Cell aggregate and method for producing cell aggregate

Abstract

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 while stirring.

Description

Cell aggregates and method for producing cell aggregates

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

[0002] In recent years, in cell transplantation therapy, in order to fully utilize the functions of transplanted cells, attention has been focused on transplanting cell aggregates (also called cell masses or cell spheroids) created by three-dimensional culture, rather than single cells cultured in a monolayer. These aggregates more closely reflect the cell-cell interactions and cell-extracellular matrix interactions in vivo.

[0003] Methods for producing cell aggregates have been reported previously, including a method in which cells are cultured on a culture plate with a non-adhesive bottom and cells suspended in the medium are allowed to adhere to each other to produce cell aggregates (non-adhesive surface cell culture method), a method in which a cell suspension is dropped onto the inside of a culture plate lid, and the resulting dome-shaped droplets are cultured upside down, causing the cells to gather below the droplets due to gravity and adhere to each other to form cell aggregates (hanging drop cell culture method), and a method in which cells are cultured in a rotating cell culture chamber, causing the cells to come into contact with each other within the chamber to form cell aggregates (rotary cell culture method). Furthermore, collagen-rich mesenchymal stem cell clumps produced in a growth medium supplemented with factors that promote collagen production, and frozen grafts for mesenchymal stem cell transplantation obtained by cryopreserving the cell clumps with a cryopreservative, have been reported (Patent Document 1).

[0004] Patent No. 7105487

[0005] In cell transplantation therapy using cell aggregates, it is necessary to prepare cell aggregates for each transplant surgery, but there is no guarantee that the cell number or cell function of the cell aggregates will be uniform. On the other hand, if cell aggregates that meet a certain quality are cryopreserved and then thawed and transplanted at the time of transplantation surgery, a stable transplant effect can be expected. However, with conventional techniques, cell viability after thawing of the cell aggregates is low, making it difficult to cryopreserve the cell aggregates. Therefore, the present invention aims to provide cell aggregates that can be cryopreserved and a method for producing the cell aggregates.

[0006] As a result of extensive research, the inventors surprisingly succeeded in obtaining cell aggregates by supporting adherent cells on a molded article having a nonwoven fiber structure made of fibers containing an ethylene-vinyl alcohol copolymer and culturing the cells under agitation. Even more surprisingly, they found that the cell aggregates had a high cell survival rate, with the cells in the center surviving even after freezing and thawing, and could be cryopreserved. Thus, the present invention was completed.

[0007] That is, the present invention provides the following: (1) A cell aggregate comprising adherent cells and an extracellular matrix, the cell aggregate comprising viable cells even at a depth of 30 μm or more from the surface of the aggregate. (2) The cell aggregate according to (1) above, comprising viable cells even at a depth of 30 μm or more from the surface of the aggregate after freeze-thawing the aggregate. (3) The cell aggregate according to (1) or (2) above, which is in a granular shape with a diameter exceeding 1.5 mm. (4) The cell aggregate according to any one of (1) to (3) above, in which less than 50% of the extracellular matrix is ​​collagen. (5) The cell aggregate according to any one of (1) to (4) above, in a frozen state. (6) A method for producing a cell aggregate, comprising supporting adherent cells on a molded article having a nonwoven fiber structure made of fibers comprising an ethylene-vinyl alcohol-based copolymer and culturing the cells under agitation. (7) A method for producing a cell aggregate according to (6), in which the fibers comprising an ethylene-vinyl alcohol-based copolymer are heat-and-moisture adhesive fibers. (8) The manufacturing method according to (6) above, wherein the fiber containing the ethylene-vinyl alcohol-based copolymer is a conjugated fiber containing a non-thermal adhesive fiber. (9) The manufacturing method according to (8) above, wherein the conjugated fiber is a core-sheath type conjugated fiber containing a sheath made of an ethylene-vinyl alcohol-based copolymer and a core made of a polyester-based resin. (10) The manufacturing method according to any one of (6) to (9) above, comprising supporting adherent 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.

[0008] According to the present invention, cell aggregates can be obtained by a simple method in which adherent cells are supported on a molded article having a nonwoven fiber structure made of fibers containing an ethylene-vinyl alcohol copolymer, and the cells are cultured under agitation. Compared to cell aggregates obtained by conventional techniques, the cell aggregates obtained by the present invention are larger in size, exhibit improved cell viability during culture, and also exhibit improved cell viability after freeze-thawing. The cell aggregates of the present invention are freezable because cells in the center of the aggregate survive even after freeze-thawing. Thus, use of the cell aggregates of the present invention eliminates the need to prepare cell aggregates for each treatment, thereby shortening the lead time of cell transplantation therapy (the time from deciding on treatment to starting treatment).

[0009] A micrograph showing the results of a live-dead assay of cell aggregates. A micrograph showing the results of a live-dead assay of cell aggregates after freezing and thawing. A micrograph showing the results of picrosirius red staining of cell aggregates.

[0010] 1. Cell Aggregates As used herein, the term "cell aggregate" refers to a mass of cells formed by the aggregation or aggregation of multiple cells. The cell aggregates of the present invention are aggregates of adherent cells, and contain extracellular matrix in addition to cells. The cell aggregates of the present invention are cell aggregates produced outside the body (in vitro or ex vivo).

[0011] As used herein, the term "adherent cells" refers to cells that grow by adhering to a culture support during cell culture. These cells may be derived from any organism, including animals (e.g., mammals and non-mammals), plants, and insects, and are not particularly limited thereto. Examples of adherent cells include, but are not limited to, osteoblasts, chondrocytes, hematopoietic cells, epithelial cells (e.g., mammary epithelial cells), endothelial cells (e.g., vascular endothelial cells), epidermal cells, fibroblasts, mesenchymal-derived cells, cardiac myocytes, myoblasts, smooth muscle cells, skeletal muscle cells derived from living organisms, human tumor cells, fibrocytes, EB virus mutant cells, hepatocytes, kidney cells, bone marrow cells, macrophages, hepatic parenchymal cells, pancreatic β cells, small intestinal cells, mammary gland cells, salivary gland cells, thyroid cells, skin cells, and somatic stem cells (e.g., mesenchymal stem cells).

