Resin membrane and cell culture vessel

Abstract

To provide a resin film in which shrinkage of a cell mass is hard to occur in culturing cells and which has excellent extensibility of cells.SOLUTION: A resin film made of a scaffold material for cell culture containing synthetic resin, in which zeta potential at pH7.0 is -40 mV or more, and a dispersion term component of surface free energy is 24.5 mJ / m2 or more and 45 mJ / m2 or less.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a resin membrane made of a scaffold material for cell culture. The present invention also relates to a cell culture vessel using the resin membrane. [Background technology]

[0002] Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are expected to be applied to drug discovery and regenerative medicine. To achieve such applications, it is essential to safely and reproducibly culture and proliferate pluripotent stem cells. In particular, for industrial use in regenerative medicine, it is necessary to handle large quantities of stem cells in an undifferentiated state. For this reason, various culture methods using natural and synthetic polymers have been investigated.

[0003] For example, Non-Patent Document 1 below proposes a poly(vinyl alcohol-vinyl acetal-itaconic acid) copolymer obtained by condensation reaction of fibronectin as a scaffold material for the proliferation of iPS cells and ES cells. This scaffold material is said to have excellent hydrophilicity and water resistance.

[0004] Furthermore, the following Patent Document 1 discloses a scaffold material using DMAEMA as a cation and acrylic acid, nucleic acid or heparin as an anion.

[0005] Furthermore, the following Patent Document 2 discloses a culture vessel having a surface covered with a hydrophilic and flexible polyrotaxane gel as a scaffold material used for culturing ES cells or iPS cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2017-70303 A [Patent Document 2] JP 2017-23008 A [Non-patent literature]

[0007] [Non-Patent Document 1] Sci.Rep.5,18136, published December 14, 2015 Summary of the Invention [Problem to be solved by the invention]

[0008] It is known that the undifferentiated and differentiated characteristics of stem cells are involved in their proliferation, and there is a demand for technology to control this. Conventionally, it has been known that the use of adhesive proteins such as laminin and vitronectin as natural polymers, or matrigel derived from mouse sarcoma, results in very high cell adhesion after seeding.

[0009] However, the methods using natural polymers or mouse sarcoma-derived Matrigel and the like tend to require complicated operations and are expensive. There are also safety issues. Therefore, various scaffold materials using synthetic polymers such as those described in Non-Patent Document 1, Patent Document 1, and Patent Document 2 have been proposed.

[0010] However, the cell scaffold material described in Non-Patent Document 1 has a problem in that when it does not contain fibronectin, that is, when it contains only a synthetic polymer, the cells do not spread but contract and float.

[0011] Furthermore, the cell scaffold material described in Patent Document 1 has a problem in that it is highly hydrophilic due to its ionic nature, which causes shrinkage of cell clusters and cell aggregation.

[0012] Furthermore, the cell scaffold material described in Patent Document 2 is highly hydrophilic and therefore easily swells, which causes the seeded cells to easily peel off from the scaffold material during culture, resulting in low cell adhesion.

[0013] An object of the present invention is to provide a resin film and a cell culture vessel made of a scaffold material for cell culture that is less susceptible to shrinkage of cell aggregates during cell culture and has excellent cell extensibility. [Means for solving the problem]

[0014] The resin film according to the present invention is a resin film made of a scaffold material for cell culture containing a synthetic resin, and has a zeta potential of -40 mV or more at pH 7.0 and a dispersion term component of the surface free energy of 24.5 mJ / m 2 More than 45mJ / m 2 The following is the result.

[0015] In a specific aspect of the resin film according to the present invention, the polar term component of the surface free energy is 1 mJ / m 2 More than 20mJ / m 2 The following is the result.

[0016] In another specific aspect of the resin membrane according to the present invention, the zeta potential at pH 7.0 is +30 mV or less.

[0017] In still another specific aspect of the resin film according to the present invention, the storage modulus at 100° C. is 1×10 4 Pa or more, 1×10 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 1×10 or more, 5 The following is the result.

[0018] In still another specific aspect of the resin film according to the present invention, the synthetic resin contains 0.2 mol % or more and 20 mol % or less of a Bronsted basic group in the constituent unit.

[0019] In still another specific aspect of the resin film according to the present invention, the water swelling ratio is 50% or less.

[0020] In still 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.

[0021] In still another specific aspect of the resin film according to the present invention, the synthetic resin includes a vinyl polymer.

