Scaffold material for stem cell culture and stem cell culture method using the scaffold material

Abstract

The present invention relates to a scaffold material for stem cell culture, which comprises a synthetic resin, wherein the storage modulus at 100°C is 1.0×10 4 Pa or above and 1.0×10 8 pa or less, the ratio of the storage modulus at 25°C to the storage modulus at 100°C ((storage modulus at 25°C) / (storage modulus at 100°C)) is 1.0×10 1 Above and 1.0×10 5 The scaffold material for stem cell culture has appropriate hydrophilicity and strength, has high fixation of stem cells after seeding, can efficiently proliferate cells, and has excellent damage resistance.

Description

Technical Field

[0001] The present invention relates to a scaffold material for stem cell culture and a stem cell culture method using the scaffold material. Prior art

[0002] The application of stem cells to drug development or regenerative medicine is expected. Stem cells are cells with self-replication and differentiation abilities, and there are: pluripotent stem cells that can differentiate into all types of cells, and tissue stem cells and tissue progenitor cells that can only differentiate into the constituent cell types of the same series of body tissues. Pluripotent stem cells, for example, can include human pluripotent stem cells (hPSC) such as human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC). Cultivating stem cells safely and with excellent reproducibility and making them proliferate has become an essential basic technology in the medical application of these cells. Especially in the industrial use of regenerative medicine, stem cells must be processed in large quantities in an undifferentiated state. Therefore, the technology of using natural and synthetic polymers or feeder cells to proliferate stem cells while maintaining pluripotency (or multi-differentiation ability) is widely studied. In particular, it is known that if adhesive proteins such as laminin and vitronectin, and matrix glue derived from mouse sarcoma are used as natural polymers, the cell fixation after inoculation is very high.

[0003] However, the following technical problems can be cited: natural polymers are expensive due to their very low productivity; since they are naturally derived substances, there are batch-to-batch variations; and there are safety concerns due to animal-derived ingredients.

[0004] In order to solve the above technical problems, a stem cell culture resin carrier using a synthetic resin is proposed. For example, in the Example column of Patent Document 1, in order to provide a hydrophilic and water-resistant scaffold material in the culture of mouse fibroblasts, a polyvinyl alcohol acetal compound with an acetalization degree of 20 to 60 mol% is disclosed. In the Example column of Patent Document 2, in the culture of mouse ES cells, a hydrogel composed of an acrylic polymer is disclosed. In the Example column of Patent Document 3, in the culture of mouse iPS cells, a hydrophilic and soft polyrotaxane gel is disclosed.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2006-314285

[0008] Patent Document 2: Japanese Patent Application Publication No. 2010-158180

[0009] Patent Document 3: Japanese Patent Application Publication No. 2017-23008 Summary of the invention

[0010] Technical Problems Solved by the Invention

[0011] However, in Patent Document 1, there is a problem that swelling occurs in the culture medium due to high hydrophilicity, and the scaffold material resin is peeled off. In addition, there is a problem that the fixation of stem cells and pluripotent stem cells after inoculation is low and they do not proliferate sufficiently. In Patent Document 2, 2-acrylamide-2-methylpropanesulfonic acid sodium salt, sodium p-styrenesulfonate, and N,N'-dimethylacrylamide are used. There is a problem that swelling occurs in the culture medium due to high hydrophilicity, and the scaffold material resin is peeled off. In Patent Document 3, there is a problem that swelling occurs in the culture medium due to high hydrophilicity, and the scaffold material resin is peeled off. Since it is a soft scaffold material, there is a problem of promoting differentiation into cardiomyocytes.

[0012] In addition, the following technical problems are cited: in the seeding step of seeding stem cells in a culture container, the coating film is peeled off due to the contact between the tip of the pipette and the bottom surface of the culture container, resulting in unevenness between test batches.

[0013] Based on the above, a scaffold material for stem cell culture having appropriate hydrophilicity and strength, and a stem cell culture method using the scaffold material are sought.

[0014] An object of the present invention is to provide a stem cell culture scaffold material having appropriate hydrophilicity and strength, high fixation of stem cells after seeding, efficient cell proliferation and excellent damage resistance, and a stem cell culture method using the scaffold material.

[0015] Technical means to solve technical problems

[0016] The present invention relates to the following contents.

[0017] (1) A scaffold material for stem cell culture, comprising a synthetic resin, wherein the storage modulus of the scaffold material for stem cell culture at 100°C is 1.0×10 4 Pa or more and 1.0×10 8 Pa or less, the ratio of the storage modulus at 25°C to the storage modulus at 100°C ((storage modulus at 25°C) / (storage modulus at 100°C)) of the scaffold material for stem cell culture is 1.0×10 1 Above and 1.0×10 5 the following.

[0018] (2) The scaffold material for stem cell culture according to (1), wherein the synthetic resin is a polyvinyl acetal resin.

[0019] (3) The scaffold material for stem cell culture according to (1) or (2), wherein the stem cells are pluripotent stem cells.

[0020] (4) A stem cell culture container comprising the stem cell culture scaffold material according to any one of (1) to (3).

[0021] Effects of the Invention

[0022] According to the present invention, there are provided a stem cell culture scaffold material having appropriate hydrophilicity and strength and high fixation of stem cells after seeding, and a stem cell culture method using the scaffold material. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A : is a three-dimensional conceptual diagram of a stem cell culture container according to an embodiment. Figure 1B This is a conceptual side cross-section diagram.

[0024] Figure 2 This is a conceptual perspective view of a 6-well stem cell culture container according to an embodiment.

