Resin film formed from scaffold material for cell culture, carrier for cell culture, and container for cell culture

Abstract

Provided is a resin film formed from a scaffold material for cell culture, which has excellent adhesion properties after cell seeding and can increase the cell proliferation rate. [Solution] A resin film formed from a scaffold material for cell culture, wherein the scaffold material for cell culture contains a synthetic resin having a polyvinyl acetal skeleton, the resin film has a phase-separated structure including at least a first phase and a second phase, and the ratio of the surface area of ​​one of the first phase and the second phase to the entire surface is 0.01 or more and 0.95 or less.

Description

[Technical Field]

[0001] The present invention relates to a resin film formed from a scaffold material for cell culture, and also to a cell culture carrier and a cell culture vessel comprising the resin film. [Background technology]

[0002] In recent years, cell therapy and next-generation medical treatments using stem cells have been attracting attention. In particular, human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), as well as differentiated cells derived from them, are expected to be applied to drug discovery and regenerative medicine. To achieve such applications, it is necessary to safely and reproducibly culture and proliferate pluripotent stem cells and differentiated cells.

[0003] In particular, in industrial applications of regenerative medicine, it is necessary to handle large quantities of stem cells, and therefore it is necessary to support the proliferation of pluripotent stem cells using natural polymeric materials, synthetic polymeric materials, or feeder cells. For this reason, various culture methods using scaffold materials such as natural polymeric materials and synthetic polymeric materials have been investigated.

[0004] For example, Patent Document 1 below discloses a carrier for cell culture that is made of a molded product made of a polyvinyl acetal compound or a molded product made of the polyvinyl acetal compound and a water-soluble polysaccharide, and the degree of acetalization of the polyvinyl acetal compound is 20 to 60 mol %.

[0005] Patent Document 2 below discloses a composition containing a first fibrous polymer scaffold, in which the fibers of the first fibrous polymer scaffold are aligned. It describes that the fibers constituting the first fibrous polymer scaffold are made of an aliphatic polyester such as polyglycolic acid or polylactic acid.

[0006] Furthermore, Patent Document 3 listed below discloses a cell culture method for maintaining the undifferentiated state of pluripotent stem cells, which includes a step of culturing the pluripotent stem cells on a culture vessel having a surface coated with a polyrotaxane block copolymer. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-314285 [Patent Document 2] Special Publication No. 2009-524507 [Patent Document 3] Japanese Patent Application Laid-Open No. 2017-23008 Summary of the Invention [Problem to be solved by the invention]

[0008] The use of natural polymeric scaffold materials can enhance cell adhesion after seeding. In particular, the use of adhesive proteins such as laminin and vitronectin, or Matrigel derived from mouse sarcoma, as natural polymeric scaffold materials, is known to significantly enhance cell adhesion after seeding. However, natural polymeric scaffold materials are expensive, have significant lot-to-lot variations due to their natural origin, and pose safety concerns due to animal-derived components.

[0009] In contrast, scaffold materials using synthetic resins are easier to handle, less expensive, have less lot-to-lot variation, and are safer than scaffold materials using natural polymer materials. However, in scaffold materials using synthetic resins such as those described in Patent Documents 1 to 3, the synthetic resins used are highly hydrophilic, which can lead to excessive swelling of the synthetic resins. Furthermore, synthetic resins have a lower affinity for cells than natural polymer materials, which can lead to detachment of cell masses during culture. As such, scaffold materials using synthetic resins can result in poor cell adhesion after seeding, leading to insufficient cell proliferation.

[0010] An object of the present invention is to provide a resin film, a cell culture carrier, and a cell culture vessel formed from a scaffold material for cell culture, which has excellent adhesion properties after cell seeding and can increase the cell proliferation rate. [Means for solving the problem]

[0011] The resin film formed by the scaffold material for cell culture according to the present invention has a phase-separated structure including at least a first phase and a second phase, and the ratio of the surface area of ​​one of the first and second phases to the entire surface area is 0.01 or more and 0.95 or less.

[0012] In a specific aspect of the resin film according to the present invention, the ratio of the perimeter to the area of ​​the second phase (perimeter / area) is 0.001 (1 / nm) or more and 0.40 (1 / nm) or less.

[0013] In another specific aspect of the resin film according to the present invention, the phase-separated structure is a sea-island structure, the first phase is a sea portion, and the second phase is an island portion.

[0014] In still another specific aspect of the resin film according to the present invention, the number of the second phases that are the island portions is 1 / μm 2 More than 5000 pieces / μm 2 The following is the result.

[0015] In yet another specific aspect of the resin film according to the present invention, the phase-separated structure is formed by a phase-separated structure within a molecule of the synthetic resin.

[0016] In yet another specific aspect of the resin film according to the present invention, the dispersion term component of the surface free energy is 25.0 mJ / m 2 More than 50.0mJ / m 2 or less, and the polar term component is 1.0 mJ / m 2 More than 20.0mJ / m 2 The following is the result.

[0017] In yet another specific aspect of the resin film according to the present invention, the synthetic resin has a cationic functional group, and the content of the cationic functional group contained in the structural unit of the synthetic resin is 0.2 mol % or more and 50 mol % or less.

[0018] In yet another specific aspect of the resin film according to the present invention, the second phase has a peptide portion.

[0019] In yet another particular aspect of the resin film according to the present invention, the peptide portion has a cell adhesive amino acid sequence.

[0020] In yet another specific aspect of the resin film according to the present invention, the water swelling ratio is 50% or less.

[0021] In still another specific aspect of the resin film according to the present invention, the storage modulus at 100°C is 1.0 × 10 4 Pa or more, 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 x 10 1 That's it, 1.0 x 10 5 The following is the result.

[0022] In yet another particular aspect of the resin film according to the present invention, the scaffold material for cell culture is substantially free of animal-derived raw materials.

[0023] In yet another specific aspect of the resin film according to the present invention, the synthetic resin includes a vinyl polymer.

[0024] In yet another specific aspect of the resin film according to the present invention, the synthetic resin contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic acid ester.

[0025] The carrier for cell culture according to the present invention comprises a carrier and a resin film configured according to the present invention, the resin film being disposed on the surface of the carrier.

[0026] The cell culture vessel according to the present invention comprises a vessel body and a resin film configured according to the present invention, with the resin film being disposed on the surface of the vessel body. [Effects of the Invention]

[0027] According to the present invention, it is possible to provide a resin film formed from a scaffold material for cell culture, which has excellent adhesion properties after cell seeding and can increase the proliferation rate of cells, a carrier for cell culture, and a container for cell culture. [Brief explanation of the drawings]

[0028] [Figure 1] 1 is a schematic front cross-sectional view showing a cell culture vessel according to one embodiment of the present invention. [Figure 2] 1 is an atomic force microscope photograph of the resin film obtained in Example 3. [Figure 3] 1 is an atomic force microscope photograph of the resin film obtained in Example 14. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be clarified below by describing specific embodiments of the present invention with reference to the drawings.

[0030] The present invention relates to a resin film formed from a scaffold material for cell culture. The scaffold material for cell culture includes a synthetic resin. The resin film of the present invention has a phase-separated structure including at least a first phase and a second phase. In the resin film of the present invention, the ratio of the surface area of ​​one of the first phase and the second phase to the entire surface area is 0.01 or more and 0.95 or less.

[0031] The resin film of the present invention has the above-mentioned configuration, and therefore has excellent adhesion properties after cell seeding, and can increase the cell proliferation rate.

[0032] Conventional scaffold materials for cell culture using natural polymer materials can enhance cell adhesion after seeding, but they are expensive, have large lot-to-lot variations due to the natural origin of the materials, and have safety concerns due to animal-derived components. On the other hand, conventional scaffold materials using synthetic resins can cause cell mass detachment during culture due to excessive swelling of the synthetic resin or low affinity for cells. Therefore, conventional scaffold materials using synthetic resins can result in poor cell adhesion after seeding, and cells may not proliferate sufficiently.