[0022] In still 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.

[0023] The cell culture vessel according to the present invention includes a resin membrane constructed according to the present invention in at least a part of a cell culture region. Effect of the Invention

[0024] According to the resin film and cell culture vessel of the present invention, during cell culture, it is possible to increase the extensibility of the cells while suppressing the shrinkage of the cell aggregates. [Brief description of the drawings]

[0025] [Figure 1] 1 is a schematic front cross-sectional view showing a cell culture vessel according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing the relationship between SF (shape factor) and the planar shape of a cell aggregate. [Diagram 3] 13 is a photograph showing the state of cell extensibility when SF≈0.2. [Figure 4] 13 is a photograph showing the state of cell extensibility when SF≈1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention will be clarified below by describing specific embodiments of the present invention with reference to the drawings.

[0027] The resin membrane according to the present invention is used for culturing cells.

[0028] The resin film according to the present invention is a resin film made of a scaffold material for cell culture containing a synthetic resin. The zeta potential of the resin film according to the present invention at pH 7.0 is -40 mV or more, and the dispersion term component of the surface free energy is 24.5 mJ / m 2 More than 45mJ / m 2 The following is the result.

[0029] In the resin film according to the present invention, since the zeta potential and the dispersion term component of the surface free energy are within the above-mentioned specific ranges, the cell aggregates are unlikely to shrink during cell culture, and the extensibility of the cells can be increased. This will be explained below.

[0030] (Zeta potential) The zeta potential of the resin film according to the present invention is the potential difference between the surface of the resin film, which is the interface, and a bulk part in the solution sufficiently distant from the surface of the resin film when the resin film is placed in the solution. The zeta potential can be determined by appropriately selecting a method such as a streaming potential method, an electrophoresis method, or an electroosmosis method according to the shape of the object to be measured.

[0031] For example, when the object to be measured is a membrane, the measurement can be performed by a streaming potential method, and when the object to be measured is a fiber or a particle, the measurement can be performed by an electroosmosis method.

[0032] Therefore, the zeta potential of the resin film according to the present invention is preferably a zeta potential measured by a streaming potential method. The streaming potential method can be measured, for example, by a zeta potential measuring device manufactured by Anton Paar. Specifically, it can be measured in a dilute KCl solution by the method described in the Examples below. As an electrophoretic method, for example, a method of measuring by light scattering in water using a zeta potential / particle size / molecular weight measuring system (manufactured by Otsuka Electronics Co., Ltd.) can be mentioned.

[0033] The resin membrane according to the present invention has a zeta potential of -40 mV or more at pH 7.0, and therefore can enhance cell extensibility.

[0034] The extensibility of cells can be evaluated by determining the shape factor (SF).

[0035] Here, the shape factor (SF) is a coefficient for evaluating the shape of the area of ​​the cell cluster in a planar view after the cells are cultured, and is expressed as SF=4×π×(planar area of ​​the cell cluster) / (length of the outer periphery of the cell cluster).2 It can be calculated as follows.

[0036] As shown in Figure 2, if the SF is 1, the planar shape of the cell cluster will be circular. The smaller the SF, the further away it is from a circle, the less likely the cell cluster will shrink and the higher the cell's extensibility. For example, in the case of the star shape shown in Figure 2, the SF is 0.3, which can be said to be more extensible than a circle with SF=1.

[0037] In the present invention, since the zeta potential is -40mV or more, the SF value can be reduced, and the extensibility of cells can be increased. The zeta potential of the resin membrane at pH 7.0 is preferably -35mV or more, more preferably -30mV or more, even more preferably -25mV or more, particularly preferably -20mV or more, and preferably +30mV or less, more preferably less than +30mV, even more preferably less than +25mV, particularly preferably less than +20mV. In this case, the SF value can be further reduced, and the extensibility of cells can be further increased.

[0038] The zeta potential can be increased by increasing the content of cationic functional groups, such as amino groups, in the synthetic resin, and can be decreased by increasing the content of anionic functional groups, such as carboxyl groups, in the synthetic resin.

[0039] (Surface Free Energy) From the viewpoint of suppressing the shrinkage of cell aggregates and enhancing the extensibility of cells, the dispersion term component of the surface free energy of the resin film according to the present invention is 24.5 mJ / m 2 More than 45mJ / m 2 The dispersion term component is 29.0 mJ / m 2 Above, 45.0mJ / m 2 Preferably, it is less than 30.0 mJ / m 2 Above, 36.0mJ / m 2 In this case, the contraction of the cell aggregates can be further suppressed, and the extensibility of the cells can be further increased.