[0012] As used herein, the term "extracellular matrix" refers to an insoluble substance present outside cells in living organisms, a non-cellular component that fills the intercellular spaces. Extracellular matrix is ​​primarily composed of water, proteins, and polysaccharides, and is known to vary in composition and form depending on the biological tissue. The extracellular matrix contained in the cell aggregate of the present invention may preferably be an extracellular matrix produced by the adherent cells contained in the cell aggregate themselves. The extracellular matrix contained in the cell aggregate of the present invention contains a relatively small amount of collagen; for example, less than about 50%, preferably about 48% or less, more preferably about 46% or less of the extracellular matrix 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, a diameter (in the case of non-spherical cells, the longest diameter, hereinafter referred to as the "major axis") of about 1.5 mm or more, preferably about 2 mm or more, and more preferably about 5 mm or more. 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 may be measured by a conventional method, for example, by observing an image under a microscope.

[0014] Furthermore, in cell aggregates of the prior art, cells in the center of the aggregate die after freezing and thawing, whereas in the cell aggregates of the present invention, cells in the center of the aggregate remain viable even after freezing and thawing, resulting in improved cell viability after freezing and thawing compared to the prior art. Furthermore, in cell aggregates of the prior art, cells in the center of large aggregates, such as those with a diameter of more than about 1.5 mm, die even without freezing and thawing. In contrast, in the cell aggregates of the present invention, cells in the center remain viable even in large aggregates, resulting in improved cell viability compared to the prior art.

[0015] Here, the central portion refers to, for example, a portion at a depth of about 30 μm or more, preferably about 40 μm or more, from the surface of the cell aggregate (i.e., an interior of the cell aggregate at a distance of about 30 μm or more, preferably about 40 μm or more, from the surface of the cell aggregate), although this depends on the size of the cell aggregate. The distance from the surface of the cell aggregate to the deep portion can be measured, for example, using a microscopic image. Furthermore, cell survival in the cell aggregate can be confirmed by microscopic observation. Thus, the cell aggregate of the present invention is a freezable cell aggregate, i.e., a cell aggregate that can be frozen and thawed.

[0016] Therefore, the cell aggregates of the present invention may be frozen cell aggregates. The cell aggregates are preferably frozen in a cryopreservation solution. Examples of cryopreservation solutions include those known in the art, such as those containing 10% DMSO (dimethyl sulfoxide), 20% FBS (fetal bovine serum), and 70% DMEM (Dulbecco's Modified Eagle Medium). Alternatively, commercially available cryopreservation solutions may be used. Examples of commercially available cryopreservation solutions include, but are not limited to, CELLBANKER1 (manufactured by Xenogen Pharma). Cell aggregates may be frozen at any temperature that allows freezing, such as 0°C or below, -70°C or below, or -80°C or below. The cell aggregates may also be stored in a frozen state, for example, but not limited to, for 24 hours or more, or even for a longer period of time, such as one month or more. Freezing and cryopreservation may be performed in a freezer or liquid nitrogen. The freezing and cryopreservation temperatures 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, cryopreservation temperature, and storage period may be appropriately selected by those skilled in the art. For example, the cell aggregates may be placed in a freezer at about -70°C to -90°C for about 24 to 72 hours, and then transferred to liquid nitrogen at about -196°C and stored for 24 hours or more.

[0017] The cell aggregate of the present invention is used, for example, as a cell transplant, and also in cell-based tests such as screening of anticancer drugs. In particular, the cell aggregate of the present invention can be frozen and thawed, and therefore can be prepared and stored in advance before use, which is very 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") comprising supporting adherent cells on a molded article having a nonwoven fiber structure and made of fibers containing an ethylene-vinyl alcohol copolymer and culturing the cells under agitation. According to the production method of the present invention, the adherent cells proliferate in the interstices of the molded article, and the cells aggregate by gathering or aggregating to form cell aggregates. The cell aggregates then naturally detach from the molded article. Furthermore, agitation promotes proliferation and detachment. That is, agitation promotes the supply of oxygen and nutrients necessary for cell survival to the interior of the nonwoven fiber structure, thereby promoting cell proliferation. Furthermore, promotion of cell proliferation promotes cell aggregate formation by providing the number of cells necessary for cell aggregate formation within the nonwoven fiber structure. Furthermore, agitation also has the effect of physically separating cell aggregates formed within the nonwoven fiber structure from the nonwoven fiber structure. Here, the fibers containing an ethylene-vinyl alcohol copolymer are preferably moist heat-bondable fibers.

[0019] As used herein, the term "nonwoven fiber structure" refers to a board-like structure formed by bonding fibers together rather than weaving them.

[0020] In this specification, the term "heat-adhesive fiber under moisture" refers to a fiber that contains a heat-adhesive resin under moisture and that can exhibit adhesive properties when exposed to high-temperature steam or hot water. In this specification, the term "heat-adhesive resin under moisture" refers to a resin that can be softened by high-temperature steam or hot water and exhibit adhesive properties.

