Resin membrane and cell culture vessel

Abstract

To provide a resin film with excellent cell fixability after inoculation.SOLUTION: The present invention provides a resin film comprising a container for cell culture, containing synthetic resin. The synthetic resin has a degree of cationic modification of 0.2 mol% or more and 50 mol% or less, and the resin film has an isoelectric point of 2.8 or more and 6.0 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] In recent years, cell medicine and next-generation medical treatments using stem cells have been attracting attention. In particular, human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are expected to be applied to drug discovery and regenerative medicine. To achieve such applications, it is essential to safely and reproducibly culture and grow 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, extensive research has been conducted on technologies that support the growth of pluripotent stem cells using natural and synthetic polymers and feeder cells while maintaining their pluripotency.

[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] 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 good cell morphology 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 medium replacement or long-term culture, resulting in low cell fixation.

[0013] An object of the present invention is to provide a resin membrane and a cell culture vessel that are excellent in terms of cell fixation properties after seeding. [Means for solving the problem]

[0014] The resin membrane of the present invention is a resin membrane made of a scaffold material for cell culture containing a synthetic resin, wherein the degree of cationic modification of the synthetic resin is 0.2 mol % or more and 50 mol % or less, and the isoelectric point of the resin membrane is 2.8 or more and 6.0 or less.

[0015] In a specific aspect of the resin film according to the present invention, the dispersion term component of the surface free energy is 24.5 mJ / m 2 Above, 45.0mJ / m 2 or less, and the polar term component is 1.0 mJ / m 2 More than 20.0mJ / m 2 The following is the result.

[0016] In another particular aspect of the resin film according to the present invention, the water swelling ratio is 50% or less.

[0017] In still another specific aspect of the resin film according to the present invention, the storage modulus at 100° C. is 1.0×10 4 Pa or more, 1.0×10 8 Pa or less, and the ratio of the storage modulus at 25°C to the storage modulus at 100°C ((storage modulus at 25°C) / (storage modulus at 100°C)) is 1.0 x 10 1 That's it, 1.0 x 10 5 The following is the result.

[0018] 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.

[0019] In still another specific aspect of the resin film according to the present invention, the synthetic resin includes a vinyl polymer.

[0020] 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.

[0021] The cell culture vessel according to the present invention comprises a vessel body and a resin film configured according to the present invention, the resin film being disposed on the surface of the vessel body. Effect of the Invention

[0022] According to the present invention, it is possible to provide a resin membrane and a cell culture vessel that are excellent in terms of cell fixation properties after seeding. [Brief description of the drawings]

[0023] [Figure 1] 1 is a schematic front cross-sectional view showing a cell culture vessel according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention will be clarified below by describing specific embodiments of the present invention with reference to the drawings.

[0025] The resin membrane according to the present invention is used for culturing cells. The resin membrane according to the present invention is a resin membrane made of a scaffold material for cell culture containing a synthetic resin. The degree of cation modification of the synthetic resin is 0.2 mol% or more and 50 mol% or less. In the present invention, the isoelectric point of the resin membrane is 2.8 or more and 6.0 or less.

[0026] The isoelectric point is the pH at which the average charge after ionization of a synthetic resin having both anionic and cationic functional groups is 0. Therefore, the isoelectric point is the pH at which the zeta potential of the resin membrane is 0.

[0027] The zeta potential of a resin film 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 in this specification 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.

[0028] 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 particle, the measurement can be performed by an electroosmosis method.

[0029] Therefore, the zeta potential of the resin film 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.

[0030] The resin film of the present invention has the above-mentioned configuration and is therefore excellent in cell fixation after seeding.

[0031] Conventional scaffold materials for cell culture using natural polymer materials can increase the fixation of cells after seeding, but they are expensive, have large lot-to-lot variations because they are naturally derived substances, and have safety concerns due to animal-derived components. On the other hand, scaffold materials using synthetic polymer materials are easier to handle, less expensive, have smaller lot-to-lot variations, and are safer than scaffold materials using natural polymer materials. However, scaffold materials using synthetic resin materials have a problem of lower adhesiveness compared to scaffold materials using natural polymer materials. The present inventors have found that ionic dissociation and changes cause cells to peel off from scaffold materials due to medium replacement or long-term culture.

