Preventive or therapeutic agent for bone tissue-related diseases

Abstract

To provide an agent for bone regeneration, and prevention or treatment of bone tissue-related disease, the agent having a spheroid containing a mesenchymal stem cell as an active ingredient.SOLUTION: An agent has a spheroid containing a mesenchymal stem cell as an active ingredient. Preferably the spheroid is a spheroid cultured on a cell culture substrate that has a plurality of recesses with openings having a diameter of 1000 μm or less, where the inside faces of the recesses include non-cell-adhesive surfaces, and the bottom faces of the recesses include cell-adhesive surfaces.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent that can be used for bone regeneration, prevention or treatment of bone tissue-related diseases, etc. More specifically, the present invention relates to an agent containing a specific cell culture as an active ingredient. [Background technology]

[0002] Clinical applications for repair and treatment of bone defects, fractures, etc. are being studied. For example, Patent Document 1 discloses that the amount of bone formation and the rate of bone maturation can be increased by adding a protein that has the effect of inducing bone formation (BMP: Bone morphogenetic protein) to a self-assembling peptide hydrogel used as a scaffold for cell culture. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2012-82180 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, according to the study by the present inventor, there are problems with protein preparations, such as side effects caused by the administered protein, complicated preparation methods, etc. Furthermore, when used as a cell preparation, the effect is insufficient in two-dimensional cell cultures, and it is difficult to find a suitable combination with a scaffold material and establish a preparation method for a three-dimensional cell culture, so there is room for further improvement.

[0005] In bone formation, it is believed that chondrocytes differentiate into hypertrophic chondrocytes, which then invade the blood vessels and calcify, and are then replaced by osteoblasts and osteoclasts that invade the blood vessels to form bone tissue, resulting in the formation of bone. In other words, it is believed that various cell groups, such as chondrocytes, osteoblasts, and osteoclasts, are involved in bone formation.

[0006] On the other hand, mesenchymal stem cells (MSCs) have excellent anti-inflammatory effects as well as self-renewal and pluripotency. When using such mesenchymal stem cells as cell preparations, it is preferable to use them in the form of spheroids (cell masses; cell aggregates) in which cultured cells form a three-dimensional network from the viewpoint of improving wound healing effects. However, among cell preparations containing spheroids as active ingredients, there are no known ones that show bone regeneration, or even a high therapeutic effect against bone tissue-related diseases.

[0007] An object of the present invention is to provide a bone regenerating agent, a preventive or therapeutic agent for bone tissue-related diseases, and the like, which contain spheroids containing mesenchymal stem cells as an active ingredient. [Means for solving the problem]

[0008] The present invention relates to the following [1] to

[12] . [1] A bone regeneration agent containing spheroids containing mesenchymal stem cells as an active ingredient. [2] A preventive or therapeutic agent for bone tissue-related diseases, comprising spheroids containing mesenchymal stem cells as an active ingredient. [3] The agent according to [1] or [2] above, which is also an anti-inflammatory agent. [4] The agent according to any one of [1] to [3] above, which is also an agent for cartilage regeneration. [5] The agent according to any one of [1] to [4] above, which is a spheroid cultured on a cell culture substrate having a plurality of recesses with an opening diameter of 1000 μm or less. [6] The agent according to any one of [1] to [5] above, wherein the spheroids are cultured on a cell culture substrate having a culture surface with a cell adhesive surface. [7] The agent according to [6] above, wherein the cell adhesive surface is composed of a substance exhibiting cell adhesiveness. [8] The agent according to [6] or [7] above, wherein the cell adhesive surface comprises a polyimide resin. [9] The agent according to any one of [1] to [8] above, which is a spheroid cultured on a cell culture substrate having a plurality of recesses with an opening diameter of 1000 μm or less, the inner side surfaces of the recesses having a non-cell-adhesive surface, and the bottom surfaces of the recesses having a cell-adhesive surface.

[10] The agent according to any one of [1] to [9] above, wherein the spheroid expresses a protein associated with anti-inflammation and / or a gene encoding the same.

[11] The agent according to

[10] above, wherein the protein involved in anti-inflammation is TSG-6.

[12] The agent according to any one of [2] to

[11] above, wherein the bone tissue-related disease is selected from the group consisting of osteoarthritis, osteochondritis dissecans, articular cartilage damage, bone fracture, intractable fracture, rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, gouty arthritis, pseudogouty arthritis, osteogenesis imperfecta, and osteoporosis. Effect of the Invention

[0009] According to the present invention, there is provided a spheroid-containing cell preparation which can exhibit high therapeutic effects on bone regeneration and further on bone tissue-related diseases. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the results of toluidine blue staining and GFP staining of repair tissues using single cells and spheroids in Test Example 1. [Diagram 2] FIG. 2 shows the results of toluidine blue staining and type II collagen staining of repair tissues using single cells and spheroids in Test Example 1. [Diagram 3]FIG. 3 shows the results of evaluation of the cartilage structures of the blank, single cells, and spheroids in Test Example 1. [Figure 4] 4 shows the results of analyzing the new bone formation increase rate of the blank, single cells, and spheroids in Test Example 1 based on the results of Masson's trichrome staining (graph: new bone formation increase rate, photograph: stained photograph). [Diagram 5] FIG. 5 shows the results of HE staining of repair tissues using single cells and spheroids in Test Example 1. [Figure 6] FIG. 6 shows the results of toluidine blue staining of repair tissues using single cells and spheroids in Test Example 1. [Figure 7] Figure 7A shows the results of comparing the amount of TSG-6 gene expressed in single cells and spheroids in Test Example 2, and Figure 7B shows the results of comparing the amount of TGF-β1 gene expressed in single cells and spheroids in Test Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] One aspect of the present invention is an agent containing spheroids containing mesenchymal stem cells as an active ingredient. Such an agent can be used, for example, as a bone regeneration (bone tissue repair) agent, and further as a preventive or therapeutic agent for bone tissue-related diseases.

[0012] The spheroids of the present invention may be any spheroid containing mesenchymal stem cells, and may be obtained by culturing mesenchymal stem cells.

[0013] The mesenchymal stem cells (MSCs) are not particularly limited as long as they are undifferentiated mesenchymal cells, and those collected according to a conventional method from mammalian bone marrow, periosteum, adipose tissue, peripheral blood, umbilical cord blood, etc. can be used. After collection, undifferentiated MSCs can also be selected based on the presence or absence of plastic adhesion. Here, it is preferable to use mesenchymal stem cells derived from adipose tissue as MSCs from the viewpoint of ease of procurement and high proliferation. It is also preferable to use MSCs derived from the same mammal as the subject of administration of the agent of the present invention, and MSCs derived from the same mammal other than the subject of administration or MSCs (autologous cells) of the subject himself can be used. The species from which these cells are derived is also not particularly limited, and various cells derived from humans and non-human mammals can be used. Examples of biological species from which cells are derived include primates such as humans, rhesus monkeys, green monkeys, cynomolgus monkeys, chimpanzees, tamarins, and marmosets, rodents such as mice, rats, hamsters, and guinea pigs, dogs, cats, rabbits, pigs, cows, goats, sheep, and horses. Note that, as MSCs, cells obtained by growing MSCs to 70 to 90% confluence (preferably 80% confluence) at zero passage may be further grown to obtain MSCs at, for example, 1st to 10th passage.