[0021] Examples of the heat-and-moisture adhesive resin include thermoplastic resins, particularly hydrophilic resins and water-soluble resins, which soften in hot water (for example, 80 to 120°C, particularly about 95 to 100°C) and are capable of self-adhesion or adhesion to other fibers. Examples include cellulose-based resins (C1-3 alkyl cellulose ethers such as methyl cellulose, hydroxy C1-3 alkyl cellulose ethers such as hydroxymethyl cellulose, carboxy C1-3 alkyl cellulose ethers such as carboxymethyl cellulose, and salts thereof), polyalkylene glycol resins (poly C2-4 alkylene oxides such as polyethylene oxide and polypropylene oxide), polyvinyl-based resins (polyvinylpyrrolidone, polyvinyl ethers, vinyl alcohol-based polymers (vinyl alcohol-based polymers containing α-C2-10 olefin units such as ethylene or propylene, particularly ethylene-vinyl alcohol copolymers), and polyvinyl acetone. Examples of suitable adhesive resins include acrylic copolymers and their salts (copolymers containing units of acrylic monomers such as (meth)acrylic acid and its salts, and (meth)acrylamide, particularly (meth)acrylic copolymers containing (meth)acrylamide units), modified vinyl copolymers (copolymers of vinyl monomers such as isobutylene, styrene, ethylene, and vinyl ether with unsaturated carboxylic acids or their anhydrides such as maleic anhydride, or their salts), polymers containing hydrophilic substituents (polyesters, polyamides, polystyrenes, or their salts containing sulfo, carboxy, or hydroxy groups), and aliphatic polyester resins (polylactic acid resins, for example, polylactic acid). Furthermore, resins that can be softened by hot water or high-temperature steam to exhibit adhesive properties, such as polyolefin resins, polyester resins, polyamide resins, polyurethane resins, thermoplastic elastomers, and rubbers (such as styrene elastomers), can also be used as heat-and-moisture adhesive resins.

[0022] The heat-and-moisture adhesive resins may be used alone or in combination of two or more.

[0023] As the heat-and-moisture 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] The ethylene-vinyl alcohol copolymer can be obtained by saponifying an ethylene-vinyl ester copolymer.

[0025] Examples of vinyl ester monomers used in the production of the 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, vinyl benzoate, etc. Among these, vinyl acetate is most preferred from the standpoint of cost.

[0026] The content (copolymerization ratio) of ethylene units in the ethylene-vinyl alcohol copolymer is, for example, 10 to 60 mol%, preferably 20 to 55 mol%, and more preferably about 30 to 50 mol%. Having the ethylene units in this range results in the unique property of having wet heat adhesiveness but not hot water solubility. If the ethylene unit ratio is too low, the ethylene-vinyl alcohol copolymer will easily swell or gel with low-temperature steam (water), and will easily change shape even when wet with water. On the other hand, if the ethylene unit ratio is too high, the hygroscopicity will decrease, making it difficult to achieve fiber adhesion under wet heat, making it difficult to produce molded articles with practical strength. When the ethylene unit ratio is particularly in the range of 30 to 50 mol%, the resin of the fibers will soften, facilitating the formation of bonded points between the fibers, resulting in particularly excellent processability of the molded article into sheet- or plate-like structures.

[0027] The degree of saponification of the vinyl alcohol units in the ethylene-vinyl alcohol copolymer is, for example, about 90 to 99.99 mol%, preferably about 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 decreases due to thermal decomposition and gelation. On the other hand, if the degree of saponification is too high, it becomes difficult to produce the fiber itself. Here, the degree of saponification of the vinyl alcohol units refers to the degree to which alkoxycarbonyl groups (also known as ester groups, RO-CO-) in the vinyl alcohol units are hydrolyzed and replaced with hydroxyl groups, and the saponification degree is expressed as the percentage of hydroxyl groups relative to the total number of carboxyl groups and hydroxyl groups. The degree of saponification is measured by sodium hydroxide titration.

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

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

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

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

[0032] Examples of the cross-sectional structure of the conjugated fiber in which the ethylene-vinyl alcohol copolymer occupies the surface include a core-sheath type, an island-in-sea type, a side-by-side type or a multi-layered type, a radially bonded type, a randomly conjugated type, etc. Among these cross-sectional structures, a core-sheath type structure in which the ethylene-vinyl alcohol copolymer occupies the entire surface continuously in the length direction (i.e., a core-sheath type structure in which the sheath portion is composed of the ethylene-vinyl alcohol copolymer) is preferred in terms of high adhesiveness.

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

[0034] Examples of non-moisture heat-sensitive 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-moisture heat-sensitive adhesive resins can be used alone or in combination of two or more. Among these non-moisture heat-sensitive adhesive resins, resins having a melting point higher than that of the moisture-sensitive adhesive resin (e.g., ethylene-vinyl alcohol copolymer) are preferred in terms of heat resistance and dimensional stability (e.g., polypropylene resins, polyester resins, and polyamide resins). Polyester resins and polyamide resins are more preferred in terms of an excellent balance between heat resistance, fiber formability, and the like.

[0035] The polyester resin is preferably an aromatic polyester resin such as a poly(C2-4 alkylene arylate) resin [e.g., polyethylene terephthalate resin such as polyethylene terephthalate (PET), poly(trimethylene terephthalate), poly(butylene terephthalate), polyethylene naphthalate, etc.], with a polyethylene terephthalate resin being more preferred. The polyethylene terephthalate resin may contain, in addition to ethylene terephthalate units, 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.) at a ratio of about 20 mol% or less.

[0036] Preferred polyamide resins include aliphatic polyamides such as polyamide 6, polyamide 66, polyamide 610, polyamide 10, polyamide 12, and polyamide 6-12, copolymers thereof, and 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 a conjugated fiber composed of an ethylene-vinyl alcohol-based copolymer and a non-thermal adhesive resin (fiber-forming polymer), the ratio (mass ratio) of the two can be appropriately selected depending on the structure (e.g., core-sheath structure). The mass ratio of the ethylene-vinyl alcohol-based copolymer to the non-thermal adhesive resin under moisture is, for example, about 90 / 10 to 10 / 90, preferably about 80 / 20 to 15 / 85, and more preferably about 60 / 40 to 20 / 80. If the proportion of the ethylene-vinyl alcohol-based copolymer is too high, it becomes difficult to ensure fiber strength. If the proportion of the ethylene-vinyl alcohol-based copolymer is too low, it becomes difficult to maintain the ethylene-vinyl alcohol-based copolymer continuously along the fiber surface in the longitudinal direction, resulting in reduced adhesiveness. This tendency also applies when the ethylene-vinyl alcohol-based copolymer is coated on the surface of a non-thermal adhesive fiber under moisture.

