Resin membranes, cell culture carriers, and cell culture containers formed from cell culture scaffold materials

Abstract

The present invention provides a resin film formed of a cell culture scaffold material, which has excellent fixation after cell inoculation and can improve the proliferation rate of cells. The present invention provides a resin film, which is a resin film formed of a cell culture scaffold material, the cell culture scaffold material contains a synthetic resin, and the resin film has a phase separation structure containing 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

[0001] This application is a divisional application of a patent application in China with application number 202080006412.X, invention name “resin membrane formed by scaffold material for cell culture, carrier for cell culture and container for cell culture” and application date May 15, 2020. Technical Field

[0002] The present invention relates to a resin film formed of a scaffold material for cell culture and also to a carrier for cell culture and a container for cell culture comprising the resin film. Background Art

[0003] In recent years, next-generation medicine using cell medicine and stem cells has attracted attention. In particular, the application of human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and artificial pluripotent stem cells (hiPSCs) or differentiated cells induced from them in pharmaceuticals and regenerative medicine is expected. In order to achieve such applications, it is necessary to culture and proliferate pluripotent stem cells and differentiated cells safely and with good reproducibility.

[0004] In particular, in the industrial application of regenerative medicine, a large number of stem cells need to be processed, and therefore, natural polymer materials, synthetic polymer materials or feeder cells are needed to support the proliferation of pluripotent stem cells. Therefore, various studies have been conducted on the cultivation methods using scaffold materials such as natural polymer materials and synthetic polymer materials.

[0005] For example, Patent Document 1 below discloses a cell culture carrier comprising a molded product containing a polyvinyl acetal compound or a molded product containing the polyvinyl acetal compound and a water-soluble polysaccharide, wherein the degree of acetalization of the polyvinyl acetal compound is 20 to 60 mol %.

[0006] Patent Document 2 below discloses a composition comprising a first fiber polymer scaffold in which fibers of the first fiber polymer scaffold are arranged, and states that the fibers constituting the first fiber polymer scaffold are composed of aliphatic polyesters such as polyglycolic acid and polylactic acid.

[0007] Furthermore, Patent Document 3 listed below discloses a method for maintaining the undifferentiated nature of pluripotent stem cells, which comprises the step of culturing the pluripotent stem cells in a culture vessel having a surface coated with a polyrotaxane block copolymer.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Application Publication No. 2006-314285

[0011] Patent Document 2: Japanese Patent Application No. 2009-524507

[0012] Patent Document 3: Japanese Patent Application Publication No. 2017-23008 Summary of the invention

[0013] Technical problem solved by the invention

[0014] However, when natural polymer materials are used as scaffold materials, the fixation of cells after inoculation can be improved. In particular, it is known that when adhesive proteins such as laminin, vitronectin, and matrigel derived from mouse sarcoma are used as natural polymer materials, the fixation of cells after inoculation is very high. On the other hand, natural polymer materials are relatively expensive, and since they are natural source materials, the unevenness between batches is relatively large, or there are risks in safety due to the ingredients of animal origin.

[0015] In contrast, when a scaffold material using a synthetic resin is used, it has good operability, low price, less batch-to-batch variation, and excellent safety compared to a scaffold material using a natural polymer material. However, in the case of the scaffold materials using a synthetic resin in Patent Documents 1 to 3, since a synthetic resin with high hydrophilicity is used, the synthetic resin swells excessively. In addition, synthetic resins have a lower affinity for cells than natural polymer materials, so cell blocks sometimes peel off during culture. Such scaffold materials using synthetic resins have low fixation after cell inoculation, and cells sometimes cannot fully proliferate.

[0016] An object of the present invention is to provide a resin film, a cell culture carrier, and a cell culture container formed of a cell culture scaffold material, which are excellent in fixation after cell seeding and can increase the cell proliferation rate.

[0017] Technical means of solving problems

[0018] In the resin film formed by the cell culture scaffold material of the present invention, the cell culture scaffold material contains a synthetic resin, and the resin film has a phase separation 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 greater than 0.01 and less than 0.95.

[0019] In a specific embodiment of the resin film of the present invention, a ratio of the perimeter to the area of ​​the second phase (perimeter / area) is not less than 0.001 (1 / nm) and not more than 0.40 (1 / nm).

[0020] In another specific embodiment of the resin film of the present invention, the phase separation structure is a sea-island structure, the first phase is a sea portion, and the second phase is an island portion.

[0021] In another specific embodiment of the resin film of the present invention, the number of the second phase as the island portion is 1 / μm 2 Above, 5000 / μm 2 the following.

[0022] In another specific embodiment of the resin film of the present invention, the phase separation structure is composed of a phase separation structure within a molecule of the synthetic resin.

[0023] In another specific embodiment of the resin film of the present invention, the dispersion term component of the surface free energy is 25.0 mJ / m 2 Above, 50.0mJ / m 2 Below, and the polar component is 1.0mJ / m 2 Above, 20.0mJ / m 2 the following.

[0024] In another specific embodiment of the resin film of 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.

[0025] In another specific embodiment of the resin film of the present invention, the second phase has a peptide part.

[0026] In another specific embodiment of the resin film of the present invention, the peptide portion has a cell-adhesive amino acid sequence.

[0027] In another specific embodiment of the resin film of the present invention, the water swelling ratio is 50% or less.

[0028] In another specific embodiment of the resin film of the present invention, the storage elastic modulus at 100°C is 1.0×10 4 Pa or above, 1.0×10 8 Pa or less, and the ratio of the storage modulus at 25°C to the storage modulus at 100°C ((storage modulus at 25°C) / (storage modulus at 100°C)) is 1.0×10 1 Above, 1.0×10 5 the following.

[0029] In another specific embodiment of the resin film of the present invention, the cell culture scaffold material contains substantially no animal-derived raw materials.

[0030] In another specific embodiment of the resin film of the present invention, the synthetic resin includes a vinyl polymer.

[0031] In another specific embodiment of the resin film of the present invention, the synthetic resin contains at least a polyvinyl alcohol derivative or a poly(meth)acrylate.

[0032] The cell culture carrier of the present invention comprises a carrier and a resin film configured according to the present invention, wherein the resin film is disposed on the surface of the carrier.

[0033] The cell culture container of the present invention comprises a container body and a resin film configured according to the present invention, wherein the resin film is disposed on a surface of the container body.

[0034] Effects of the Invention

[0035] According to the present invention, a resin film, a cell culture carrier, and a cell culture container formed of a cell culture scaffold material which are excellent in fixation after cell seeding and can improve the cell proliferation rate can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] [ Figure 1 ] is a schematic front cross-sectional view of a cell culture container according to one embodiment of the present invention.

[0037] [ Figure 2 ] is an atomic force microscope photograph of the resin film obtained in Example 3.

[0038] [ Figure 3 ] is an atomic force microscope photograph of the resin film obtained in Example 14.

[0039] Specific embodiments of the present invention

[0040] Hereinafter, specific embodiments of the present invention will be described with reference to the accompanying drawings to clarify the present invention.

[0041] The present invention relates to a resin film formed of a cell culture scaffold material. The cell culture scaffold material comprises a synthetic resin. The resin film of the present invention has a phase separation structure comprising 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 is greater than 0.01 and less than 0.95.

[0042] The resin film of the present invention has the above-mentioned structure, and thus has excellent fixation after cell seeding, and can increase the cell proliferation rate.