[0025] Figure 3 This is a graph showing the evaluation criteria for cell proliferation 5 days after cell inoculation.

[0026] Figure 4A as well as Figure 4B These are phase contrast microscope photographs of the cells of the Example and the Comparative Example 5 days after inoculation.

[0027] Implementation

[0028] Hereinafter, the present invention will be described with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0029] [Scaffold materials for stem cell culture]

[0030] In order to solve the above technical problems, the inventors have made intensive research from the aspect of the physical properties of synthetic resins. As a result, they have found that the above technical problems can be solved by using synthetic resins with specific storage modulus, thereby completing the present invention. That is, the present invention relates to a scaffold material for stem cell culture, which contains a synthetic resin, wherein the storage modulus of the scaffold material for stem cell culture at 100°C is 1.0×10 4 Pa or more and 1.0×10 8 Pa or less, and the ratio of the storage modulus at 25°C to the storage modulus at 100°C ((storage modulus at 25°C) / (storage modulus at 100°C)) is 1.0×10 1 Above and 1.0×10 5 the following.

[0031] The stem cell culture scaffold material has appropriate hydrophilicity and strength, so the fixation of stem cells after seeding is improved. In particular, the initial fixation rate of stem cells after seeding is improved in serum-free medium culture without feeder cells or adhesive proteins.

[0032] From the viewpoint of more appropriately adhering the stem cells, the 100°C storage modulus of the resin film is more preferably 1.0×10 5 Pa or more and 1.0×10 7 Pa or less, more preferably 2.0×10 5 Pa or more and 8.0×10 6 Pa or less, and more preferably 3.0×10 5 Pa or more and 5.0×10 6 Below Pa.

[0033] From the viewpoint of allowing stem cells to adhere more appropriately, the 25°C storage modulus of the resin film is preferably 1.0×10 8 Pa or more and 1.0×10 10 Pa or less, more preferably 3.0×10 8 Pa or more and 8.0×10 9 Pa or less, more preferably 5.0×10 8 Pa or more and 5.0×10 9 Below Pa.

[0034] The ratio of the storage modulus at 25°C to the storage modulus at 100°C of the resin film is preferably 1.0×10 1 Above and 1.0×10 5 Below, more preferably 1.0×10 2 Above and 1.0×10 4 If the values ​​of the storage modulus at 25°C and the storage modulus at 100°C, and the ratio of the storage modulus at 25°C to the storage modulus at 100°C are within the above ranges, the bottom surface of the culture container will not be damaged by the tip of the pipette during the inoculation step, and the elastic modulus changes under the culture condition of 37°C, allowing stem cells to adhere appropriately.

[0035] From the viewpoint of allowing stem cells to adhere appropriately, the glass transition point of the resin film is preferably -30°C to 95°C, more preferably 0°C to 90°C, and even more preferably 20°C to 90°C.

[0036] In addition, the storage modulus and the glass transition point can be measured by the following method.

[0037] Specifically,

[0038] 1. The samples were stacked using a heat press to obtain a sheet with a thickness of 500 μm.

[0039] 2. The obtained sheet was measured by a dynamic viscoelasticity measuring device (DVA-200 manufactured by IT Instruments Co., Ltd.) under tension conditions at a frequency of 10 Hz and a heating rate of 5°C / min in a temperature range of -150°C to 150°C. The storage moduli at 25°C and 100°C were obtained from the obtained tensile storage modulus graph, and the 25°C storage modulus / 100°C storage modulus was calculated.

[0040] 3. From the graph obtained by the storage modulus measurement, the peak temperature of the loss tangent was determined and defined as the glass transition temperature Tg.

[0041] The storage modulus at 100° C. and 25° C. can be improved by, for example, introducing a rigid functional group or a highly crystalline functional group into the main chain and the side chain to reduce the mobility of the polymer chain.

[0042] On the other hand, the storage modulus can be reduced by introducing a soft functional group or a functional group with low crystallinity into the main chain and the side chain to reduce the mobility of the polymer chain.

[0043] The components of the scaffold material for stem cell culture are not particularly limited as long as the above-mentioned technical characteristics are met, but it preferably contains a synthetic resin.

[0044] A synthetic resin is a resin having as a main component a polymer (hereinafter also simply referred to as a "polymer") obtained by polymerizing (including polycondensation) a polymerizable monomer (hereinafter also simply referred to as a "monomer"). The polymer also includes a copolymer of one or more polymerizable monomers.

[0045] Examples of the polymer include polymers composed of one or more polymerizable monomers selected from (unsaturated hydrocarbons, aromatic hydrocarbons, (unsaturated fatty acids, aromatic carboxylic acids, (unsaturated ketones, aromatic ketones, (unsaturated alcohols, aromatic alcohols, (unsaturated amines, aromatic amines, (unsaturated thiols, aromatic thiols and organosilicon compounds.

[0046] Specific examples of the polymer include polyolefins, polyethers, polyvinyl alcohols, polyvinyl acetals, polyesters, poly(meth)acrylates, epoxy resins, polyamides, polyimides, polyurethanes, polycarbonates, celluloses, and polypeptides, etc. Among them, poly(meth)acrylates and polyvinyl acetals are preferred, and polyvinyl acetals are more preferred, from the viewpoint of the fixation of stem cells.

[0047] It should be noted that these polymers can use one, and two or more combinations can also be used. In the case of combining two or more polymers, two or more polymers can be mixed and used, and can also be used as a polymer that the skeleton of two or more polymers is chemically bonded. In addition, the copolymer structure can also be a graft copolymer, a block copolymer and a combination thereof. Therefore, in the case of combining multiple polymers as a synthetic resin, it is preferred that poly (methyl) acrylate and polyvinyl acetal are combined.