[0038] Preferred examples of the composite fiber include a composite fiber formed from an ethylene-vinyl alcohol copolymer and a polyester resin, and a composite fiber formed from an ethylene-vinyl alcohol copolymer and a polyamide resin, and more preferred examples include a composite fiber formed from an ethylene-vinyl alcohol copolymer and a polyester resin.

[0039] Examples of the fiber of the present invention that is a sheath-core type composite fiber include a sheath-core type composite fiber formed of a sheath made of an ethylene-vinyl alcohol-based copolymer and a core made of a non-thermal adhesive resin, preferably a sheath-core type composite fiber formed of a sheath made of an ethylene-vinyl alcohol-based copolymer and a core made of a polyester-based resin, and a sheath-core type composite fiber formed of a sheath made of an ethylene-vinyl alcohol-based copolymer and a core made of a polyamide-based resin, and more preferably a sheath-core type composite fiber formed of a sheath made of an ethylene-vinyl alcohol-based copolymer and a core made of a polyester-based resin.

[0040] The average fineness of the present fiber is, for example, 0.01 to 100 dtex, preferably 0.1 to 50 dtex, more preferably 0.5 to 30 dtex, and particularly preferably about 1 to 10 dtex. When the average fineness is within this range, an excellent balance between fiber strength and adhesiveness is achieved. The average fineness is measured by the method of JIS L 1015.

[0041] The average fiber length of the present fiber 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 are sufficiently entangled, improving the mechanical strength of the molded article. The average fiber length is measured according to the method of JIS L 1015.

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

[0043] The above-mentioned "molded article having a nonwoven fiber structure and made of fibers containing an ethylene-vinyl alcohol-based copolymer" may further contain non-thermal adhesive fibers. In this specification, "non-thermal adhesive fibers" refers to fibers that cannot exhibit adhesive function even with high-temperature steam or hot water.

[0044] Examples of non-heat-adhesive fibers that can be bonded under moisture include polyester fibers (aromatic polyester fibers such as polyethylene terephthalate fibers, polytrimethylene terephthalate fibers, polybutylene terephthalate fibers, and polyethylene naphthalate fibers), polyamide fibers (aliphatic polyamide fibers such as polyamide 6, polyamide 66, polyamide 11, polyamide 12, polyamide 610, and polyamide 612, semi-aromatic polyamide fibers, and aromatic polyamide fibers such as polyphenylene isophthalamide, polyhexamethylene terephthalamide, and poly-p-phenylene terephthalamide), and polyolefin fibers (polyethylene, polypropylene, and the like). C2-4 olefin fibers, etc.), acrylic fibers (acrylonitrile fibers having acrylonitrile units such as acrylonitrile-vinyl chloride copolymers, etc.), polyvinyl fibers (polyvinyl acetal fibers, etc.), polyvinyl chloride fibers (polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, vinyl chloride-acrylonitrile copolymer fibers, etc.), polyvinylidene chloride fibers (vinylidene chloride-vinyl chloride copolymer fibers, vinylidene chloride-vinyl acetate copolymer fibers, etc.), polyparaphenylene benzobisoxazole fibers, polyphenylene sulfide fibers, cellulose fibers (for example, rayon fibers, acetate fibers, etc.). These non-thermal adhesive fibers under moisture can be used alone or in combination of two or more.

[0045] The average fineness and average fiber length of the non-thermal adhesive fiber under moisture are the same as those of the thermal adhesive fiber under moisture.

[0046] The ratio of the present fiber to the non-thermal adhesive fiber contained in the molded article (mass ratio of the present fiber / non-thermal adhesive fiber under moisture) is, for example, 20 / 80 to 100 / 0, preferably 30 / 70 to 100 / 0. When producing a hard molded article, a higher proportion of the present fiber is preferred, and the mass ratio of the present fiber / non-thermal adhesive fiber is more preferably about 80 / 20 to 100 / 0, even more preferably 90 / 10 to 100 / 0, and particularly preferably about 95 / 5 to 100 / 0. When the proportion of the present fiber is within this range, a molded article can be obtained that ensures high surface hardness and bending behavior. When producing a molded article utilizing the properties of the non-thermal adhesive fiber, the mass ratio of the present fiber / non-thermal adhesive fiber is more preferably about 20 / 80 to 99 / 1, even more preferably 30 / 70 to 90 / 10, and particularly preferably about 40 / 60 to 80 / 20.

[0047] The fiber bonding ratio of the molded article is preferably 10 to 85%, more preferably 10 to 70%, even more preferably 10 to 60%, and particularly preferably about 10 to 35%. In this specification, "fiber bonding ratio" refers to the ratio of the number of fibers in which two or more fibers are bonded to the total number of fibers in the cross section of the molded article having a nonwoven fiber structure (i.e., fiber bonding ratio = 100 × number of fibers in which two or more fibers are bonded in the cross section of the molded article / total number of fibers in the cross section of the molded article). Therefore, a low fiber bonding ratio means that the ratio of multiple fibers bonded together (the ratio of fibers bonded together in a bundle) is low.

[0048] The fibers constituting the nonwoven fiber structure of the molded article are bonded at their contact points, and in order to generate a large bending stress with as few contact points as possible, it is preferable that these bond points be uniformly distributed along the thickness direction from the surface to the interior (center) and back of the molded article. If the bond points are concentrated on the surface or interior, not only will it be difficult to ensure sufficient bending stress, but shape stability will also be reduced in areas with fewer bond points.

[0049] Because the molded article is formed from the present fiber, it has excellent shape stability and is easy to handle during cell culture. The molded article can constantly maintain the voids necessary for cell adhesion and proliferation throughout the culture period, and therefore has a high ability to maintain cell morphology during cell culture. Therefore, using the molded article as a culture carrier enables the immobilization of cells, particularly adherent cells, and the three-dimensional culture of adherent cells.