[0043] The cell culture scaffold materials using the conventional natural polymer materials can improve the fixation of cells after inoculation, but the price is relatively high, and the batch-to-batch variability is large due to the natural source material, or there are safety risks due to the animal-derived components. On the other hand, in the case of conventional scaffold materials using synthetic resins, the synthetic resins swell excessively, or the affinity with cells is reduced, so that cell blocks sometimes peel off during culture. Therefore, in the case of conventional scaffold materials using synthetic resins, the fixation of cells after inoculation is low, and cells sometimes cannot fully proliferate.

[0044] The present inventors focused on the phase separation structure of the resin film formed by the cell culture scaffold material and found that: by adopting a phase separation structure in which the ratio of the surface area of ​​one of the first phase and the second phase to the entire surface is set to the specific range, the affinity with the cells can be improved, thereby improving the adhesion after inoculation and further improving the cell proliferation rate. The reason is not clear, but it is believed that in the case of having such a phase separation structure, energy distribution can be smoothly performed, and the existence position and existence ratio of the first phase and the second phase with different affinity and strength can be adjusted, so that the affinity is improved regardless of the type of cells, and the aggregation and adsorption effect of cells or cell surface proteins is achieved.

[0045] Therefore, the resin film of the present invention can improve the adhesion to cells after seeding and can increase the cell growth rate.

[0046] Furthermore, in the present invention, since synthetic resins can be used as described above, the present invention has good handleability, low price, less batch-to-batch variation, and excellent safety compared to scaffold materials using natural polymer materials.

[0047] It should be noted that, in the present invention, as the synthetic resin, a synthetic resin having a peptide portion can be used. The details of the synthetic resin having a peptide portion are as follows.

[0048] In the present invention, the ratio of the surface area of ​​one of the first phase and the second phase to the entire surface (surface area fraction) 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 fraction is within the above range, the adhesion to cells after inoculation can be further improved, and the cell proliferation rate can be further improved.

[0049] In the present invention, as the phase separation structure, for example, microphase separation structures such as sea island structure, cylindrical structure, gyro structure and layered structure can be cited. In the sea island structure, for example, the first phase can be set as the sea part, and the second phase can be set as the island part. In the cylindrical structure, gyro structure or layered structure, for example, the phase with the largest surface area can be set as the first phase, and the phase with the second largest surface area can be set as the second phase. Among these, the sea island structure is preferred as the phase separation structure. Thus, by having a continuous phase and a discontinuous phase, the affinity with cells is improved, and the adhesion with cells after inoculation can be further improved, thereby further improving the proliferation rate of cells.

[0050] When the phase separation structure is a sea-island structure, the surface area fraction of the second phase relative to the entire surface is 0.01 or more, preferably 0.1 or more, more preferably 0.2 or more, and is 0.95 or less, preferably 0.9 or less, and more preferably 0.8 or less. When the surface area fraction is within the above range, the adhesion to the cells after inoculation can be further improved, and the cell proliferation rate can be further improved.

[0051] The ratio of the perimeter of the second phase to the area (perimeter / area) is preferably 0.001 (1 / nm) or more, more preferably 0.0015 (1 / nm) or more, and further preferably 0.008 (1 / nm) or more. The ratio of the perimeter of the second phase to the area (perimeter / area) is preferably 0.40 (1 / nm) or less, more preferably 0.20 (1 / nm) or less, further preferably 0.08 (1 / nm) or less, and particularly preferably 0.013 (1 / nm) or less. When the ratio (perimeter / area) is within the range, the adhesion to the cells after inoculation can be further improved, and the proliferation rate of the cells can be further improved.

[0052] When the synthetic resin does not have a peptide portion, the ratio of the perimeter of the second phase to the area (perimeter / area) is preferably 0.001 (1 / nm) or more, more preferably 0.0015 (1 / nm) or more, 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, the adhesion to the cells after inoculation can be further improved, and the proliferation rate of the cells can be further improved.

[0053] When the synthetic resin has a peptide portion, the ratio of the perimeter of the second phase to the area (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 further preferably 0.10 (1 / nm) or less. When the ratio (perimeter / area) is within the above range, the adhesion to the cells after inoculation can be further improved, and the proliferation rate of the cells can be further improved.

[0054] The number of the second phase as the island portion is preferably 1 / μm 2 More than 2 pieces / μm 2 More than 10 / μm is more preferable 2 More than 5000 pieces / μm is preferred 2 Below, more preferably 1000 pieces / μm 2 Less than 500 / μm, more preferably less than 500 / μm 2 , particularly preferably 300 / μm 2 In this case, the adhesion to the cells after seeding can be further improved, and the cell proliferation rate can be further improved.

[0055] The average diameter of the second phase as the island is preferably 20 nm or more, more preferably 30 nm or more, further preferably 50 nm or more, particularly preferably 80 nm or more, preferably 3.5 μm or less, more preferably 3.0 μm or less, and further preferably 1.5 μm or less. When the average diameter of the second phase is within the above range, the adhesion to the cells after inoculation can be further improved, and the proliferation rate of the cells can be further improved.

[0056] When the synthetic resin does not have a peptide portion, the average diameter of the second phase as the island portion is preferably 50 nm or more, more preferably 100 nm or more, further preferably 120 nm or more, particularly preferably 200 nm or more, preferably 1 μm or less, more preferably 300 nm or less, and further preferably 250 nm or less. When the average diameter of the second phase is within the above range, the adhesion to the cells after inoculation can be further improved, and the proliferation rate of the cells can be further improved.

[0057] When the synthetic resin has a peptide portion, the average diameter of the second phase as the island portion is preferably 10 nm or more, more preferably 20 nm or more, further preferably 40 nm or more, preferably 1 μm or less, more preferably 300 nm or less, further preferably 100 nm or less. When the average diameter of the second phase is within the above range, the adhesion to the cells after inoculation can be further improved, and the proliferation rate of the cells can be further improved.

[0058] The presence or absence of a phase separation structure and parameters representing the phase separation structure can be confirmed, for example, by an atomic force microscope (AFM), a transmission electron microscope (TEM), a scanning electron microscope (SEM), or the like.

[0059] Specifically, the ratio of the surface area of ​​one of the first phase and the second phase to the entire surface (surface area fraction), the ratio of the circumference and area of ​​the second phase (circumference / area), the number of the second phase as an island and its average diameter size can be obtained based on the microscope observation image and using image analysis software such as ImageJ.

[0060] The ratio of the surface area of ​​one of the first phase and the second phase to the entire surface (surface area fraction) can be calculated as follows: within the observation area (30 μm×30 μm), the surface area occupied by one of the first phase and the second phase is divided by the area of ​​the observation area.

[0061] The ratio of the perimeter to the area of ​​the second phase (perimeter / area) can be obtained by dividing the total perimeter of the second phase by the total area of ​​the second phase in the observation region (30 μm×30 μm).

[0062] When the phase separation structure is a sea-island structure, the number of the second phase as the island portion can be obtained by dividing the number of the second phase in the observation area (30 μm×30 μm) by the area of ​​the observation area. In addition, the average diameter of the second phase as the island portion is obtained as the average diameter of a circle of the same area.

[0063] In addition, the phase separation structure can be obtained, for example, by mixing, copolymerizing or graft copolymerizing at least two different polymers, or using a synthetic resin with a peptide portion to form a phase separation structure between or within the molecules of the synthetic resin. In particular, from the viewpoint of further improving the adhesion of cells, it is preferred that the phase separation structure is formed by an intramolecular phase separation structure. That is, the synthetic resin is preferably a copolymer of at least two different polymers or a synthetic resin with a peptide portion, more preferably a graft copolymer or a synthetic resin with a peptide portion.