[0014] The spheroids are not particularly limited and any known spheroids may be used. However, from the viewpoint of the extracellular matrix content and uniformity of the obtained spheroids, the spheroids may be spheroids cultured on a cell culture substrate having multiple recesses with an opening diameter of 1000 μm or less.

[0015] For example, when preparing spheroids in wells, the number of wells in the substrate cannot be determined in general, depending on the substrate area, the type of cells to be cultured, etc., and can be appropriately set according to the common technical knowledge. For example, 2The lower limit number per recess may be 1, but may be 10, 20, 30, or 50, and the upper limit number may be 1000, 500, 300, 200, or 100. The total number of recesses on the surface of the substrate may be set as appropriate, for example, 10 or more, 100 or more, 1000 or more, 10000 or more, or 50000 or more.

[0016] The shape of the opening of the recess is not limited to a circle, and may be, for example, a polygon or an ellipse. The diameter of the opening may be 1000 μm or less, and may be appropriately set according to the common general technical knowledge depending on the size of the cells to be cultured. In the present invention, the diameter refers to the diameter (maximum length) of a circle formed so as to include the target site, regardless of the shape of the target site. The diameter of the opening is, for example, within the range of 10 to 1000 μm, 10 to 700 μm, 10 to 600 μm, or 10 to 500 μm.

[0017] The shape of the bottom surface of the recess is not limited to a circle, and may be, for example, a polygon or an ellipse, and may be the same as or different from the shape of the opening. The bottom surface may be flat (planar), curved, or smooth, but is more preferably flat and / or smooth. The diameter (length) of the bottom surface may be the same as or different from the diameter of the opening, and the diameter of the bottom surface may be smaller than or larger than the diameter of the opening. The diameter of the bottom surface may be, for example, within the range of 10 to 1000 μm, 10 to 700 μm, 10 to 600 μm, 10 to 500 μm, 10 to 400 μm, or 10 to 300 μm. For example, when the diameter of the bottom surface is smaller than the diameter of the opening, the shape of the recess is a tapered shape toward the bottom surface side of the recess.

[0018] The ratio of the bottom aperture to the opening aperture (bottom aperture / opening aperture) is not particularly limited, but examples of such a ratio include 5 / 1 to 1 / 5, 3 / 1 to 1 / 3, and 1 / 1 to 1 / 2 from the viewpoint of ease of seeding and recovering cells.

[0019] In addition, the distance (gap) between an opening and an adjacent opening is not particularly limited, but examples of the distance (gap) include a range of, for example, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 300 μm or less, 200 μm or less, 100 μm or less, depending on the desired cell culture, and may be any finite value.

[0020] The depth of the recesses can be appropriately set according to the size of the cells to be cultured and in accordance with common general technical knowledge, and is, for example, within the range of 10 to 1000 μm, or 10 to 300 μm.

[0021] The ratio of the aperture diameter to the depth of the recess (aperture diameter of the opening / depth of the recess) is not particularly limited, but from the viewpoint of ease of seeding and recovery of cells, examples include 5 / 1 to 1 / 5, 3 / 1 to 1 / 3, and 2 / 1 to 1 / 2. When within the above range, cells are unlikely to jump out of the recess, and operations such as cell recovery and degassing treatment are easy to perform.

[0022] The thickness of the bottom surface of the recess is not particularly limited and can be appropriately set in accordance with common technical knowledge.

[0023] The inner surface of the well is not particularly limited as long as it can form spheroids. From the viewpoint of forming spheroids in the well and improving the adhesiveness of the obtained spheroids, a cell culture substrate may be used in which the culture surface of the spheroids has a cell adhesive surface. In this case, the cell adhesive surface may form a part of the inner surface of the well or may be the entire surface, for example, a part of or the entire surface of the inner side or bottom surface of the well.

[0024] A cell-adhesive surface is, for example, a surface on which cells adhere with a certain number of adhesion points when the cells settle on the surface in a solution used for culture. Alternatively, it is a surface on which the cells adhere so as to be fixed to such an extent that they can be peeled off by a liquid flow such as pipetting. In addition, a surface on which cells adhere to such an extent that they can form a three-dimensional or three-dimensional tissue such as a layer or spheroid, rather than a surface on which the cells adhere and are maintained or grown two-dimensionally, can be mentioned. Such a surface may be, for example, a surface formed by physically or chemically fixing or arranging a substance exhibiting cell adhesiveness on the surface of a substrate constituting the bottom surface of a recess, and may be a surface on which a substance exhibiting cell adhesiveness is arranged outside the recess, or the substrate itself may be made of a substance exhibiting cell adhesiveness.

[0025] The substance exhibiting cell adhesiveness is not particularly limited and may be used as long as the cells used for culture adhere to it or the substance can bind to cell surface molecules such as proteins and sugar chains present in the cell membrane of the cells used. The substance may be hydrophilic or hydrophobic, but from the viewpoint of cell adhesiveness and spheroid formability, hydrophilic (particularly, hydrophilic but not superhydrophilic) or hydrophobic (particularly, hydrophobic but not superhydrophobic) substances are preferred, and more preferably, hydrophobic substances are preferred. The degree of adhesiveness of the substance exhibiting cell adhesiveness may be such that the cells do not jump out of the recess. Examples of such substances include substances obtained from living organisms or synthesized, such as proteins (collagen, fibronectin, laminin, etc.) and synthetic resins (fluororesins, polyimide resins, polysulfones, polyethersulfones, polydimethylsiloxanes, mixtures thereof, etc.). When a synthetic resin is selected, a cell culture substrate with excellent handling properties can be obtained due to the strength and heat resistance of the synthetic resin itself. In addition, it is preferable to select a synthetic resin such as a polyimide resin from the viewpoint of biocompatibility, obtaining adhesive spheroids, improving the uniformity of the obtained spheroids, or facilitating medium replacement by moderately adhering to various cells. By selecting a non-biological component such as a polyimide resin, the spheroids obtained from the cell culture substrate containing the polyimide resin can be easily applied to fields such as regenerative medicine and drug discovery.