[0033] The present inventors focused on the phase-separated structure of a resin film formed from a cell culture scaffold material and discovered that by creating a phase-separated structure in which the ratio of the surface area of ​​one of the first and second phases to the entire surface falls within the above-mentioned specific range, affinity with cells can be increased, thereby improving adhesion after seeding and ultimately increasing the cell proliferation rate. Although the reason for this is unclear, it is thought that such a phase-separated structure allows for smooth energy distribution and allows for adjustment of the locations and proportions of the first and second phases, which have different affinities and strengths, thereby increasing affinity regardless of cell type and achieving the accumulation and adsorption effects of cells or cell surface proteins.

[0034] Therefore, the resin film of the present invention can enhance adhesiveness to cells after seeding, thereby increasing the cell proliferation rate.

[0035] Furthermore, in the present invention, since synthetic resins can be used as described above, compared to scaffolding materials using natural polymer materials, they are easy to handle, inexpensive, have little variation between lots, and are safe.

[0036] In the present invention, a synthetic resin having a peptide moiety may be used as the synthetic resin. Details of the synthetic resin having a peptide moiety will be described later.

[0037] In the present invention, the ratio of the surface area of ​​one of the first and second phases to the entire surface (surface area ratio) is 0.01 or more, preferably 0.10 or more, and 0.95 or less, more preferably 0.90 or less. When the surface area ratio is within the above range, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0038] In the present invention, examples of the phase-separated structure include microphase-separated structures such as a sea-island structure, a cylindrical structure, a gyroid structure, or a lamellar structure. In the sea-island structure, for example, the first phase can be the sea portion, and the second phase can be the island portion. In the cylindrical structure, gyroid structure, or lamellar structure, for example, the phase with the largest surface area can be the first phase, and the phase with the second largest surface area can be the second phase. Among these, the sea-island structure is preferred as the phase-separated structure. In this way, having a continuous phase and a discontinuous phase can increase affinity with cells and further increase adhesiveness with cells after seeding, thereby further increasing the cell proliferation rate.

[0039] When the phase-separated structure is an island-sea structure, the surface area ratio of the second phase to the entire surface is 0.01 or more, preferably 0.1 or more, more preferably 0.2 or more, and 0.95 or less, preferably 0.9 or less, more preferably 0.8 or less. When the surface area ratio is within the above range, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0040] The ratio of the perimeter to the area of ​​the second phase (perimeter / area) is preferably 0.001 (1 / nm) or more, more preferably 0.0015 (1 / nm) or more, and even more preferably 0.008 (1 / nm) or more. The ratio of the perimeter to the area of ​​the second phase (perimeter / area) is preferably 0.40 (1 / nm) or less, more preferably 0.20 (1 / nm) or less, even more preferably 0.08 (1 / nm) or less, and particularly preferably 0.013 (1 / nm) or less. When the ratio (perimeter / area) is within the above range, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0041] When the synthetic resin does not have a peptide portion, the ratio of the perimeter to the area of ​​the second phase (perimeter / area) is preferably 0.001 (1 / nm) or more, more preferably 0.0015 (1 / nm) or more, and preferably 0.08 (1 / nm) or less, more preferably 0.013 (1 / nm) or less. When the ratio (perimeter / area) is within the above range, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0042] When the synthetic resin has a peptide portion, the ratio of the perimeter to the area of ​​the second phase (perimeter / area) is preferably 0.008 (1 / nm) or more, more preferably 0.013 (1 / nm) or more, preferably 0.40 (1 / nm) or less, more preferably 0.20 (1 / nm) or less, and even more preferably 0.10 (1 / nm) or less. When the ratio (perimeter / area) is within the above range, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0043] The number of the second phases, which are islands, is preferably 1 / μm 2 More preferably, 2 particles / μm 2 More preferably, 10 particles / μm 2 More than 5000 particles / μm 2 Less than or equal to 1000 particles / μm, more preferably 1000 particles / μm 2 or less, more preferably 500 or less / μm 2 , and particularly preferably 300 pieces / μm2 In this case, the adhesiveness of the cells after seeding can be further increased, and the cell proliferation rate can be further increased.

[0044] The average diameter of the second phase, which is an island portion, is preferably 20 nm or more, more preferably 30 nm or more, even more preferably 50 nm or more, particularly preferably 80 nm or more, and preferably 3.5 μm or less, more preferably 3.0 μm or less, and even more preferably 1.5 μm or less. When the average diameter of the second phase is within the above range, adhesiveness to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0045] When the synthetic resin does not have a peptide portion, the average diameter of the second phase, which is the island portion, is preferably 50 nm or more, more preferably 100 nm or more, even more preferably 120 nm or more, particularly preferably 200 nm or more, and preferably 1 μm or less, more preferably 300 nm or less, and even more preferably 250 nm or less. When the average diameter of the second phase is within the above range, adhesiveness to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0046] When the synthetic resin has a peptide portion, the average diameter of the second phase, which is the island portion, is preferably 10 nm or more, more preferably 20 nm or more, even more preferably 40 nm or more, and preferably 1 μm or less, more preferably 300 nm or less, and even more preferably 100 nm or less. When the average diameter of the second phase is within the above range, adhesiveness to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0047] The presence or absence of a phase-separated structure and parameters indicating the above-described phase-separated structure can be confirmed using, for example, an atomic force microscope (AFM), a transmission electron microscope (TEM), a scanning electron microscope (SEM), or the like.

[0048] Specifically, the ratio of the surface area of ​​one of the first and second phases to the entire surface (surface area fraction), the ratio of the perimeter to the area of ​​the second phase (perimeter / area), the number of the second phase islands, and their average diameter size can be determined from the above-mentioned microscope observation image using image analysis software such as ImageJ.

[0049] The ratio of the surface area of ​​one of the first and second phases to the entire surface (surface area ratio) can be determined by dividing the surface area occupied by one of the first and second phases within an observation area (30 μm × 30 μm) by the area of ​​the observation area.

[0050] The ratio of the perimeter to the area of ​​the second phase (perimeter / area) is determined by dividing the total perimeter of the second phase by the total area of ​​the second phase within the observation area (30 μm × 30 μm).

[0051] When the phase separation structure is an island-sea structure, the number of the second phase islands is calculated by dividing the number of the second phase islands in an observation area (30 μm × 30 μm) by the area of ​​the observation area. The average diameter of the second phase islands is calculated as the average diameter of a circle with the same area.

[0052] Furthermore, the above-described phase-separated structure can be obtained by, for example, blending, copolymerizing, or graft-copolymerizing at least two different polymers, or by using a synthetic resin having a peptide moiety, thereby forming an inter- or intramolecular phase-separated structure in the synthetic resin. Among these, from the viewpoint of further enhancing cell adhesiveness, it is preferable that the above-described phase-separated structure is formed by an intramolecular phase-separated structure. That is, the above-described synthetic resin is preferably a copolymer of at least two different polymers or a synthetic resin having a peptide moiety, and more preferably a graft copolymer or a synthetic resin having a peptide moiety.

[0053] The phase-separated structure as described above is preferably obtained by copolymerizing two or more polymers (monomers) that differ in solubility parameter (SP value) of 0.1 or more, preferably 0.5 or more, and more preferably 1 or more. In this case, the sea-island structure can be formed more easily.

[0054] The SP value is a measure of the intermolecular force acting between a solvent and a solute, and is a measure of the affinity between substances. The SP value can be calculated based on the theory of regular solutions by Hidebrand. The SP value can be obtained from literature information, or by the calculation methods of Hansen and Hoy, the estimation method of Fedors, etc. In this specification, the Fedors formula δ 2 =ΣE / ΣV (δ is the SP value, E is the evaporation energy, and V is the molar volume). The unit of the SP value is (cal / cm 3 ) 0.5 The Fedors method is described in the Journal of the Japan Adhesion Association, Vol. 22, p. 566, 1986.