[0064] The phase separation structure is preferably obtained by copolymerizing two or more different polymers (monomers) having solubility parameters (SP values) 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.

[0065] The SP value is a measure of the molecular force acting between the solvent and the solute, and is a measure of the affinity between substances. The SP value can be obtained based on the theory of canonical solution of Hidebrand. In addition, the SP value can be obtained based on literature information, and can be obtained by the calculation method of Hansen and Hoy, the estimation method of Fedors, etc. In this specification, it refers to the formula δ obtained by Fedors. 2=ΣE / ΣV (δ represents SP value, E represents evaporation energy, and V represents molar volume). It should be noted that the unit of SP value is (cal / cm 3 ) 0.5 The method of Fedors is recorded in the Journal of the Japan Adhesion Society, Vol. 22, 1986, p. 566.

[0066] It should be noted that phase separation parameters such as surface area fraction, which represent the phase separation structure, can be adjusted by controlling the mixing ratio of two polymers, the structure of the polymer, or the content of the peptide part, for example.

[0067] It should be noted that in the present invention, other phases different from the first phase and the second phase may exist. The other phase may be one phase or multiple phases. Such a phase can be obtained, for example, by copolymerizing another polymer (monomer) having a different SP value by grafting. In this case, the two phases occupying the larger area on the surface of the resin film are set as the first phase and the second phase.

[0068] When the synthetic resin does not have a peptide portion, the dispersion term component of the surface free energy in the resin film formed of the cell culture scaffold material is preferably 25.0 mJ / m 2 Above, 50.0mJ / m 2 In this case, the hydrophilicity of the cell culture scaffold material can be appropriately adjusted, and the interfacial adhesion with cells after inoculation can be further improved through the synergistic effect with the phase separation structure, which can further improve the cell proliferation rate. The dispersion term component is more preferably 30.0 mJ / m 2 More preferably, 35.0 mJ / m 2 More preferably, 47.0 mJ / m 2 Below, more preferably 45.5 mJ / m 2 the following.

[0069] When the synthetic resin does not have a peptide portion, the polar component of the surface free energy in the resin film formed of the cell culture scaffold material is preferably 1.0 mJ / m 2 Above, 20.0mJ / m 2 In this case, the adhesion to cells after inoculation is further improved, and the cell proliferation rate can be further increased. The polar term component is more preferably 2.0 mJ / m 2 More preferably, 3.0 mJ / m 2 More preferably, 10.0 mJ / m 2 Below, more preferably 5.0 mJ / m 2 the following.

[0070] It should be noted that the dispersion component γ of the surface free energyd and the dipole component γ as the polar term p , calculated using the Kaelble-Uy theory. The Kaelble-Uy theory, as shown in the following formula (1), is based on the total surface free energy γ as the dispersion term component γ d and the dipole component γ p The assumed theoretical formula for the sum.

[0071] [Mathematical formula 1]

[0072] γ=γ d +γ p (1)

[0073] In addition, in the Kaelble-Uy theory, the surface free energy of the liquid is set to γ l (mJ / m 2 ), the surface free energy of the solid is set to γ s (mJ / m 2 ), when the contact angle is θ(°), the following equation (2) holds true.

[0074] [Mathematical formula 2]

[0075]

[0076] Therefore, the surface free energy γ of the liquid can be used l The contact angle θ of two known liquids on the resin film formed by the cell culture scaffold material is measured, and γ is solved. s d and γ s p The dispersion term component γ of the surface free energy of the resin film formed by the cell culture scaffold material is obtained by using the simultaneous equations of d and the dipole component γ p .

[0077] It should be noted that, in this specification, the surface free energy γ l As two known liquids, pure water and diiodomethane are used.

[0078] The contact angle θ is measured in the following manner using a contact angle meter (for example, "DMo-701" manufactured by Kyowa Interface Chemical Co., Ltd.).

[0079] 1 μL of pure water or diiodomethane was dripped onto the surface of the resin film formed by the cell culture scaffold material. The angle formed by the pure water and the resin film 30 seconds after the dripping was set as the contact angle θ for pure water. Similarly, the angle formed by the diiodomethane and the resin film 30 seconds after the dripping was set as the contact angle θ for diiodomethane.

[0080] The dispersion term component γ of the surface free energy can be reduced by increasing the content of hydrophobic functional groups in the synthetic resin, increasing the content of functional groups having a cyclic structure, or reducing the content of butyl groups. d In addition, by increasing the content of hydrophilic functional groups in the synthetic resin or increasing the content of butyl groups, the dipole component γ of the surface free energy can be reduced. p .

[0081] The resin film formed of the cell culture scaffold material of the present invention preferably has a storage elastic modulus at 100°C of 0.6×10 4 Pa or more, 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, more preferably 0.8×10 8 Pa or less, more preferably 1.0×10 7 Below Pa.

[0082] In particular, the ratio of the storage elastic modulus at 25°C to the storage elastic modulus at 100°C ((storage elastic modulus at 25°C) / (storage elastic modulus at 100°C)) of the resin film formed of the cell culture scaffold material of the present invention is preferably 1.0×10 1 More preferably, 5.0×10 1 More preferably, 8.0×10 2 More than, and preferably 1.0×10 5 Below, more preferably 0.75×10 5 Below, more preferably 0.5×10 5 By setting the storage elastic modulus within the above range, the fixation of cells after seeding can be further improved.

[0083] It should be noted that the storage modulus at 25°C and 100°C can be measured, for example, by a dynamic viscoelasticity measuring device (manufactured by IT Measurement and Control Co., Ltd., DVA-200) under tensile conditions at a frequency of 10 Hz and a heating rate of 5°C / min in a temperature range of -150°C to 150°C. According to the obtained graph of the tensile storage modulus, the storage modulus at 25°C and 100°C is obtained, and the 25°C storage modulus / 100°C storage modulus is calculated. 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 is used, and the measurement is carried out under the conditions of 10 Hz, a strain of 0.1%, a temperature of -150°C to 150°C, and a heating rate of 5°C / min.

[0084] The storage elastic modulus at 25°C and 100°C can be increased, for example, by increasing the degree of crosslinking in the synthetic resin, stretching the synthetic resin, etc. In addition, the storage elastic modulus at 25°C and 100°C can be reduced by reducing the number average molecular weight in the synthetic resin, reducing the glass transition temperature, etc.

[0085] The resin film formed by the scaffold material for cell culture of the present invention preferably has a water swelling ratio of 50% or less, and more preferably 40% or less. In this case, the fixation of the cells after inoculation can be further improved. It should be noted that the lower limit of the water swelling ratio is not particularly limited, for example, it can be set to 0.5%. The water swelling ratio can be measured in the following manner. For example, a resin film (measurement sample) formed by a scaffold material for cell culture with a length of 50 mm, a width of 10 mm, and a thickness of 0.05 mm to 0.15 mm is immersed in water at 25°C for 24 hours. The weight of the sample before and after immersion is measured, and the water swelling ratio is calculated = (sample weight after immersion - sample weight before immersion) / (sample weight before immersion) × 100 (%).

[0086] The water swelling ratio can be reduced, for example, by increasing the hydrophobic functional groups of the synthetic resin, reducing the number average molecular weight, and the like.

[0087] (Synthetic resin)

[0088] The cell culture scaffold material comprises a synthetic resin (hereinafter, sometimes described as synthetic resin X). The main chain of synthetic resin X is preferably a carbon chain. It should be noted that in this specification, "structural unit" refers to a repeating unit of a monomer constituting the synthetic resin. It should be noted that when the synthetic resin has a graft chain, it includes a repeating unit of a monomer constituting the graft chain.

