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. Among them, the dispersive component γ<supgt;d< / supgt> of the surface free energy of the scaffold material for stem cell culture is 24.5 or more and less than 45.0, and the dipole component γ<supgt>p< / supgt> of its surface free energy is 1 or more and less than 20.0. According to this scaffold material for stem cell culture, it has moderate hydrophilicity and strength, and has a high fixation after inoculation of stem cells, and cell proliferation can be carried out efficiently.

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. In addition, since it is a soft scaffold material, there is a problem that differentiation into cardiomyocytes is promoted.

[0012] 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.

[0013] 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, and capable of efficient cell proliferation, and a stem cell culture method using the scaffold material.

[0014] Technical means to solve technical problems

[0015] The present invention relates to the following contents.

[0016] (1) A scaffold material for stem cell culture, wherein the dispersion component γ of the surface free energy d is greater than 24.5 and less than 45.0, and the dipole component γ of its surface free energy p It is 1.0 or more and less than 20.0.

[0017] (2) The scaffold material for stem cell culture according to (1), wherein the scaffold material for stem cell culture contains a synthetic resin.

[0018] (3) The scaffold material for stem cell culture according to (2), wherein the synthetic resin contains at least one of a polyvinyl acetal skeleton and a poly(meth)acrylate skeleton.

[0019] (4) The scaffold material for stem cell culture according to (2), wherein the synthetic resin is a polyvinyl acetal resin.

[0020] (5) A scaffold material for stem cell culture, comprising a synthetic resin, wherein the synthetic resin comprises a polyvinyl acetal resin, and the degree of acetalization of the polyvinyl acetal resin is higher than 60 mol %.

[0021] (6) The scaffold material for stem cell culture according to (4) or (5), wherein the polyvinyl acetal resin contains at least one structural unit selected from a structural unit having an amine structure, a structural unit having an imine structure, and a structural unit having an amide structure.

[0022] (7) The scaffold material for stem cell culture according to (6), wherein the total content of the structural unit having an amine structure, the structural unit having an imine structure, and the structural unit having an amide structure in the polyvinyl acetal resin is 0.1 mol% or more and 20 mol% or less.

[0023] (8) The scaffold material for stem cell culture according to any one of (1) to (7), wherein the stem cells are pluripotent stem cells.

[0024] (9) A stem cell culture container comprising the stem cell culture scaffold material according to any one of (1) to (8) in at least a part of a cell culture region.

[0025] (10) A fiber for stem cell culture, comprising the scaffold material for stem cell culture according to any one of (1) to (8).

[0026] (11) A method for culturing stem cells, comprising using the scaffold material according to any one of (1) to (8).

[0027] (12) The method for culturing stem cells according to (11), comprising the step of seeding the cell mass on a scaffold material.

[0028] Effects of the Invention

[0029] 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

[0030] Figure 1 The main synthetic resin is γ p Relative to γ d A diagram summarizing the relationships.

[0031] Figure 2 yes Figure 1 A partial enlarged view of .

[0032] Figure 3 yes Figure 1A partial enlarged view of .

[0033] Figure 4 This is a graph showing the evaluation criteria for initial adhesion 24 hours after cell inoculation.

[0034] Figure 5 This is a phase contrast microscope photograph of the cells in the stem cell culture scaffold material of the example 24 hours after seeding.

[0035] Figure 6 This is a phase contrast microscope photograph taken 24 hours after the cells were seeded in the stem cell culture scaffold material of the comparative example.

[0036] Figure 7 This is a graph showing the evaluation criteria for cell proliferation 5 days after cell inoculation.

[0037] Figure 8 This is a phase contrast microscope photograph of the stem cell culture scaffold material of the example 5 days after the cells were seeded.

[0038] Fig. 9 This is a phase contrast microscope photograph taken 5 days after the cells were seeded in the stem cell culture scaffold material of the comparative example. DETAILED DESCRIPTION

[0039] Hereinafter, the present invention will be described with reference to embodiments, but the present invention is not limited to the following embodiments.

[0040] In addition, the terms used in this specification will be described.

[0041] "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."

[0042] 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 (multilineage differentiating stress enduring cells), embryonic cancer cells (embryonic germ cells), embryonic germ stem cells (embryonic germ cells), and m GS cells (multipotent germ stem cells).

[0043] 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."

[0044] As tissue stem cells and tissue progenitor cells, for example, 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 epithelial cells, skeletal muscle stem cells, adipose stem cells, renal progenitor cells, sperm stem cells, etc. As the stem cell, for example, the stem cells described in "More Fully Understand! Stem Cells and Regenerative Medicine" (Yangtu Company, Kenji Nagafune) can be cited.

[0045] [Scaffold materials for stem cell culture 1]

[0046] The inventors of the present invention have found that the above technical problems can be solved by controlling the surface free energy of the scaffold material for stem cell culture, thereby completing the present invention. That is, the first embodiment of the present invention relates to a dispersion component γ of the surface free energy. d and the dipole component γ p Scaffold materials for stem cell culture within a given range.