[0050] Furthermore, the molded article has a density of, for example, 0.02 to 0.7 g / cm from the viewpoint of permeability of medium components (nutrient supply) and oxygen for maintaining cells in cell culture and removal of waste products. ３ , preferably 0.025 to 0.3 g / cm ３ , more preferably 0.03 to 0.15 g / cm ３ , more preferably 0.075 to 0.125 g / cm ３ The apparent density is 0.015 to 0.015 mm. By adjusting the apparent density to the above range, cell proliferation can be continued for a long period of time. Here, "apparent density" refers to 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. 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 molded article is substantially composed of the present fiber without being impregnated with resin. Furthermore, its nonwoven fiber structure is formed by bonding the present fiber. The molded article 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 molded article as a cell culture carrier in a bioreactor or the like, efficient cell proliferation is possible.

[0052] The shape of the molded body is not particularly limited, and may be, for example, spherical, plate-like, rod-like, angular, elliptical, disk-like, cylindrical, columnar, etc. Among these shapes, spherical and disk-like shapes are preferred. When the molded body is used in a bioreactor as a cell culture carrier, a spherical shape is preferred. In particular, when used in a fluidized-bed bioreactor, the fluidity of spherical carriers is superior to that of carriers with other shapes. When used in a fixed-bed bioreactor, spherical carriers are easy to pack, have small contact points between carriers, and are less likely to aggregate, making them easy to handle.

[0053] When 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 a commonly available culture plate. On the other hand, when 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 separation and recovery of the carrier, and is 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 a vernier caliper and averaging the measured values.

[0054] The molded article has a nonwoven fiber structure in which the fibers are appropriately bonded, resulting in high bending stress despite its light weight and low density. The molded article is breathable and insulating, as well as having high hardness, excellent folding resistance, and toughness. Specifically, the molded article is shaped like a plate, and even when a load is applied to its surface, it is resistant to localized deformation. It absorbs the applied stress by bending and deforming, resulting in high impact resistance and resistance to breakage or fracture, even when subjected to a strong impact. Furthermore, the molded article can be constructed essentially from fibers alone, eliminating the need for the addition of chemical binders or special chemicals. Therefore, it does not use components that generate harmful substances (such as volatile organic compounds, such as formaldehyde), and is inactive against cells.

[0055] Components other than the constituent fibers adhering to the molded body may be removed by washing or the like. Examples of such components include oils. Oils are used to reduce fiber friction during the entanglement process in a typical method for producing nonwoven fiber structures. The removal method is not particularly limited, and cleaning may be performed using, for example, a liquid component containing water or an organic solvent, an acid or alkali component, a surfactant, or the like. The cleaning method is also not particularly limited, and the molded body may be immersed in the liquid component. When performing continuous cleaning, the molded body may be placed on a mesh conveyor and conveyed while passing the liquid component through the interior of the molded body. This cleaning may be performed multiple times. The molded body may be washed in stages with different liquid components. Finally, the molded body may be washed one or more times with water to prevent cleaning components from remaining in the molded body. After cleaning, the liquid components may be removed by suction, compression, or the like, and the liquid remaining in the molded body may be evaporated and dried using hot air or the like.

[0056] When the molded body is used for long-term cell culture, a difference in cell density occurs between the center and the outer periphery of the molded body as cell growth continues over a long period of time. To avoid such uneven cell growth, one or more through-holes may be provided in the molded body. The provision of through-holes allows for efficient supply of nutrients to the interior (center) of the molded body and removal of waste products, resulting in uniform cell growth in the molded body. Cell growth is also observed within the through-holes. The size and number of through-holes provided in the molded body may be determined appropriately depending on the size of the molded body and are not particularly limited. Depending on the size of the molded body, for example, 1 to 4 through-holes, preferably 1 or 2 through-holes, each measuring approximately 0.5 mm to 2 mm, preferably approximately 0.7 μm to 1.5 mm, may be provided.

[0057] The molded article is produced by forming a nonwoven fiber structure using the present fiber. Any method known in the art may be used for this production. For example, when the present fiber is a heat-and-moisture adhesive fiber, it can be produced by the following method. First, fibers containing the present fiber are formed into a web. Conventional web formation methods, such as direct methods such as spunbonding and meltblowing, carding using staple fibers, and dry methods such as air-laying, can be used. Among these methods, carding using staple fibers is particularly widely used. Examples of webs obtained using staple fibers include random webs, semi-random webs, parallel webs, and cross-lap webs. Among these webs, semi-random webs and parallel webs are preferred when the proportion of bundled fusible fibers is to be increased. The resulting fiber web is then transported to the next process on a mesh conveyor and exposed to a stream of superheated steam or high-temperature steam (high-pressure steam) to produce a molded article having a nonwoven fiber structure. That is, when the fiber web transported by the mesh conveyor passes through a high-temperature steam flow sprayed from the nozzle of the steam spraying 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 in processing to the desired density while performing high-temperature steam treatment, and an endless mesh conveyor is preferably used. A typical single mesh conveyor may be used, or, if necessary, two mesh conveyors may be combined to transport the web sandwiched between them. By transporting the web in this manner, deformation of the transported web due to external forces such as water used in processing, high-temperature steam, and vibration of the mesh conveyor can be suppressed during web processing. Furthermore, the density and thickness of the processed nonwoven fiber structure molded article can be controlled by adjusting the belt spacing.

[0059] When two mesh conveyors are combined, a steam sprayer 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 out excess steam that has passed through the web. Furthermore, in order to steam both the front and back sides of the web at the same time, a suction box may be installed downstream of the mesh conveyor on the side where the steam sprayer is installed, and a steam sprayer may be installed inside the mesh conveyor on the opposite side to the suction box. If there is no downstream steam sprayer or suction box, the front and back sides of the fiber web can be steam treated by turning the treated fiber web over 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 interfere with 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 the shape appropriately depending on the application. In particular, when a molded product with a flat surface is desired, a fine mesh net may be used. Note that the upper limit is about 90 mesh, and nets with mesh finer than this have low breathability and make it difficult for water vapor to pass through. As for the material of the mesh conveyor, from the viewpoint of heat resistance to steam treatment, metals, heat-resistant resins such as heat-treated polyester resins, polyphenylene sulfide resins, polyarylate resins (wholly aromatic polyester resins), and aromatic polyamide resins are preferred.