[0026] Examples of polyimide resins include polyimide resins containing a structural unit represented by the following formula (I). From the viewpoint of good spheroid formation, resins having fluorine atoms in the molecule are preferred, and fluorine-containing polyimides (fluorine-containing polyimide resins) are more preferred. The polyimide resin used in the present invention is typically obtained by imidizing polyamic acid obtained by polymerizing at least one acid dianhydride and at least one diamine. The polyimide resin may contain polyamic acid as a part of its chemical structure. The polyimide resin may be produced by a known method. As an example, a two-stage synthesis method can be used. The two-stage synthesis method of polyimide resins is a method in which polyamic acid is synthesized as a precursor and the polyamic acid is converted into polyimide acid. The polyamic acid as the precursor may be a polyamic acid derivative. Examples of polyamic acid derivatives include polyamic acid salts, polyamic acid alkyl esters, polyamic acid amides, polyamic acid derivatives from bis-methylidene pyromelitides, polyamic acid silyl esters, and polyamic acid isoimides. Examples of polyimides include polyimides made of an acid anhydride such as pyromellitic dianhydride, biphenyltetracarboxylic dianhydride, or benzophenonetetracarboxylic dianhydride, and a diamine such as oxydiamine, paraphenylenediamine, metaphenylenediamine, or benzophenonediamine.Examples of the resin having fluorine atoms include 4,4'-hexafluoroisopropylidenediphthalic anhydride (6FDA) / 1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, 6FDA / 1,3-bis(4-aminophenoxy)benzene (TPER) copolymer, 6FDA / 4,4'-oxydiphthalic anhydride (ODPA) / TPEQ copolymer, 4,4'-(4,4'-isopropylidenediphenoxy)diphthalic acid (BPADA) / 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoroisopropylidenediphthalic anhydride (6FDA) / 1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, Examples of such fluorine-containing polyimide resins include fluorine-containing polyimide resins containing a structural unit represented by the following formula (I), such as 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)propane (HFBAPP), 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)propane (BAPP) copolymer, 6FDA / 2,2'-bis(trifluoromethyl)benzidine (TFMB) copolymer, 6FDA / 4,4'-diaminodiphenyl ether (ODA) copolymer, and 6FDA / 4,4'-bis(4-aminophenoxy)biphenyl (BAPB) copolymer; ethylene-tetrafluoroethylene copolymer, etc.

[0027] [ka]

[0028] In the above formula (I), X 0 represents either an oxygen atom, a sulfur atom, or a divalent organic group; Y represents a divalent organic group; Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 each independently represents a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, p is 0 or 1. In the polyimide resin, the chemical structure represented by formula (I) may be different for each structural unit of the resin, or may be the same. 0 , Y, Z 1 , Z 2 , Z 3 , Z 4 , Z5 , and Z 6 At least one of them preferably contains one or more fluorine atoms.

[0029] In the above formula (I), when p=0, X 0 may not be present (in other words, the left and right benzene rings may be directly bonded), but when p=1, the left and right benzene rings are 0 Bind via.

[0030] X 0 Specific examples of the divalent organic group represented by the formula include an alkylene group, an arylene group, an aryleneoxy group, and an arylenethio group, and among these, an alkylene group, an aryleneoxy group, and an arylenethio group are preferred, and an alkylene group and an aryleneoxy group are more preferred, and these may be substituted with a fluorine atom. The number of carbon atoms in the alkylene group is, for example, 1 to 12, and preferably 1 to 6.

[0031] X 0 Examples of the alkylene group substituted with a fluorine atom include -C(CF3)2- and -C(CF3)2-C(CF3)2-. 0 Among the above-mentioned alkylene groups which are examples of the alkylene groups, -C(CF3)2- is preferred.

[0032] X 0 Examples of the arylene group include the following:

[0033] [ka]

[0034] X 0 Examples of the aryleneoxy group include the following.

[0035] [ka]

[0036] X 0 Examples of the arylene thio group include the following.

[0037] [ka]

[0038] From the viewpoint of good formation of spheroids on the substrate, X 0 The divalent organic group represented by the formula (I) may be one selected from the group consisting of b-2 to b-10 and c-2 to c-10 above, may be one selected from the group consisting of b-7 to b-9 and c-7 to c-9 above, or may be a structure represented by b-8.

[0039] X 0 The above-mentioned arylene group, aryleneoxy group and arylenethio group, which are examples of the above, may each be independently substituted with a group selected from the group consisting of a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom), a methyl group and a trifluoromethyl group. There may be a plurality of these substituents, and in this case, the types of the substituents may be the same or different. The preferred substituents substituted on the arylene group, aryleneoxy group and arylenethio group are a fluorine atom and / or a trifluoromethyl group, and preferably a fluorine atom. When Y does not contain a fluorine atom, the arylene group, aryleneoxy group and arylenethio group are preferably substituted with at least one or more fluorine atoms.

[0040] In the above formula (I), the divalent organic group represented by Y is not particularly limited, but may be, for example, a divalent organic group having an aromatic ring. More specifically, it may be a group consisting of one benzene ring, or a group having a structure in which two or more benzene rings are bonded directly or via a carbon atom (i.e., a single bond or an alkylene group), an oxygen atom, or a sulfur atom. Specifically, the following groups may be exemplified.

[0041] [ka]

[0042] [ka]

[0043] [ka]

[0044] [ka]

[0045] The divalent organic group having an aromatic ring, which is an example of Y, may be substituted with a group selected from the group consisting of a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom), a methyl group, and a trifluoromethyl group, if possible. There may be a plurality of these substituents, and in that case, the types of the substituents may be the same or different. Suitable substituents substituted on the divalent organic group having an aromatic ring are particularly X 0 When does not contain a fluorine atom, it is preferably a fluorine atom and / or a trifluoromethyl group, and more preferably a fluorine atom.

[0046] From the viewpoint of spheroid formation, in the above formula (I), Y is preferably a structure selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7, and more preferably a structure of e-1, e-3 or e-4.

[0047] In the above formula (I), Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 may be the same or different, and are each independently selected from a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom; X 0 and Y do not contain a fluorine atom, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 At least one of these is preferably a fluorine atom.

[0048] In a preferred embodiment of the present invention, in terms of spheroid formation, X 0 is selected from the group consisting of -C(CF3)2-, the above b-2 to b-10, and c-2 to c-10; and Y is selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7. In a more preferred embodiment of the present invention, in the above formula (I), X 0 is selected from the group consisting of -C(CF3)2-, b-7 to b-9, and c-7 to c-9; and Y is selected from the group consisting of e-1, e-3, and e-4.

[0049] The polyimide resin consisting of the structural unit represented by the above formula (I) can be obtained by a method of baking a polyamic acid obtained by polymerization of an acid dianhydride and a diamine. The imidization rate of the "polyimide resin consisting of the structural unit represented by the formula (I)" does not have to be 100%. That is, the polyimide resin consisting of the structural unit represented by the formula (I) may be composed only of the structural unit represented by the above formula (I), but may also contain a structural unit in which the cyclic imide structure does not undergo dehydration ring closure and remains as an amic acid, as long as the objective effect of the present invention is not impaired.

[0050] The polyamic acid synthesis reaction is preferably carried out in an organic solvent. The organic solvent used in the polyamic acid synthesis reaction is not particularly limited as long as it can efficiently proceed the reaction between the raw materials, acid dianhydride and diamine, and is inactive to these raw materials. For example, polar solvents such as N-methylpyrrolidone (NMP), N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, sulfolane, methyl isobutyl ketone, acetonitrile, benzonitrile, nitrobenzene, nitromethane, acetone, methyl ethyl ketone, isobutyl ketone, and methanol; non-polar solvents such as toluene and xylene, etc. are included. Among them, it is preferable to use a polar solvent. These organic solvents may be used alone or as a mixture of two or more kinds. The reaction mixture after the amidation reaction may be directly subjected to thermal imidization. The concentration of the polyamic acid in the polyamic acid solution is not particularly limited, but is preferably 5% by weight or more, more preferably 10% by weight or more, and preferably 50% by weight or less, more preferably 40% by weight or less, in consideration of the polymerization reactivity of the resulting resin composition, the viscosity after polymerization, and ease of handling in the subsequent film formation and baking. The viscosity of the polyamic acid is not particularly limited, but can be measured according to a known measurement method, and is, for example, within the range of 1 to 20 Pa·s, preferably 3 to 15 Pa·s at 23°C.