[0047] It should be noted that the dispersion component γ of the surface free energy in this specification is d and the dipole component γ p The Kaelble-Uy theoretical formula can be used for determination.

[0048] Here, the Kaelble-Uy theory is shown in equation (1), based on the total surface free energy γ being derived from the dispersion component γ d With the dipole component γ p The assumption consisting of the sum of .

[0049] γ=γ d +γ p (1)

[0050] In addition, if γ l represents the surface free energy of the liquid surface, expressed as γ s If θ represents the surface free energy of the solid and θ represents the contact angle, then the following equation (2) holds true.

[0051]

[0052] Therefore, using γ lTwo liquids of known composition (pure water and diiodomethane in the present invention) are used to measure the contact angle θ of the two liquids with respect to the scaffold material for stem cell culture, and the contact angle γ s d , γ s p The relevant simultaneous equations are solved to obtain the dispersion component γ of the stem cell culture scaffold material. d , dipole component γ p .

[0053] The contact angle of pure water can be obtained by dropping 1 μL of pure water on the scaffold material and taking an image of the droplet 30 seconds later using a contact angle meter (manufactured by Kyowa Interface Chemical Co., Ltd., DMo-701). In addition, the contact angle of diiodomethane can be obtained by dropping 1 μL of diiodomethane on the scaffold material and taking an image of the droplet 30 seconds later in the same manner.

[0054] The dispersion component γ of the surface free energy can be optimally adjusted d and the dipole component γ p From the viewpoint of the invention, the stem cell culture scaffold material preferably contains a synthetic resin. In addition, the dispersion component γ of the surface free energy can be preferably adjusted. d and the dipole component γ p From the viewpoint of the present invention, the synthetic resin preferably contains at least one of a polyvinyl acetal skeleton and a poly(meth)acrylate skeleton.

[0055] Figure 1 The dipole component γ of the surface free energy of the main synthetic resin p Relative to the dispersion component γ d A diagram summarizing the relationships. Figure 2 , Figure 3 They are Figure 1 A partial enlarged view of .

[0056] The dispersion component γ of the surface free energy of the scaffold material for stem cell culture of the present invention d is greater than 24.5 and less than 45.0. d It is more preferably 28.0 or more and 38.0 or less, and still more preferably 32.8 or more and 36.0 or less.

[0057] The dipole component γ of the surface free energy of the scaffold material for stem cell culture of the present invention p is greater than 1.0 and less than 20.0. p It is more preferably 1.0 or more and 10.0 or less, and still more preferably 2.5 or more and 5.0 or less.

[0058] The dispersion component γ dand the dipole component γ p , for example, it can be controlled by appropriately changing the skeleton of the synthetic resin described below.

[0059] The dispersion component γ d , for example, it can be increased by increasing the amount of non-polar functional groups in the synthetic resin skeleton or introducing functional groups having a cyclic structure, and can be reduced by reducing the amount of butyl components in the synthetic resin. The synthetic resin preferably contains at least one of a polyvinyl acetal skeleton and a poly(meth)acrylate skeleton.

[0060] Dipolar component γ p , for example, it can be increased by increasing the amount of polar functional groups in the synthetic resin skeleton or the functional groups containing ether structures, and can be decreased by increasing the amount of butyl groups as non-polar functional groups.

[0061] [Synthetic resin]

[0062] 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.

[0063] Examples of the polymer include polymers composed of one or more polymerizable monomers such as (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 organic silicon compounds.

[0064] Specific examples of the polymer include polyolefins, polyethers, polyvinyl alcohols, polyvinyl acetals, polyesters, poly(meth)acrylates, epoxy resins, polyamides, polyimides, polyurethanes, polycarbonates, celluloses, 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.

[0065] It should be noted that these polymers can only use one, or two or more can be used in combination. In the case of combining two or more polymers, two or more polymers can be mixed and used, or the form of a polymer formed by chemically bonding the skeleton of two or more polymers can be used. As a synthetic resin, in the case of combining two or more polymers, preferably poly (methyl) acrylate and polyvinyl acetal are combined.

[0066] The term "(meth)acrylic acid" as used herein refers to at least one selected from the group consisting of (meth)acrylate and (meth)acrylic acid. In addition, poly(meth)acrylic acid is a polymer obtained by polymerizing (meth)acrylate or (meth)acrylic acid as a monomer thereof, and also includes a polymer obtained by copolymerizing a monomer other than (meth)acrylate or (meth)acrylic acid.

[0067] The (meth)acrylate is not particularly limited, and examples thereof include alkyl (meth)acrylates, cyclic alkyl (meth)acrylates, aryl (meth)acrylates, (meth)acrylamides, polyethylene glycol (meth)acrylates, phosphorylcholine (meth)acrylates, and the like.

[0068] 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.