[0040] The polar term component of the surface free energy of the resin film according to the present invention is 1 mJ / m 2 More than 20mJ / m 2 It is preferable that the concentration is less than 1.0 mJ / m 2 More than 10.0mJ / m 2 More preferably, it is less than 1.5 mJ / m 2 More than 6.0mJ / m 2 In this case, the contraction of the cell aggregates can be further suppressed, and the extensibility of the cells can be further increased.

[0041] The dispersion term component γ of the above surface free energy d and the polar term 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

[0042]

number

[0043] In addition, in the Kaelble-Uy theoretical formula, the surface free energy of a liquid is expressed as γ l (mJ / m 2 ) and the surface free energy of the solid is γ s (mJ / m 2 ) and the contact angle is θ (°), the following equation (2) is established.

[0044]

number

[0045] 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 was measured, and γ s d and γs p By solving the simultaneous equations, the dispersion term component γ of the surface free energy of the resin film is obtained. d and the dipole component γ p can be sought.

[0046] In this specification, the surface free energy γ l Two such liquids for which the properties are known are pure water and diiodomethane.

[0047] The contact angle θ is measured using a contact angle meter (for example, "DMo-701" manufactured by Kyowa Interface Science Co., Ltd.) as follows.

[0048] 1 μL of pure water or diiodomethane is dropped onto the surface of the resin film. 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 the 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 the diiodomethane.

[0049] 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 above-mentioned surface free energy can be reduced. d In 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 p can be made smaller.

[0050] (Storage modulus) The storage modulus of the resin film 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, more preferably 0.8×10 8 Pa or less, more preferably 1.0×10 7 Pa or less.

[0051] The ratio of the storage modulus of the resin film at 25° C. to the storage modulus at 100° C. ((storage modulus at 25° C.) / (storage modulus at 100° C.)) is preferably 1.0×10 or more, more preferably 5.0×10 1 More preferably, 8.0×10 2 More than 1.0×10 5 Less than or equal to 0.75×10 5 Less than 0.5×10, more preferably 5 By setting the ratio within the above range, the fixation of cells after seeding can be further improved.

[0052] The storage modulus at 25°C and 100°C can be determined, for example, by measuring using a dynamic viscoelasticity measuring device (DVA-200, manufactured by IT Measurement & Control Co., Ltd.) under tensile conditions, with a frequency of 10Hz, a strain of 0.1%, a temperature range of -150°C to 150°C, and a temperature rise rate of 5°C / min. The storage modulus at 25°C and 100°C is determined from the obtained tensile storage modulus graph, and the ratio (storage modulus at 25°C / storage modulus at 100°C) is calculated. The measurement sample has a length of 50mm, a width of 5mm to 20mm, and a thickness of 0.1mm to 1.0mm.

[0053] The storage 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. The storage modulus at 25° C. and 100° C. can be decreased, for example, by decreasing the number average molecular weight of the synthetic resin, decreasing the glass transition temperature, etc.

[0054] (Water swelling ratio) The water swelling ratio of the resin film is preferably 50% or less, more preferably 40% or less, further preferably 30% or less, and particularly preferably 20% or less. In this case, the contraction of the cell aggregates can be further suppressed, the extensibility of the cells can be further increased, and the fixation of the cells after seeding can be further increased.

[0055] The water swelling ratio can be measured as follows. For example, a resin film (measurement sample) made of a scaffold material for cell culture, 50 mm long, 10 mm wide, and 0.05 mm to 0.15 mm thick, 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 as follows: (sample weight after immersion-sample weight before immersion) / (sample weight before immersion)×100(%).

[0056] The water swelling ratio can be reduced, for example, by increasing the hydrophobic functional groups of the synthetic resin, decreasing the number average molecular weight, or the like.

[0057] [Synthetic resin] The synthetic resin contained in the scaffold material for cell culture (hereinafter, sometimes referred to as synthetic resin X) is not particularly limited as long as it has the specific zeta potential and the specific range of surface free energy. In this specification, the term "structural unit" refers to a repeating unit of a monomer constituting synthetic resin X. In addition, when synthetic resin X has a graft chain, it includes the repeating unit of the monomer constituting the graft chain.