[0061] Because the high-temperature steam injected from the steam injection device is an airflow, it penetrates into the web without significantly displacing the fibers in the web, unlike hydroentanglement or needlepunching. It is believed that the penetration of the steam flow into the web and the moist heat effect allow the steam flow to efficiently cover the surface of each fiber in the web in a moist, heated state, enabling uniform thermal bonding. Furthermore, because this treatment is carried out under a high-speed airflow in an extremely short time, sufficient heat conduction from the steam to the fiber surface is achieved, but the treatment ends before sufficient heat conduction to the interior of the fibers is achieved. As a result, the pressure and heat of the high-temperature steam are unlikely to cause deformation that would cause the entire fiber web to collapse or reduce its thickness. As a result, moist-thermal bonding is completed without significant deformation of the fiber web, resulting in a generally uniform degree of bonding on the surface and in the thickness direction.

[0062] Furthermore, when obtaining a molded product with high surface hardness and bending strength, it is important to expose the web to high-temperature steam while compressed to the desired apparent density between a mesh conveyor or rollers. In particular, when attempting to obtain a molded product with a relatively high density, it is necessary to compress the fiber web with sufficient pressure when treating with high-temperature steam. Furthermore, by ensuring an appropriate clearance between the mesh conveyor or rollers, it is possible to adjust the desired thickness and density. Since it is difficult to compress the web all at once using a mesh conveyor, it is preferable to set the tension of the mesh conveyor as high as possible and gradually narrow the clearance from upstream of the steam treatment point. Furthermore, by adjusting the steam pressure and treatment speed, molded products with the desired bending hardness, surface hardness, lightness, and breathability can be obtained.

[0063] If it is desired to increase the hardness at this time, the back side of the mesh conveyor opposite the nozzle, sandwiching the web, can be made of a stainless steel plate or the like, so that water vapor cannot pass through.The water vapor that passes through the web to be treated is reflected here, and the heat-retaining effect of the water vapor firmly bonds the fiber.On the other hand, if a light bond is required, a suction box can be installed to exhaust excess water vapor outside the room.

[0064] The nozzle for spraying high-temperature steam may be a plate or die with predetermined orifices arranged continuously in the width direction, and may be arranged so that the orifices are arranged in the width direction of the web being fed. There may be one or more rows of orifices, and multiple rows may be arranged in parallel. Alternatively, multiple nozzle dies each having a single row of orifices may be installed in parallel.

[0065] When using a nozzle with orifices in a plate, the plate thickness may be, for example, about 0.5 to 1 mm. There are no particular limitations on the diameter and pitch of the orifices, as long as they allow for the desired fiber fixation. However, the orifice diameter is typically about 0.05 to 2 mm, preferably about 0.1 to 1 mm, and more preferably about 0.2 to 0.5 mm. The orifice pitch is typically about 0.5 to 3 mm, preferably about 1 to 2.5 mm, and more preferably about 1 to 1.5 mm. If the orifice diameter is too small, the nozzle processing accuracy will be reduced, making processing difficult, which will lead to equipment problems, and clogging will be a common operational problem. 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 be too dense, reducing the strength of the nozzle itself. On the other hand, if the pitch is too large, there will be cases where the high-temperature steam does not sufficiently reach the entire surface of the web, resulting in uneven adhesion between the fibers in the web and reduced strength of the molded product.

[0066] The pressure of the high-temperature steam is also not particularly limited as long as it can achieve the desired fixation of the fibers, and can be set depending on the material and shape of the fibers used. The pressure is, for example, about 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 disruption of the formation, or the fibers may melt too much and lose their fiber shape in some places. If the pressure is too low, the amount of heat required to fuse the fibers may not be imparted to the web, or the steam may not penetrate the web, resulting in uneven fiber fusion in the thickness direction, making it difficult to control the uniform spraying of steam from the nozzle.

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

[0068] If necessary, it is possible to impart a predetermined uneven shape to the mesh conveyor and then transfer the uneven shape to the resulting board product.The board may also be laminated with other materials to form a laminate, or may be processed by molding into a desired shape (such as a cylindrical, rectangular prism, sphere, or ellipsoid).

[0069] After the fibers of the fiber web are partially wet-heat bonded in this manner, moisture may remain in the resulting molded product, so the web may be dried as needed. Regarding drying, it is necessary that the surface of the molded product that has come into contact with the drying heater does not lose its fiber form after drying due to melting of the fibers, and any conventional method can be used as long as the fiber form can be maintained. For example, large drying equipment such as a cylinder dryer or tenter used for drying nonwoven fabrics may be used, but since the remaining moisture is often so small that it can be dried using relatively mild drying means, non-contact methods such as far-infrared irradiation, microwave irradiation, or electron beam irradiation, or methods using hot air, are preferred.

[0070] Furthermore, although the molded article is obtained by bonding the present fibers with high-temperature steam as described above, the fibers may be bonded by other conventional methods, such as heat-pressure fusion (such as hot embossing), mechanical compression (such as needle punching), etc. Furthermore, other conventional methods may be used to bond the obtained molded articles together.

[0071] When the present fiber is a heat-and-moisture bondable fiber, it can also be bonded by immersing the fiber web in hot water. However, using a method of immersion in hot water makes it difficult to control the fiber bonding rate and to obtain a molded product with a highly uniform fiber bonding rate. The reasons for this are presumably due to differences in heat-and-moisture bonding depending on the position due to the influence of air inevitably contained in the fiber web, the effect on the structure caused by the air being pushed out of the fiber web, deformation of the internal fiber microstructure caused by the take-up roller when the heat-and-moisture bonded fiber web is removed from hot water, and differences in vertical deformation of the microstructure caused by the weight of the hot water contained in the removed fiber web.

[0072] The molded article obtained as described above can be further processed by a known method (for example, pressing, cutting) to form it into a desired shape.

