream section is bent so that it is parallel to the lower conveyor. When the upper conveyor moves up and down, it maintains this parallel relationship. These mesh conveyors rotate at the same speed and in the same direction, and the structure allows for pressurization while maintaining a predetermined clearance between the two conveyor belts and between the web thickness adjustment rolls. This is designed to adjust the web thickness before steam treatment, similar to a calendering process. In other words, the card web fed from the upstream side travels on the lower conveyor, but the gap between it and the upper conveyor gradually narrows as it reaches the web thickness adjustment roll. When this gap becomes narrower than the web thickness, the web is sandwiched between the upper and lower conveyor belts and travels while being gradually compressed. This web is compressed to a thickness approximately equal to the clearance provided in the web thickness adjustment roll, then subjected to steam treatment at that thickness, and continues to travel downstream on the conveyor while maintaining that thickness. In this case, the web thickness adjustment roll was adjusted to a linear pressure of 50 kg / cm.

[0081] Next, the card web was introduced into a steam injection device installed on the lower conveyor, and high-temperature steam at 0.4 MPa was injected from this device (vertically) in the thickness direction of the card web to perform steam treatment and obtain a molded body having a nonwoven fiber structure. In this steam injection device, a nozzle was installed in the lower conveyor to blow high-temperature steam towards the web via a conveyor net, and a suction device was installed on the upper conveyor. In addition, another injection device with the nozzle and suction device in the reversed configuration was installed downstream of this injection device in the direction of web movement, and steam treatment was performed on both the front and back surfaces of the web.

[0082] The steam injection nozzles had a diameter of 0.3 mm, and a steam injection system was used in which the nozzles were arranged in a single row at 1 mm intervals along the width direction of the conveyor. The processing speed was 3 m / min, and the distance between the upper and lower conveyor belts on the nozzle side and the suction side was 10 mm. The nozzles were positioned on the underside of the conveyor belt so as to be in close contact with the belt.

[0083] The molded material obtained in this way had a board-like shape, was much harder than general nonwoven fabrics, and exhibited excellent shape stability. Furthermore, the fiber adhesion rate of the obtained molded material was 26.7%. The molded material obtained in this way was processed into a 2mm thick plate, and then further cut into a 6mm diameter disc shape.

[0084] Two through holes, approximately 1 mm in diameter, were made in the resulting disc-shaped molded body.

[0085] (2) Cell culture under stirring using a molded body Cells were seeded onto the molded bodies prepared in (1) above. Human immortalized adipose-derived mesenchymal stem cells (ADSC (ASC52telo)) were used. Cell suspensions of 6 × 10^6 cells / mL were prepared by suspending the cells in culture medium, and cells were seeded by dropping 45 μL of this suspension onto each molded body using a micropipette. The ADSCs seeded on the molded bodies were cultured in a spinner flask (50 mL of medium) at 37°C for 20 days with stirring (70 rpm). The culture medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin. Half of the culture medium was replaced twice every 7 days.

[0086] (3) Cell aggregates The culture described in (2) above resulted in the formation of cell aggregates. Granular cell aggregates suspended in the culture medium were observed. The cell aggregates were collected by filtration. The size of the obtained cell aggregates was measured by dimensional measurement of the microscopic images. As a result, cell aggregates with a diameter or longest axis of approximately 2 mm were obtained.

[0087] Example 2: Production of cell aggregates by the manufacturing method of the present invention 2 (4) Fabrication of molded body A disc-shaped molded body without through holes was fabricated using the method described in (1) of Example 1.

[0088] (5) Cell culture under stirring using a molded body Cells were seeded onto the molded bodies prepared in (4) above. Human immortalized adipose-derived mesenchymal stem cells (ADSCs) were used. Cells were suspended in culture medium to prepare a cell suspension of 6 × 10^6 cells / mL, and cells were seeded by dropping 45 μL of this suspension onto each molded body using a micropipette. The ADSCs seeded on the molded bodies were cultured in a spinner flask (50 mL of medium) at 37°C for 25 days with stirring (70 rpm). The culture medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin. Half of the culture medium was replaced twice every 7 days.

[0089] (6) Cell aggregates The culture described in (5) above resulted in the formation of cell aggregates. Granular cell aggregates suspended in the culture medium were observed. The cell aggregates were collected by filtration. The size of the obtained cell aggregates was measured by dimensional measurement of the microscopic images. As a result, cell aggregates with a diameter or longest axis of approximately 2 mm were obtained.

[0090] Comparative Example 1: Production of cell aggregates by cell culture without stirring Cells were seeded onto the molded body prepared in Example 1 in the same manner as in Example 1. ADSC cells were used. The ADSCs seeded on the molded body were cultured in a 12-well dish (1 mL of medium) at 37°C for 20 days under static conditions. The medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin. Half of the medium was replaced twice every 7 days. No cell aggregates were observed after 20 days of culture.

[0091] Comparative Example 2: Preparation of cell aggregates by conventional method 1 ADSCs were suspended in culture medium to prepare a cell suspension of 2.5 × 10^5 cells / mL. The culture medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin. 1 mL of the cell suspension was placed in a 5 mL centrifuge tube and cultured at 37°C for 45 days. Half of the culture medium was replaced twice every 7 days. After 45 days of culture, cell aggregates were observed. The size of the obtained cell aggregates was measured using the same method as in Example 1. The diameter or longest axis was approximately 1.5 mm.