[0058] The synthetic resin X preferably contains 0.2 mol% or more of the Bronsted basic group in the structural unit, more preferably 2 mol% or more, more preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 15 mol% or less. In this case, the effect of the present invention can be more effectively exhibited. The Bronsted basic group and the like will be described later.

[0059] (vinyl polymer) The synthetic resin X preferably contains a vinyl polymer, and more preferably is a vinyl polymer. The 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, it is possible to more easily suppress the swelling of the scaffold material for cell culture in water. Examples of the vinyl polymer include polyvinyl alcohol derivatives, poly(meth)acrylic acid esters, polyvinylpyrrolidone, polystyrene, and ethylene-vinyl acetate copolymers. From the viewpoint of further enhancing the adhesiveness to cells, the vinyl polymer is preferably a polyvinyl alcohol derivative or a poly(meth)acrylic acid ester. The synthetic resin X preferably contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic acid ester. The synthetic resin X is preferably a polyvinyl alcohol derivative, and is also preferably a poly(meth)acrylic acid ester.

[0060] (Synthetic resin X having a polyvinyl acetal skeleton) The cell culture scaffold material preferably contains a synthetic resin X having a polyvinyl acetal skeleton. The synthetic resin X preferably contains a synthetic resin having a polyvinyl acetal skeleton, and more preferably is a synthetic resin having a polyvinyl acetal skeleton.

[0061] In this specification, "synthetic resin X having a polyvinyl acetal skeleton" may be referred to as "polyvinyl acetal resin X".

[0062] The polyvinyl acetal resin X has an acetal group, an acetyl group, and a hydroxyl group on the side chain.

[0063] The synthesis of the polyvinyl acetal resin X includes at least a step of acetalizing polyvinyl alcohol with an aldehyde.

[0064] The aldehyde used in the acetalization of polyvinyl alcohol to obtain the polyvinyl acetal resin X is not particularly limited. The aldehyde may be, for example, an aldehyde 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.

[0065] 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. The aldehydes may be used alone or in combination of two or more.

[0066] 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 X is preferably a polyvinyl butyral resin.

[0067] A vinyl compound may be copolymerized in the polyvinyl acetal resin X. That is, the polyvinyl acetal resin X may be a copolymer of a structural unit of the polyvinyl acetal resin and a vinyl compound. In the present invention, the polyvinyl acetal resin copolymerized with a vinyl compound is also referred to as the polyvinyl acetal resin.

[0068] The vinyl compound is a compound having a vinyl group (H2C=CH-). The vinyl compound may be a polymer having a structural unit having a vinyl group.

[0069] The copolymer may be a block copolymer of a polyvinyl acetal resin and a vinyl compound, or a graft copolymer in which a vinyl compound is grafted onto a polyvinyl acetal resin. The copolymer is preferably a graft copolymer.

[0070] Examples of the vinyl compound include ethylene, allylamine, vinylpyrrolidone, vinylimidazole, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, and (meth)acrylic acid ester, etc. These vinyl compounds may be used alone or in combination of two or more.

[0071] From the viewpoint of further enhancing the adhesiveness of cells, the polyvinyl acetal resin X preferably has a Brønsted basic group or a Brønsted acidic group, more preferably has a Brønsted basic group. That is, it is preferable that a part of the polyvinyl acetal resin X is modified with a Brønsted basic group or a Brønsted acidic group, and more preferably that a part of the polyvinyl acetal resin X is modified with a Brønsted basic group.

[0072] The polyvinyl acetal resin X preferably has a Brønsted basic group or a Brønsted acidic group, more preferably a Brønsted basic group. In that case, the extensibility of the cells can be further increased. The Brønsted basic group or the Brønsted acidic group may be present in a part of the polyvinyl acetal resin X, and in that case, a monomer having a Brønsted basic group or a Brønsted acidic group may be copolymerized or grafted. The degree of modification by the Brønsted basic group is preferably 0.2 mol% or more, more preferably 2 mol% or more, preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 15 mol% or less. If the degree of modification by the Brønsted basic group is within the above specific range, the extensibility of the cells can be further increased.

[0073] 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, and a substituent having an amide structure. Examples of the functional group 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, azatropyridene, 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, chlorine, and choline, and derivatives thereof.

[0074] Examples of the Bronsted acid 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 acid group is preferably a carboxyl group.