[0032] Therefore, the inventors focused on the degree of cationic modification of the synthetic resin and the isoelectric point of the resin film made of the scaffold material, and found that by setting the degree of cationic modification of the synthetic resin and the isoelectric point of the resin film within specific ranges, respectively, it is possible to control the ionic dissociation and change, thereby enhancing adhesion to cells after seeding.

[0033] Therefore, according to the present invention, it is possible to enhance adhesion of cells after seeding, and to suppress detachment of cells from the resin film due to medium exchange or long-term culture.

[0034] In addition, since the resin film of the present invention contains a synthetic resin, it is easier to handle, less expensive, has less variation between lots, and is safer than scaffold materials using natural polymeric materials.

[0035] In the present invention, the isoelectric point of the resin membrane is 2.8 or more, more preferably 3.0 or more and 6.0 or less, more preferably 5.0 or less, in which case the fixation rate of cells after seeding can be further increased.

[0036] The isoelectric point 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.

[0037] In the present invention, the dispersion term component of the surface free energy in the resin film is preferably 24.5 mJ / m 2 Above, 45.0mJ / m 2 This makes it possible to further increase the adhesiveness of cells after seeding. The dispersion term component is more preferably 30.0 mJ / m or less. 2 More preferably, 33.0 mJ / m 2 More preferably, 40.0 mJ / m 2 Less than 38.0 mJ / m 2 The following is the result.

[0038] The polar term component of the surface free energy is preferably 1.0 mJ / m 2 More than 20.0mJ / m 2 In this case, the adhesiveness to the cells after seeding can be further improved. The polar term component is more preferably 1.5 mJ / m or less. 2 More preferably, 2.0 mJ / m 2More preferably, 10.0 mJ / m 2 Less than or equal to 7.5 mJ / m 2 The following is the result.

[0039] In addition, the dispersion term component of the surface free energy γ 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

[0040]

number

[0041] 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.

[0042]

number

[0043] Therefore, the surface free energy of the liquid, γ l Using two types of liquids with known contact angles, θ and γ s d and γ s p By solving the simultaneous equations, the dispersion term component γ of the surface free energy of the resin film is obtained. d and the dipole component γ p can be sought.

[0044] In this specification, the surface free energy γ lTwo such liquids for which the properties are known are pure water and diiodomethane.

[0045] The contact angle θ is measured using a contact angle meter (for example, "DMo-701" manufactured by Kyowa Interface Science Co., Ltd.) as follows.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In particular, 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 1 More preferably, 5.0×10 1 More preferably, 8.0×10 2 More than 1.0×105 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.

[0050] The storage modulus at 25°C and 100°C can be determined, for example, by measuring using a dynamic viscoelasticity measuring device (IT Measurement and Control, DVA-200) under tensile conditions, under the following measurement conditions: frequency 10Hz, strain 0.1%, temperature range -150°C to 150°C, and heating rate 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.

[0051] 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.

[0052] The water swelling ratio of the resin film is preferably 50% or less, more preferably 40% or less, even more preferably 30% or less, and particularly preferably 20% or less. In this case, the fixation of cells after seeding can be further improved. The lower limit of the water swelling ratio is not particularly limited, but can be, for example, 0.5%.

[0053] 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(%).

[0054] 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.

[0055] [Synthetic resin] The cell culture scaffold material used in the present invention contains a synthetic resin (hereinafter, may be referred to as synthetic resin X). In this specification, the term "structural unit" refers to a repeating unit of a monomer that constitutes the synthetic resin. In addition, when the synthetic resin has a graft chain, it includes a repeating unit of a monomer that constitutes the graft chain.

[0056] The main chain of the synthetic resin X is preferably a carbon chain. In addition, the synthetic resin X preferably contains a cationic functional group and an anionic functional group.

[0057] Examples of the cationic functional group contained in the synthetic resin X include substituents having a structure such as an amino group, an imino group, or an amide group. Examples of the cationic 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. These cationic functional groups may be used alone or in combination of two or more kinds.