[0089] When the synthetic resin X does not have a peptide portion, the synthetic resin X preferably has a cationic functional group. When the synthetic resin X has a peptide portion, the synthetic resin X having the 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 structures such as an amino group, an imino group, and an amide group. For example, there are no particular limitations, and examples thereof include conjugated amine functional groups such as hydroxyamino, urea, guanidine, and biguanide, heterocyclic amino functional groups such as piperazine, piperidine, tetrahydropyrrole, 1,4-diazabicyclo[2.2.2]octane, hexamethylenetetramine, morpholine, pyridine, pyridazine, pyrimidine, pyrazine, pyrrole, azacycloheptatriene, 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. These cationic functional groups may be used alone or in combination of two or more.

[0090] In the present invention, the content of the cationic functional group contained in the structural unit of the synthetic resin X is preferably 0.2 mol% or more, preferably 2 mol% or more, more preferably 3 mol% or more, and is 50 mol% or less, preferably 10 mol% or less, more preferably 7 mol% or less. By using the synthetic resin X containing the cationic functional group within such a range, the fixation of the cells after inoculation can be further improved, and the cell proliferation rate can be further improved. It should be noted that the content of the cationic functional group can be, for example, 1 H-NMR (nuclear magnetic resonance spectroscopy) was used for measurement.

[0091] (Vinyl polymer)

[0092] The synthetic resin X preferably comprises a vinyl polymer, more preferably a vinyl polymer. It should be noted that a vinyl polymer refers to a polymer of a compound having a vinyl or vinylidene group. When the synthetic resin X is a vinyl polymer, the swelling of the cell culture scaffold material in water can be further suppressed. As the vinyl polymer, for example, polyvinyl alcohol derivatives, poly (meth) acrylates, polyvinyl pyrrolidone, polystyrene, ethylene-vinyl acetate copolymers, etc. can be cited. In addition, as the vinyl polymer, from the viewpoint of being easy to improve the adhesion to the cell, preferably a polyvinyl alcohol derivative or poly (meth) acrylates.

[0093] (Synthetic resin X having a polyvinyl acetal skeleton)

[0094] The scaffold material for cell culture preferably comprises 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 and a vinyl compound and / or a vinylidene compound. A vinyl compound is a compound having a vinyl group (H2C=CH-). A vinylidene compound is a compound having a vinylidene group (H2C=CR-). A vinyl compound or a vinylidene compound may be a vinyl polymer as a polymer thereof. It should be noted that in the following description, vinyl compounds, vinylidene compounds and vinyl polymers copolymerized with polyvinyl acetal resins are sometimes collectively referred to as "vinyl compound A".

[0095] 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 obtained by grafting a vinyl compound A to a polyvinyl acetal resin. The copolymer is preferably a graft copolymer. In this case, a phase separation structure can be further easily formed.

[0096] As the vinyl compound and vinylidene compound, ethylene, allylamine, vinyl pyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth) acrylic acid, vinylamine and (meth) acrylate, etc., can be mentioned. These vinyl compounds can be used alone or in combination of two or more. Therefore, it can be a vinyl polymer obtained by copolymerizing these vinyl compounds.

[0097] 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 separation 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. It should be noted that the upper limit of the difference in SP value is not particularly limited, and for example, can be set to 10.0.

[0098] In the copolymer, the first phase is preferably a polyvinyl acetal resin, and the second phase is preferably a vinyl compound A. It is preferred that the first phase is formed by the polyvinyl acetal resin portion of the copolymer, and the second phase is formed by the vinyl compound A portion. In this case, it is preferred that the first phase of the polyvinyl acetal resin is a sea portion, and the second phase of the vinyl compound A is 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.

[0099] The content fraction (mol / mol) of the 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 further preferably 0.70 or less. When the content fraction is above the lower limit, a phase separation structure can be further easily formed. When the content fraction is below the upper limit, the proliferation rate of cells can be further increased.

[0100] <Polyvinyl acetal resin>

[0101] Hereinafter, the polyvinyl acetal resin (the polyvinyl acetal resin portion of the copolymer) will be described in more detail.

[0102] The polyvinyl acetal resin has an acetal group, an acetyl group, and a hydroxyl group in a side chain.

[0103] The method for synthesizing a polyvinyl acetal resin at least comprises a step of acetalizing polyvinyl alcohol with an aldehyde.

[0104] The aldehyde used in the acetalization of polyvinyl alcohol for obtaining the polyvinyl acetal resin is not particularly limited. As the aldehyde, for example, an aldehyde having 1 to 10 carbon atoms can be mentioned. 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.

[0105] Examples of the aldehyde include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, hexanal, heptaldehyde, octanal, nonanal, decanal, acrolein, benzaldehyde, cinnamaldehyde, perillaldehyde, formylpyridine, formylimidazole, formylpyrrole, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formylisoindole, formylpurine, formylbenzimidazole, formylbenzotriazole, formylquinoline, formylisoquinoline, formylquinoxaline, formylcinnamylline, formylpteridine, formylfuran, formyloxolane, formyloxane, formylthiophene, formyltetrahydrothiophene, formylcyclopentanesulfide, formyladenine, formylguanine, formylcytosine, formylthymine or formyluracil. The aldehydes may be used alone or in combination of two or more.

[0106] The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde or valeraldehyde, more preferably butyraldehyde. Therefore, the polyvinyl acetal skeleton is preferably a polyvinyl butyral skeleton. The polyvinyl acetal resin is preferably a polyvinyl butyral resin.

[0107] From the viewpoint of further improving the cell adhesion, the polyvinyl acetal resin preferably has a Bronsted basic group or a Bronsted acidic group, and more preferably has a Bronsted basic group. 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.

[0108] Brønsted basic groups are those that can accept hydrogen ions H from other substances. + Examples of the Brønsted basic group include amine basic groups such as a substituent having an imide structure, a substituent having an imide structure, a substituent having an amine structure, or a substituent having an amide structure. The Brønsted basic group is not particularly limited, and examples thereof include conjugated amine functional groups such as hydroxyamino, urea, guanidine, and biguanide, heterocyclic amino functional groups such as piperazine, piperidine, tetrahydropyrrole, 1,4-diazabicyclo[2.2.2]octane, hexamethylenetetramine, morpholine, pyridine, pyridazine, pyrimidine, pyrazine, pyrrole, azacycloheptatriene, 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.

[0109] 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. The Bronsted acidic group is preferably a carboxyl group.

[0110] The polyvinyl acetal resin preferably contains a structural unit having an imide structure, a structural unit having an imide structure, a structural unit having an amine structure, or a structural unit having an amide structure. In this case, the polyvinyl acetal resin may contain only one of these structural units or two or more of them.

[0111] The polyvinyl acetal resin may contain 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.

[0112] The polyvinyl acetal resin may contain a structural unit having an imide structure. The structural unit having an imide structure is preferably a structural unit having an imino group (=NH).

[0113] 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.

[0114] The polyvinyl acetal resin may contain a structural unit having an amine structure. 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.

[0115] The structural unit having an amine structure may be a structural unit having an amide structure. The amide structure refers to a structure having -C(=O)-NH-.

[0116] 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.

[0117] It should be noted that the content of the structural unit having an imide 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 can be obtained by 1 H-NMR (nuclear magnetic resonance spectroscopy) determination.

[0118] <Vinyl Compound A>

[0119] Hereinafter, the vinyl compound A will be described in more detail.