[0048] The poly(meth)acrylate in the present specification is a polymer obtained by polymerizing a (meth)acrylate as a monomer, but includes a polymer obtained by copolymerizing a monomer other than a (meth)acrylate.

[0049] The (meth)acrylate is not particularly limited, but preferably contains at least one selected from alkyl (meth)acrylates, cyclic alkyl (meth)acrylates, aryl (meth)acrylates, (meth)acrylamides, polyethylene glycol (meth)acrylates, and phosphorylcholine (meth)acrylates.

[0050] Examples of the alkyl (meth)acrylate include methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, isopropyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, tert-butyl (meth)acrylate, n-octyl (meth)acrylate, isooctyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, isononyl (meth)acrylate, decyl (meth)acrylate, isodecyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, and isotetradecyl (meth)acrylate.

[0051] It should be noted that these alkyl (meth)acrylates are not particularly limited, and hydrogen atoms may be substituted with various substituents such as an alkoxy group having 1 to 3 carbon atoms and a tetrahydrofurfuryl group. Examples of alkyl (meth)acrylates in which hydrogen atoms are substituted with the above substituents include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0052] Examples of the cyclic alkyl (meth)acrylate include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0053] As said aryl (meth)acrylate, phenyl (meth)acrylate, benzyl (meth)acrylate, etc. are mentioned, for example.

[0054] Examples of the acrylamides include (meth)acrylamide, N-isopropyl (meth)acrylamide, N-tert-butyl (meth)acrylamide, N,N'-dimethyl (meth)acrylamide, (3-(meth)acrylamidepropyl)trimethylammonium chloride, 4-(meth)acryloylmorpholine, 3-(meth)acryloyl-2-oxazolidinone, N-[3-(dimethylamino)propyl] (meth)acrylamide, N-(2-hydroxyethyl) (meth)acrylamide, N-hydroxymethyl (meth)acrylamide, and 6-(meth)acrylamidocaproic acid.

[0055] Examples of the polyethylene glycol (meth)acrylates include methoxy-polyethylene glycol (meth)acrylate, ethoxy-polyethylene glycol (meth)acrylate, hydroxy-polyethylene glycol (meth)acrylate, methoxy-diethylene glycol (meth)acrylate, ethoxy-diethylene glycol (meth)acrylate, hydroxy-diethylene glycol (meth)acrylate, methoxy-triethylene glycol (meth)acrylate, ethoxy-triethylene glycol (meth)acrylate, and hydroxy-triethylene glycol (meth)acrylate.

[0056] Examples of the (meth)acrylphosphocholine include 2-(meth)acryloyloxyethylphosphocholine.

[0057] The monomer other than the (meth)acrylic acid ester is not particularly limited, and examples thereof include (meth)acrylic acid, ethylene, and vinyl ester.

[0058] The (meth)acrylates may be used alone or in combination of two or more. It should be noted that in the present specification, the (meth)acrylic acid is a general term for acrylic acid and methacrylic acid, and the (meth)acrylates are a general term for acrylic acid and methacrylic acid.

[0059] Among synthetic resins, polyvinyl acetal resin is preferably used. Hereinafter, polyvinyl acetal resin will be described.

[0060] (Polyvinyl acetal resin)

[0061] The polyvinyl acetal resin is a resin obtained by acetalizing polyvinyl alcohol with an aldehyde, and has an acetyl group, a hydroxyl group, and an acetal group in a side chain.

[0062] Examples of the aldehyde used in the acetalization include aldehydes having a chain aliphatic group, a cyclic aliphatic group, or an aromatic group having 1 to 10 carbon atoms. As these aldehydes, conventionally known aldehydes can be used.

[0063] The type of the aldehyde is not particularly limited, and examples thereof include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, hexanal, heptaldehyde, octanal, nonanal, decanal, acrolein, benzaldehyde, cinnamaldehyde, perillaldehyde, formylpyridine, formylimidazole, formylpyrrole, formylpiperidine, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formylisoindole, formylpurine, formylpurine, Formylbenzimidazole, formylbenzotriazole, formylquinoline, formylisoquinoline, formylquinoxaline, formylcinnamyl, formylpteridine, formylfuran, formyloxolane, formyloxane, formylthiophene, formyltetrahydrothiophene, formylcyclopentanesulfide, formyladenine, formylguanine, formylcytosine, formylthymine, formyluracil, etc. The aldehyde may be chain or cyclic.

[0064] The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, and more preferably butyraldehyde.

[0065] The polyvinyl alcohol may be a copolymer with a vinyl compound. Examples of the vinyl compound include ethylene, allylamine, vinyl pyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, and (meth)acrylic acid esters.

[0066] The polyvinyl acetal resin preferably has a Bronsted basic group or a Bronsted acidic group in part thereof. The reason for this is that when a part of the polyvinyl acetal resin is modified with a Bronsted basic group or a Bronsted acidic group, the initial fixation rate after the stem cells are seeded is improved in the serum-free medium culture without feeder cells or adhesive proteins, and the stem cells can be easily cultured.

[0067] The Brownian basic group can accept hydrogen ions H from other substances. + Examples of the functional group include amino, imino, acylamino, and acylimino.

[0068] Therefore, as the polyvinyl acetal resin, a polyvinyl acetal resin containing at least one selected from the group consisting of a structural unit having an amine structure, a structural unit having an imine structure, and a structural unit having an amide structure in its skeleton is preferred.