[0069] It should be noted that these alkyl (meth)acrylates are not particularly limited and may be substituted with various substituents such as an alkoxy group having 1 to 3 carbon atoms and a tetrahydrofurfuryl group. Examples thereof include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0070] Examples of the cyclic alkyl (meth)acrylate include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0071] As said aryl (meth)acrylate, phenyl (meth)acrylate, benzyl (meth)acrylate, etc. are mentioned, for example.

[0072] 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)acrylamide caproic acid.

[0073] 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.

[0074] Examples of the (meth)acrylphosphocholine include 2-(meth)acryloyloxyethylphosphocholine.

[0075] The monomer other than the (meth)acrylic acid ester is not particularly limited, and examples thereof include (meth)acrylic acid, ethylene, and vinyl ester.

[0076] 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.

[0077] In addition, from the viewpoint of improving the fixation of stem cells, it is more preferable to combine the first aspect of the present invention with the following second aspect.

[0078] [Scaffold materials for stem cell culture 2]

[0079] The present inventors have conducted intensive studies and have found that the above-mentioned technical problems can be solved by using a synthetic resin containing a polyvinyl acetal resin, thereby completing the present invention.

[0080] A second aspect of the present invention relates to a stem cell culture scaffold material comprising a synthetic resin, wherein the synthetic resin comprises a polyvinyl acetal resin, and the degree of acetalization of the polyvinyl acetal resin is higher than 60 mol%. It should be noted that the stem cell culture scaffold material of the present invention includes an aspect consisting only of a synthetic resin.

[0081] 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.

[0082] It has not been disclosed that, when using a synthetic resin as a scaffold material for stem cell culture, the acetalization degree of the synthetic resin is set to be higher than 60 mol%. The reason is that the proportion of hydroxyl groups decreases with the increase of the acetalization degree, resulting in a decrease in the hydrophilicity of the resin, and therefore there is a risk of reduced fixation after the scaffold material for stem cell culture is inoculated with stem cells or reduced penetration of polysaccharides required for cell culture. However, the inventors and others learned that strength is more important than hydrophilicity, and found that by setting the acetalization degree to be higher than 60 mol% to improve the strength of the scaffold material for stem cell culture, the fixation of the stem cells after inoculation is improved, thereby completing the present invention. Below, the polyvinyl alcohol acetal resin is described in more detail.

[0083] (Polyvinyl acetal resin)

[0084] 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.

[0085] The preferred lower limit of the acetalization degree of the polyvinyl acetal resin is 60 mol%, and the preferred upper limit is 90 mol%. If the acetalization degree is 60 mol% or more, the fixation of stem cells is excellent, and cell proliferation can be carried out efficiently. In addition, if the acetalization degree is 90 mol% or less, it can be used as a material with good solubility in a solvent. A more preferred lower limit is 65 mol%, and a more preferred upper limit is 85 mol%.

[0086] The acetal degree of the polyvinyl acetal resin can be 1 The measurement was carried out by H-NMR measurement.

[0087] 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.

[0088] 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.

[0089] The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, and more preferably butyraldehyde.

[0090] The lower limit of the degree of polymerization of the polyvinyl acetal resin is preferably 100, more preferably 200, further preferably 500, and further preferably 1500. If the degree of polymerization is within the range, even if swelling occurs in the culture medium for cell culture, the scaffold material strength can be appropriately maintained, and therefore, cell proliferation is 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 range, the operability is good, and the scaffold material can be appropriately formed.

[0091] The polyvinyl acetal may also be a copolymer formed with a vinyl compound. Examples of the vinyl compound include ethylene, allylamine, vinyl pyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, (meth)acrylate, etc. It should be noted that, as the (meth)acrylate, for example, the (meth)acrylate may be used.

[0092] The polyvinyl acetal resin may be a graft copolymer with a vinyl compound. Examples of the vinyl compound include the compounds listed above.

[0093] The graft copolymer includes a graft copolymer having "units composed of polyvinyl acetal" and "units composed of a vinyl compound" (hereinafter also referred to as "graft copolymer"). The vinyl compound refers to a compound containing a structural unit having a vinyl group (H2C=CH-).

[0094] In the present invention, "units composed of polyvinyl acetal" and "units composed of a vinyl compound" refer to "polyvinyl acetal" and "units composed of a vinyl compound" present in the graft copolymer. In addition, a graft copolymer having units including units composed of polyvinyl acetal and units composed of a vinyl compound refers to a branched copolymer in which "units composed of polyvinyl acetal" or "units composed of a vinyl compound" constituting a side chain different from the main chain are bonded to "units composed of polyvinyl acetal" or "units composed of a vinyl compound" constituting a main chain.

[0095] The molecular weight of the graft copolymer is not particularly limited, but preferably the number average molecular weight (Mn) is 10,000 to 600,000, the weight average molecular weight (Mw) is 20,000 to 1,200,000, and the ratio (Mw / Mn) thereof is 2.0 to 40. When Mn, Mw, and Mw / Mn are within this range, the strength of the stem cell scaffold material can be appropriately maintained.