[0055] Phase separation parameters that indicate the phase separation structure, such as surface area fraction, can be adjusted, for example, by controlling the blending ratio of the two types of polymers, the polymer structure, or the content of the peptide portion.

[0056] In the present invention, other phases different from the first and second phases may be present. The other phases may be one phase or multiple phases. Such phases can be obtained, for example, by copolymerizing, for example, by grafting, still another polymer (monomer) having a different SP value. In this case, the two phases occupying a large area on the surface of the resin film are referred to as the first phase and the second phase.

[0057] When the synthetic resin does not have a peptide portion, the dispersion term component of the surface free energy in the resin film formed by the scaffold material for cell culture is preferably 25.0 mJ / m 2 More than 50.0mJ / m 2In this case, the hydrophilicity of the cell culture scaffold material can be appropriately adjusted, and the synergistic effect with the phase-separated structure can further increase the interfacial adhesion with cells after seeding, thereby further increasing the cell proliferation rate. The dispersion term component is more preferably 30.0 mJ / m or less. 2 More preferably, 35.0 mJ / m 2 More preferably, 47.0 mJ / m 2 or less, more preferably 45.5 mJ / m 2 The following is the result.

[0058] When the synthetic resin does not have a peptide portion, the polar component of the surface free energy of the resin film formed by the scaffold material for cell culture is preferably 1.0 mJ / m 2 More than 20.0mJ / m 2 In this case, the adhesiveness to the cells after seeding can be further increased, and the cell proliferation rate can be further increased. The polar term component is more preferably 2.0 mJ / m or less. 2 More preferably, 3.0 mJ / m 2 More preferably, 10.0 mJ / m 2 or less, more preferably 5.0 mJ / m 2 The following is the result.

[0059] The dispersion term component of the surface free energy γ d and the polar term component, the dipole component γ p is calculated using the Kaelble-Uy theoretical formula. The Kaelble-Uy theoretical formula is expressed as follows: the total surface free energy γ is calculated by dividing the dispersion term component γ d and the dipole component γ p This is a theoretical formula based on the assumption that it is the sum of

[0060]

number

[0061] In addition, in the Kaelble-Uy theoretical formula, the surface free energy of the liquid is expressed as γ l (mJ / m2 ) and the surface free energy of the solid is γ s (mJ / m 2 ) and the contact angle is θ (°), the following formula (2) is established.

[0062]

number

[0063] Therefore, the surface free energy of the liquid γ l Using two types of liquids with known contact angles, the contact angle θ of each liquid with respect to the resin film formed by the scaffold material for cell culture was measured, and γ s d and γ s p By solving the simultaneous equations, the dispersion term component γ of the surface free energy of the resin film formed by the scaffold material for cell culture was obtained. d and the dipole component γ p can be obtained.

[0064] In this specification, the surface free energy γ l Two such liquids for which the properties are known are pure water and diiodomethane.

[0065] The contact angle θ is measured using a contact angle meter (for example, "DMo-701" manufactured by Kyowa Interface Science Co., Ltd.) as follows.

[0066] 1 μL of pure water or diiodomethane is dropped onto the surface of a resin film formed from a scaffold material for cell culture. The angle between the pure water and the resin film 30 seconds after the drop is defined as the contact angle θ with respect to pure water. Similarly, the angle between the diiodomethane and the resin film 30 seconds after the drop is defined as the contact angle θ with respect to diiodomethane.

[0067] By increasing the content of hydrophobic functional groups in the synthetic resin, increasing the content of functional groups having a cyclic structure, or decreasing the content of butyl groups, the dispersion term component γ of the surface free energy can be reduced. dIn addition, by increasing the content of hydrophilic functional groups in the synthetic resin or by increasing the content of butyl groups, the dipole component γ of the surface free energy can be reduced. p can be made smaller.

[0068] In the resin film formed from the scaffold material for cell culture of the present invention, the storage modulus at 100°C is preferably 0.6 × 10 4 Pa or more, preferably 0.8×10 4 Pa or more, more preferably 1.0 × 10 4 Pa or more, preferably 1.0 × 10 8 Pa or less, preferably 0.8×10 8 Pa or less, more preferably 1.0 × 10 7 Pa or less.

[0069] In particular, the resin film formed from the scaffold material for cell culture of the present invention has a 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 preferably 1.0 x 10 1 More preferably, 5.0 × 10 1 More preferably, 8.0 × 10 2 or more, preferably 1.0 × 10 5 Less than or equal to 0.75 × 10 5 or less, more preferably 0.5 × 10 5 By setting the storage modulus within the above range, the fixation of cells after seeding can be further improved.

[0070] The storage moduli at 25°C and 100°C are measured, for example, using a dynamic viscoelasticity measuring device (IT Measurement & Control, DVA-200) under tensile conditions at a frequency of 10 Hz in the temperature range of -150°C to 150°C at a heating rate of 5°C / min. The storage moduli at 25°C and 100°C are obtained from the resulting tensile storage modulus graph, and the 25°C storage modulus / 100°C storage modulus is calculated. Using a measurement sample with a length of 50 mm, a width of 5 to 20 mm, and a thickness of 0.1 to 1.0 mm, the measurement is performed under conditions of 10 Hz, a strain of 0.1%, a temperature range of -150°C to 150°C, and a heating rate of 5°C / min.

[0071] The storage moduli at 25°C and 100°C can be increased by, for example, increasing the degree of crosslinking in the synthetic resin, stretching the synthetic resin, etc. The storage moduli at 25°C and 100°C can be decreased by, for example, decreasing the number average molecular weight of the synthetic resin, decreasing the glass transition temperature, etc.

[0072] The resin film formed from the scaffold material for cell culture of the present invention preferably has a water swelling ratio of 50% or less, more preferably 40% or less. In this case, the fixation of cells after seeding can be further improved. The lower limit of the water swelling ratio is not particularly limited, but can be, for example, 0.5%. The water swelling ratio can be measured as follows. For example, a resin film (measurement sample) formed from the scaffold material for cell culture, measuring 50 mm in length, 10 mm in width, and 0.05 to 0.15 mm in thickness, is immersed in water at 25°C for 24 hours. The weights of the sample before and after immersion are measured, and the water swelling ratio = (sample weight after immersion - sample weight before immersion) / (sample weight before immersion) × 100 (%) is calculated.

[0073] The water swelling ratio can be reduced by, for example, increasing the hydrophobic functional groups of the synthetic resin or decreasing the number average molecular weight.

[0074] (synthetic resin) The scaffold material for cell culture contains a synthetic resin (hereinafter, sometimes referred to as synthetic resin X). The main chain of synthetic resin X is preferably a carbon chain. In this specification, the term "structural unit" refers to a repeating unit of a monomer that constitutes the synthetic resin. In addition, when the synthetic resin has a graft chain, it includes a repeating unit of a monomer that constitutes the graft chain.

[0075] When synthetic resin X does not have a peptide portion, synthetic resin X preferably has a cationic functional group. When synthetic resin X has a peptide portion, synthetic resin X having a peptide portion may or may not have a cationic functional group in a structural portion other than the peptide portion. Examples of the cationic functional group include substituents having a structure such as an amino group, an imino group, or an amide group. Examples of such functional groups include, but are not limited to, conjugated amine functional groups such as hydroxyamino group, urea group, guanidine, and biguanide; heterocyclic amino functional groups such as piperazine, piperidine, pyrrolidine, 1,4-diazabicyclo[2.2.2]octane, hexamethylenetetraamine, morpholine, pyridine, pyridazine, pyrimidine, pyrazine, pyrrole, azatropylidene, pyridone, imidazole, benzimidazole, benzotriazole, pyrazole, oxazole, imidazoline, triazole, thiazole, thiazine, tetrazole, indole, isoindole, purine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, carbazole, acridine, adenine, guanine, cytosine, thymine, uracil, and melamine; and cyclic pyrrole functional groups such as porphyrin, chlorin, and choline, as well as derivatives thereof. These cationic functional groups may be used alone or in combination of two or more.