[0069] The Bronsted acidic group is a group that can convert hydrogen ions H + A general term for substances that transfer to other substances.

[0070] Examples of the Bronsted acidic group include a carboxyl group, a sulfonic acid group, a maleic acid group, a sulfinic acid group, a sulfenic acid group, a phosphoric acid group, a phosphonic acid group, and salts thereof, etc. Among them, a carboxyl group is preferred.

[0071] The method of modifying the polyvinyl acetal resin with the Bronsted acidic group is not particularly limited, and the polyvinyl acetal resin may be obtained by copolymerization or grafting with itaconic acid or (meth)acrylic acid.

[0072] In the present invention, the imine structure refers to a structure having a C=N bond. The modified polyvinyl acetal resin preferably has an imine structure in the side chain. In addition, the imine structure may be directly bonded to the carbon constituting the main chain of the modified polyvinyl acetal resin, or may be bonded through a linking group such as an alkylene group. It should be noted that the imine structure in the side chain includes the imine structure in the graft chain of the modified polyvinyl acetal resin. As a structural unit having the imine structure, for example, the structural unit represented by the following formula (1) can be cited.

[0073] [Chemical formula 1]

[0074]

[0075] In formula (1), R 1 represents a single bond or an alkylene group, R 2 It represents a group having an imine structure.

[0076] In the formula (1), R 1 In the case of an alkylene group, the preferred lower limit of the number of carbon atoms of the alkylene group is 1, and the preferred upper limit is 12. If the number of carbon atoms of the alkylene group exceeds 12, the optimum strength may not be obtained. 1 In the case of an alkylene group, a more preferred upper limit of the number of carbon atoms of the alkylene group is 5.

[0077] In the formula (1), R 1 In the case of an alkylene group, examples of the alkylene group include straight-chain alkylene groups such as methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, octamethylene, and decamethylene; branched-chain alkylene groups such as methylmethylene, methylethylene, 1-methylpentylene, and 1,4-dimethylbutylene; and cyclic alkylene groups such as cyclopropylene, cyclobutylene, and cyclohexylene. Among them, straight-chain alkyl groups such as methylene, ethylene, trimethylene, and tetramethylene are preferred, and methylene and ethylene are more preferred.

[0078] As the R 2 , a functional group represented by the following formula (2) can be mentioned.

[0079] [Chemical formula 2]

[0080]

[0081] In formula (2), R 3 represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R4 It represents a hydrocarbon group having 1 to 18 carbon atoms.

[0082] Examples of the hydrocarbon group include saturated hydrocarbon groups, unsaturated hydrocarbon groups, aromatic hydrocarbon groups, etc. It should be noted that the hydrocarbon group may be a hydrocarbon group consisting of only saturated hydrocarbon groups, unsaturated hydrocarbon groups, and aromatic hydrocarbon groups, or a hydrocarbon group using two or more hydrocarbon groups.

[0083] Examples of the saturated hydrocarbon group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, 2-ethylhexyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, octadecyl, etc. Among them, methyl, ethyl, n-propyl, and n-butyl are preferred.

[0084] Examples of the aromatic hydrocarbon group include a phenyl group, a tolyl group, a xylyl group, a tert-butylphenyl group, and a benzyl group.

[0085] Preferably, in the modified polyvinyl acetal resin, in the structural unit having the imine structure, R 1 is a single bond, R 3 is a hydrogen atom, a methyl group or an ethyl group, R 4 It is methyl or ethyl.

[0086] In the modified polyvinyl acetal resin, the preferred lower limit of the content of the structural unit having an imine structure is 0.1 mol%, and the preferred upper limit is 20.0 mol%. If the content of the structural unit having the imine structure is 0.1 mol% or more, it becomes a material with good viscosity stability over time. If the content of the structural unit having the imine structure is 20.0 mol% or less, acetalization can be fully performed. The more preferred lower limit of the content of the structural unit having the imine structure is 1.0 mol%, and the more preferred upper limit is 15.0 mol%.

[0087] In the modified polyvinyl acetal resin, the ratio of the content of the structural unit having an imine structure to the following acetalization degree (content of the structural unit having an imine structure / acetalization degree) is preferably 0.001 to 0.5. By setting it within the above range, high strength and excellent adhesion can be achieved at the same time, and the durability after adhesion can be improved.

[0088] The modified polyvinyl acetal resin preferably contains a structural unit having an imino (=NH) structure.

[0089] The modified polyvinyl acetal resin preferably has the imino group in a side chain. The imino group may be directly bonded to the carbon constituting the main chain of the modified polyvinyl acetal resin or may be bonded via a linking group such as an alkylene group.

[0090] The modified polyvinyl acetal resin preferably contains a structural unit having an amino group or an amide structure.

[0091] The modified polyvinyl acetal resin preferably has the amino group or amide structure in the side chain. In addition, the amino group or amide structure may be directly bonded to the carbon of the main chain constituting the modified polyvinyl acetal resin, or may be bonded through a linking group such as an alkylene group. In addition, the amino group may be a primary amine, a secondary amine, a tertiary amine, or a quaternary amine. Among these, primary amines are preferred from the viewpoint of cell fixation.

[0092] It should be noted that the amino group or amide structure is present in the side chain means that the amino group or amide structure is present in the graft chain of the modified polyvinyl acetal resin.

[0093] In particular, the amino group is preferably -NH2. It should be noted that, in the present invention, the amide structure refers to a structure having -C(=O)-NH-. Among them, the structural unit having the amino group is preferably a structure represented by the following formula (3). In addition, the structural unit having the amide structure is preferably a structure represented by the following formula (4).