[0120] The vinyl compound A is preferably a (meth)acrylate or a poly(meth)acrylate resin. In particular, the synthetic resin X is preferably a copolymer obtained by graft-copolymerizing a (meth)acrylate or a poly(meth)acrylate resin as a polymer thereof with a polyvinyl acetal resin.

[0121] The poly(meth)acrylate resin can be obtained by polymerization of (meth)acrylate or polymerization of (meth)acrylate and the above-mentioned other monomers.

[0122] Examples of the (meth)acrylate include alkyl (meth)acrylates, cyclic alkyl (meth)acrylates, aryl (meth)acrylates, (meth)acrylamides, polyethylene glycol (meth)acrylates, and phosphorylcholine (meth)acrylates.

[0123] 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.

[0124] The alkyl (meth)acrylate may be substituted with various substituents such as an alkoxy group having 1 to 3 carbon atoms and a tetrahydrofurfuryl group. Examples of such alkyl (meth)acrylate include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0125] Examples of the cycloalkyl (meth)acrylate include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0126] As the aryl (meth)acrylate, there can be mentioned phenyl (meth)acrylate, benzyl (meth)acrylate, and the like.

[0127] Examples of the (meth)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.

[0128] 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.

[0129] Examples of (meth)acrylphosphocholine include 2-(meth)acryloyloxyethylphosphocholine.

[0130] As other monomers copolymerized with (meth)acrylate, vinyl compounds are preferably used. Examples of the vinyl compound include ethylene, allylamine, vinyl pyrrolidone, vinyl imidazole, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, or (meth)acrylate. The vinyl compound may be used alone or in combination of two or more.

[0131] In addition, in this specification, "(meth)acrylic acid" means "acrylic acid" or "methacrylic acid", and "(meth)acrylate" means "acrylate" or "methacrylate".

[0132] It should be noted that in the present invention, the synthetic resin X may be a copolymer of a resin having a poly(meth)acrylate skeleton and other vinyl compounds as long as the phase separation structure of the present invention can be formed.

[0133] As other vinyl compounds in this case, ethylene, allylamine, vinyl pyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, or other (meth)acrylates having different SP values ​​may be used.

[0134] (Synthetic resin X having a peptide moiety)

[0135] The cell culture scaffold material preferably comprises a synthetic resin X having a peptide portion. The synthetic resin X having a peptide portion can be obtained by reacting a synthetic resin X, a linker, and a peptide. The synthetic resin X having a peptide portion is preferably a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion, a linker portion, and a peptide portion, and more preferably a peptide-containing polyvinyl butyral resin having a polyvinyl butyral resin portion, a linker portion, and a peptide portion. The synthetic resin X having a peptide portion can be used alone or in combination of two or more.

[0136] The peptide portion is preferably composed of 3 or more amino acids, more preferably 4 or more amino acids, further preferably 5 or more amino acids, and preferably 10 or less amino acids, more preferably 6 or less amino acids. When the number of amino acids constituting the peptide portion is above the lower limit and below the upper limit, the adhesion to cells after inoculation can be further improved, and the cell proliferation rate can be further improved.

[0137] The peptide portion preferably has an amino acid sequence with cell adhesion. It should be noted that the amino acid sequence with cell adhesion refers to an amino acid sequence whose cell adhesion activity has been confirmed by a phage display method, an agarose bead method or a plate coating method. As the phage display method, for example, the method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995 1189-1196" can be used. As the agarose bead method, for example, the method described in "Protein Nuclease Vol.45No.15(2000)2477" can be used. As the plate coating method, for example, the method described in "Protein Nuclease Vol.45No.15(2000)2477" can be used.

[0138] As the cell adhesive amino acid sequence, for example, there can be mentioned: 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). In addition, examples of the cell adhesive amino acid sequence include sequences described in "Patient Physiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Journal of Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, pp. 58-66, 1992". The peptide portion may have only one cell adhesive amino acid sequence or may have two or more.

[0139] 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 further preferably has at least an RGD sequence represented by the following formula (1). In this case, the adhesion to cells after inoculation can be further improved, and the cell proliferation rate can be further improved.

[0140] Arg-Gly-Asp-X… Formula (1)

[0141] In the formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro or Asn.

[0142] The peptide portion may be linear or may have a cyclic peptide backbone. The cyclic peptide backbone refers to a cyclic backbone composed of a plurality of amino acids. From the viewpoint of further effectively exerting the effect of the present invention, the cyclic peptide backbone is preferably composed of 4 or more amino acids, preferably composed of 5 or more amino acids, and preferably composed of 10 or less amino acids.

[0143] In the synthetic resin X with a peptide portion, the content of the peptide portion is preferably 0.1 mol% or more, more preferably 1 mol% or more, further preferably 5 mol% or more, and particularly preferably 10 mol% or more. In the synthetic resin X with a peptide portion, the content of the peptide portion is preferably 60 mol% or less, more preferably 50 mol% or less, further preferably 35 mol% or less, and particularly preferably 25 mol% or less. When the content of the peptide portion is above the lower limit, a phase separation structure can be further easily formed. When the content of the peptide portion is above the lower limit, the adhesion to the cell after inoculation can be further improved, and the proliferation rate of the cell can be further improved. In addition, when the content of the peptide portion is below the upper limit, the manufacturing cost can be suppressed. It should be noted that the content (mol%) of the peptide portion is the ratio of the amount of the peptide portion relative to the sum of the amount of each structural unit constituting the synthetic resin X with a peptide portion.

[0144] The content of the peptide portion can be measured by FT-IR or LC-MS.

[0145] From the viewpoint of further improving the adhesion to cells after inoculation and the viewpoint of further improving the proliferation rate of cells, in the synthetic resin X having a peptide portion, it is preferred that the second phase has a peptide portion, and more preferably the peptide portion has an amino acid sequence of the cell adhesion. It is preferred that the second phase is formed by the peptide portion of the synthetic resin X having a peptide portion. In this case, the second phase having a peptide portion is preferably 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.

[0146] 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. The linker may be used alone or in combination of two or more.

[0147] The linker is preferably a compound having a functional group that can condense with the carboxyl group or amino group of the peptide. As the functional group that can condense with the carboxyl group or amino group of the peptide, a carboxyl group, a thiol group and an amino group can be mentioned. From the viewpoint of good reaction with the peptide, the linker is preferably a compound having a carboxyl group. As the linker, the vinyl compound A can be used.

[0148] As the linker having a carboxyl group, (meth) acrylic acid and carboxyl-containing acrylamide, etc. can be cited. 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 reacts with the synthetic resin X, thereby increasing the number of carboxyl groups that can react with the peptide.

[0149] [Scaffold materials for cell culture]

[0150] The cell culture scaffold material contains the synthetic resin X. From the viewpoint of effectively exerting the effect of the present invention and the viewpoint of improving productivity, the content of the synthetic resin X in 100% by weight of the cell culture scaffold material is preferably 90% by weight or more, more preferably 95% by weight or more, further preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). Therefore, the cell culture scaffold material is most preferably the synthetic resin X. When the content of the synthetic resin X is above the lower limit, the effect of the present invention can be further effectively exerted.

[0151] The cell culture scaffold material may contain components other than the synthetic resin X. 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, synthetic peptides, and the like.

[0152] From the viewpoint of effectively exerting the effects of the present invention, the less the content of the components other than the synthetic resin X, the better. In 100% by weight of the cell culture scaffold material, the content of the component is preferably 10% by weight or less, more preferably 5% by weight or less, further preferably 2.5% by weight or less, particularly preferably 1% by weight or less, and most preferably 0% by weight (no content). Therefore, the cell culture scaffold material most preferably does not contain components other than the synthetic resin X.