[0073] In the manufacturing method of the present invention, the molded body is used as a cell culture carrier to culture adherent cells. That is, the adherent cells are supported on the molded body and cultured. The cells can be supported by any method as long as the cells are ultimately supported on the molded body. Since the cells are adherent cells, the cells easily adhere to the molded body by contacting the molded body and are supported on or in the molded body. For example, the adherent cells can be supported on the molded body by a method such as dropping a cell suspension onto the molded body or immersing the molded body in the cell suspension. The cell suspension is, for example, in a liquid such as water or a culture medium, preferably at a concentration of 10 ３ ~10 ８ cells / ml, more preferably 10 ３ ~10 ６ The adherent cells may be in the form of a suspension having a concentration of about 100 cells / ml. The adherent cells are preferably supported in the gaps between the fibers of the molded article.

[0074] The adherent cells thus supported on the molded article are then cultured under agitation. Culture conditions, such as a culture medium and culture temperature, suitable for the cells to be cultured (i.e., the adherent cells supported on the molded article) may be selected. Examples of culture conditions include those described in "Cell Culture Handbook: Basics and Analysis of Cell Culture," edited by Toshio Kuroki and Nam-ho Kyo, Yodosha, published in July 2004.

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

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

[0077] In the production method of the present invention, agitation promotes aggregation of adherent cells in the molded body, resulting in efficient formation of cell aggregates. Furthermore, agitation causes the cell aggregates formed in the molded body to separate from the molded body and become liberated into the culture medium. The cell aggregates thus produced can be easily recovered from the culture medium by, for example, 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.

[0079] Example 1: Production of cell aggregates by the production method of the present invention 1 (1) Production of molded body 0.1 g / cm ３A disk-shaped molded article having a thickness of 2 mm and a diameter of 6 mm and an apparent density of 100 g / m was prepared. Two through-holes with a diameter of approximately 1 mm were formed in the molded article. The fiber in question was 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-heat-adhesive resin = 50 / 50, number of crimps 22.9 / inch, crimp rate 12.2%), which had a core component of polyethylene terephthalate and a sheath component of ethylene-vinyl alcohol copolymer (ethylene content 44 mol%, saponification degree 98.4 mol%, MI (temperature 190°C and load 2160 g) 5.5 g / 10 min). The fiber was cut by a carding method to a basis weight of approximately 100 g / m. ２ Seven sheets of this web were stacked to give a total basis weight of 700 g / m ２ This carded web was transferred to a mesh conveyor equipped with a 50 mesh, 500 mm 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 backside of the belts of both conveyors, allowing high-temperature steam to be injected through the belts onto the passing web. Furthermore, metal rolls for adjusting the web thickness (hereinafter sometimes abbreviated as "web thickness adjustment rolls") are installed upstream of the nozzles. The lower conveyor has a flat upper surface (i.e., the surface through which the web passes), while the upper conveyor has a curved lower surface that conforms to the web thickness adjustment roll. The web thickness adjustment roll of the upper conveyor is arranged to form a pair with the web thickness adjustment roll of the lower conveyor. The upper conveyor is also movable up and down, allowing the distance between the web thickness adjustment rolls of the upper and lower conveyors to be adjusted to a predetermined value. Furthermore, the upstream side of the upper conveyor is inclined at a 30-degree angle (relative to the underside of the downstream side of the upper conveyor) relative to the downstream portion, with the web thickness adjustment roll as the base point, and the downstream portion is bent so as to be parallel to the lower conveyor. When the upper conveyor moves up and down, it moves while maintaining this parallel relationship. These mesh conveyors rotate at the same speed and in the same direction, and these two conveyor belts and the web thickness adjustment rolls are configured to apply pressure while maintaining a predetermined clearance. This operates like a so-called calendering process to adjust the thickness of the web before steam treatment. That is, the card web fed from the upstream side runs on the lower conveyor, and the gap between it and the upper conveyor gradually narrows before reaching 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 gradually compressed as it travels. The web is compressed to a thickness approximately equal to the clearance provided in the web thickness adjusting roll, and is steamed at that thickness. After that, the web continues to run downstream of the conveyor while maintaining the same thickness. Here, the web thickness adjusting roll was adjusted to a linear pressure of 50 kg / cm.

[0081] The carded web was then introduced into a steam injection device attached to the lower conveyor, and high-temperature steam at 0.4 MPa was injected from this device so as to pass through the carded web in the thickness direction (vertically) to subject it to steam treatment, thereby obtaining a molded article having a nonwoven fiber structure. This steam injection device had a nozzle installed in the lower conveyor so as to spray high-temperature steam toward the web through a conveyor net, and a suction device installed on the upper conveyor. In addition, another injection device was installed downstream in the web traveling direction from this injection device, where the nozzle and suction device were combined in a reversed position, and steam treatment was performed on both the front and back sides of the web.

[0082] The water vapor injection nozzle had a hole diameter of 0.3 mm, and the nozzles were arranged in a row at 1 mm intervals along the width direction of the conveyor. The processing speed was 3 m / min, and the gap (distance) between the upper and lower conveyor belts on the nozzle side and suction side was 10 mm. The nozzle was placed on the back side of the conveyor belt so that it was almost in contact with the belt.

[0083] The molded article thus obtained had a board-like shape and was much harder than ordinary nonwoven fabrics, with excellent shape stability. The fiber adhesion rate of the molded article was 26.7%. The molded article thus obtained was processed into a 2 mm thick plate, which was then hollowed out into a 6 mm diameter disk.

[0084] Two through holes with a diameter of about 1 mm were formed in the obtained disk-shaped molded body.

[0085] (2) Cell Culture Using Molded Body Under Agitation Cells were seeded onto the molded body prepared in (1) above. Human immortalized adipose-derived mesenchymal stem cells (Adipose-Derived Stem Cells: ADSCs (ASC52telo)) were used. Cells were suspended in culture medium to prepare a cell suspension at 6 × 10^6 cells / mL, and seeded by dropping 45 μL per molded body using a micropipette. The ADSCs seeded onto the molded body were cultured in a spinner flask (50 mL medium volume) under agitation (70 rpm) at 37°C for 20 days. The culture medium used was Dulbecco's Modified Eagle's 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.