[0094] [Chemical formula 3]

[0095]

[0096] [Chemical formula 4]

[0097]

[0098] In formula (4), R 5 represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms. Examples of the hydrocarbon group include an alkyl group, an alkenyl group, a cycloalkyl group, and a cycloalkenyl group.

[0099] The preferred lower limit of the content of the structural unit with the amino group or amide structure is 0.1 mol%, and the preferred upper limit is 20 mol%. If the content of the structural unit with the amino group or amide structure is more than 0.1 mol%, the addition characteristics can be made sufficient. If the content is below 20 mol%, the solubility will not rise excessively, and it becomes easy to obtain the modified polyvinyl acetal resin powder by precipitation method. The more preferred lower limit of the content is 0.5 mol%, and the more preferred upper limit is 10 mol%. It should be noted that the content of the structural unit with the amino group or amide structure can be measured by NMR etc. In addition, the preferred lower limit of the total content of the structural unit with the amino group or amide structure and the structural unit with imino group is 0.1 mol%, and the preferred upper limit is 20 mol%. The more preferred lower limit of the content is 0.5 mol%, and the more preferred upper limit is 10 mol%.

[0100] In the modified polyvinyl acetal resin, the ratio of the content of the structural unit having an imino group to the structural unit having an amino group or an amide structure (structural unit having an imino group / structural unit having an amino group or an amide structure) is preferably 0.5 / 99.5 to 99.5 / 0.5. If the ratio is 0.5 / 99.5 or more, the viscosity stability over time can be sufficient, and if the ratio is 99.5 / 0.5 or less, the crosslinking performance can be fully exerted from the viewpoint of fixation. The more preferred lower limit of the ratio is 5 / 95, and the more preferred upper limit is 75 / 25.

[0101] The degree of acetalization of the modified polyvinyl acetal resin is not particularly limited, and the preferred lower limit is 60 mol%, and the preferred upper limit is 90 mol%. If the degree of acetalization is 60 mol% or more, the fixation of stem cells is excellent, and cell proliferation can be carried out efficiently. In addition, if the degree of acetalization is 90 mol% or less, the solubility in the solvent can be good. More preferably, it is higher than 60 mol%, more preferably 65 mol% or more, and more preferably 85 mol% or less, and more preferably 80 mol% or less. The degree of acetalization of the modified polyvinyl acetal resin can be measured by NMR, etc.

[0102] The amount of acetyl groups in the modified polyvinyl acetal resin is not particularly limited, but the preferred lower limit is 0.0001 mol % and the preferred upper limit is 5 mol %.

[0103] From the viewpoint of cell adhesion immediately after seeding, the polyvinyl acetal resin skeleton preferably contains 0.1 to 30 mol % of at least one functional group selected from structural units having an amine structure, structural units having an imino group, and structural units having an amide structure, and more preferably contains 1 to 10 mol %.

[0104] Here, the terms used in this specification are explained.

[0105] "Stem cells" refer to cells that have the ability to self-replicate and differentiate. Among stem cells, those that have the ability to self-replicate and can differentiate from one cell into all cells of the endoderm, mesoderm, and ectoderm are called "pluripotent stem cells."

[0106] Examples of pluripotent stem cells include induced pluripotent stem cells (hereinafter referred to as "iPS cells"), embryonic stem cells (hereinafter referred to as "ES cells"), Muse cells (multilinege differentiating stress enduring cells), embryonic cancer cells (embryonic germ cells), embryonic germ stem cells (embryonic germ cells), and mGS cells (multipotentgerm stem cells).

[0107] Among stem cells, cells that have the ability to self-replicate, belong to any of the ectoderm tissue, endoderm tissue, mesoderm tissue, and reproductive system tissue, and show limited differentiation ability into the constituent cell type of the organ to which they belong are called "tissue stem cells" and "tissue progenitor cells."

[0108] Examples of tissue stem cells and tissue progenitor cells include neural stem cells, neural crest stem cells, retinal stem cells, corneal stem cells, keratinocyte epidermal stem cells, melanocyte stem cells, mammary stem cells, liver stem cells, intestinal stem cells, tracheal stem cells, hematopoietic stem cells, mesenchymal stem cells, cardiac stem cells, endothelial progenitor cells, vascular pericytes, skeletal muscle stem cells, adipose stem cells, renal progenitor cells, sperm stem cells, and the like.

[0109] By using the stem cell scaffold material of one embodiment of the present invention, the stem cell type is not particularly limited, and all can be used as a stem cell scaffold material. Among them, it is preferably used for the culture of pluripotent stem cells, especially iPS cells. In the serum-free medium culture without feeder cells and adhesive proteins, the initial fixation rate after stem cell inoculation is improved, and stem cell culture can be appropriately performed.

[0110] Examples of such stem cells include those described in "A Better Understanding! Stem Cells and Regenerative Medicine" (Yoto Co., Ltd., written by Kenji Osafune).

[0111] The lower limit of the degree of polymerization of the polyvinyl acetal resin is preferably 100, more preferably 200, further preferably 500, and further more preferably 1500. If the degree of polymerization is within the above range, even if swelling occurs in a culture medium for cell culture, the strength of the scaffold material can be preferably maintained, and cell proliferation can be improved. The upper limit of the degree of polymerization is preferably 6000, more preferably 3000, and further preferably 2500. If the degree of polymerization is within the above range, the operability is good, and the scaffold material can be appropriately formed.