[0076] In the present invention, the content of cationic functional groups contained in the structural units of synthetic resin X is preferably 0.2 mol% or more, preferably 2 mol% or more, more preferably 3 mol% or more, and 50 mol% or less, preferably 10 mol% or less, and more preferably 7 mol% or less. By using synthetic resin X containing cationic functional groups within such a range, the fixation of cells after seeding can be further improved, and the cell proliferation rate can be further increased. The content of cationic functional groups can be, for example, 1 It can be measured by H-NMR (nuclear magnetic resonance spectroscopy).

[0077] (vinyl polymer) The synthetic resin X preferably contains a vinyl polymer, and more preferably is a vinyl polymer. A vinyl polymer is a polymer of a compound having a vinyl group or a vinylidene group. When the synthetic resin X is a vinyl polymer, swelling of the scaffold material for cell culture in water can be more easily suppressed. Examples of vinyl polymers include polyvinyl alcohol derivatives, poly(meth)acrylic acid esters, polyvinylpyrrolidone, polystyrene, and ethylene-vinyl acetate copolymers. Furthermore, the vinyl polymer is preferably a polyvinyl alcohol derivative or a poly(meth)acrylic acid ester, from the viewpoint of more easily enhancing adhesiveness to cells.

[0078] (Synthetic resin X having a polyvinyl acetal skeleton) The scaffold material for cell culture preferably contains a synthetic resin X having a polyvinyl acetal skeleton. In the present invention, the synthetic resin X having a polyvinyl acetal skeleton is preferably a copolymer of a structural unit of a polyvinyl acetal resin with a vinyl compound and / or a vinylidene compound. The vinyl compound is a compound having a vinyl group (HC=CH-). The vinylidene compound is a compound having a vinylidene group (HC=CR-). The vinyl compound or vinylidene compound may be a vinyl polymer, which is a polymer thereof. In the following description, the vinyl compound, vinylidene compound, and vinyl polymer copolymerized into the polyvinyl acetal resin may be collectively referred to as "vinyl compound A."

[0079] In the present invention, the copolymer may be a block copolymer of a polyvinyl acetal resin and a vinyl compound A, or a graft copolymer in which a vinyl compound A is grafted onto a polyvinyl acetal resin. The copolymer is preferably a graft copolymer. In this case, a phase-separated structure can be formed more easily.

[0080] Examples of the vinyl compounds and vinylidene compounds include ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, and (meth)acrylic acid esters. These vinyl compounds may be used alone or in combination of two or more. Therefore, the vinyl compounds may be copolymerized to form vinyl polymers.

[0081] In the copolymer, the difference in SP value between the polyvinyl acetal resin and the vinyl compound A is preferably 0.5 or more. In this case, a phase-separated structure can be formed more easily. The difference in SP value between the polyvinyl acetal resin and the vinyl compound A is more preferably 1.0 or more. The upper limit of the difference in SP value is not particularly limited, but can be, for example, 10.0.

[0082] The copolymer preferably has a first phase consisting of a polyvinyl acetal resin and a second phase consisting of a vinyl compound A. The first phase is preferably formed by the polyvinyl acetal resin portion of the copolymer, and the second phase is preferably formed by the vinyl compound A portion. In this case, the first phase of the polyvinyl acetal resin is preferably a sea portion, and the second phase of the vinyl compound A is preferably an island portion. However, the first phase of the vinyl compound A may be a sea portion, and the second phase of the polyvinyl acetal resin may be an island portion.

[0083] The content (mol / mol) of vinyl compound A in the copolymer is preferably 0.015 or more, more preferably 0.3 or more, preferably 0.95 or less, more preferably 0.90 or less, and even more preferably 0.70 or less. When the content is above the lower limit, a phase-separated structure can be formed more easily. When the content is below the upper limit, the cell proliferation rate can be further increased.

[0084] <Polyvinyl acetal resin> The polyvinyl acetal resin (the polyvinyl acetal resin portion of the copolymer) will be described in more detail below.

[0085] The polyvinyl acetal resin has an acetal group, an acetyl group, and a hydroxyl group on the side chain.

[0086] The method for synthesizing a polyvinyl acetal resin includes at least a step of acetalizing polyvinyl alcohol with an aldehyde.

[0087] The aldehyde used in the acetalization of polyvinyl alcohol to obtain a polyvinyl acetal resin is not particularly limited. Examples of the aldehyde include aldehydes having 1 to 10 carbon atoms. The aldehyde may have a chain aliphatic group, a cyclic aliphatic group, or an aromatic group. The aldehyde may be a chain aldehyde or a cyclic aldehyde.

[0088] Examples of the aldehyde include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal, acrolein, benzaldehyde, cinnamaldehyde, perillaldehyde, formylpyridine, formylimidazole, formylpyrrole, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formylisoindole, formylpurine, formylbenzimidazole, formylbenzotriazole, formylquinoline, formylisoquinoline, formylquinoxaline, formylcinnoline, formylpteridine, formylfuran, formyloxolane, formyloxane, formylthiophene, formylthiolane, formylthiane, formyladenine, formylguanine, formylcytosine, formylthymine, and formyluracil. These aldehydes may be used alone or in combination of two or more.

[0089] The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, or pentanal, and more preferably butylaldehyde. Therefore, the polyvinyl acetal skeleton is preferably a polyvinyl butyral skeleton. The polyvinyl acetal resin is preferably a polyvinyl butyral resin.

[0090] From the viewpoint of further enhancing cell adhesiveness, the polyvinyl acetal resin preferably has a Brønsted basic group or a Brønsted acidic group, more preferably a Brønsted basic group. That is, it is preferable that a portion of the polyvinyl acetal resin is modified with a Brønsted basic group or a Brønsted acidic group, more preferably a portion of the polyvinyl acetal resin is modified with a Brønsted basic group.

[0091] The Bronsted basic group is a hydrogen ion, H +The Bronsted basic group is a general term for a functional group that can accept a substituent from another substance. Examples of the Bronsted basic group include amine-based basic groups such as a substituent having an imine structure, a substituent having an imide structure, a substituent having an amine structure, or a substituent having an amide structure. The Bronsted basic group is not particularly limited, and examples thereof include conjugated amine functional groups such as a hydroxyamino group, a urea group, guanidine, and biguanide; heterocyclic amino functional groups such as piperazine, piperidine, pyrrolidine, 1,4-diazabicyclo[2.2.2]octane, hexamethylenetetraamine, morpholine, pyridine, pyridazine, pyrimidine, pyrazine, pyrrole, azatropylidene, pyridone, imidazole, benzimidazole, benzotriazole, pyrazole, oxazole, imidazoline, triazole, thiazole, thiazine, tetrazole, indole, isoindole, purine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, carbazole, acridine, adenine, guanine, cytosine, thymine, uracil, and melamine; cyclic pyrrole functional groups such as porphyrin, chlorin, and choline; and derivatives thereof.

[0092] 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, or salts thereof. The Bronsted acidic group is preferably a carboxyl group.

[0093] The polyvinyl acetal resin preferably has a structural unit having an imine structure, an imide structure, an amine structure, or an amide structure, and may have only one or more of these structural units.

[0094] The polyvinyl acetal resin may have a structural unit having an imine structure. The imine structure refers to a structure having a C=N bond. In particular, the polyvinyl acetal resin preferably has an imine structure in a side chain.

[0095] The polyvinyl acetal resin may have a structural unit having an imide structure. The structural unit having an imide structure is preferably a structural unit having an imino group (=NH).

[0096] The polyvinyl acetal resin preferably has an imino group in a side chain. In this case, the imino group may be directly bonded to a carbon atom constituting the main chain of the polyvinyl acetal resin, or may be bonded to the main chain via a linking group such as an alkylene group.