[0096] The degree of acetalization in the graft copolymer can be measured, for example, by dissolving a xylene-soluble component of the graft copolymer in deuterated dimethyl sulfoxide and evaporating the mixture. 1 The degree of acetalization was measured by HN MR measurement.

[0097] The polyvinyl acetal resin preferably has a Bronsted basic group or a Bronsted acidic group in a part thereof. That is, preferably a part of the polyvinyl acetal resin is modified by a Bronsted basic group or a Bronsted acidic group, and more preferably a part of the polyvinyl acetal resin is modified by a Bronsted basic group. When a part of the polyvinyl acetal resin is modified by 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.

[0098] In addition, in this specification, the polyvinyl acetal resin which has a Bronsted basic group or a Bronsted acidic group in a part of a polyvinyl acetal resin is called a modified polyvinyl acetal resin.

[0099] The Brønsted basic group is a group that can accept hydrogen ions H from other substances. + Examples of the Bronsted basic group include amine basic groups such as a substituent having an amine structure, a substituent having an imine structure, a substituent having an amide structure, and a substituent having an imide structure.

[0100] Therefore, as such a polyvinyl acetal resin, it is preferred that the polyvinyl acetal resin contains at least one selected from the group consisting of a structural unit having an amine structure, a structural unit having an imine structure, a structural unit having an amide structure, and a structural unit having an imide structure. The total content of the structural unit having an amine structure, the structural unit having an imine structure, the structural unit having an amide structure, and the structural unit having an imide structure in the polyvinyl acetal resin is preferably 0.1 mol% to 30 mol%, and more preferably 1 mol% to 10 mol% from the viewpoint of cell adhesion immediately after inoculation.

[0101] In the present invention, the imine structure refers to a structure having a C=N bond. The polyvinyl acetal resin preferably has an imine structure in a side chain. In addition, the imine structure may be directly bonded to the carbon constituting the main chain of the polyvinyl acetal resin, or may be bonded via a linking group such as an alkylene group. It should be noted that having the imine structure in the side chain includes having the imine structure on a graft chain of the 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.

[0102] [Chemical formula 1]

[0103]

[0104] In formula (1), R 1 represents a single bond or an alkylene group, R 2 It represents a group having an imine structure.

[0105] 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.

[0106] 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.

[0107] As the R 2 , and the functional group may be a functional group represented by the following formula (2).

[0108] [Chemical formula 2]

[0109]

[0110] In formula (2), R 3 represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, R 4 It represents a hydrocarbon group having 1 to 18 carbon atoms.

[0111] 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 any one of saturated hydrocarbon groups, unsaturated hydrocarbon groups, and aromatic hydrocarbon groups, or a hydrocarbon group using two or more hydrocarbon groups.

[0112] 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.

[0113] Examples of the aromatic hydrocarbon group include a phenyl group, a tolyl group, a xylyl group, a tert-butylphenyl group, and a benzyl group.

[0114] 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, ethyl or propyl.

[0115] In the 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, the viscosity stability over time becomes good. 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%.

[0116] It should be noted that the content of the structural unit having the imine structure can be 1 The measurement was carried out by H-NMR measurement.

[0117] In the 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 bonding can be improved.

[0118] The polyvinyl acetal resin preferably contains a structural unit having an imino group (=NH) structure.

[0119] The 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 polyvinyl acetal resin or may be bonded via a linking group such as an alkylene group.

[0120] The modified polyvinyl acetal resin preferably contains a structural unit having an amine structure or a structural unit having an amide structure.

[0121] The modified polyvinyl acetal resin preferably has the amine structure or amide structure in the side chain. In addition, the amine structure or amide 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. In addition, the amine structure may be a primary amine, a secondary amine, a tertiary amine, or a quaternary amine. Among these, primary amines are preferred from the perspective of improving the fixation of stem cells.

[0122] It should be noted that the “having the amine structure or amide structure in the side chain” means that the amine structure or amide structure is present in the graft chain of the modified polyvinyl acetal resin.

[0123] In particular, the amine structure 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 amine structure 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).

[0124] [Chemical formula 3]

[0125]

[0126] [Chemical formula 4]

[0127]

[0128] 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.

[0129] The preferred lower limit of the content of the structural unit having the amine structure or amide structure is 0.1 mol%, and the preferred upper limit is 20 mol%. If the content of the structural unit having the amine structure or amide structure is 0.1 mol% or more, the addition characteristics can be sufficient. If the content is 20 mol% or less, the solubility will not increase excessively, and it becomes easy to obtain the modified polyvinyl acetal resin powder by the 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 having the amine structure or amide structure can be adjusted by 1H-NMR measurement is used for measurement. In addition, the preferred lower limit of the total content of the structural unit having the amine structure or amide structure and the structural unit having the imine structure 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 %.