[0112] [Methods for culturing stem cells]

[0113] According to the scaffold material for stem cell culture, various stem cells can be cultured, but considering its characteristics, it is preferably used for the culture of pluripotent stem cells among stem cells. The reason is that it is generally believed that the fixation rate of pluripotent stem cells after inoculation is low, but the scaffold material for stem cell culture is not easily swollen by the water in the culture medium, and can maintain appropriate hydrophilicity and strength, so the fixation rate of pluripotent stem cells after inoculation is improved.

[0114] [Container for stem cell culture]

[0115] The present invention also relates to a stem cell culture container using the stem cell culture scaffold material, that is, to a stem cell culture container having a resin film containing the stem cell scaffold material in at least a part of a stem cell culture region.

[0116] Figure 1A : is a three-dimensional conceptual diagram of a stem cell culture container according to an embodiment. Figure 1B The shape of the container for stem cell culture is not particularly limited, and examples thereof include the following forms: Figure 1A The bottom cylindrical culture dish 1 shown in the figure has a stem cell culture area on its bottom surface. Figure 1B As shown, a resin film 12 is provided.

[0117] In the culture of stem cells, scaffold materials for stem cell culture can be used not only for flat culture (two-dimensional culture method) but also for culture of stem cells on substrates such as porous membranes and hydrogels that are closer to the state in vivo (three-dimensional culture method). The reason is that by using scaffold materials for cell culture in bioreactors, stem cells can be efficiently proliferated.

[0118] Since the cell culture scaffold material has appropriate hydrophilicity and strength, it is preferably used in a two-dimensional culture method.

[0119] The shape and size of the container for planar culture (two-dimensional culture method) are not particularly limited, and examples thereof include a cell culture test plate or a cell culture flask having one or more holes. Figure 2 The stem cell culture container shown has 6 wells. The number of wells in the microplate is not limited, and examples thereof include 2, 4, 6, 12, 24, 48, 96, 384, and the like.

[0120] The shape of the hole is not particularly limited, and examples thereof include a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, etc. The shape of the bottom of the hole is not particularly limited, and examples thereof include a flat bottom, a round bottom, a concave-convex bottom, etc.

[0121] The material of the test plate for cell culture and the flask for cell culture having one or more holes is not particularly limited, and examples thereof include polymer resins, metals, and inorganic materials. Examples of the polymer resin include polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymer, polyimide, polyamide, polyamideimide, (meth)acrylic resin, epoxy resin, polysiloxane, etc. Examples of the metal include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, platinum, etc. Examples of the inorganic material include silicon oxide (glass), aluminum oxide, titanium oxide, zirconium oxide, iron oxide, silicon nitride, etc.

[0122] In addition to the above, the cell culture scaffold material can also be used for a suspension culture method in which stem cells are grown in free suspension in a culture medium.

[0123] [Other embodiments]

[0124] In the present invention, in addition to the above-mentioned scaffold material for stem cell culture, an invention using a scaffold material for stem cell culture is provided as another embodiment.

[0125] For example, a stem cell culture carrier (medium) containing the stem cell culture scaffold material and a polysaccharide is provided. The polysaccharide is not particularly limited, and various polysaccharides can be used. Among them, water-soluble polysaccharides are preferred.

[0126] In addition, a stem cell culture fiber having a stem cell culture scaffold material is also provided. In this case, the stem cell culture scaffold material is preferably coated on the fiber. In addition, the stem cell culture scaffold material can also be in the form of being impregnated or mixed in the fiber. Although the stem cell culture fiber is difficult to adhere to a planar structure such as a flask, it is suitable for a three-dimensional culture method of stem cells that are easily adhered to a three-dimensional structure such as a fiber (fibril) structure. Suitable for the culture of stem cells, especially adipose stem cells.

[0127] The stem cell culture scaffold material may also be cross-linked. The reason is that water swelling can be suppressed by cross-linking, and strength can be appropriately improved. A cross-linking agent may also be further added to the stem cell culture scaffold material for cross-linking.

[0128] The cross-linking agent is not particularly limited, and examples thereof include polyols, polycarboxylic acids, hydroxycarboxylic acids, metal soaps, and polysaccharides.

[0129] The polyol is not particularly limited, and examples thereof include ethylene glycol, propylene glycol, butylene glycol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, dodecanediol, undecanediol, diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, catechol, pyrogallol, diboric acid, methylene diboric acid, ethylene diboric acid, propylene diboric acid, phenylene diboric acid, biphenyl diboric acid, and bisphenol derivatives.

[0130] The polycarboxylic acid is not particularly limited, and examples thereof include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, phthalic acid, and poly(meth)acrylic acid.

[0131] The hydroxycarboxylic acid is not particularly limited, and examples thereof include glycolic acid, lactic acid, malonic acid, glyceric acid, hydroxybutyric acid, malic acid, tartaric acid, citramalic acid, citric acid, isocitric acid, leucine, mevalonic acid, pantoic acid, ricinoleic acid, ricinoleic acid, hydroxyethyl acid, quinic acid, shikimic acid, hydroxybenzoic acid, salicylic acid, creosote acid, vanillic acid, syringic acid, pyrocatechuic acid, dihydroxybenzoic acid, protocatechuic acid, gentisic acid, orsuccinic acid, gallic acid, mandelic acid, benzilic acid, atrolactic acid, myristic acid, phlorothalic acid, coumaric acid, umbelliferous acid, caffeic acid, ferulic acid, sinapic acid, and hydroxystearic acid.

[0132] The metal soap is not particularly limited, and examples thereof include salts of fatty acids such as stearic acid, lauric acid, ricinoleic acid, and caprylic acid, and metals such as lithium, sodium, magnesium, calcium, barium, zinc, and aluminum.