[0051] The polyamic acid is imidized by either thermal imidization or chemical imidization to obtain a resin composition containing a fluorine-containing polyimide. In a specific embodiment, the polyamic acid is imidized by heat treatment (thermal imidization) to obtain a resin composition containing a fluorine-containing polyimide. Polyimides obtained by thermal imidization are less likely to contain residual catalyst and are more preferable for cell culture applications.

[0052] In the case of imidization by thermal imidization, for example, the polyamic acid is baked in air, or more preferably in an inert gas atmosphere such as nitrogen, helium, argon or the like, or in vacuum, preferably at a temperature of 50 to 400°C, more preferably 100 to 380°C, for a time of preferably 0.1 to 10 hours, more preferably 0.2 to 5 hours, to carry out an imidization reaction, thereby obtaining a resin composition containing a polyimide.

[0053] The polyamic acid to be subjected to the thermal imidization reaction is preferably in a form dissolved in a suitable solvent. The solvent may be any solvent capable of dissolving polyamic acid, and the solvents mentioned above with respect to the polyamic acid synthesis reaction may also be used.

[0054] In the case of imidization by chemical imidization, polyamic acid can be directly imidized in a suitable solvent by using a dehydration / cyclization agent described below.

[0055] The dehydration / cyclization reagent can be used without any particular limitation as long as it has the effect of chemically dehydrating and cyclizing polyamic acid to form a polyimide. As such a dehydration / cyclization reagent, it is preferable to use a tertiary amine compound alone or a tertiary amine compound and a carboxylic acid anhydride in combination, in that imidization can be efficiently promoted.

[0056] Examples of the tertiary amine compound include trimethylamine, triethylamine, tripropylamine, tributylamine, pyridine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, N,N,N',N'-tetramethyldiaminomethane, N,N,N',N'-tetramethylethylenediamine, N,N,N',N'-tetramethyl-1,3-propanediamine, N,N,N',N'-tetramethyl-1,4-phenylenediamine, N,N,N',N'-tetramethyl-1,6-hexanediamine, N,N,N',N'-tetraethylmethylenediamine, and N,N,N',N'-tetraethylethylenediamine. Among these, pyridine, DABCO, and N,N,N',N'-tetramethyldiaminomethane are particularly preferred, and DABCO is more preferred. The tertiary amine compound may be used alone or in combination of two or more kinds.

[0057] Examples of carboxylic acid anhydrides include acetic anhydride, trifluoroacetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, succinic anhydride, and maleic anhydride. Among these, acetic anhydride and trifluoroacetic anhydride are particularly preferred, and acetic anhydride is more preferred. The carboxylic acid anhydride may be one type or two or more types.

[0058] In the chemical imidization, a polar solvent having excellent solubility is suitable as a solvent for dissolving polyamic acid. For example, tetrahydrofuran, N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, etc. are listed, and among these, one or more selected from the group consisting of N,N-dimethylacetamide, N,N-dimethylformamide, and N-methylpyrrolidone are particularly preferred from the viewpoint of uniform reaction. When these solvents are used as the solvent for the amidation reaction, the polyamic acid can be used for chemical imidization without being separated from the reaction mixture after the amidation reaction.

[0059] The weight average molecular weight of the polyimide resin is, for example, 5,000 to 2,000,000, preferably 8,000 to 1,000,000, and more preferably 20,000 to 500,000. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the polyimide resin after baking are substantially the same, and in this specification, the weight average molecular weight of the resin is a value measured by the following method. When the weight average molecular weight is in the above range, the synthesis and handling of the polyimide resin, film formation, and spheroid formability are improved.

[0060] (Measurement of weight average molecular weight) Equipment: Tosoh Corporation HCL-8220GPC Column: TSKgel Super AWM-H Eluent (LiBr·H2O, NMP containing phosphoric acid): 0.01 mol / L Measurement method: A 0.5% by weight solution is prepared using an eluent, and the molecular weight is calculated based on a calibration curve prepared using polystyrene.

[0061] A cell adhesive substance or cell adhesive surface (a hydrophobic (particularly, a hydrophobic but not superhydrophobic) cell adhesive substance or surface) may preferably have a static water contact angle of 70° or more and a sliding angle of 15° or more, or may have a static water contact angle of 70° or more and a sliding angle of 15° or more. When the cell adhesive substance or cell adhesive surface satisfies such conditions, spheroid formation is further promoted. From the viewpoint of spheroid adhesion and spheroid formability, the static water contact angle is more preferably 75° or more (e.g., greater than 75°), even more preferably 77° or more, even more preferably 79° or more, and even more preferably 80° or more (e.g., greater than 80°), and the upper limit of the static water contact angle is, for example, less than 150°, preferably 120° or less (e.g., less than 120°), more preferably 110° or less, and even more preferably 100° or less (e.g., less than 99°, 98° or less, 97° or less, 95° or less, etc.). On the other hand, a cell adhesive substance or a cell adhesive surface [hydrophilic (particularly, hydrophilic but not superhydrophilic) cell adhesive substance or surface] may have a static water contact angle of 65° or less, more preferably 55° or less, and even more preferably 50° or less. The lower limit may be 0° or more, preferably 5° or more, and more preferably 10° or more. From the viewpoint of spheroid formability, the higher the sliding angle, the more preferable it is in the following order: 18° or more, 19° or more, 20° or more, 22° or more, 24° or more, 26° or more, 28° or more, and 30° or more. The upper limit of the sliding angle is, for example, less than 80°, preferably 70° or less (e.g., less than 70°), more preferably 60° or less (e.g., less than 60°), and even more preferably 50° or less (e.g., less than 50°). The static water contact angle and the sliding angle may be values ​​measured by the following method.

[0062] (Method for measuring static water contact angle) Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500) Measurement method: Measure the adhesion angle of the droplet immediately after dropping 2 μL of water onto a surface (non-cell adhesive surface or cell adhesive surface) or film (film made of non-cell adhesive or cell adhesive substance) (measurement temperature: 25°C).

[0063] (Method of measuring fall angle) Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500) Measurement method: After dropping 25 μL of water onto a surface (non-cell-adhesive surface or cell-adhesive surface) or film (film made of non-cell-adhesive or cell-adhesive substance), the substrate is continuously tilted and the angle at which the water falls is taken as the sliding angle (measurement temperature: 25°C).

[0064] The resin constituting the cell adhesive surface may further contain additive components such as a plasticizer and an antioxidant.

[0065] The proportion of the inner surface of the recess that is occupied by the surface exhibiting cell adhesiveness is not particularly limited, but it is preferable that the surface occupies, for example, 90% or more, 95% or more, 99% or more of the bottom surface of the recess, or essentially the entire bottom surface.

[0066] The well may have a non-cell-adhesive surface in addition to the cell-adhesive surface. For example, the inside surface of the well may have a non-cell-adhesive surface, and the bottom surface may have a cell-adhesive surface. Such a configuration can improve the uniformity of the resulting spheroids.