[0075] The polyvinyl acetal resin X preferably has a structural unit having an imine structure, a structural unit having an imide structure, a structural unit having an amine structure, or a structural unit having an amide structure, and more preferably has a structural unit having an imine structure or a structural unit having an amine structure. In this case, the polyvinyl acetal resin X may have only one type of these structural units, or two or more types of these structural units.

[0076] The polyvinyl acetal resin X 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 X preferably has an imine structure in a side chain.

[0077] The polyvinyl acetal resin X 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).

[0078] The polyvinyl acetal resin X 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 X, or may be bonded to the main chain via a linking group such as an alkylene group.

[0079] The polyvinyl acetal resin X may have 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.

[0080] 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-.

[0081] The polyvinyl acetal resin X preferably has an amine structure or an imine structure in a side chain. In this case, the amine structure or the imine structure may be directly bonded to a carbon atom constituting the main chain of the polyvinyl acetal resin X, or may be bonded to the main chain via a linking group such as an alkylene group.

[0082] 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 1 It can be measured by H-NMR (nuclear magnetic resonance spectroscopy).

[0083] (Synthetic resin X having a poly(meth)acrylic acid ester skeleton) The cell culture scaffold material preferably contains a synthetic resin X having a poly(meth)acrylic acid ester skeleton. The synthetic resin X preferably contains a synthetic resin having a poly(meth)acrylic acid ester skeleton, and more preferably is a synthetic resin having a poly(meth)acrylic acid ester skeleton.

[0084] In this specification, "synthetic resin X having a poly(meth)acrylic acid ester skeleton" may be referred to as "poly(meth)acrylic acid ester resin X".

[0085] Therefore, the poly(meth)acrylic acid ester resin X is a resin having a poly(meth)acrylic acid ester skeleton.

[0086] The poly(meth)acrylic acid ester resin X can be obtained by polymerization of a (meth)acrylic acid ester or by copolymerization of a (meth)acrylic acid ester with another monomer.

[0087] Examples of the (meth)acrylic acid ester include (meth)acrylic acid alkyl esters, (meth)acrylic acid cyclic alkyl esters, (meth)acrylic acid aryl esters, (meth)acrylamides, (meth)acrylic acid polyethylene glycols, and (meth)acrylic acid phosphorylcholine.

[0088] Examples of (meth)acrylic acid alkyl esters 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.

[0089] 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.

[0090] Examples of the (meth)acrylic acid cyclic alkyl ester include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0091] Examples of the (meth)acrylic acid aryl ester include phenyl (meth)acrylate and benzyl (meth)acrylate.

[0092] 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.

[0093] 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.

[0094] Examples of the (meth)acrylate phosphorylcholine include 2-(meth)acryloyloxyethyl phosphorylcholine.

[0095] 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, 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.

[0096] In this specification, "(meth)acrylic" means "acrylic" or "methacrylic", and "(meth)acrylate" means "acrylate" or "methacrylate".

[0097] The poly(meth)acrylic acid ester resin X preferably has a Bronsted basic group or a Bronsted acidic group, similar to the synthetic resin X having a polyvinyl acetal skeleton. In that case, the extensibility of the cells can be further improved. The Bronsted basic group may be present in a part of the poly(meth)acrylic acid ester resin X, and in that case, a monomer having a Bronsted basic group may be copolymerized or grafted. The degree of modification by the Bronsted basic group is preferably 0.2 mol% or more, more preferably 2 mol% or more, preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 15 mol% or less. If the degree of modification by the Bronsted basic group is within the above specific range, the extensibility of the cells can be further improved.

[0098] (Other resins) The scaffold material for cell culture may contain a polymer other than the above-mentioned synthetic resins, such as polyolefin resin, polyether resin, polyvinyl alcohol resin, polyester, epoxy resin, polyamide resin, polyimide resin, polyurethane resin, and polycarbonate resin.

[0099] [Scaffolding materials 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.

[0100] The above-mentioned cell culture scaffold material may contain components other than the above-mentioned synthetic resin X. Examples of the components other than the above-mentioned synthetic resin include polysaccharides, cellulose, synthetic peptides, and polypeptides.

[0101] 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 small as possible. In 100% by weight of the scaffold material for cell culture, the content of the 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.