[0133] The polysaccharide is not particularly limited, and examples thereof include pectin, guar gum, tannin gum, tamarind gum, carrageenan, propylene glycol, carboxymethyl cellulose, amylose, amylopectin, glycogen, cellulose, chitin, agarose, carrageenan, heparin, hyaluronic acid, xyloglucan, and glucomannan acid. Example

[0134] The present invention is described below with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples. It should be noted that the structural units of the obtained synthetic resin, such as the content (mol %) of the structural unit having an amino group, the content (mol %) of the structural unit having an imine structure, the degree of acetalization (mol %), the amount of acetyl groups (mol %), the amount of hydroxyl groups (mol %), etc., are obtained by dissolving the synthetic resin in DMSO-d6 (dimethyl sulfoxide) and using 1 H-NMR (nuclear magnetic resonance spectroscopy) was used for measurement.

[0135] [Example 1]

[0136] (Preparation of polyvinyl butyral)

[0137] In a reactor equipped with a stirring device, 2700mL of ion exchange water, 300 average polymerization degrees, and 300g of polyvinyl alcohol having a saponification degree of 99 mol% are added, and heated and dissolved while stirring to obtain a solution. Next, 35% by mass hydrochloric acid as a catalyst is added to the solution in a manner that makes the hydrochloric acid concentration 0.2% by mass, and after adjusting the temperature to 15°C, 20g of n-butyraldehyde (n-BA) is added while stirring. Thereafter, 130g of n-butyraldehyde (n-BA) is added, and as a result, white granular polyvinyl butyral resin is precipitated. 15 minutes after precipitation, 35% by mass hydrochloric acid is added in a manner that makes the hydrochloric acid concentration 1.8% by mass, heated to 50°C, and aged for 2 hours at 50°C. Subsequently, the solution is cooled, and after neutralization, the polyvinyl butyral resin is washed with water and dried to obtain polyvinyl butyral.

[0138] The obtained polyvinyl butyral had an average degree of polymerization of 300, a hydroxyl group content of 36 mol%, an acetyl group content of 1 mol%, and an acetalization degree of 63 mol%.

[0139] (Preparation of a cell culture vessel)

[0140] A polyvinyl butyral solution was obtained by dissolving 1 g of the obtained polyvinyl butyral in 19 g of butanol. 150 μL of the obtained polyvinyl butyral solution was sprayed onto a φ22 mm cover glass and spun at 2000 rpm for 20 seconds using a spin coater to obtain a smooth resin film. The obtained resin film and the cover glass were placed in a φ22 mm polystyrene culture dish to obtain a cell culture container.

[0141] The cell culture container equipped with the resin membrane was tested under the following conditions.

[0142] (Cell culture test method)

[0143] 1 mL of phosphate-buffered saline was added to the obtained cell culture container and the container was left to stand in a 37°C incubator for 1 hour. After removing the phosphate-buffered saline from the culture dish, 1.5×10 4 h-iPS cells 253G1 were cultured in the presence of 1 mL of culture medium TeSR E8 (manufactured by STEM CELL) and 10 μM ROCK-Inhibitor (Y27632) at 37°C in an incubator with a CO2 concentration of 5%. Every 24 hours, 750 μL of the culture medium was removed and 250 μL of new TeSR E8 was added to adjust the concentration of ROCK-Inhibitor (Y27632) to 10 μM, thereby replacing the culture medium.

[0144] (Evaluation method of physical properties of film)

[0145] (1) Storage modulus

[0146] The obtained resin films were overlapped by a hot press to obtain a sheet with a thickness of 500 μm. The obtained sheet was measured by a dynamic viscoelasticity measuring device (manufactured by IT Measurement Control Co., Ltd., DVA-200) under tensile conditions at a frequency of 10 Hz and a heating rate of 5°C / min in a temperature range of -150°C to 150°C. Based on the obtained tensile storage modulus chart, the storage moduli at 25°C and 100°C were obtained, and the 25°C storage modulus / 100°C storage modulus was calculated.

[0147] (2)Tg

[0148] The peak temperature of the loss tangent was determined from the graph obtained by the storage modulus measurement, and this was defined as the glass transition temperature Tg.

[0149] (Method of Cell Block Culture Test)

[0150] 1 mL of phosphate-buffered saline was added to the obtained cell culture container and allowed to stand in an incubator at 37°C for 1 hour, after which the phosphate-buffered saline in the culture container was removed. A colony of h-iPS cells 252G1 in a confluent state was added to a 35 mm culture dish, followed by 1 mL of 0.5 mM ethylenediamine / phosphate buffer solution, which was allowed to stand at room temperature for 2 minutes. The ethylenediamine / phosphate buffer solution was then removed and 1.0 × 10 5 The cells were broken into 50-200 μm cell clumps by pipetting in 1 mL of TeSR E8 medium and inoculated into a culture container. The cells were cultured in an incubator at 37°C and 5% CO2 concentration in the presence of 1 mL of TeSR E8 medium (manufactured by STEM CELL) and 10 μM of ROCK-Inhibitor (Y27632). The medium was replaced every 24 hours by removing 750 μL of the medium and adding 250 μL of new TeSR E8.

[0151] (Cultivation and evaluation methods)

[0152] (1) Drop evaluation (damage resistance evaluation)

[0153] When a 10 mm φ zirconia ball (YTZ-10) was dropped from a height of 1 cm onto the stent material, the presence or absence of a drop mark was visually observed and evaluated. The evaluation was performed based on the following criteria.