[0067] A non-cell-adhesive surface is, for example, a surface on which, when cells are settled in a solution used for culture, the cells hardly change their shape and do not adhere at all, or, even if they adhere weakly temporarily, they naturally detach. Such a surface may be formed, for example, by physically or chemically fixing a substance exhibiting non-cell adhesiveness to the surface of a substrate constituting a recess, and the substrate itself may be made of a substance exhibiting non-cell adhesiveness.

[0068] The substance exhibiting cell non-adhesiveness is not particularly limited as long as the cells used for culture do not adhere to it or do not bind to cell surface molecules such as proteins and sugar chains present in the cell membrane of the cells used, and may be biocompatible or not. In addition, the substance may be hydrophobic or hydrophilic, for example, super-hydrophobic or super-hydrophilic. From the viewpoint of cell non-adhesiveness, uniformity and formability of spheroids, etc., a substance exhibiting hydrophobicity (particularly super-hydrophobicity) [e.g., more hydrophobic (particularly super-hydrophobic) to a hydrophobic or hydrophilic (particularly hydrophobic) cell adhesive surface or a resin constituting the surface (and further its contact angle)] is particularly preferred, but a substance exhibiting hydrophilicity (particularly super-hydrophilicity) [e.g., more hydrophilic (particularly super-hydrophilic) to a hydrophilic or hydrophobic (e.g. hydrophobic) cell adhesive surface or a resin constituting the surface (and further its contact angle)] is also preferred. Examples of such substances include ethylene glycol and its derivatives, MPC (2-methacryloyloxyethyl phosphorylcholine) and its derivatives, compounds containing HEMA (hydroxyethyl methacrylate) and its derivatives, or polymers of these compounds, compounds containing SPC (segmented polyurethane) and its derivatives, proteins obtained from living organisms (albumin, etc.), and sugar chains to which cells do not adhere (agarose, cellulose, etc.). Among these, MPC and its derivatives, or polymers thereof, are preferred from the viewpoint of adhesion to cell-adhesive surfaces, from the viewpoint of simplifying the manufacturing process of the cell culture substrate, or from the viewpoint of improving the uniformity of the obtained cells or spheroids. In addition, the substance may be appropriately modified depending on the ease of handling, the desired degree of hydrophobicity (e.g., superhydrophobicity) / hydrophilicity (e.g., superhydrophilicity), etc. For example, a hydrophilic substance may be crosslinked to achieve both hydrophilicity and low solubility in water. In addition, a raw material substance (e.g., hydrophobic or hydrophilic) may be appropriately hydrophobized or hydrophilized (e.g., introduction of a hydrophobic group or a hydrophilic group) to obtain a material with the desired hydrophobicity or hydrophilicity.

[0069] The immobilization of a substance exhibiting non-cell adhesiveness can be performed on the surface of a substrate by drying a solution containing the substance on the surface of the substrate, melting the substance and pressing it, curing the substance applied to the substrate with energy rays such as UV, forming a covalent bond by chemically reacting the functional group of the substance with the functional group on the substrate (e.g., condensation reaction between functional groups such as carboxyl groups and amino groups), or bonding the thiol group of the substance with a thin metal (platinum, gold, etc.) film formed on the substrate in advance. The thickness of the immobilization is not particularly limited, and is exemplified as 0.01 to 1000 μm.

[0070] The proportion of the inner surface of the well that is occupied by the surface exhibiting non-adhesive properties against cells is not particularly limited, but for example, the proportion of the inner surface of the well that is occupied by the surface is preferably such that the adhesion of cultured cells to the inner surface of the well is reduced, preferably 90% or more, more preferably 95% or more, and particularly preferably the entirety of the inner surface area.

[0071] A surface that exhibits non-cell adhesiveness can be determined, for example, by using the static water contact angle as an index of surface properties from the viewpoint of making the size of the formed spheroids uniform and improving the circularity. For example, in the case of a hydrophobic surface formed of the above-mentioned material, the static water contact angle is preferably 90° or more, more preferably 93° or more, and even more preferably 95° or more. It may also be 150° or less, preferably 130° or less, and more preferably 120° or less. On the other hand, in the case of a hydrophilic surface, the static water contact angle is preferably 65° or less, more preferably 55° or less, and even more preferably 50° or less. It may be 0° or more, and is preferably 5° or more, and more preferably 10° or more. For example, a highly hydrophobic MPC polymer (or a surface formed from the MPC) may achieve a static water contact angle of, for example, 90° or more, or 100° or more. Such a static water contact angle may be a value on a non-cell-adhesive surface, or may be a value on a substance that exhibits non-cell adhesiveness (or a substance that constitutes a non-cell-adhesive surface).

[0072] In addition, in terms of improving the size uniformity and circularity of the obtained spheroids, it is also important to balance the degree of adhesion between the cell adhesive surface and the cell non-adhesive surface. Therefore, even if the cell non-adhesive surface exhibits adhesiveness, it is sufficient if it has lower adhesiveness than the cell adhesive surface. For example, when the static water contact angle is used as an index, the difference (or its absolute value) between the static water contact angle between the cell adhesive surface (or cell adhesive substance) and the cell non-adhesive surface (or cell non-adhesive substance) is preferably 3° or more (e.g., 5° or more), more preferably 10° or more (e.g., 12° or more), and even more preferably 15° or more. In addition, the upper limit can be appropriately selected depending on the combination of hydrophobicity and hydrophilicity of the cell non-adhesive substance (surface) and the cell adhesive substance (surface), and is not particularly limited, and may be, for example, 100°, 90°, 80°, 70°, 60°, 50°, 40°, 30°, etc.

[0073] The recess may have the above-mentioned structure, but the substrate surface, which is also the peripheral portion of the recess, may also have a cell non-adhesive surface from the viewpoint of simplifying the production of the cell culture sheet. With such a structure, cells seeded in the peripheral portion also tend to form spheroids in the recess. The cell non-adhesive surface of the substrate surface may be the same cell non-adhesive surface as the inner surface of the recess, or a different cell non-adhesive surface. In the case of the same cell non-adhesive surface, the substrate surface and the inner surface of the recess may have a continuous surface. The proportion of the substrate surface that is also the peripheral portion of the recess that is the surface that exhibits cell non-adhesive properties is not particularly limited, but it is preferable that the surface that exhibits cell non-adhesive properties occupies 90% or more, 95% or more, 99% or more of the peripheral portion of the recess, or substantially the entire peripheral portion. The proportion of the surface that exhibits cell non-adhesive properties occupying the total of the peripheral portion of the recess and the inner surface of the recess is not particularly limited, but it is preferable that the surface that exhibits cell non-adhesive properties occupying 90% or more, 95% or more, 99% or more of the total of the peripheral portion of the recess and the inner surface of the recess, or substantially the entire peripheral portion and the inner surface.

[0074] In the present invention, the cell culture substrate used for preparing spheroids can be, for example, a cell culture substrate having a plurality of recesses with an opening diameter of 1000 μm or less, the inner side of the recess having a cell non-adhesive surface, and the bottom of the recess having a cell adhesive surface, in terms of improving the size uniformity and adhesiveness of the obtained spheroids. The cell culture substrate can also be a layered structure including a layer including the bottom of the recess and a layer including the inner side of the recess. Here, the layer including the inner side of the recess constitutes a layer having through holes. One embodiment of the cell culture substrate that can be used in the present invention can be, for example, a cell culture sheet that is a laminate of a layer having a cell non-adhesive surface with through holes and a layer having a cell adhesive surface.