[0092] Comparative Example 3: Preparation of Cell Aggregates by Conventional Method 2 ADSCs were suspended in culture medium to prepare a cell suspension of 1.0 × 10^4 cells / mL. The culture medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin. 1 mL of the cell suspension was placed in a 96-well U-bottom plate and cultured at 37°C for 2 days. After 2 days of culture, cell aggregates were observed. The size of the obtained spheroids was measured using the same method as in Example 1. The diameter or longest axis was approximately 500 μm.

[0093] Evaluation of cell viability in cell aggregates Cell viability was evaluated in the cell aggregates obtained in Example 1 and Comparative Example 2. Live-Dead Assay was performed using a confocal laser microscope for evaluation. The Live or Dead™ Cell Viability Assay Kit (Thermo Fisher Scientific) was used. This kit uses two dyes: Calcein AM (for living cells) and a cell-impermeable DNA-binding dye. Non-fluorescent Calcein AM, when hydrolyzed by endogenous esterases, produces hydrophilic Calcein that easily accumulates in the cytoplasm. Esterase activity is proportional to the number of living cells. The DNA-binding dye does not penetrate living cells with intact membranes, but binds only to the DNA of dead cells. This allows for Live / Dead evaluation using green / red colors.

[0094] As a result, cell survival was observed in the cell aggregates obtained in Example 1, whereas cell survival was not observed in the cell aggregates obtained in Comparative Example 2 (Figure 1). This is thought to be because the molded body containing moist heat-adhesive fibers and having a nonwoven fiber structure has appropriate hydrophilicity, which promotes the production of extracellular matrix, thereby suppressing excessive cell density and improving viability.

[0095] Evaluation of cell viability in cell aggregates after freeze-thaw cycles. The cell viability after freeze-thawing was evaluated for the cell aggregates obtained in Example 1 and Comparative Example 3. After rinsing the cell aggregates with PBS (phosphate-buffered saline), the cell aggregates were placed in a microtube containing 1 mL of the cryopreservation agent CELLBANKER1 (manufactured by Xenogen Pharma Co., Ltd.), and the tube was shaken in an orbital shaker for 30 seconds. The CELLBANKER1 in the microtube was removed, and the same amount of new CELLBANKER1 was added. The microtube was placed in a cell freezing container, Mr. Frosty (manufactured by Thermo Fisher Scientific), and frozen in a deep freezer at -90°C for 24 hours. After that, it was kept in liquid nitrogen for 24 hours. The microtube was removed from the liquid nitrogen and thawed in a water bath at 37°C. The cell aggregates in the microtube were removed, and a Live-Dead Assay was performed in the same manner as above.

[0096] As a result, the cell aggregates obtained in Example 1 showed cell survival even after freeze-thaw cycles (Figure 2). On the other hand, the cell aggregates obtained in Comparative Example 3 did not show cell survival (Figure 2). Therefore, it was found that the cell aggregates of the present invention showed improved cell viability after freeze-thaw cycles compared to conventional cell aggregates. Normally, freezing causes cell death due to the destruction of cell membranes by ice, but in the cell aggregates of the present invention, it is thought that the presence of extracellular matrix inhibited ice growth, thus reducing damage to cells.

[0097] Confirmation of extracellular matrix in cell aggregates Picrosilius red staining was performed on the cell aggregates obtained in Example 1 and Comparative Example 2. Staining was carried out using a Picrosilius red staining kit (manufactured by ScyTek Laboratories). In Picrosilius red staining, collagen is stained red, and cytoplasm and other proteins are stained yellow. After staining, the red and yellow regions were identified visually, and the area of ​​these regions was analyzed using WinROOF image analysis software (manufactured by Mitani Corporation).

[0098] As a result, in the cell aggregates obtained in Example 1, the area stained red accounted for 45.3%, indicating that components other than collagen made up more than 50% of the total (Figure 3). On the other hand, in the cell aggregates of Comparative Example 2, almost 100% was stained red, indicating that collagen was the main component of the extracellular matrix (Figure 3).

Claims

1. A cell aggregate comprising adherent cells and extracellular matrix, wherein the aggregate contains viable cells even at a depth of 30 μm or more from the surface of the aggregate after freeze-thawing, the aggregate is granular with a diameter of 1.5 mm or more, and the adherent cells are mesenchymal stem cells.

2. The cell aggregate according to claim 1, wherein less than 50% of the extracellular matrix is ​​collagen.

3. A cell aggregate according to claim 1 or 2, which is in a frozen state.

4. It consists of fibers containing an ethylene-vinyl alcohol copolymer and has a nonwoven fiber structure, and has a concentration of 0.025 to 0.3 g / cm³. ３ A method for producing cell aggregates, comprising supporting adherent cells on a molded body having an apparent density and culturing the cells under stirring, wherein the adherent cells are mesenchymal stem cells.

5. The manufacturing method according to claim 4, wherein the fiber containing the ethylene-vinyl alcohol copolymer is a moist heat adhesive fiber.

6. The manufacturing method according to claim 4, wherein the fiber containing the ethylene-vinyl alcohol copolymer is a composite fiber containing a non-moist heat-adhering fiber.

7. The manufacturing method according to claim 6, wherein the composite fiber is a core-sheath type composite fiber comprising a sheath portion made of an ethylene-vinyl alcohol copolymer and a core portion made of a polyester resin.

8. The manufacturing method according to any one of claims 4 to 7, comprising culturing the adherent cells supported on the molded body under stirring at 30 to 140 rpm.

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