[0102] It is preferable that the scaffold material for cell culture is substantially free of animal-derived raw materials. By not including raw materials of animal origin, 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 raw materials of animal origin" means that the amount of raw materials of animal origin in the scaffold material for cell culture is 3% by weight or less. It is preferable that the amount of raw materials of animal origin in the scaffold material for cell culture is 1% by weight or less, and more preferably 0% by weight. In other words, it is more preferable that the scaffold material for cell culture is completely free of raw materials of animal origin.

[0103] (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, the resin film according to the present invention is used for culturing cells, and is used as a scaffold for cells when culturing the cells.

[0104] The above cells include animal cells such as human, mouse, rat, pig, cow, and monkey cells. The above cells also include somatic cells, such as stem cells, progenitor cells, and mature cells. The above somatic cells may be cancer cells.

[0105] 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.

[0106] Examples of the mature cells include nerve cells, cardiomyocytes, retinal cells, and hepatic cells.

[0107] (Shape of scaffold material for cell culture) The resin film according to the present invention is formed by a scaffold material for cell culture. The resin film is formed by 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 the scaffold material for cell culture. The thickness of the resin film is not particularly limited.

[0108] The present specification also provides a particle, fiber, porous body, or film containing the above-mentioned scaffold material for cell culture. In this case, the shape of the above-mentioned scaffold material for cell culture is not particularly limited, and may be a particle, fiber, porous body, or film. The above-mentioned particle, fiber, porous body, or film may contain a component other than the above-mentioned scaffold material for cell culture.

[0109] The film containing the scaffold material for cell culture is preferably used for flat culture (two-dimensional culture) of cells. Also, the particles, fibers, or porous bodies containing the scaffold material for cell culture are preferably used for three-dimensional culture of cells.

[0110] (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.. Fig. 1 is a front cross-sectional view that typically shows a cell culture vessel according to one embodiment of the present invention.

[0111] The cell culture vessel 1 includes a vessel body 2 and a resin film 3. 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, the cells can be cultured on a plate.

[0112] The container body may include 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, the scaffold material for cell culture may be disposed on the surface of the second container body.

[0113] The container body may be a conventionally known container body (container). The shape and size of the container body are not particularly limited.

[0114] The container body may be a cell culture plate or a cell culture flask having one or more wells (holes). The number of wells in the plate is not particularly limited. The number of wells may be, but is not particularly limited to, 2, 4, 6, 12, 24, 48, 96, 384, etc. The shape of the well may be, but is not particularly limited to, a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, etc. The shape of the bottom of the well may be, but is not particularly limited to, a flat bottom, a round bottom, an uneven bottom, etc.

[0115] 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, polyimide, polyamide, polyamideimide, (meth)acrylic resins, epoxy resins, and silicone. 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. EXAMPLES

[0116] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0117] The following synthetic resin X1 was synthesized as a raw material for a scaffold material for cell culture.

[0118] In a reactor equipped with a stirrer, 2700 mL of ion-exchanged water and 300 parts by weight of amine-modified polyvinyl alcohol with an average degree of polymerization of 1600, an amine modification degree of 2.0 mol%, and a saponification degree of 97.5 mol% were added, and the mixture was heated and dissolved while stirring to obtain a solution. To the obtained solution, 35% by weight hydrochloric acid was added as a catalyst so that the hydrochloric acid concentration was 0.2% by weight. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butyl aldehyde were added while stirring. Next, 148 parts by weight of n-butyl aldehyde were added to precipitate a white particulate polyvinyl butyral resin. 15 minutes after the precipitation, 35% by weight hydrochloric acid was added so that the hydrochloric acid concentration was 1.8% by weight, and then the mixture was heated to 50°C and held at 50°C for 2 hours. Next, the solution was cooled and neutralized, and the polyvinyl butyral resin was washed with water and dried to obtain a synthetic resin X1, which is a polyvinyl butyral resin.

[0119] The obtained polyvinyl butyral resin (synthetic resin X1) had an average degree of polymerization of 1600, a hydroxyl group amount of 21 mol %, an amine group amount of 2 mol %, an acetylation degree of 1 mol %, and an acetalization degree (butyralization degree) of 76 mol %.

[0120] The content of structural units in the obtained synthetic resin was measured by dissolving the synthetic resin in DMSO-D6 (dimethyl sulfoxide) and then 1 The measurements were performed using H-NMR (nuclear magnetic resonance spectroscopy).

[0121] (Examples 1 to 6 and Comparative Examples 1 to 4) In Example 1, the synthetic resin X1 was used, and in Examples 2 to 6, the synthetic resins X2 to X6 were used, respectively.