[0097] The polyvinyl acetal resin may have a structural unit having an amine structure, wherein the amine group in the amine structure may be a primary amine group, a secondary amine group, a tertiary amine group, or a quaternary amine group.

[0098] The structural unit having an amine structure may be a structural unit having an amide structure, which refers to a structure having —C(═O)—NH—.

[0099] The polyvinyl acetal resin preferably has an amine structure or an amide structure in a side chain. In this case, the amine structure or the amide structure may be directly bonded to a carbon atom constituting the main chain of the polyvinyl acetal resin, or may be bonded to the main chain via a linking group such as an alkylene group.

[0100] The content of the structural unit having an imine structure, the content of the structural unit having an imide structure, the content of the structural unit having an amine structure, and the content of the structural unit having an amide structure are as follows: 1 It can be measured by H-NMR (nuclear magnetic resonance spectroscopy).

[0101] <Vinyl compound A> The vinyl compound A will be described in more detail below.

[0102] The vinyl compound A is preferably a (meth)acrylic acid ester or a poly(meth)acrylic acid ester resin. In particular, the synthetic resin X is preferably a copolymer in which a (meth)acrylic acid ester or a poly(meth)acrylic acid ester resin, which is a polymer thereof, is graft-copolymerized onto a polyvinyl acetal resin.

[0103] The poly(meth)acrylic acid ester resin can be obtained by polymerizing a (meth)acrylic acid ester or by polymerizing a (meth)acrylic acid ester with the other monomers described above.

[0104] Examples of the (meth)acrylic acid ester include (meth)acrylic acid alkyl ester, (meth)acrylic acid cyclic alkyl ester, (meth)acrylic acid aryl ester, (meth)acrylamide, (meth)acrylic acid polyethylene glycol, and (meth)acrylic acid phosphorylcholine.

[0105] Examples of the (meth)acrylic acid alkyl ester include methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, isopropyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, t-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.

[0106] The (meth)acrylic acid alkyl ester may be substituted with a substituent such as an alkoxy group having 1 to 3 carbon atoms and a tetrahydrofurfuryl group. Examples of such (meth)acrylic acid alkyl ester include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0107] Examples of the (meth)acrylic acid cyclic alkyl ester include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0108] Examples of the (meth)acrylic acid aryl ester include phenyl (meth)acrylate and benzyl (meth)acrylate.

[0109] Examples of (meth)acrylamides include (meth)acrylamide, N-isopropyl(meth)acrylamide, N-tert-butyl(meth)acrylamide, N,N'-dimethyl(meth)acrylamide, (3-(meth)acrylamidopropyl)trimethylammonium chloride, 4-(meth)acryloylmorpholine, 3-(meth)acryloyl-2-oxazolidinone, N-[3-(dimethylamino)propyl](meth)acrylamide, N-(2-hydroxyethyl)(meth)acrylamide, N-methylol(meth)acrylamide, and 6-(meth)acrylamidohexanoic acid.

[0110] Examples of 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.

[0111] Examples of the (meth)acrylate phosphorylcholine include 2-(meth)acryloyloxyethyl phosphorylcholine.

[0112] As another monomer to be copolymerized with the (meth)acrylic acid ester, a vinyl compound is preferably used. Examples of the vinyl compound include ethylene, allylamine, vinylpyrrolidone, vinylimidazole, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, and (meth)acrylic acid ester. Only one type of vinyl compound may be used, or two or more types may be used in combination.

[0113] In this specification, "(meth)acrylic" means "acrylic" or "methacrylic", and "(meth)acrylate" means "acrylate" or "methacrylate".

[0114] In the present invention, the synthetic resin X may be a copolymer of a resin having a poly(meth)acrylic acid ester skeleton and another vinyl compound, as long as it can form the phase-separated structure of the present invention.

[0115] In this case, the other vinyl compound may be ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, or other (meth)acrylic acid esters with different SP values.

[0116] (Synthetic resin X having a peptide moiety) The scaffold material for cell culture preferably contains a synthetic resin X having a peptide moiety. The synthetic resin X having a peptide moiety can be obtained by reacting the synthetic resin X with a linker and a peptide. The synthetic resin X having a peptide moiety is preferably a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin moiety, a linker moiety and a peptide moiety, and more preferably a peptide-containing polyvinyl butyral resin having a polyvinyl butyral resin moiety, a linker moiety and a peptide moiety. Only one type of synthetic resin X having a peptide moiety may be used, or two or more types may be used in combination.

[0117] The peptide portion is preferably composed of 3 or more amino acids, more preferably 4 or more amino acids, even more preferably 5 or more amino acids, and preferably 10 or less amino acids, and more preferably 6 or less amino acids. When the number of amino acids constituting the peptide portion is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, adhesiveness to cells after seeding can be further improved, and the cell proliferation rate can be further increased.

[0118] The peptide moiety preferably has a cell-adhesive amino acid sequence. The cell-adhesive amino acid sequence refers to an amino acid sequence whose cell-adhesive activity has been confirmed by phage display, Sepharose bead, or plate coating. The phage display method can be, for example, the method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995, pp. 1189-1196." The Sepharose bead method can be, for example, the method described in "Protein, Nucleic Acid, Enzyme, Vol. 45, No. 15 (2000) 2477." The plate coating method can be, for example, the method described in "Protein, Nucleic Acid, Enzyme, Vol. 45, No. 15 (2000) 2477."

[0119] Examples of the cell adhesive amino acid sequences include the RGD sequence (Arg-Gly-Asp), YIGSR sequence (Tyr-Ile-Gly-Ser-Arg), PDSGR sequence (Pro-Asp-Ser-Gly-Arg), HAV sequence (His-Ala-Val), ADT sequence (Ala-Asp-Thr), QAV sequence (Gln-Ala-Val), LDV sequence (Leu-Asp-Val), IDS sequence (Ile-Asp-Ser), REDV sequence (Arg-Glu-Asp-Val), IDAPS sequence (Ile-Asp-Ala-Pro-Ser), KQAGDV sequence (Lys-Gln-Ala-Gly-Asp-Val), and TDE sequence (Thr-Asp-Glu). Further examples of the cell adhesive amino acid sequence include those described in "Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Medical Center Journal, Vol. 8, No. 1, pp. 58-66, 1992." The peptide portion may have only one type of cell adhesive amino acid sequence, or two or more types.

[0120] The cell adhesive amino acid sequence preferably has at least one of the above-mentioned cell adhesive amino acid sequences, more preferably has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, and even more preferably has at least an RGD sequence represented by the following formula (1): In this case, adhesiveness to cells after seeding can be further increased, and the cell proliferation rate can be further increased.

[0121] Arg-Gly-Asp-X...Formula (1)

[0122] In the above formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0123] The peptide portion may be linear or may have a cyclic peptide backbone. The cyclic peptide backbone is a cyclic backbone composed of a plurality of amino acids. From the viewpoint of more effectively exerting the effects of the present invention, the cyclic peptide backbone is preferably composed of four or more amino acids, more preferably five or more amino acids, and more preferably ten or less amino acids.

[0124] In synthetic resin X having a peptide moiety, the content of the peptide moiety is preferably 0.1 mol% or more, more preferably 1 mol% or more, even more preferably 5 mol% or more, and particularly preferably 10 mol% or more. In synthetic resin X having a peptide moiety, the content of the peptide moiety is preferably 60 mol% or less, more preferably 50 mol% or less, even more preferably 35 mol% or less, and particularly preferably 25 mol% or less. When the content of the peptide moiety is equal to or greater than the lower limit, a phase-separated structure can be formed more easily. When the content of the peptide moiety is equal to or greater than the lower limit, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased. Furthermore, when the content of the peptide moiety is equal to or less than the upper limit, production costs can be reduced. The content (mol%) of the peptide moiety is the amount of substance of the peptide moiety relative to the sum of the amounts of substance of each structural unit constituting synthetic resin X having a peptide moiety.