[0075] The layer having a cell non-adhesive surface may be a layer of a substance exhibiting cell non-adhesiveness immobilized on a layered substrate, or a layer of a substance exhibiting cell non-adhesiveness, but from the viewpoint of forming through-holes or simplifying the production of the cell culture sheet, a layer of a substance exhibiting cell non-adhesiveness immobilized on a layered substrate is preferred. By adopting such a form, the production of the cell culture sheet becomes easier, and further, for example, by forming through-holes or recesses in the substrate and then immobilizing a substance exhibiting cell non-adhesiveness, it is possible to prevent the cell non-adhesive surface from being damaged due to the formation of recesses in the cell culture sheet, which is also preferred from the viewpoint of improving the cell culture properties of the cell culture sheet.

[0076] As the layered substrate, any substrate known in the art can be used. For example, a plate-shaped body made of a synthetic resin such as polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (polyethylene terephthalate, etc.), polyurethane, polysulfone, polyacrylate, polymethacrylate, polyvinyl, polycycloolefin, polyether ketone, polyether ether ketone, polyimide, silicon, synthetic rubber such as EPDM (Ethylene Propylene Diene Monomer), natural rubber, glass, ceramic, metal material such as stainless steel, etc. can be mentioned. A transparent substrate is also one of the preferred forms.

[0077] The immobilization of the substance exhibiting non-cell adhesiveness to the layered substrate can be carried out in the same manner as the method for immobilizing the substance exhibiting non-cell adhesiveness to the inner surface of the recessed portion described above. From the viewpoint of workability, it is preferable to carry out the immobilization after forming the through-holes described below.

[0078] The layer having a non-adhesive surface preferably includes the sheet surface of the cell culture sheet and through-holes. The walls of the through-holes preferably correspond to the inner surface of the recesses described above, and the diameter and shape of the openings and the opposite ends thereof can be set in the same manner as the recesses described above. The depth of the through-holes corresponds to the thickness of the layer having a non-adhesive surface, but also corresponds to the depth of the recesses described above, and can be set in the same manner as the recesses. When a substance exhibiting non-adhesive properties is immobilized on a layered substrate, the layer of the substance exhibiting non-adhesive properties is preferably, for example, 1 nm or more, more preferably 10 nm or more, and the thickness of the entire layer including the layer of the substance exhibiting non-adhesive properties and the substrate can be appropriately set as long as it is within the range of the thickness of the layer having a non-adhesive surface described above.

[0079] The formation of the through-holes is not particularly limited as long as the through-holes can be formed in the above-mentioned size, and can be carried out. For example, the through-holes can be formed by perforation processing (drilling, etc.), optical microfabrication (laser (e.g., CO2 laser, excimer laser, semiconductor laser, YAG laser), etc.), etching processing, embossing processing, etc. In the above processing, the shape of the through-hole may be tapered, and in this case, the periphery of the end may be deformed, and a structure may be formed in which the layer thickness of the edge of the opening and the layer thickness of the part located in the middle region between the opening and the adjacent opening are different.

[0080] The layer having a cell adhesive surface may be a layer of a substrate having a substance exhibiting cell adhesiveness immobilized thereon, or may be a layer made of a substance exhibiting cell adhesiveness.

[0081] The thickness of the layer having a cell adhesive surface is, for example, 1 nm or more and 4 mm or less, preferably 1 μm or more and 1 mm or less, and may be the same as the thickness of the bottom surface of the recess.

[0082] The cell culture sheet also includes an embodiment in which an adhesive layer (sticking layer) is further included between the layer having a cell adhesive surface and the layer having a non-cell adhesive surface.

[0083] The adhesive layer may be any known material in the art. Examples include silicone resins, synthetic rubber, and natural rubber. Preferably, an adhesive layer with low elution properties can be used. Preferably, a commercially available double-sided tape or the like can be used.

[0084] The thickness of the adhesive layer is not particularly limited and can be appropriately set within a range that does not impair the effects of the present invention, for example, 0.5 to 100 μm.

[0085] The cell culture sheet may have layers other than those mentioned above laminated thereon, and may have a layer having cavities laminated thereon.

[0086] The thickness of the cell culture sheet is not particularly limited, but from the viewpoint of ease of handling, it is preferably 10 to 5000 μm, more preferably 100 to 2000 μm. The sheet area is also not particularly limited, and may be, for example, 0.01 to 10000 cm. 2 , preferably 0.03 to 5000 cm 2 Examples include:

[0087] The cell culture sheet may be directly installed in a known cell culture device. Alternatively, it may be appropriately sized according to the size of the target device. For example, the sheet may be accommodated and fixed in various cell culture containers such as culture dishes, flasks, culture bags, etc., and culture may be performed by adding a medium containing cells to the container. Alternatively, the cell culture device itself on which the cell culture sheet is installed may be in the form of various cell culture containers such as culture plates such as single or multi-well plates, culture petri dishes, culture dishes, flasks, culture bags, etc.

[0088] The medium and conditions used for culturing cells can be appropriately set depending on the cells used. When using the cell culture sheet or the cell culture instrument on which the sheet is placed, it is preferable to perform a degassing treatment in advance as necessary. The degassing treatment is not particularly limited, and may be a general treatment such as spraying, pipetting, shaking, temperature change such as heating and cooling, centrifugation, vacuum degassing, or ultrasonic treatment, and is preferably spraying, pipetting, or temperature change.

[0089] By using the cell culture sheet, the seeded cells are sorted by the divided shape (shape formed by a plurality of recesses), and the substrate exhibits an appropriate adhesive property, improving the uniformity of the obtained spheroids. In addition, the appropriate adhesive property makes it easy to handle during the culture operation, and the spheroids can be collected by simply shaking or gently pipetting the cell culture sheet or cell culture tool. Although the cell culture sheet has minute recesses with an opening diameter of 1000 μm or less, a special adhesive environment is formed in each recess by the cell adhesive bottom surface and the cell non-adhesive inner surface surrounding it, and it is presumed that the effect of improving not only the size uniformity and circularity of the obtained spheroids but also the yield is improved. However, the present invention is not bound by these speculations.

[0090] The diameter of the obtained spheroid is not particularly limited, but is, for example, 10 to 1000 μm, preferably 10 to 800 μm. Here, the diameter of the spheroid can be measured by a conventional method (for example, image analysis software, particle size distribution meter), and can be expressed, for example, as a fluid diameter or a circle equivalent diameter. The circularity of the obtained spheroid is, for example, 0.5 to 1.0, preferably 0.7 to 1.0.

[0091] The agent according to the present invention, which contains the above-mentioned spheroids as an active ingredient, can be used for repairing, restoring, or regenerating defective or damaged areas in bone tissue or periodontal tissue, and as described above, can be used, for example, for bone regeneration (bone regeneration agent, bone filling agent), etc. The application field of the agent according to the present invention is not particularly limited, and can be applied, for example, to fields such as bone regenerative medicine, as well as skull, jaw, and facial reconstruction surgery, cosmetic surgery, plastic surgery, orthopedics, oral surgery, otolaryngology, dentistry, etc.