[0130] In the polyvinyl acetal resin, the ratio of the content of the structural unit having an imine structure to the structural unit having an amine structure or an amide structure (structural unit having an imine structure / structural unit having an amine or 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 cross-linking performance can be fully exerted from the perspective of improving the fixation of stem cells. The more preferred lower limit of the ratio is 5 / 95, and the more preferred upper limit is 90 / 10.

[0131] The Bronsted acidic group is a group that can convert hydrogen ions H + A general term for functional groups that are transferred to other substances.

[0132] 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. Among them, the Bronsted acidic group is preferably a carboxyl group.

[0133] The method for modifying the polyvinyl acetal resin by the Bronsted acidic group is not particularly limited, and the method may be: copolymerizing the polyvinyl alcohol with itaconic acid and (meth)acrylic acid; introducing a Bronsted acidic group into the side chain of the polyvinyl alcohol; or the like.

[0134] The acetalization degree of the 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 acetalization degree is 60 mol% or more, the fixation of stem cells is excellent, and cell proliferation can be carried out efficiently. In addition, if the acetalization degree is 90 mol% or less, the solubility in the solvent can be improved. The more preferred lower limit is 65 mol%, and the more preferred upper limit is 85 mol%. The acetalization degree of the polyvinyl acetal resin can be obtained by 1 The measurement was carried out by H-NMR measurement.

[0135] The amount of acetyl groups in the polyvinyl acetal resin is not particularly limited, but the preferred lower limit is 0.0001 mol % and the preferred upper limit is 5 mol %.

[0136] As a method for preparing the polyvinyl acetal resin, for example, the following method can be cited: polyvinyl alcohol obtained by saponifying polyvinyl acetate obtained by copolymerizing a monomer having the imine structure with vinyl acetate is acetalized using a conventionally known method. In addition, a method of introducing an imine structure by acetalizing a polyvinyl alcohol containing a structural unit having an amine group or an amide structure using a conventionally known method can also be used. The following method can also be used: a modified polyvinyl alcohol having an imine structure obtained by post-modifying a polyvinyl alcohol containing a structural unit having an amine group or an amide structure is acetalized using a conventionally known method. In addition, an imine structure can also be introduced by post-modifying an unmodified polyvinyl acetal resin. That is, the modified polyvinyl acetal resin can be an acetalized product of a polyvinyl alcohol containing a structural unit having an amine group or an amide structure. Among these, the following method is preferred: a modified polyvinyl acetal resin having an imine structure is obtained by acetalizing a polyvinyl alcohol containing a structural unit having an amine group or an amide structure. In particular, when this method is used, an imine structure can be obtained by adding an excess amount of the aldehyde or the acid catalyst used in the acetalization.

[0137] In the method of adding an excess amount of aldehyde, preferably 70 to 150 parts by weight of aldehyde is added relative to 100 parts by weight of polyvinyl alcohol containing a structural unit having an amino group or an amide structure. As aldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, isobutyraldehyde, n-valeraldehyde, and benzaldehyde are particularly preferred.

[0138] In the method of adding an acid catalyst in excess, it is preferred to add an acid catalyst in an amount of 0.5% by weight or more of the total amount. In addition, it is preferred to add 5.0 to 70.0 parts by weight of an acid catalyst relative to 100 parts by weight of polyvinyl alcohol containing a structural unit having an amine group or an amide structure. As the acid catalyst, hydrochloric acid, nitric acid, sulfuric acid, and p-toluenesulfonic acid are particularly preferred. It should be noted that when this method is used, as a method for confirming a structural unit having an amine group or an amide structure, a structural unit having an imine structure, for example, 1 H-NMR confirmation method, etc.

[0139] The acetalization can be carried out using a known method, preferably in an aqueous solvent, a mixed solvent of water and an organic solvent having compatibility with water, or an organic solvent. As the organic solvent having compatibility with water, for example, an alcohol organic solvent can be used. As the organic solvent, for example, alcohol organic solvents, aromatic organic solvents, aliphatic ester solvents, ketone solvents, low-grade paraffin solvents, ether solvents, amine solvents, etc. can be mentioned. As the alcohol organic solvent, for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol, etc. can be mentioned. As the aromatic organic solvent, for example, xylene, toluene, ethylbenzene, methyl benzoate, etc. can be mentioned.

[0140] Examples of the aliphatic ester solvent include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, ethyl propionate, methyl butyrate, ethyl butyrate, methyl acetoacetate, and ethyl acetoacetate.

[0141] Examples of the ketone solvent include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, methylcyclohexanone, benzophenone, acetophenone, etc. Examples of the low-grade paraffin solvent include hexane, pentane, octane, cyclohexane, decane, etc. Examples of the ether solvent include diethyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol diethyl ether, etc. Examples of the amide solvent include N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetanilide, etc.