[0125] The content of the peptide moiety can be measured by FT-IR or LC-MS.

[0126] From the viewpoint of further enhancing adhesiveness to cells after seeding and further increasing the cell proliferation rate, in synthetic resin X having a peptide portion, it is preferable that the second phase has a peptide portion, and it is more preferable that the peptide portion has the above-mentioned cell-adhesive amino acid sequence. It is preferable that the second phase is formed by the peptide portion of synthetic resin X having a peptide portion. In this case, it is preferable that the second phase having a peptide portion is an island portion. However, the first phase may have a peptide portion, and the second phase having a peptide portion may be a sea portion.

[0127] In the synthetic resin X having a peptide portion, the synthetic resin X portion and the peptide portion are preferably bonded via a linker. That is, the synthetic resin X having a peptide portion is preferably a synthetic resin X having a peptide portion and a linker portion. Only one type of the above linker may be used, or two or more types may be used in combination.

[0128] The linker is preferably a compound having a functional group capable of condensing with a carboxyl group or an amino group of the peptide. Examples of the functional group capable of condensing with a carboxyl group or an amino group of the peptide include a carboxyl group, a thiol group, and an amino group. From the viewpoint of favorable reaction with the peptide, the linker is preferably a compound having a carboxyl group. The above-mentioned vinyl compound A can also be used as the linker.

[0129] Examples of the linker having a carboxyl group include (meth)acrylic acid and carboxyl group-containing acrylamide, etc. By using a carboxylic acid (carboxylic acid monomer) having a polymerizable unsaturated group as the linker having a carboxyl group, the carboxylic acid monomer can be polymerized by graft polymerization when the linker is reacted with synthetic resin X, thereby increasing the number of carboxyl groups that can react with peptides.

[0130] [Scaffold material for cell culture] The scaffold material for cell culture contains the synthetic resin X. From the viewpoint of effectively exerting the effects of the present invention and increasing productivity, the content of the synthetic resin X in 100% by weight of the scaffold material for cell culture is preferably 90% by weight or more, more preferably 95% by weight or more, even more preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). Therefore, the scaffold material for cell culture is most preferably the synthetic resin X. When the content of the synthetic resin X is equal to or more than the lower limit, the effects of the present invention can be exerted even more effectively.

[0131] The cell culture scaffold material may contain components other than the synthetic resin X. Examples of components other than the synthetic resin X include polyolefin resins, polyether resins, polyvinyl alcohol resins, polyesters, epoxy resins, polyamide resins, polyimide resins, polyurethane resins, polycarbonate resins, polysaccharides, cellulose, polypeptides, and synthetic peptides.

[0132] From the viewpoint of effectively exerting the effects of the present invention, the content of components other than the synthetic resin X is preferably as low as possible. In 100% by weight of the scaffold material for cell culture, the content of these components is preferably 10% by weight or less, more preferably 5% by weight or less, even more preferably 2.5% by weight or less, particularly preferably 1% by weight or less, and most preferably 0% by weight (not contained). Therefore, it is most preferable that the scaffold material for cell culture does not contain any components other than the synthetic resin X.

[0133] The scaffold material for cell culture preferably contains substantially no animal-derived raw materials. By not containing any animal-derived raw materials, it is possible to provide a scaffold material for cell culture that is highly safe and has little variation in quality during production. Here, "substantially free of animal-derived raw materials" means that the content of animal-derived raw materials in the scaffold material for cell culture is 3% by weight or less. The scaffold material for cell culture preferably contains 1% by weight or less, and more preferably 0% by weight, of animal-derived raw materials. In other words, the scaffold material for cell culture more preferably contains no animal-derived raw materials at all.

[0134] (Cell culture using scaffold materials for cell culture) The scaffold material for cell culture is used for culturing cells. The scaffold material for cell culture is used as a scaffold for cells when culturing the cells. Therefore, a resin film formed from the scaffold material for cell culture of the present invention is used for culturing cells and as a scaffold for the cells when culturing the cells.

[0135] The cells include animal cells from humans, mice, rats, pigs, cows, monkeys, etc. The cells also include somatic cells, such as stem cells, progenitor cells, and mature cells. The somatic cells may be cancer cells.

[0136] Examples of the mature cells include nerve cells, cardiac muscle cells, retinal cells, and hepatic cells.

[0137] Examples of the stem cells include mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0138] (Shape of scaffold material for cell culture) The resin film of the present invention is formed from a scaffold material for cell culture. The resin film is formed using a scaffold material for cell culture. The resin film is preferably a membrane-like scaffold material for cell culture. The resin film is preferably a membrane-like product of a scaffold material for cell culture.

[0139] The present specification also provides particles, fibers, porous bodies, or films containing the above-mentioned scaffold material for cell culture. In this case, the shape of the scaffold material for cell culture is not particularly limited, and may be particles, fibers, porous bodies, or films. Note that the particles, fibers, porous bodies, or films may contain components other than the above-mentioned scaffold material for cell culture.

[0140] A film containing the scaffold material for cell culture is preferably used for flat culture (two-dimensional culture) of cells, and particles, fibers, or porous bodies containing the scaffold material for cell culture are preferably used for three-dimensional culture of cells.

[0141] (Cell culture carrier) The present invention also relates to a carrier for cell culture, in which the resin film is disposed on the surface of the carrier. The carrier for cell culture of the present invention can be obtained, for example, by disposing the resin film on the surface of the carrier by coating or the like. The shape of the carrier may be particles, fibers, a porous body, or a film. That is, the carrier for cell culture of the present invention may be in the shape of particles, fibers, a porous body, or a film. Note that the carrier for cell culture of the present invention may contain components other than the carrier and the resin film.

[0142] (Cell culture container) The present invention also relates to a cell culture vessel having the above-mentioned resin film in at least a part of a cell culture region. Figure 1 is a cross-sectional view schematically showing a cell culture vessel according to one embodiment of the present invention.

[0143] The cell culture vessel 1 comprises a vessel body 2 and a resin film 3 formed from a scaffold material for cell culture. The resin film 3 is disposed on a surface 2a of the vessel body 2. The resin film 3 is disposed on a bottom surface of the vessel body 2. By adding a liquid medium to the cell culture vessel 1 and seeding cells on the surface of the resin film 3, cells can be cultured on a plate.

[0144] The container body may include a first container body and a second container body such as a cover glass on the bottom surface of the first container body. The first container body and the second container body may be separable. In this case, a resin film formed from the cell culture scaffold material may be disposed on the surface of the second container body.

[0145] The container body may be a conventionally known container body (container). The shape and size of the container body are not particularly limited.

[0146] Examples of the container body include cell culture plates and cell culture flasks having one or more wells (holes). The number of wells in the plate is not particularly limited. Examples of the number of wells include, but are not limited to, 2, 4, 6, 12, 24, 48, 96, and 384. Examples of the shape of the well include, but are not limited to, a perfect circle, an ellipse, a triangle, a square, a rectangle, and a pentagon. Examples of the shape of the well bottom include, but are not limited to, a flat bottom, a round bottom, and an uneven bottom.

[0147] The material of the container body is not particularly limited, but examples thereof include resins, metals, and inorganic materials. Examples of the resins include polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymers, polyimides, polyamides, polyamideimides, (meth)acrylic resins, epoxy resins, and silicones. Examples of the metals include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, and platinum. Examples of the inorganic materials include silicon oxide (glass), aluminum oxide, titanium oxide, zirconium oxide, iron oxide, and silicon nitride. [Example]

[0148] Next, the present invention will be clarified by showing specific examples and comparative examples of the present invention, but the present invention is not limited to the following examples.

[0149] The following synthetic resins were synthesized as raw materials for cell culture scaffold materials.