[0153] The cell culture scaffold material preferably does not actually contain raw materials of animal origin. Since it does not contain raw materials of animal origin, it is possible to provide a cell culture scaffold material with high safety and less uneven quality during manufacturing. It should be noted that "does not actually contain raw materials of animal origin" means that the raw materials of animal origin in the cell culture scaffold material are 3% by weight or less. In the cell culture scaffold material, the raw materials of animal origin in the cell culture scaffold material are preferably 1% by weight or less, and more preferably 0% by weight. That is, the cell culture scaffold material, more preferably, does not contain raw materials of animal origin at all.

[0154] (Cell culture using cell culture scaffold material)

[0155] The cell culture scaffold material is used for culturing cells. The cell culture scaffold material is used as a scaffold for the cells when culturing cells. Therefore, the resin film formed by the cell culture scaffold material of the present invention is used for culturing cells and is used as a scaffold for the cells when culturing cells.

[0156] Examples of the cells include animal cells of humans, mice, rats, pigs, cattle, monkeys, etc. In addition, examples of the cells include somatic cells, such as stem cells, progenitor cells, and mature cells. The somatic cells may be cancer cells.

[0157] Examples of the mature cells include nerve cells, cardiomyocytes, retinal cells, and hepatocytes.

[0158] Examples of the stem cells include mesenchymal stem cells (MSC), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0159] (Shape of scaffold material for cell culture)

[0160] The resin film of the present invention is formed by a cell culture scaffold material. The resin film is formed using a cell culture scaffold material. The resin film is preferably a film-shaped cell culture scaffold material. The resin film is preferably a film-shaped object of a cell culture scaffold material.

[0161] In this specification, particles, fibers, porous bodies or films containing the cell culture scaffold material are provided. In this case, the shape of the cell culture scaffold material is not particularly limited, and can be particles, fibers, porous bodies, or films. It should be noted that the particles, fibers, porous bodies or films may contain constituent elements other than the cell culture scaffold material.

[0162] The membrane containing the cell culture scaffold material is preferably used for planar culture (two-dimensional culture) of cells. In addition, the particles, fibers or porous bodies containing the cell culture scaffold material are preferably used for three-dimensional culture of cells.

[0163] (Carrier for cell culture)

[0164] The present invention relates to a cell culture carrier in which the resin film is arranged on the surface of the carrier. The cell culture carrier of the present invention can be obtained, for example, by arranging the resin film on the surface of the carrier by coating or the like. The shape of the carrier can be particles, fibers, porous bodies or films. That is, the cell culture carrier of the present invention can have the shape of particles, fibers, porous bodies or films. It should be noted that the cell culture carrier of the present invention may include components other than the carrier and the resin film.

[0165] (Cell culture container)

[0166] The present invention relates to a cell culture container including the resin film in at least a part of a cell culture region. Figure 1 This is a cross-sectional view schematically showing a cell culture container according to one embodiment of the present invention.

[0167] The cell culture container 1 includes a container body 2 and a resin film 3 formed of a cell culture scaffold material. The resin film 3 is arranged on a surface 2a of the container body 2. The resin film 3 is arranged on the bottom surface of the container body 2. Liquid culture medium is added to the cell culture container 1, and cells are seeded on the surface of the resin film 3, so that cells can be cultured in a flat surface.

[0168] It should be noted that 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 of the cell culture scaffold material may be arranged on the surface of the second container body.

[0169] As the container body, a conventionally known container body (container) can be used. The shape and size of the container body are not particularly limited.

[0170] As the container body, a cell culture plate and a cell culture flask having one or more holes (holes) can be cited. The number of holes in the plate is not particularly limited. As the number of holes, there is no particular limitation, for example, 2, 4, 6, 12, 24, 48, 96, 384, etc. can be cited. As the shape of the hole, there is no particular limitation, and the shape can be a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, etc. As the shape of the bottom surface of the hole, there is no particular limitation, and the shape can be a flat bottom, a round bottom, a concave-convex, etc.

[0171] The material of the container body is not particularly limited, and examples thereof include resins, metals, and inorganic materials. Examples of the 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. Example

[0172] Next, the present invention will be explained by giving specific examples and comparative examples of the present invention. It should be noted that the present invention is not limited to the following examples.

[0173] As raw materials for the cell culture scaffold material, the following synthetic resins were synthesized.

[0174] (Example 1)

[0175] Into a reactor equipped with a stirring device, 2700 mL of ion exchange water, 300 parts by weight of polyvinyl alcohol having an average degree of polymerization of 1700 and a saponification degree of 98 mol% are added, and heated to dissolve while stirring to obtain a solution. To the obtained solution, 35% by weight of hydrochloric acid is added as a catalyst so that the hydrochloric acid concentration is 0.2% by weight. Next, the temperature is adjusted to 15°C, and 22 parts by weight of n-butyraldehyde is added while stirring. Next, 148 parts by weight of n-butyraldehyde is added to precipitate a polyvinyl butyral resin in the form of white particles. After precipitation for 15 minutes, 35% by weight of hydrochloric acid is added so that the hydrochloric acid concentration is 1.8% by weight, and then heated to 50°C and maintained at 50°C for 2 hours. Next, the solution is cooled, neutralized, washed with water, and dried to obtain a polyvinyl butyral resin (PVB, SP value: 9.9) as a polyvinyl alcohol acetal resin. 90 parts by weight of the obtained polyvinyl butyral resin was dissolved in tetrahydrofuran to form a 1% by weight solution, and 5 parts by weight of Irgacure 184, 2 parts by weight of N-vinyl pyrrolidone (SP value: 11.7) and 8 parts by weight of n-lauryl methacrylate (SP value: 8.2) as an initiator were added to carry out graft polymerization to obtain a synthetic resin. The obtained synthetic resin had an acetalization degree (butyralization degree) of 69 mol%, a hydroxyl group amount of 27.5 mol%, an acetylation degree of 2.0 mol%, a vinyl pyrrolidone group content of 0.3 mol%, and a n-lauryl methacrylate moiety content of 1.2 mol%.

[0176] (Examples 2 to 11 and Comparative Example 1)

[0177] Except for changing the weight ratio of polyvinyl butyral resin, N-vinyl pyrrolidone and n-lauryl methacrylate, synthetic resins were obtained in the same manner as in Example 1. The acetalization degree (butyralization degree), hydroxyl amount, acetylation degree, vinyl pyrrolidone 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.

[0178] (Example 12)

[0179] Into a reactor equipped with a stirring device, 2700 mL of ion exchange water, 300 parts by weight of polyvinyl alcohol having an average degree of polymerization of 1700 and a saponification degree of 99 mol% are added, and heated to dissolve while stirring to obtain a solution. To the obtained solution, 35% by weight of hydrochloric acid is added as a catalyst so that the hydrochloric acid concentration is 0.2% by weight. Next, the temperature is adjusted to 15°C, and 22 parts by weight of n-butyraldehyde are added while stirring. Next, 148 parts by weight of n-butyraldehyde is added to precipitate white granular polyvinyl alcohol acetal resin (polyvinyl alcohol butyral resin). After precipitation for 15 minutes, 35% by weight of hydrochloric acid is added so that the hydrochloric acid concentration is 1.8% by weight, and then heated to 50°C and kept at 50°C for 2 hours. Next, the solution was cooled, neutralized, and 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 (butyralization degree) 70 mol%, hydroxyl content 27 mol%, acetylation degree 3 mol%).