[0122] Synthetic resin X2 is a resin obtained by blending 2 mol % of dimethylaminoacrylamide as a monomer into polyvinyl acetal resin having a butyralization degree of 71 mol %, an acetylation degree of 1 mol %, and a hydroxyl group amount of 28 mol %, and has the composition shown in Table 1. The synthesis conditions were as follows: That is, the polyvinyl acetal resin was dissolved in THF at a concentration of 20 wt %, 2 parts by weight of dimethylaminoacrylamide was added, and then 3 parts by weight of an initiator was dissolved therein, and the mixture was irradiated with UV for 20 minutes using a UV polymerization tester.

[0123] Moreover, as shown in Table 1, synthetic resin X3 and synthetic resin X4 are similar to synthetic resin X2, except that the content of dimethylaminoacrylamide in the synthetic resin is changed from 2 mol % to 5 mol % and 21 mol %.

[0124] Synthetic resin X5 is similar to synthetic resin X2, except that diethylaminoacrylamide is used as a monomer and its content is 28 mol%. Synthetic resin X6 is similar to synthetic resin X2, except that vinylimidazole is used as a monomer and its content is 16 mol%.

[0125] In Comparative Examples 2 to 4, polyvinyl acetal resins having the compositions shown in Table 2 were used.

[0126] Fabrication of cell culture vessels; 0.1 parts by weight of the synthetic resins shown in Tables 1 and 2 were dissolved in 19.9 parts by weight of a butanol / methanol mixed solvent (9:1). 200 μL of the resulting solution was cast onto a polystyrene dish and heated at 60°C for 120 minutes to obtain a cell culture vessel with a smooth surface resin film (a resin film made of a scaffold material for cell culture).

[0127] In Comparative Example 1, a polystyrene dish itself was used as the cell culture vessel.

[0128] Zeta potential measurement at pH 7.0; The zeta potential of the resin films obtained in Examples 1 to 6 and Comparative Examples 2 to 4 was measured using a zeta potential measuring device (manufactured by Anton Paar, model number SURPASS 3). First, a 0.1 mM KCl solution was prepared. Next, the resin film of the scaffold material was set in a cell, and then a pH electrode and a conductivity meter were set. The zeta potential at each pH was measured by titrating from pH 9 to 3 using HCl and NaOH solutions.

[0129] The zeta potential of the polystyrene dish was determined in Comparative Example 1. In Comparative Examples 2 and 3, the zeta potential was below the lower limit of measurement, and therefore measurement was not possible.

[0130] Measurement of surface free energy; The surface free energy of the resin films obtained in Examples 1 to 6 and Comparative Examples 2 to 4 was measured using a contact angle meter (Kyowa Interface Science Co., Ltd., DMo-701). 1 μL of pure water was deposited on the resin film, and an image of the droplet was taken after 30 seconds to obtain the contact angle of pure water. In addition, 1 μL of diiodomethane was deposited on the resin film, and an image of the droplet was taken after 30 seconds to obtain the contact angle of diiodomethane. The surface free energy γ and dispersion term component γ d , the polar term component, the dipole component γ p In Comparative Example 1, the surface free energy of a polystyrene dish was determined.

[0131] Water swelling ratio; Resin films (measurement samples) made of each scaffold material, 50 mm long, 10 mm wide, and 0.05 mm to 0.15 mm thick, 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) x 100 (%).

[0132] Storage modulus; The storage modulus of each resin film at 25°C and 100°C was measured by a dynamic viscoelasticity measuring device (DVA-200, manufactured by IT Measurement & Control Co., Ltd.). The measurement by the dynamic viscoelasticity measuring device was performed using a measurement sample with a length of 50 mm, a width of 5 mm to 20 mm, and a thickness of 0.1 mm to 1 mm under the conditions of a frequency of 10 Hz, a strain of 0.1%, a temperature of -150°C to 150°C, and a temperature rise rate of 5°C / min. In Comparative Example 1, the storage modulus of a polystyrene dish was obtained. In addition, the ratio ((storage modulus at 25°C) / (storage modulus at 100°C)) was calculated from the obtained storage modulus at 25°C and 100°C.

[0133] Cell seeding and culture; The following liquid medium and ROCK (Rho-associated kinase) specific inhibitor were prepared.