[0142] Examples of the amine solvent include ammonia, trimethylamine, triethylamine, n-butylamine, di-n-butylamine, tri-n-butylamine, aniline, N-methylaniline, N,N-dimethylaniline, and pyridine.

[0143] These solvents may be used alone or in combination of two or more thereof. Among these, ethanol, n-propanol, isopropanol, and tetrahydrofuran are particularly preferred from the viewpoint of solubility in the resin and ease of purification.

[0144] The acetalization is preferably carried out in the presence of an acid catalyst. The acid catalyst is not particularly limited, and examples thereof include: inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, and phosphoric acid; carboxylic acids such as formic acid, acetic acid, and propionic acid; or sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. These acid catalysts may be used alone or in combination of two or more compounds. Among them, hydrochloric acid, nitric acid, and sulfuric acid are preferred, and hydrochloric acid is particularly preferred.

[0145] By using the stem cell scaffold material 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.

[0146] [Methods for culturing stem cells]

[0147] 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.

[0148] 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.

[0149] Since the cell culture scaffold material has appropriate hydrophilicity and strength, it is preferably used in a two-dimensional culture method.

[0150] As a container for plane culture (two-dimensional culture method), the shape or size is not particularly limited, and examples include a cell culture test plate with one or more holes, a cell culture flask, etc. The number of holes in the microplate is not limited, and examples include: 2, 4, 6, 12, 24, 48, 96, 384, etc. The shape of the hole is not particularly limited, and examples include: a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, etc. The shape of the bottom surface of the hole is not particularly limited, and examples include: a flat bottom, a round bottom, a concave-convex, etc.

[0151] 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.

[0152] 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.

[0153] [Method for culturing pluripotent stem cells]

[0154] In the method for culturing pluripotent stem cells, it is preferred to seed the cell masses on a stem cell culture scaffold material containing a synthetic resin.

[0155] The cell mass can be obtained by adding a cell detachment agent to a culture vessel in which confluence is formed and uniformly disrupting the cells by pipetting. The cell detachment agent is not particularly limited, but is preferably an ethylenediamine / phosphate buffer solution. The size of the cell mass is preferably 50 to 200 μm.

[0156] [Other embodiments]

[0157] 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.

[0158] 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.

[0159] The present invention provides a stem cell culture container, which has a resin film in at least a part of the cell culture region, and uses the stem cell culture scaffold material as the resin film. As a container, there is no particular limitation as long as it is a container having a resin film in at least a part of the cell culture region, and various containers can be used. As a container, the planar culture container or bioreactor can be used.

[0160] 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.

[0161] 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.

[0162] The cross-linking agent is not particularly limited, and examples thereof include polyols, polycarboxylic acids, hydroxycarboxylic acids, metal soaps, and polysaccharides.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] Example

[0169] 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 content of structural units in the obtained synthetic resin and modified polyvinyl acetal resin, such as the content of structural units having an amine structure (mol%), the content of structural units having an imine structure (mol%), the content of structural units having an amide structure (mol%), the degree of acetalization (mol%), the amount of acetyl groups (mol%), the amount of hydroxyl groups (mol%), and the amount of (meth)acrylate groups (mol%), are determined by dissolving the synthetic resin in DMSO-d6 (dimethyl sulfoxide) and using 1 H-NMR (nuclear magnetic resonance spectroscopy) was used for measurement.

[0170] [Example 1]

[0171] (Preparation of polyvinyl butyral)

[0172] In a reactor equipped with a stirring device, 2700 mL of ion exchange water, 250 average polymerization degrees, and 300 g of polyvinyl alcohol having a saponification degree of 99 mol% were added, and heated and dissolved while stirring to obtain a solution. Next, 35 wt % hydrochloric acid as a catalyst was added to the solution in a manner such that the hydrochloric acid concentration became 0.2 wt %, and after adjusting the temperature to 15 ° C, 22 g of n-butyraldehyde (n-BA) was added while stirring. Thereafter, 148 g of n-butyraldehyde (n-BA) was added, and as a result, polyvinyl butyral in the form of white particles was precipitated. 15 minutes after precipitation, 35 wt % hydrochloric acid was added in a manner such that the hydrochloric acid concentration became 1.8 wt %, heated to 50 ° C, and aged for 2 hours at 50 ° C. Subsequently, the solution was cooled, neutralized, and the polyvinyl butyral was washed with water and dried to obtain polyvinyl butyral.

[0173] The obtained polyvinyl butyral had an average degree of polymerization of 250, a hydroxyl group content of 28 mol%, an acetyl group content of 1 mol%, and an acetalization degree of 71 mol%.

[0174] (Preparation of a cell culture vessel)

[0175] 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 (manufactured by Matsunami Co., Ltd., 22 pellets No. 1 were dusted by an air dust collector and used), and a smooth resin film was obtained by rotating at 2000 rpm for 20 seconds using a spin coater. The obtained resin film was placed in a φ22 mm polystyrene culture dish together with the cover glass to obtain a cell culture container.