[0092] The agent of the present invention can also be used as a preventive or therapeutic agent for bone tissue-related diseases, etc. As described above, the agent of the present invention can involve bone regeneration, and is therefore suitable for preventing or treating bone tissue-related diseases, etc.

[0093] The bone tissue-related disease means any disease related to bone tissue, the symptoms of which can be improved by bone regeneration. Examples of the bone tissue-related disease include osteoarthritis, osteochondritis dissecans, articular cartilage damage, fracture, intractable fracture, rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, gouty arthritis, pseudogouty arthritis, osteogenesis imperfecta, osteoporosis, etc.

[0094] The present invention also includes the following aspects. A method for bone regeneration comprising administering to a patient in need thereof a spheroid comprising mesenchymal stem cells. · Spheroids containing mesenchymal stem cells for bone regeneration. Use of spheroids comprising mesenchymal stem cells in the manufacture of a medicament for bone regeneration.

[0095] The present invention also includes the following aspects. A method for preventing or treating a bone tissue-related disease, comprising administering to a patient in need thereof a spheroid comprising mesenchymal stem cells. A spheroid comprising mesenchymal stem cells for use in the prevention or treatment of bone tissue-related diseases. Use of spheroids comprising mesenchymal stem cells in the manufacture of a medicament for the prevention or treatment of a bone tissue-related disease.

[0096] The spheroid according to the present invention contains mesenchymal stem cells as an active ingredient, and therefore predominantly expresses the TGFβ1 (Transforming Growth Factor-β1) gene. TGFβ1 is a member of the TGF-β superfamily, and mammalian TGF-β has three isoforms, β1, β2, and β3. TGFβ1 is responsible for regulating growth, differentiation, and motility in addition to cell proliferation, and is also involved in physiological functions such as embryogenesis, tissue reconstruction, and wound healing. By increasing the expression level of the gene, it can also act on chondrocytes, osteoblasts, and the like, and exhibit some effect on bone formation. In addition, the spheroid expresses a protein related to anti-inflammation and / or a gene encoding the same. The protein related to anti-inflammation is not particularly limited, but can be evaluated using, for example, TSG-6 (Tumor necrosis factor (TNF)-stimulated gene 6) as an indicator. TSG-6 is a type of hyaluronic acid-binding protein that has a hyaluronic acid-binding domain, and the hyaluronic acid-binding domain is involved in the stability of the extracellular matrix and cell migration. In addition, when TSG-6 forms a covalent and / or non-covalent complex with inter-alpha-inhibitor (IαI), it enhances the serine protease activity of IαI. It is presumed that the expression of the protein related to anti-inflammation exerts bone regeneration and anti-inflammatory effects, and therefore has excellent preventive and therapeutic effects for bone tissue-related diseases. In other words, it can be said that the spheroid according to the present invention has the effect or mechanism of action of bone regeneration and anti-inflammation.

[0097] The agent according to the present invention can be prepared, stored, and administered in the same manner, while referring to the findings of conventionally known cell preparations. The agent according to the present invention is usually in the form of an injection. When preparing an injection, a pH regulator, a buffer, a stabilizer, an isotonicity agent, a local anesthetic, and the like can be added to the active ingredient, and a subcutaneous, intramuscular, and intravenous injection can be produced by a conventional method. In this case, examples of the pH regulator and buffer include sodium citrate, sodium acetate, sodium phosphate, and saline phosphate. Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride. Examples of the isotonicity agent include sodium chloride and glucose.

[0098] The agent according to the present invention may further contain, in addition to the active ingredient, various commonly used additive ingredients, if necessary.

[0099] The amount of the active ingredient contained in the agent according to the present invention can be appropriately determined depending on the dosage range of the active ingredient, the number of doses, and the like.

[0100] Methods for administering the agent according to the present invention include, but are not limited to, local administration to the injured area, as well as injection into an articular cavity, the spinal cavity, intravenously, into subcutaneous tissue, or into a muscle.

[0101] The dosage range is not particularly limited, and can be set appropriately depending on the effectiveness of the ingredients contained therein, the dosage form, the administration route, the type of disease, the characteristics of the subject (body weight, age, condition, use of other medications, etc.), and the judgment of the attending physician. EXAMPLES

[0102] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. In the following examples, room temperature means 20 to 30°C.

[0103] Preparation Example 1: Cell culture sheet <Preparation of a layer having a cell adhesive surface (preparation of a fluorine-containing polyimide film)> In a 100 mL three-neck flask, 2.976 g (10.2 mmol) of 1,4-bis(aminophenoxy)benzene, 4.524 g (10.2 mmol) of 4,4'-hexafluoroisopropylidenediphthalic anhydride, and 42.5 g of N-methylpyrrolidone were charged. After stirring at room temperature under a nitrogen atmosphere, the mixture was kept for 5 days to obtain a fluorine-containing polyamic acid resin composition (solid content concentration 15.0 mass%, 6FDA / TPEQ polyamic acid). The viscosity of the polyamic acid was 6 Pa s.

[0104] The fluorine-containing polyamic acid resin composition obtained above was applied to a glass substrate using a die coater so that the thickness of the fluorine-containing polyimide film after baking was 40 μm, forming a coating film.The coating film was then baked at 360°C for 1 hour under a nitrogen atmosphere.The baked product was then peeled off from the glass substrate to obtain a fluorine-containing polyimide film.The static water contact angle of this fluorine-containing polyimide film was 80.8°, and the sliding angle was 23.8°.

[0105] The methods for measuring the above physical properties are as follows. (Viscosity measurement) Equipment: AS ONE Viscometer TV-22 Setting: VI RANGE: H ROTOR No.6 SPEED: 10 rpm Viscometer calibration standard solution: Nippon Grease Co., Ltd. JS 14000 Measurement method: After calibrating with a standard solution for calibrating the viscometer, measure using 0.3 g of varnish. (Measurement temperature: 23°C) (Static water contact angle measurement) Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500) Measurement method: 2 μL of water is dropped onto the film, and the adhesion angle of the droplet is measured immediately afterwards (measurement temperature: 25° C.). (Measurement of fall angle) Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500) Measurement method: After dropping 25 μL of water onto the film, the substrate is continuously tilted and the angle at which the water falls is taken as the falling angle (measurement temperature: 25°C).

[0106] <Preparation of layer having non-cell adhesive surface> After peeling off the release tape on one side of a double-sided tape (thickness 25 μm), the tape was attached to a transparent PET film (thickness 250 μm). A CO2 laser was used to form staggered through holes with a diameter of 300 μm and a pitch of 500 μm (formed through holes: 400 / cm). 2 (Laser entrance hole diameter: 500 μm, laser exit hole diameter: 300 μm) Then, the surface of the PET film was coated with an MPC polymer solution (0.5% ethanol solution, hydrophobic MPC polymer) to a thickness of 0.05 μm, and dried in a dryer at 50°C for 2 hours to obtain a layer with a cell non-adhesive surface [static water contact angle of 107.5° on the PET film coating layer (MPC polymer coating layer) side].

[0107] <Preparation of cell culture sheets and cell culture vessels> Next, the layer having a cell adhesive surface prepared above was attached to the surface of the layer having a non-cell adhesive surface from which the release tape had been removed, to prepare a cell culture sheet (sheet thickness: 315 μm). The obtained cell culture sheet was placed in a culture plate to complete a container for cell culture.