[0150] Example 1 2700 mL of ion-exchanged water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 98 mol% were added to a reactor equipped with a stirrer, and the mixture was heated and dissolved while stirring to obtain a solution. 35 wt% hydrochloric acid was added as a catalyst to the obtained solution so that the hydrochloric acid concentration was 0.2 wt%. The temperature was then adjusted to 15°C, and 22 parts by weight of n-butylaldehyde was added while stirring. 148 parts by weight of n-butylaldehyde was then added to precipitate a white particulate polyvinyl butyral resin. 15 minutes after precipitation, 35 wt% hydrochloric acid was added so that the hydrochloric acid concentration was 1.8 wt%, and the mixture was then heated to 50°C and maintained at 50°C for 2 hours. The solution was then cooled and neutralized, washed with water, and dried to obtain a polyvinyl butyral resin (PVB, SP value: 9.9) as a polyvinyl acetal resin. 90 parts by weight of the resulting polyvinyl butyral resin was dissolved in tetrahydrofuran to form a 1% by weight solution, and 5 parts by weight of Irgacure 184 as an initiator, 2 parts by weight of N-vinylpyrrolidone (SP value: 11.7), and 8 parts by weight of n-lauryl methacrylate (SP value: 8.2) were added, followed by graft polymerization to obtain a synthetic resin. The resulting synthetic resin had an acetalization degree (butyralization degree) of 69 mol%, a hydroxyl group content of 27.5 mol%, an acetylation degree of 2.0 mol%, a vinylpyrrolidone group content of 0.3 mol%, and an n-lauryl methacrylate content of 1.2 mol%.

[0151] (Examples 2 to 11 and Comparative Example 1) Except for changing the weight ratio of polyvinyl butyral resin, N-vinylpyrrolidone, and n-lauryl methacrylate, synthetic resins were obtained in the same manner as in Example 1. The acetalization degree (butyralization degree), hydroxyl group amount, acetylation degree, vinylpyrrolidone group content, and n-lauryl methacrylate moiety content of the synthetic resins obtained in Examples 2 to 11 and Comparative Example 1 are shown in Tables 1, 2, and 4.

[0152] Example 12 2700 mL of ion-exchanged water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 99 mol% were added to a reactor equipped with a stirrer, and the mixture was heated and dissolved while stirring to obtain a solution. 35 wt% hydrochloric acid was added as a catalyst to the obtained solution so that the hydrochloric acid concentration was 0.2 wt%. The temperature was then adjusted to 15°C, and 22 parts by weight of n-butylaldehyde was added while stirring. Next, 148 parts by weight of n-butylaldehyde was added, causing the precipitation of a white particulate polyvinyl acetal resin (polyvinyl butyral resin). 15 minutes after the precipitation, 35 wt% hydrochloric acid was added so that the hydrochloric acid concentration was 1.8 wt%, and the mixture was then heated to 50°C and maintained at 50°C for 2 hours. Next, the solution was cooled and neutralized, and then the polyvinyl butyral resin was washed with water and dried to obtain a polyvinyl acetal resin (polyvinyl butyral resin, average degree of polymerization 1700, degree of acetalization (degree of butyralization) 70 mol%, amount of hydroxyl groups 27 mol%, degree of acetylation 3 mol%).

[0153] Linker installation: 99 parts by weight of the resulting polyvinyl acetal resin and 1 part by weight of acrylic acid (linker) were dissolved in 300 parts by weight of THF and reacted for 20 minutes under UV irradiation in the presence of a photopolymerization initiator to graft copolymerize the polyvinyl acetal resin and acrylic acid, thereby introducing the linker. One part by weight of the linker-introduced polyvinyl acetal resin was dissolved in 19 parts by weight of butanol. 150 μL of the resulting solution was dispensed onto the surface of a 22 mm diameter cover glass (Matsunami Co., Ltd., "22-maru No. 1") that had been dusted with an air duster. The glass was then spun at 2000 rpm for 20 seconds using a spin coater, and then heated at 60°C for 60 minutes to obtain a resin film with a smooth surface.

[0154] Formation of the peptide part: A linear peptide (5 amino acid residues, designated GRGDS in the table) having the amino acid sequence Gly-Arg-Gly-Asp-Ser was prepared. Ten parts by weight of this peptide and 1 part by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensation agent) were added to calcium- and magnesium-free phosphate-buffered saline to a final peptide concentration of 1 mM to prepare a peptide-containing solution. One part by weight of this peptide-containing solution was added to a spin-coated resin film (polyvinyl acetal resin with a linker formed thereon) and allowed to react, resulting in dehydration condensation between the carboxyl group of the linker and the amino group of the Gly of the peptide. In this way, a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion, a linker portion, and a peptide portion was prepared.

[0155] The obtained peptide-containing polyvinyl acetal resin had an acetalization degree (butyralization degree) of 69 mol%, a hydroxyl group content of 27 mol%, an acetylation degree of 3 mol%, a carboxyl group content of 0.1 mol%, and a peptide moiety content of 1.0 mol%.

[0156] Example 13 A peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that 85 parts by weight of polyvinyl acetal resin and 15 parts by weight of acrylic acid (linker) were used for the introduction of the linker, and the amount of peptide added was changed to 15 parts by weight for the formation of the peptide.

[0157] Example 14 A peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that 70 parts by weight of polyvinyl acetal resin and 30 parts by weight of acrylic acid (linker) were used for the introduction of the linker, and the amount of peptide added was changed to 30 parts by weight for the formation of the peptide.

[0158] Example 15 A peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that 67 parts by weight of polyvinyl acetal resin and 33 parts by weight of acrylic acid (linker) were used for the introduction of the linker, and the amount of peptide added was changed to 33 parts by weight for the formation of the peptide.

[0159] Example 16 A peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that 63 parts by weight of polyvinyl acetal resin and 37 parts by weight of acrylic acid (linker) were used for the introduction of the linker, and the amount of peptide added was changed to 37 parts by weight for the formation of the peptide.

[0160] Example 17 A peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that 30 parts by weight of polyvinyl acetal resin and 70 parts by weight of acrylic acid (linker) were used for the introduction of the linker, and the amount of peptide added was changed to 70 parts by weight for the formation of the peptide.

[0161] (Comparative Example 2) As the synthetic resin, polystyrene resin was used as it was.

[0162] (Comparative Example 3) 2700 mL of ion-exchanged water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 98 mol% were added to a reactor equipped with a stirrer and heated to dissolve while stirring to obtain a solution. 35 wt% hydrochloric acid was added to the resulting solution as a catalyst to obtain a hydrochloric acid concentration of 0.2 wt%. The temperature was then adjusted to 15°C, and 22 parts by weight of n-butylaldehyde was added while stirring. 148 parts by weight of n-butylaldehyde was then added to precipitate a white granular polyvinyl butyral resin. 15 minutes after precipitation, 35 wt% hydrochloric acid was added to obtain a hydrochloric acid concentration of 1.8 wt%, followed by heating to 50°C and maintaining at 50°C for 2 hours. The solution was then cooled, neutralized, washed with water, and dried to obtain a polyvinyl butyral resin (SP value: 9.9). In other words, a polyvinyl butyral resin (synthetic resin) without a vinyl compound copolymerized therein was obtained.

[0163] Comparative Example 4 17 parts by weight of N-vinylpyrrolidone and 83 parts by weight of n-lauryl methacrylate were mixed to obtain a (meth)acrylic monomer solution. 1 part by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained (meth)acrylic monomer solution, and the solution was applied to a PET film. The coated surface was irradiated with light at a wavelength of 365 nm at an integrated light intensity of 2000 mJ / cm using a UV conveyor device "ECS301G1" manufactured by Eye Graphics Co., Ltd. at 25°C. 2 A poly(meth)acrylic acid ester resin solution was obtained by irradiating the solution with light at 1000 W at 1000 W. The resulting poly(meth)acrylic acid ester resin solution was dried in a vacuum at 80°C for 3 hours to obtain a synthetic resin in the form of a poly(meth)acrylic acid ester resin.