[0154] ○: No drop marks were observed

[0155] ×: Dropping traces were observed

[0156] (2) Cell proliferation

[0157] In the cell culture test, a phase contrast microscope with a magnification of 10×4 (manufactured by Olympus, IX73) was used to obtain cell images 5 days after cell inoculation. At this time, an image of the field of view showing the most average adhesion morphology in the culture container was obtained. Figure 3 The cell proliferation was evaluated by comparing samples 1 to 10. Figure 3 The more the colony grows due to cell proliferation, the higher the evaluation is. It should be noted that if the colony grows excessively in the horizontal direction (horizontal and vertical directions of the screen), it will begin to accumulate in the vertical direction (direction near the front of the screen), so there is a tendency for light penetration to decrease. The results of the examples and comparative examples obtained are summarized and shown in Figure 4A , Figure 4B .

[0158] [Example 2]

[0159] The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 250 was used and the charged amounts of n-butylaldehyde were changed to 22 g and 148 g.

[0160] [Example 3]

[0161] The test was carried out in the same manner as in Example 2 except that polyvinyl alcohol having an average degree of polymerization of 850 was used.

[0162] [Example 4]

[0163] The test was carried out in the same manner as in Example 2 except that polyvinyl alcohol having an average degree of polymerization of 1,700 was used.

[0164] [Example 5]

[0165] The test was conducted in the same manner as in Example 2 except that polyvinyl alcohol having an average degree of polymerization of 2400 was used and acetaldehyde was used instead of n-butyraldehyde (n-BA).

[0166] [Example 6]

[0167] The test was conducted in the same manner as in Example 2 except that polyvinyl alcohol having an average degree of polymerization of 850, a degree of saponification of 98 mol % and a degree of ethylene modification of 4 mol % was used.

[0168] [Example 7]

[0169] The test was conducted in the same manner as in Example 2 except that polyvinyl alcohol having an average degree of polymerization of 250, a degree of saponification of 99 mol % and containing 2 mol % of the structural unit having an amino group represented by the formula (3) was used.

[0170] [Example 8]

[0171] The test was conducted in the same manner as in Example 2 except that polyvinyl alcohol having an average degree of polymerization of 1600, a degree of saponification of 99 mol % and containing 2 mol % of the structural unit having an amino group represented by the formula (3) was used.

[0172] [Example 9]

[0173] 48 parts by weight of methyl methacrylate, 45 parts by weight of butyl methacrylate and 7 parts by weight of methoxymethyl acrylate were dissolved in 300 parts by weight of tetrahydrofuran to obtain an acrylic monomer solution. 2 parts by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained acrylic monomer solution and applied on a PET film. The coated article was subjected to a UV conveyor device "ECS301G1" manufactured by EYE GRAPHICS at 25°C with a cumulative light intensity of 2000 mJ / cm 2 The acrylic resin solution was obtained by irradiating light with a wavelength of 365 nm. The obtained acrylic resin solution was vacuum dried at 80° C. for 3 hours to obtain an acrylic resin. The obtained acrylic resin was adjusted to a 3 wt % butanol solution and tested in the same manner as in Example 1. The weight average molecular weight of the obtained resin was about 70,000.

[0174] [Comparative Example 1]

[0175] The test was carried out in the same manner as in Example 1 using only a polystyrene culture dish without using the scaffold material resin.

[0176] [Comparative Example 2]

[0177] The test was conducted in the same manner as in Example 9 except that 100 parts by weight of N-isopropylacrylamide was used as the acrylic monomer. The weight average molecular weight of the obtained resin was about 100,000.

[0178] [Comparative Example 3]

[0179] The test was conducted in the same manner as in Example 9 except that 100 parts by weight of methyl methacrylate was used as the acrylic monomer. The weight average molecular weight of the obtained resin was about 90,000.

[0180] [Comparative Example 4]

[0181] The test was conducted in the same manner as in Example 9 except that 100 parts by weight of butyl acrylate was used as the acrylic monomer. The weight average molecular weight of the obtained resin was about 120,000.

[0182] [Comparative Example 5]

[0183] The test was carried out in the same manner as in Example 9 except that 100 parts by weight of both-terminal Silaplane FM-7711 (manufactured by JNC Corporation) was used as the acrylic monomer. The weight average molecular weight of the obtained resin could not be measured.

[0184] The obtained results are summarized in Table 1. In addition, in any of the Examples and Comparative Examples, differentiated cells were not observed.

[0185]

Claims

1. A resin film formed of a cell culture scaffold material, which is a resin film formed of a cell culture scaffold material containing a synthetic resin, wherein: The synthetic resin comprises polyvinyl acetal resin, The storage modulus of the cell culture scaffold material at 100°C is 3.0×10 5 Pa or more and 5.0×10 6 Below Pa, The storage modulus of the cell culture scaffold material at 25°C is 5.0×10 8 Pa or more and 5.0×10 9 Below Pa, The polyvinyl acetal resin includes a structural unit having an amino group or an amide structure.

2. The resin film according to claim 1, wherein The synthetic resin is polyvinyl acetal resin.

3. The resin film according to claim 1 or 2, wherein The polyvinyl acetal resin contains 0.1 mol % to 20 mol % of a structural unit having an amino group or an amide structure.

4. The resin film according to claim 1 or 2, wherein The degree of polymerization of the polyvinyl acetal resin is 100 or more and 6000 or less.

5. The resin film according to claim 1 or 2, wherein The cells are stem cells. The resin membrane according to claim 1 or 2, which is used for culture in a serum-free medium. 7 . A cell culture container comprising the resin film according to claim 1 .

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