[0108] Example 1 The cells used were adipose-derived stem cells extracted from GFP gene-transduced Lewis rats.

[0109] <Defoaming treatment> The cell culture vessel of Preparation Example 1 was subjected to a degassing treatment. Specifically, about 1 mL of PBS was added to the vessel, pipetting was performed, and the vessel was left standing for 15 minutes in a 5% (v / v) CO2 incubator at 37°C. Next, the PBS was aspirated with an aspirator, 0.2 mL of KBM ADSC-2 medium containing 1% antibiotics was added, and the vessel was left standing overnight in a 5% (v / v) CO2 incubator at 37°C.

[0110] <Spheroid Creation> Remove the medium from the culture flask in which the stem cells were previously cultured, and add the cell detachment solution Accutase TM After adding 5 mL of PBS (Promocell), the cells were detached by placing them in a 5% (v / v) CO2 incubator at 37°C for about 5 minutes. The detachment solution was then collected, washed with 10 mL of PBS, and transferred to a tube. The cells were centrifuged at 210 × g for 5 minutes, suspended in 1 mL of KBM ADSC-2 medium containing 1% antibiotics, and the number of cells was counted. After that, 1.0 × 10 6 The concentration was adjusted to cells / mL. The medium was removed from the cell culture vessel, which had been left overnight in a 5% (v / v) CO2 incubator at 37°C to degasify, and cells were seeded at 500 cells / well. After leaving the vessel in a safety cabinet for 15 minutes, the vessel was placed in a 5% (v / v) CO2 incubator at 37°C and left for 4 hours. Next, KBM ADSC-2 medium containing 1% antibiotics was added, and the vessel was placed in a 5% (v / v) CO2 incubator at 37°C again for 3 days of culture, yielding spheroids with a diameter of approximately 150 μm.

[0111] Test Example 1 (in vivo experiment) Ten 8-week-old Lewis rats were used to create osteochondral defects measuring φ1.6 mm in the trochlea of ​​both femurs. 20 spheroids of the adipose-derived stem cells of Example 1 were placed in the right knee, and 1.0 × 10 single cells of the adipose-derived stem cells were placed in the left knee. 4 Each rat was administered 100 mg of ...

[0112] As a result, in the spheroid-administered group, repair images in cartilage-like tissue were macroscopically observed at 4 weeks, and metachromatism was confirmed histologically by toluidine blue staining (Figure 1). At the same time, type II collagen was also positive (Figure 2), and the Wakitani score evaluation showed significant improvement in cartilage structure in the spheroid-administered group compared to the single cell-administered group (Table 1, Figure 3). In addition, GFP immunostaining showed positivity in the chondrocytes in the repair tissue after 4 weeks (Figure 1), suggesting that the repaired chondrocytes may have been directly differentiated from adipose-derived stem cells. Furthermore, in the spheroid-administered group, cartilage repair (Figure 1: Photograph of cartilage above the defect) and Masson's trichrome subchondral bone repair were also confirmed to be good (Figure 4: Evaluation of new bone increase rate). In Masson's trichrome staining, the darkly stained areas were ossified areas, and the lightly stained or white areas were unossified areas and granulation tissue. The rate of new bone increase was analyzed by selecting the defective areas excluding cartilage, binarizing them with ImageJ, detecting the dark areas in the relevant areas, and calculating the area ratio. In HE staining, bone repair was confirmed from the bottom of the defect hole at 2 weeks in the spheroid-administered group, and the formation of chondrocytes was confirmed at 4 weeks. Furthermore, subchondral bone repair was confirmed in both the spheroid-administered group and the single cell-administered group at 12 weeks, and the spheroid-administered group exhibited a more solid bone structure than the single cell-administered group (Figure 5). In addition, in the spheroid-administered group, toluidine blue staining confirmed the presence of vigorous chondrocytes at 4 weeks, and in addition to the regeneration of the cartilage matrix, it was also confirmed that cancellous bone regeneration ability was promoted (Figure 6).

[0113] [Table 1]

[0114] Test Example 2 (in vitro experiment) PCR evaluation was performed on spheroid and single cell types of adipose-derived stem cells isolated and cultured from Lewis rats. The expression of therapeutic-related genes (identification of properties) and gene expression of anti-inflammatory cytokines (identification of the basis of therapeutic effects) were evaluated. The spheroids obtained in Example 1 were used for the spheroid type of adipose-derived stem cells.

[0115] PCR results showed that the anti-inflammatory cytokines TSG-6 and TGF-β1 were highly expressed in the spheroid-type adipose-derived stem cells (Figure 7).

[0116] Spheroids of adipose-derived stem cells expressed more anti-inflammatory cytokine TSG-6 and TGF-β1, which promotes osteoblast proliferation, than single cells, and were effective in repairing bone and cartilage. This also suggests that the repair may involve differentiation of the administered adipose-derived stem cells themselves. [Industrial Applicability]

[0117] The agent of the present invention can be easily prepared and enables the cell culture process itself to be carried out efficiently, and therefore can be applied, for example, to fields such as bone regenerative medicine, as well as fields where an anti-inflammatory effect is expected, such as skull, jaw, and facial reconstruction surgery, cosmetic surgery, plastic surgery, orthopedics, oral surgery, otorhinolaryngology, and dentistry.

Claims

1. A method for producing a bone regenerating agent, comprising a step of preparing spheroids by culturing mesenchymal stem cells on a cell culture substrate having a plurality of recesses with an opening diameter of 1000 μm or less, wherein the inner surface of the recesses of the cell culture substrate has a non-cell-adhesive surface and the bottom surface of the recesses has a cell-adhesive surface, and the bone regenerating agent contains spheroids containing mesenchymal stem cells as an active ingredient.

2. A method for producing a preventive or therapeutic agent for a bone tissue-related disease, the method comprising a step of preparing spheroids by culturing mesenchymal stem cells on a cell culture substrate having a plurality of recesses with an opening diameter of 1000 μm or less, wherein the inner side of the recesses of the cell culture substrate has a non-cell-adhesive surface and the bottom surface of the recesses has a cell-adhesive surface, the method contains spheroids containing mesenchymal stem cells as an active ingredient, and the bone tissue-related disease is selected from the group consisting of osteoarthritis, osteochondritis dissecans, articular cartilage damage, fractures, intractable fractures, rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, gouty arthritis, pseudogouty arthritis, osteogenesis imperfecta, and osteoporosis.

3. The method according to claim 1 or 2, wherein the agent is an anti-inflammatory agent.

4. The method according to any one of claims 1 to 3, wherein the agent is an agent for cartilage regeneration.

5. The method according to any one of claims 1 to 4, wherein the culture surface of the cell culture substrate has a cell adhesive surface.

6. The method according to claim 5, wherein the cell adhesive surface is made of a material that exhibits cell adhesiveness.

7. The method according to claim 5 or 6, wherein the cell adhesive surface comprises a polyimide resin.

8. The method according to any one of claims 1 to 7, wherein the spheroids express an anti-inflammation-related protein and / or a gene encoding the same.

9. The method according to claim 8, wherein the anti-inflammation-related protein is TSG-6.

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