[0164] (Reference example A) Fabrication of natural scaffolds: One ml of a 5 μg / ml solution of Vitronectin (Corning) in phosphate buffer (PBS) was added to a φ35 mm dish. A φ22 mm cover glass (Matsunami Co., Ltd., "22 Maru No. 1") was immersed in the solution and cured at 37°C for 1 hour to obtain a naturally derived scaffold with Vitronectin smoothly adsorbed onto its surface (referred to as VTN in the table).

[0165] Preparation of cell culture vessels: The cell culture vessel was prepared by placing the laminate of Vitronectin and cover glass in a 22 mm diameter polystyrene dish. Because Vitronectin denatures when dried, significantly reducing its adhesive properties, the cell culture vessel was immediately immersed in PBS solution after preparation.

[0166] [evaluation] (Degree of acetalization and degree of cationic group modification) The degree of acetalization and the degree of cationic group modification of the synthetic resins obtained in the examples and comparative examples were determined by dissolving the synthetic resins in DMSO-d6 (dimethyl sulfoxide) and then 1 Measurement was performed by 1 H-NMR (nuclear magnetic resonance spectroscopy).

[0167] (storage modulus) The storage modulus of each scaffold material at 25°C and 100°C was measured under tensile conditions using a dynamic viscoelasticity analyzer (IT Measurement & Control, DVA-200) at a frequency of 10 Hz and a temperature range of -150°C to 150°C at a heating rate of 5°C / min. The storage moduli at 25°C and 100°C were determined from the resulting tensile storage modulus graph, and the 25°C storage modulus / 100°C storage modulus was calculated. Measurement samples were 50 mm long, 5-20 mm wide, and 0.1-1.0 mm thick, and were measured under conditions of 10 Hz, 0.1% strain, -150°C to 150°C, and a heating rate of 5°C / min.

[0168] (Water swelling ratio) Resin films (measurement samples) made from each scaffold material, measuring 50 mm in length, 10 mm in width, and 0.05 mm to 0.15 mm in thickness, were immersed in water at 25°C for 24 hours. The weights of the samples before and after immersion were measured, and the water swelling ratio was calculated as follows: (sample weight after immersion - sample weight before immersion) / (sample weight before immersion) × 100 (%).

[0169] (Production of cell culture vessels) In Examples 1 to 11 and Comparative Examples 1 to 4, a resin solution was obtained by dissolving 1 g of the resulting synthetic resin in 19 g of butanol. 150 μL of the resulting resin solution was dispensed onto a 22 mm diameter cover glass (Matsunami Co., Ltd., 22-round No. 1, dusted with an air duster before use) and spun at 2000 rpm for 20 seconds using a spin coater to obtain a smooth resin film. The resulting resin film, along with the cover glass, was placed in a 22 mm diameter polystyrene dish to obtain a cell culture vessel. In Examples 12 to 17, a laminate of the resulting peptide-containing polyvinyl acetal resin and cover glass was placed in a 22 mm diameter polystyrene dish to obtain a cell culture vessel.

[0170] (surface free energy) The surface free energy of the resin film obtained in the section on preparation of cell culture vessels was measured using a contact angle meter (Kyowa Interface Science Co., Ltd., DMo-701). 1 μL of pure water was dropped onto the resin film, and an image of the droplet was taken after 30 seconds to obtain the contact angle of pure water. 1 μL of diiodomethane was also dropped onto the resin film, and an image of the droplet was taken after 30 seconds to obtain the contact angle of diiodomethane. From the obtained contact angles, the dispersion term component of the surface free energy γ was calculated using the Kaelble-Uy theoretical formula. d (dSFE) and the polar dipole component γ p (pSFE) was calculated.

[0171] (Phase separation parameters) The resin film obtained in the section on preparation of cell culture vessels was observed using an atomic force microscope (AFM, manufactured by Bruker, product number "Dimension XR"). A SCAN ASYST AIR cantilever was used. As a result, as shown in Figure 2, a sea-island structure was observed in the resin film of Example 3, in which the polyvinyl butyral resin portion as the first phase formed a sea portion, and the resin portion containing a (meth)acrylic acid ester and a vinyl compound (a copolymer portion of N-vinylpyrrolidone and n-lauryl methacrylate) as the second phase formed island portions. Similarly, a sea-island structure was observed in Examples 1 to 2 and 4 to 11, in which the polyvinyl butyral resin portion as the first phase formed a sea portion, and the resin portion containing a (meth)acrylic acid ester and a vinyl compound (a copolymer portion of N-vinylpyrrolidone and n-lauryl methacrylate) as the second phase formed island portions. Furthermore, a sea-island structure in which the polyvinyl butyral resin portion as the first phase formed a sea portion and the peptide portion as the second phase formed islands was also observed in Examples 12 to 17. On the other hand, no phase-separated structure was observed in Comparative Examples 1 to 4.

[0172] In addition, using image analysis software (ImageJ) from the images obtained by the atomic force microscope, the ratio of the surface area of ​​the second phase to the entire surface (surface area ratio of the phase-separated structure), the ratio of the perimeter to the area of ​​the second phase (perimeter / area), the number of island portions of the second phase (island number), and the average diameter of the islands (average island size) were determined using the methods described above.

[0173] (cell proliferation rate) 1 mL of phosphate-buffered saline was added to the resulting cell culture vessel, which was then left to stand in a 37°C incubator for 1 hour. The phosphate-buffered saline was then removed from the culture vessel. A confluent colony of h-iPS cell 253G1 was added to a 35 mm dish, followed by 1 mL of 0.5 mM ethylenediamine / phosphate buffer solution and the dish was left to stand at room temperature for 2 minutes. The ethylenediamine / phosphate buffer solution was then removed, and the cell clumps (0.5 x 10) were crushed to 50-200 μm by pipetting in 1 mL of TeSRE8 medium. 5Cells were seeded into a culture vessel. Culture was performed in an incubator at 37°C with 5% CO2 in the presence of 1.7 mL of TeSR E8 medium (STEM CELL) and 10 μM ROCK inhibitor (Y27632). Medium was replaced every 24 hours by removing 1 mL of medium and adding 1 mL of fresh TeSR E8. After 5 days, the established cell mass was detached using 1.0 mL of TryPLE Express detachment solution, and the cell number was counted using a cell counter (Nucleocounter NC-3000, Chemometec).

[0174] The cell proliferation rate relative to Reference Example A was calculated using the following formula.

[0175] Cell proliferation rate (%) relative to Reference Example A = (cell number in Examples and Comparative Examples) / (cell number in Reference Example A) × 100

[0176] The cell proliferation rate was evaluated according to the following criteria.

[0177] [Evaluation criteria] AAA: Cell proliferation rate is 70% or more compared to Reference Example A AA: Cell proliferation rate compared to Reference Example A is 60% or more but less than 70% A: Cell proliferation rate compared to Reference Example A is 50% or more but less than 60% B: Cell proliferation rate compared to Reference Example A is 40% or more but less than 50% C: Cell proliferation rate compared to Reference Example A is 30% or more but less than 40% D: Cell proliferation rate compared to Reference Example A is less than 30%

[0178] The results are shown in Tables 1 to 4 below.

[0179] [Table 1]

[0180] [Table 2]

[0181] [Table 3]

[0182] [Table 4] [Explanation of symbols]

[0183] 1...Cell culture container 2...Container body 2a…Surface 3...Resin film

Claims

[Claim 1] A resin film formed from a scaffold material for cell culture, the scaffold material for cell culture contains a synthetic resin having a polyvinyl acetal skeleton; the resin film has a phase-separated structure including at least a first phase and a second phase; A resin film, wherein the ratio of the surface area of ​​one of the first phase and the second phase to the entire surface is 0.01 or more and 0.95 or less.

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