[0180] Import of connector:

[0181] 99 parts by weight of the obtained polyvinyl acetal resin and 1 part by weight of acrylic acid (joint) were dissolved in 300 parts by weight of THF, and reacted for 20 minutes under ultraviolet irradiation in the presence of a photo-radical polymerization initiator to graft copolymerize the polyvinyl acetal resin and acrylic acid, thereby introducing a joint. 1 part by weight of the polyvinyl acetal resin with the joint introduced was dissolved in 19 parts by weight of butanol. 150 μL of the obtained solution was cast on the surface of a φ22 mm cover glass ("22 pellets No. 1" manufactured by Matsunami Co., Ltd.) that had been dusted with a dust collector, and after rotating at 2000 rpm for 20 seconds using a spin coater, it was heated at 60°C for 60 minutes to obtain a resin film with a smooth surface.

[0182] Formation of the peptide part:

[0183] Prepare a linear peptide having an amino acid sequence of Gly-Arg-Gly-Asp-Ser (5 amino acid residues, recorded as GRGDS in the table). Add 10 parts by weight of the peptide and 1 part by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) to a phosphate-buffered saline solution that does not contain calcium and magnesium so that the final concentration of the peptide is 1 mM to prepare a peptide-containing solution. Add 1 part by weight of the peptide-containing solution to the resin film obtained by spin coating (polyvinyl acetal resin with a linker formed therein) to react so that the carboxyl group of the linker and the amino group of Gly of the peptide undergo dehydration condensation. Thus, a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion, a linker portion, and a peptide portion is prepared.

[0184] 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%.

[0185] (Example 13)

[0186] In the introduction of the linker, 85 parts by weight of polyvinyl acetal resin and 15 parts by weight of acrylic acid (linker) were used. In the formation of the peptide, the peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that the amount of peptide added was changed to 15 parts by weight.

[0187] (Example 14)

[0188] In the introduction of the linker, 70 parts by weight of polyvinyl acetal resin and 30 parts by weight of acrylic acid (linker) were used. In the formation of the peptide, the peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that the amount of peptide added was changed to 30 parts by weight.

[0189] (Example 15)

[0190] In the introduction of the linker, 67 parts by weight of polyvinyl acetal resin and 33 parts by weight of acrylic acid (linker) were used. In the formation of the peptide, the peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that the amount of peptide added was changed to 33 parts by weight.

[0191] (Example 16)

[0192] In the introduction of the linker, 63 parts by weight of polyvinyl acetal resin and 37 parts by weight of acrylic acid (linker) were used. In the formation of the peptide, the peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that the amount of peptide added was changed to 37 parts by weight.

[0193] (Example 17)

[0194] In the introduction of the linker, 30 parts by weight of polyvinyl acetal resin and 70 parts by weight of acrylic acid (linker) were used. In the formation of the peptide, the peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 12, except that the amount of peptide added was changed to 70 parts by weight.

[0195] (Comparative Example 2)

[0196] As the synthetic resin, polystyrene resin was used as it was.

[0197] (Comparative Example 3)

[0198] Into a reactor equipped with a stirring device, 2700 mL of ion exchange water, 300 parts by weight of polyvinyl alcohol having an average degree of polymerization of 1700 and a saponification degree of 98 mol% are added, and heated to dissolve while stirring to obtain a solution. To the obtained solution, 35% by weight of hydrochloric acid is added as a catalyst so that the hydrochloric acid concentration is 0.2% by weight. Next, the temperature is adjusted to 15°C, and 22 parts by weight of n-butyraldehyde is added while stirring. Next, 148 parts by weight of n-butyraldehyde is added to precipitate a polyvinyl butyral resin in the form of white particles. After precipitation for 15 minutes, 35% by weight of hydrochloric acid is added so that the hydrochloric acid concentration is 1.8% by weight, and then heated to 50°C and maintained at 50°C for 2 hours. Next, the solution is cooled, neutralized, washed with water, and dried to obtain a polyvinyl butyral resin (SP value: 9.9). That is, a polyvinyl butyral resin (synthetic resin) without copolymerization of a vinyl compound is obtained.

[0199] (Comparative Example 4)

[0200] 17 parts by weight of N-vinyl pyrrolidone and 83 parts by weight of n-lauryl methacrylate were mixed to obtain a (meth)acrylic acid monomer solution. 1 part by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained (meth)acrylic acid monomer solution and applied on a PET film. The coated material 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 poly(meth)acrylate resin solution was obtained by irradiating with light having a wavelength of 365 nm. The obtained poly(meth)acrylate resin solution was vacuum dried at 80° C. for 3 hours to obtain a synthetic resin as a poly(meth)acrylate resin.

[0201] (Reference Example A)

[0202] Preparation of scaffold materials from natural substances:

[0203] 1 ml of a solution of Vitronectin (Corning) adjusted to 5 μg / ml in phosphate buffer (PBS) was added to a φ35 mm dish. A φ22 mm cover glass (Matsunami "22 pills No. 1") was immersed therein, and the mixture was cured at 37°C for 1 hour to obtain a natural material-derived scaffold material (recorded as VTN in the table) with Vitronectin smoothly adsorbed on the surface.

[0204] Preparation of cell culture vessels:

[0205] The laminate of Vitronectin and the cover glass was placed in a φ22 mm polystyrene dish to obtain a cell culture container. It should be noted that Vitronectin undergoes modification during drying, which significantly reduces the adhesiveness, so the cell culture container was immersed in a PBS solution immediately after preparation.

[0206] [evaluate]

[0207] (Degree of acetalization and degree of cationic group modification)

[0208] The acetalization degree and cationic group modification degree of the synthetic resins obtained in the examples and comparative examples can be determined by dissolving the synthetic resins in DMSO-d6 (dimethyl sulfoxide) and then using 1 H-NMR (nuclear magnetic resonance spectroscopy) determination.

[0209] (Storage elastic modulus)

[0210] The storage elastic modulus of each scaffold material at 25°C and 100°C was measured by a dynamic viscoelasticity measuring device (manufactured by IT Measurement and Control Co., Ltd., DVA-200) under tensile conditions at a frequency of 10 Hz, in a temperature range of -150°C to 150°C, and a heating rate of 5°C / min. Based on the obtained graph of the tensile storage elastic modulus, the storage elastic modulus at 25°C and 100°C was obtained, and the 25°C storage elastic modulus / 100°C storage elastic modulus was calculated. 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 was used, and the conditions were 10 Hz, a strain of 0.1%, a temperature of -150°C to 150°C, and a heating rate of 5°C / min.

[0211] (Water swelling ratio)

[0212] A resin film (measurement sample) of each scaffold material having a length of 50 mm, a width of 10 mm, and a thickness of 0.05 mm to 0.15 mm was immersed in water at 25° C. for 24 hours. The weight of the sample before and after immersion was measured, and the water swelling ratio was calculated as follows: (weight of the sample after immersion - weight of the sample before immersion) / (weight of the sample before immersion)×100(%).

[0213] (Preparation of a cell culture vessel)

[0214] In the case of Examples 1 to 11 and Comparative Examples 1 to 4, 1 g of the obtained synthetic resin was dissolved in 19 g of butanol to obtain a resin solution. 150 μL of the obtained resin solution was discharged onto a φ22 mm cover glass (manufactured by Matsunami Co., Ltd., 22 pellets No. 1 were dusted with a 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 and cover glass were arranged in a φ22 mm polystyrene dish to obtain a cell culture container. In the case of Examples 12 to 17, the obtained laminate of the peptide-containing polyvinyl alcohol acetal resin and the cover glass was arranged in a φ22 mm polystyrene dish to obtain a cell culture container.