[0086] (3) Cell Aggregates The formation of cell aggregates was observed by the culture described in (2) above. Granular cell aggregates floating in the medium were observed. The cell aggregates were collected by filtration. The size of the obtained cell aggregates was measured by measuring the dimensions in a microscopic photograph. As a result, cell aggregates with a diameter or major axis of approximately 2 mm were obtained.

[0087] Example 2: Production of cell aggregates by the production method of the present invention 2 (4) Production of molded body A disk-shaped molded body without through holes was produced by the method of Example 1 (1).

[0088] (5) Cell Culture Using Molded Body Under Agitation Cells were seeded onto the molded body 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 at 6 x 10^6 cells / mL, and 45 μL per molded body was added dropwise using a micropipette to seed the cells. The ADSCs seeded onto the molded body were cultured in a spinner flask (50 mL medium volume) at 37°C under agitation (70 rpm) for 25 days. The culture medium used was Dulbecco's modified Eagle's 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.

[0089] (6) Cell Aggregates The formation of cell aggregates was observed by the culture in (5) above. Granular cell aggregates floating in the medium were observed. The cell aggregates were collected by filtration. The size of the obtained cell aggregates was measured by measuring the dimensions in a microscopic photograph. As a result, cell aggregates with a diameter or major axis of approximately 2 mm were obtained.

[0090] Comparative Example 1: Production of cell aggregates by cell culture without stirring Cells were seeded on the molded body prepared in Example 1 in the same manner as in Example 1. ADSCs were used as the cells. 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's 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. After 20 days of culture, no formation of cell aggregates was observed.

[0091] Comparative Example 2: Preparation of Cell Aggregates by Conventional Method 1 ADSCs were suspended in culture medium to prepare a cell suspension at 2.5 x 10^5 cells / mL. The culture medium used was Dulbecco's Modified Eagle's 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, the formation of cell aggregates was observed. The size of the resulting cell aggregates was measured using the same method as in Example 1. The diameter or major 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 at 1.0 x 10^4 cells / mL. The culture medium used was Dulbecco's Modified Eagle's 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 two days. After two days of culture, the formation of cell aggregates was observed. The size of the resulting spheroids was measured using the same method as in Example 1. The diameter or major 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. A Live-Dead Assay was performed using a confocal laser microscope. The Live or Dead™ Cell Viability Assay Kit (Thermo Fisher Scientific) was used. The kit uses two dyes: Calcein AM (for live cells) and a cell-impermeable DNA-binding dye. When non-fluorescent Calcein AM is hydrolyzed by endogenous esterases, it produces hydrophilic calcein, which tends to accumulate in the cytoplasm. Esterase activity is proportional to the number of live cells. The DNA-binding dye does not penetrate live cells with intact membranes and binds only to the DNA of dead cells. This allows for a green / red color to be used to evaluate Live / Dead status.

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

[0095] Evaluation of Cell Viability in Cell Aggregates After Freezing and Thawing Cell viability after freezing and 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 cryopreservative CELLBANKER1 (Xenogen Pharma Co., Ltd.) and shaken for 30 seconds using an orbital shaker. CELLBANKER1 was removed from the microtube, and the same amount of CELLBANKER1 was added. The microtube was placed in a cell freezing container, Mr. Frosty (Thermo Fisher Scientific), and frozen in a deep freezer at -90°C for 24 hours. The microtube was then stored 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 as described above.

[0096] As a result, the survival of cells inside the cell aggregates obtained in Example 1 was confirmed even after freezing and thawing (Figure 2). On the other hand, the survival of cells inside the cell aggregates obtained in Comparative Example 3 was not confirmed (Figure 2). Therefore, the cell aggregates of the present invention were confirmed to have improved cell survival after freezing and thawing compared to conventional cell aggregates. Normally, freezing destroys cell membranes with ice, leading to cell death. However, in the cell aggregates of the present invention, the presence of an extracellular matrix inhibits ice growth, which is thought to reduce damage to the cells.

[0097] Confirmation of Extracellular Matrix in Cell Aggregates Picrosirius Red staining was performed on the cell aggregates obtained in Example 1 and Comparative Example 2. Staining was performed using a Picrosirius Red staining kit (manufactured by ScyTek Laboratories). Picrosirius Red staining stains collagen red and cytoplasm and other proteins yellow. After staining, red and yellow regions were visually identified, and the areas of these regions were analyzed using image analysis software WinROOF (manufactured by Mitani Corporation).

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

Claims

1. A cell aggregate comprising adherent cells and an extracellular matrix, the cell aggregate comprising viable cells even at a depth of 30 μm or more from the surface of the aggregate.

2. The cell aggregate described in claim 1, which contains viable cells even at a depth of 30 μm or more from the surface of the aggregate after freezing and thawing the aggregate.

3. The cell aggregate described in claim 1 or 2, which is in the form of a granule having a diameter of more than 1.5 mm.

4. A cell aggregate described in any one of claims 1 to 3, wherein less than 50% of the extracellular matrix is ​​collagen.

5. A cell aggregate described in any one of claims 1 to 4, which is in a frozen state.

6. A method for producing a cell aggregate, comprising supporting adherent cells on a molded article having a nonwoven fiber structure made of fibers containing an ethylene-vinyl alcohol copolymer and culturing the cells under agitation.

7. The method according to claim 6, wherein the fiber containing the ethylene-vinyl alcohol copolymer is a heat-adhesive fiber under moisture.

8. The method according to claim 6, wherein the fiber containing the ethylene-vinyl alcohol copolymer is a composite fiber containing a non-thermal adhesive fiber under moisture.

9. The method according to claim 8, wherein the composite fiber is a sheath-core type composite fiber having a sheath portion made of an ethylene-vinyl alcohol copolymer and a core portion made of a polyester resin.

10. 0.02~0.7g / cm ３ The method according to any one of claims 6 to 9, comprising supporting adherent cells on the molded body having an apparent density of 0.01 to 0.1 mm and culturing the cells under stirring at 30 to 140 rpm.

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