[0134] TeSR E8 medium (STEM CELL) ROCK Inhibitor (Y27632)

[0135] 1 mL of phosphate buffered saline was added to the obtained cell culture vessel, and the vessel was allowed to stand in an incubator at 37° C. for 1 hour, after which the phosphate buffered saline was removed from the cell culture vessel.

[0136] A colony of h-iPS cells 253G1 that had reached a confluent state was placed in a φ35 mm dish, 1 mL of 0.5 mM ethylenediamine / phosphate buffer solution was added, and the dish was left to stand at room temperature for 2 minutes. After removing the ethylenediamine / phosphate buffer solution, cell clusters were obtained that were broken into pieces of 50 μm to 200 μm in size by pipetting with 1 mL of TeSR E8 medium. The obtained cell clusters (cell number 0.5 × 10 5 The cells were clamp-seeded in the above cell culture vessel.

[0137] At the time of seeding, 1.5 mL of liquid medium and a ROCK-specific inhibitor were added to the cell culture vessel so as to give a final concentration of 10 μM, and the cells were cultured for one day in an incubator at 37° C. and a CO 2 concentration of 5%.

[0138] (evaluation) SF evaluation; After culturing for one day, the shape factor (SF) was evaluated as described above. For the evaluation, images were obtained at 4x magnification using a phase contrast microscope. The perimeter and area of ​​the cell cluster were calculated using image analysis software (Image J), ​​and the formula was SF=4×π×(cell cluster area / cell cluster peripheral edge length). 2 The SF value was calculated by: Figure 3 is a photograph showing the planar shape of the cell aggregate when SF ≈ 0.2, and Figure 4 is a photograph showing the planar shape of the cell aggregate when SF ≈ 1.

[0139] In addition, SF was evaluated according to the following criteria. XXX: SF is less than 0.2 〇〇: SF is 0.2 or more and less than 0.4 ○: SF is 0.4 or more and less than 0.5 ×: SF is 0.5 or more

[0140] The results are shown in Tables 1 and 2 below.

[0141] [Table 1]

[0142] [Table 2]

[0143] As is clear from Tables 1 and 2, in Comparative Example 1, the SF value is large at 0.82, indicating low cell extensibility. In Comparative Examples 2 to 4, the SF values ​​are also large at 0.56 or more, indicating low cell extensibility. In contrast, in Examples 1 to 6, the SF values ​​are 0.41 or less, indicating significantly superior cell extensibility. In particular, in Examples 1, 2, and 3, the SF values ​​are 0.25 or less, which is more preferable, and in Examples 2 and 3, the SF values ​​are 0.17 or less, which is even more preferable. Furthermore, in Examples 1 to 6, no shrinkage of the cell aggregates was observed. [Explanation of symbols]

[0144] 1... Container for cell culture 2... Container body 2a... Surface 3... Resin film

Claims

1. A resin film made of a scaffold material for cell culture containing a synthetic resin, The zeta potential at pH 7.0 is −40 mV or more and +30 mV or less; The dispersion term component of the surface free energy is 30.0 mJ / m ２ Above, 36.0mJ / m ２ is less than The polar term component of the surface free energy is 1.5 mJ / m ２ Above, 6.0mJ / m ２ is less than the synthetic resin includes a synthetic resin having a polyvinyl acetal skeleton, the synthetic resin having a polyvinyl acetal skeleton has a Bronsted basic group, the degree of modification with a Bronsted basic group in the synthetic resin having a polyvinyl acetal skeleton is 2 mol % or more and 30 mol % or less; A resin film, wherein the synthetic resin having a polyvinyl acetal skeleton has a structural unit having an imine structure, a structural unit having an imide structure, a structural unit having an amine structure, or a structural unit having an amide structure.

2. Storage modulus at 100°C is 1 x 10 ４ Pa or more, 1×10 ８ Pa or less, The ratio of the storage modulus at 25° C. to the storage modulus at 100° C. ((storage modulus at 25° C.) / (storage modulus at 100° C.)) is 1×10 or more, ５ The resin film according to claim 1, wherein:

3. The resin film according to claim 1 or 2, which has a water swelling ratio of 50% or less.

4. The resin film according to any one of claims 1 to 3, wherein the content of animal-derived raw materials in the scaffold material for cell culture is 0% by weight or more and 3% by weight or less.

5. A cell culture vessel comprising the resin film according to any one of claims 1 to 4 in at least a part of a cell culture region.

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