[0176] (Surface free energy)

[0177] The surface free energy of the resin film was measured using a contact angle meter (manufactured by Kyowa Interface Chemical Co., Ltd., DMo-701). 1 μL of pure water was dripped onto the resin film, and an image of the droplet was taken 30 seconds later to obtain the contact angle of pure water. In addition, 1 μL of diiodomethane was dripped onto the resin film, and an image of the droplet was taken 30 seconds later to obtain the contact angle of diiodomethane. The surface free energy γ and the dispersion component γ were derived from the obtained contact angle using the Kaelble-Uy theory. d , dipole component γ p .

[0178] The cell culture container equipped with the resin membrane was tested under the following conditions.

[0179] (Cell culture test method)

[0180] 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 TeSR E8 (manufactured by STEM CELL) and 10 μM of ROCK-I 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-I inhibitor (Y27632) to 10 μM, thereby replacing the culture medium.

[0181] (Method of Cell Block Culture Test)

[0182] 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.

[0183] (Cultivation and evaluation methods)

[0184] (1) Initial adhesion

[0185] In the cell culture test, a phase contrast microscope with a magnification of 10×10 (manufactured by Olympus, IX73) was used to obtain cell images 24 hours 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. The obtained image was compared with Figure 4 The initial adhesion was evaluated by comparing the number of adhesive cells and the morphology of adhesive cells with samples 1 to 10. Figure 4 It is shown in Figure 1 that the number of cells increases as one moves from sample 1 to sample 8. In addition, it is shown that the pseudopodia of the cells become more elongated and the adhesion state becomes better as one moves from sample 8 to sample 10. The obtained results are summarized and shown in Figure 10. Figure 5 , 6 .

[0186] (2) Cell proliferation

[0187] 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 7 The cell proliferation was evaluated by comparing samples 1 to 10. Figure 7 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 obtained are summarized and displayed in Figure 8 , 9 .

[0188] (3) Adhesion maintenance

[0189] In the cell mass culture test, the time for which the cell mass can maintain adhesion was evaluated according to the following criteria.

[0190] 0: All cells detached within 30 minutes after medium change.

[0191] 1: After the medium is changed, adhesion is maintained for more than 30 minutes, but all cells are detached within less than 1 hour.

[0192] 2: After the culture medium is changed, adhesion is maintained for more than 1 hour, but all cells are detached within less than 24 hours.

[0193] 3: Maintain adhesion for more than 24 hours after changing the culture medium.

[0194] The obtained cell masses were confirmed to remain undifferentiated by alkaline phosphatase (ALP) staining test.

[0195] [Example 2]

[0196] The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 850 and a degree of saponification of 99 mol % was used.

[0197] [Example 3]

[0198] The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 1700 and a degree of saponification of 99 mol % was used.

[0199] [Example 4]

[0200] The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 2400 and a degree of saponification of 99 mol % was used and acetaldehyde was used instead of n-butyraldehyde (n-BA).

[0201] [Example 5]

[0202] The test was conducted in the same manner as in Example 1 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.

[0203] [Example 6]

[0204] The test was conducted in the same manner as in Example 1 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 amine group represented by the formula (3) was used.

[0205] [Example 7]

[0206] The test was conducted in the same manner as in Example 1 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 amine group represented by the formula (3) was used.

[0207] [Example 8]

[0208] 100 parts by weight of polyvinyl acetal with a polymerization degree of about 250 obtained in Example 1 and 1 part by weight of N-vinyl pyrrolidone were dissolved in 500 parts by weight of tetrahydrofuran to obtain a graft copolymer resin solution. 0.05 parts by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained resin 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 composite resin solution was obtained by irradiating light of a wavelength of 365 nm. The composite resin solution was vacuum dried at 80° C. for 3 hours to obtain a composite resin. The weight average molecular weight of the obtained resin was measured by GPC method using "2690 Separationsmodel" manufactured by Waters as a column, and the result was about 40,000 in terms of polystyrene. The obtained composite resin was adjusted to a 3 wt % butanol solution, and the test was carried out in the same manner as Example 1.

[0209] [Example 9]

[0210] The test was conducted in the same manner as in Example 8 except that 10 parts by weight of N-vinyl pyrrolidone was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 60,000.

[0211] [Example 10]

[0212] The test was conducted in the same manner as in Example 8 except that 30 parts by weight of N-vinyl pyrrolidone was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 50,000.

[0213] [Example 11]

[0214] The test was conducted in the same manner as in Example 8 except that 5 parts by weight of tetrahydrofurfuryl acrylate was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 60,000.

[0215] [Example 12]

[0216] The test was conducted in the same manner as in Example 8 except that 5 parts by weight of methoxyethyl acrylate was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 70,000.

[0217] [Example 13]

[0218] The test was conducted in the same manner as in Example 8 except that 5 parts by weight of butyl methacrylate was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 60,000.