[0215] (Surface free energy)

[0216] The surface free energy of the resin film obtained in the column "Preparation of a cell culture container" was measured using a contact angle meter (DMo-701, manufactured by Kyowa Interface Chemical Co., Ltd.). 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. Based on the obtained contact angle, the dispersion term component γ of the surface free energy was calculated using the Kaelble-Uy theoretical formula. d (dSFE) and the dipole component γ as the polar term p (pSFE).

[0217] (Phase separation parameters)

[0218] The resin film obtained in the prepared column of the cell culture container was observed by an atomic force microscope (AFM, manufactured by BRUKER, product number "Dimension XR"). The cantilever was SCAN ASYST AIR. The results were as follows. Figure 2As shown, in the case of the resin film of Example 3, it was observed that the polyvinyl butyral resin portion as the first phase formed a sea portion, and the resin portion having (meth)acrylate and vinyl compound (copolymer portion of N-vinyl pyrrolidone and n-lauryl methacrylate) as the second phase formed an island structure of the island portion. Similarly, in the cases of Examples 1 to 2 and 4 to 11, it was also observed that the polyvinyl butyral resin portion as the first phase formed a sea portion, and the resin portion having (meth)acrylate and vinyl compound (copolymer portion of N-vinyl pyrrolidone and n-lauryl methacrylate) as the second phase formed an island structure of the island portion. In addition, in the cases of Examples 12 to 17, it was also observed that the polyvinyl butyral resin portion as the first phase formed a sea portion, and the peptide portion as the second phase formed an island structure of the island portion. On the other hand, in the case of Comparative Examples 1 to 4, no phase separation structure was observed.

[0219] In addition, based on the images obtained by atomic force microscopy, image analysis software (ImageJ) was used to obtain the ratio of the surface area of ​​the second phase to the entire surface (surface area fraction of the phase separation structure), the ratio of the circumference to the area of ​​the second phase (circumference / area), the number of the second phase as islands (number of islands) and the average diameter of the islands (average island size) by the method described above.

[0220] (Cell proliferation rate)

[0221] After adding 1 mL of phosphate-buffered saline to the obtained cell culture container and standing in an incubator at 37°C for 1 hour, the phosphate-buffered saline in the culture container was removed. A confluent h-iPS cell 253G1 colony was added to a 35 mm dish, followed by 1 mL of 0.5 mM ethylenediamine / phosphate buffer solution, and standing at room temperature for 2 minutes. Then, the ethylenediamine / phosphate buffer solution was removed, and the cell clumps (0.5×10 5 cells) were inoculated into the culture container. In the presence of 1.7 mL of culture medium TeSR E8 (manufactured by STEMCELL) and 10 μM of ROCK-Inhibitor (Y27632), the cells were cultured in an incubator at 37°C and a CO2 concentration of 5%. Every 24 hours, 1 mL of culture medium was removed and 1 mL of new TeSR E8 was added to exchange the culture medium. After 5 days, 1.0 mL of TryPLE Express stripping solution was used to strip the fixed cell mass, and the number of cells was counted using a cell counter (Nucleocounter NC-3000, manufactured by Chemometec).

[0222] The cell proliferation rate relative to Reference Example A was determined using the following formula.

[0223] Cell proliferation rate relative to reference example A (%) = (number of cells in the examples and comparative examples) / (number of cells in reference example A) × 100

[0224] The cell proliferation rate was evaluated based on the following criteria.

[0225] [Evaluation criteria]

[0226] AAA…cell proliferation rate was 70% or more compared to reference example A

[0227] AA ... Cell proliferation rate relative to Reference Example A is 60% or more and less than 70%

[0228] A: The cell proliferation rate is 50% or more and less than 60% relative to that of Reference Example A.

[0229] B: The cell proliferation rate is 40% or more and less than 50% relative to that of Reference Example A.

[0230] C: The cell proliferation rate is 30% or more and less than 40% relative to that of Reference Example A.

[0231] D: Cell proliferation rate less than 30% compared to reference example A

[0232] The results are shown in Tables 1 to 4 below.

[0233] [Table 1]

[0234]

[0235] [Table 2]

[0236]

[0237] [Table 3]

[0238]

[0239] [Table 4]

[0240]

[0241] Explanation of symbols

[0242] 1…Cell culture container

[0243] 2…Container body

[0244] 2a…Surface

[0245] 3…Resin film

Claims

1. A resin film, which is a resin film formed of a scaffold material for cell culture, wherein: The cell culture scaffold material comprises a synthetic resin having a polyvinyl acetal skeleton. The synthetic resin is a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion, a linker portion, and a peptide portion. The joint of the joint part is (meth) acrylic acid, In the synthetic resin, the content of the peptide portion is 0.1 mol% or more and 15.0 mol% or less, the content of the peptide portion being the ratio of the amount of the peptide portion to the sum of the amounts of the structural units constituting the synthetic resin having the peptide portion, The resin film has a phase separation structure including at least a first phase and a second phase, and the phase separation structure is a sea-island structure. The ratio of the surface area of ​​the second phase to the entire surface is greater than 0.01 and less than 0.95, and the ratio of the surface area of ​​the second phase to the entire surface is obtained by dividing the surface area occupied by the second phase in an observation area of ​​30 μm×30 μm by the area of ​​the observation area.

2. The resin film according to claim 1, wherein The ratio of the perimeter to the area of ​​the second phase, that is, perimeter / area, is greater than or equal to 0.001 and less than or equal to 0.40, and the unit of the perimeter / area ratio is 1 / nm.

3. The resin film according to claim 1 or 2, wherein The first phase is a sea portion, and the second phase is an island portion.

4. The resin film according to claim 3, wherein The number of the second phase as the island portion is 1 / μm 2 Above, 5000 / μm 2 Hereinafter, the number of the second phase is calculated by dividing the number of the second phase in an observation region of 30 μm×30 μm by the area of ​​the observation region.

5. The resin film according to claim 1 or 2, wherein The phase separation structure is constituted by a phase separation structure within the molecules of the synthetic resin.

6. The resin film according to claim 1 or 2, wherein The second phase has the peptide portion.

7. The resin film according to claim 1 or 2, wherein The peptide portion has a cell-adhesive amino acid sequence, The cell adhesive amino acid sequence has at least an RGD sequence, a YIGSR sequence, a PDS GR sequence, a HAV sequence, an ADT sequence, a QAV sequence, a LDV sequence, an IDS sequence, a RED V sequence, an IDAPS sequence, a KQAGDV sequence or a TDE sequence. 8 . The resin film according to claim 1 , wherein the water swelling ratio is 50% or less.

9. The resin film according to claim 1 or 2, wherein The storage elastic modulus of the resin film at 100°C is 1.0×10 4 Pa or above, 1.0×10 8 Below Pa, The ratio of the storage elastic modulus of the resin film at 25°C to the storage elastic modulus at 100°C, i.e., the storage elastic modulus at 25°C / the storage elastic modulus at 100°C is 1.0×10 1 Above, 1.0×10 5 the following.

10. The resin film according to claim 1 or 2, wherein The cell culture scaffold material contains virtually no raw materials of animal origin.

11. A cell culture carrier, comprising: carrier, and The resin film according to any one of claims 1 to 10, The resin film is disposed on the surface of the carrier.

12. A cell culture container comprising: The container body, and The resin film according to any one of claims 1 to 10, The resin film is disposed on a surface of the container body.

Images