[0219] [Example 14]

[0220] 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate 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 of 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 acrylic resin was about 100,000.

[0221] [Example 15]

[0222] An acrylic resin was obtained in the same manner as in Example 14 except that 90 parts by weight of methoxyethyl acrylate and 10 parts by weight of butyl methacrylate were used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. The obtained acrylic resin was adjusted to a 3% by weight butanol solution and tested in the same manner as in Example 1. The weight average molecular weight of the obtained acrylic resin was about 80,000.

[0223] [Example 16]

[0224] An acrylic resin was obtained in the same manner as in Example 14 except that 75 parts by weight of methoxyethyl acrylate and 25 parts by weight of butyl methacrylate were used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. The obtained acrylic resin was adjusted to a 3% by weight butanol solution and tested in the same manner as in Example 1. The weight average molecular weight of the obtained resin was about 90,000.

[0225] [Example 17]

[0226] An acrylic resin was obtained in the same manner as in Example 14 except that 2 parts by weight of butyl methacrylate and 98 parts by weight of ethyl acrylate were used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. The obtained acrylic resin was adjusted to a 3% by weight butanol solution and tested in the same manner as in Example 1. The weight average molecular weight of the obtained acrylic resin was about 80,000.

[0227] [Comparative Example 1]

[0228] The test was carried out in the same manner as in Example 1 using only a polystyrene culture dish without using a scaffold material.

[0229] [Comparative Example 2]

[0230] The test was conducted in the same manner as in Example 1, except that the amount of n-butyraldehyde (n-BA) added the second time was changed from 148 g to 89 g.

[0231] [Comparative Example 3]

[0232] The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 1000 and a degree of saponification of 98 mol % was used as the synthetic resin.

[0233] [Comparative Example 4]

[0234] 100 parts by weight of N-isopropylacrylamide, 75 parts by weight of ethyl acetate, and 0.5 parts by weight of azobisisobutyronitrile were mixed and polymerized at 65°C for 8 hours under a nitrogen atmosphere to obtain a polyacrylamide resin. The obtained resin was measured by GPC method using "2690Separations model" manufactured by Waters as a column, and the weight average molecular weight in terms of polystyrene was about 90,000 (polymerization degree of about 800). Other operations were tested in the same manner as in Example 1.

[0235] [Comparative Example 5]

[0236] The test was conducted in the same manner as in Comparative Example 4, except that 100 parts by weight of ethyl acrylate was used instead of 100 parts by weight of N-isopropylacrylamide.

[0237] [Comparative Example 6]

[0238] The test was conducted in the same manner as in Comparative Example 4 except that 100 parts by weight of butyl methacrylate was used instead of 100 parts by weight of N-isopropylacrylamide. The weight average molecular weight of the obtained resin was about 90,000.

[0239] [Comparative Example 7]

[0240] The test was conducted in the same manner as in Example 8 except that 70 parts by weight of N-vinyl pyrrolidone was added to 30 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 90,000.

[0241] The obtained results are summarized in Table 1 and Table 2. Figure 5 , Figure 6 The phase contrast microscopy image of cells 24 hours after inoculation is shown in the figure. Figure 8 , Fig. 9 5 shows a phase contrast microscopic photograph of cells 5 days after inoculation. It should be noted that no differentiated cells were observed in any of the Examples and Comparative Examples.

[0242]

[0243]

Claims

1. A resin film containing a cell culture scaffold material, which is a resin film containing a cell culture scaffold material containing a synthetic resin, wherein: The dispersion component γ of the surface free energy of the cell culture scaffold material d 31.3mJ / m 2 Above and 36.2mJ / m 2 the following, The dipole component γ of the surface free energy of the cell culture scaffold material p 2.2mJ / m 2 Above and 5.0mJ / m 2 the following, The synthetic resin comprises polyvinyl acetal resin, The polyvinyl acetal resin includes at least one selected from a structural unit having an amine structure, a structural unit having an imine structure, and a structural unit having an amide structure as a structural unit.

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

3. The resin film according to claim 1, wherein The acetalization degree of the polyvinyl acetal resin is higher than 60 mol%, The polyvinyl acetal resin is polyvinyl butyral.

4. The resin film according to any one of claims 1 to 3, wherein The dispersion component of the surface free energy γ d 32.8mJ / m 2 above.

5. The resin film according to any one of claims 1 to 3, wherein In the polyvinyl acetal resin, the total content of the structural unit having an amine structure, the structural unit having an imine structure, and the structural unit having an amide structure is 0.1 mol % or more and 30 mol % or less.

6. The resin film according to any one of claims 1 to 3, wherein The cells are stem cells. 7 . A cell culture container comprising the resin film according to claim 1 in at least a part of a cell culture region. 8 . A cell culture method using the resin film according to claim 1 . 9 . The cell culture method according to claim 8 , comprising the step of seeding the cell mass on the resin membrane.

Images