[0058] The content of cationic functional groups contained in the structural units of synthetic resin X, i.e., the cationic modification degree, is 0.2 mol% or more, preferably 2 mol% or more, more preferably 3 mol% or more, and 50 mol% or less, preferably 10 mol% or less, more preferably 7 mol% or less. By using synthetic resin X containing cationic functional groups within such a range, the fixation of cells after seeding can be further improved.

[0059] The anionic functional group contained in the synthetic resin X is not particularly limited, but examples thereof include a hydroxyl group, 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 derivatives thereof. An example of the Bronsted acid group is a carboxyl group. These anionic functional groups may be used alone or in combination.

[0060] The content of anionic functional groups contained in the structural units of synthetic resin X, i.e., the degree of anionic modification, is preferably 1 mol% or more, more preferably 5 mol% or more, and preferably 20 mol% or less, more preferably 10 mol% or less. By using synthetic resin X containing anionic functional groups within such a range, the fixation of cells after seeding can be further improved.

[0061] The content of the structural unit having a cationic functional group and the structural unit having an anionic functional group is, for example, 1 It can be measured by H-NMR (nuclear magnetic resonance spectroscopy).

[0062] The synthetic resin X contained in the scaffold material for cell culture used in the present invention is not particularly limited as long as it has the specific cationic modification degree and the isoelectric point in the specific range.

[0063] (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, the swelling of the scaffold material for cell culture in water can be more easily suppressed. 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.

[0064] (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.

[0065] In this specification, "synthetic resin X having a polyvinyl acetal skeleton" may be referred to as "polyvinyl acetal resin X".

[0066] Therefore, the polyvinyl acetal resin X is a resin having a polyvinyl acetal skeleton.

[0067] The polyvinyl acetal resin X has an acetal group, an acetyl group, and a hydroxyl group on the side chain.

[0068] The method for synthesizing the polyvinyl acetal resin X includes at least a step of acetalizing polyvinyl alcohol with an aldehyde.

[0069] 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.

[0070] Examples of the aldehyde include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal, acrolein, benzaldehyde, cinnamaldehyde, perillaldehyde, formylpyridine, formylimidazole, formylpyrrole, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formylisoindole, formylpurine, formylbenzimidazole, formylbenzotriazole, formylquinoline, formylisoquinoline, formylquinoxaline, formylcinnoline, formylpteridine, formylfuran, formyloxolane, formyloxane, formylthiophene, formylthiolane, formylthiane, formyladenine, formylguanine, formylcytosine, formylthymine, and formyluracil. These aldehydes may be used alone or in combination of two or more.

[0071] 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.

[0072] 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 group is also referred to as the polyvinyl acetal resin.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] The Bronsted basic group is the hydrogen ion H +A Brønsted basic group is a general term for a functional group that can receive from another substance. Examples of the Brønsted basic group include amine-based basic groups such as a substituent having an imine structure, a substituent having an imide structure, a substituent having an amine structure, or a substituent having an amide structure. The Bronsted basic group is not particularly limited, but examples thereof include 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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).

[0082] 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.

[0083] 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.

[0084] 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-.

[0085] 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.

[0086] 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).

[0087] (Synthetic resin X having a poly(meth)acrylic acid ester skeleton) The cell culture scaffold material used in the present invention 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.

[0088] 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".

[0089] Therefore, the poly(meth)acrylic acid ester resin X is a resin having a poly(meth)acrylic acid ester skeleton.

[0090] The poly(meth)acrylic acid ester resin X can be obtained by polymerization of a (meth)acrylic acid ester, or by polymerization of a (meth)acrylic acid ester and the other monomers described above.

[0091] 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.

[0092] Examples of the (meth)acrylic acid alkyl ester include methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, isopropyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, t-butyl (meth)acrylate, n-octyl (meth)acrylate, isooctyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, isononyl (meth)acrylate, decyl (meth)acrylate, isodecyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, and isotetradecyl (meth)acrylate.

[0093] 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.

[0094] Examples of the (meth)acrylic acid cyclic alkyl ester include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0095] Examples of the (meth)acrylic acid aryl ester include phenyl (meth)acrylate and benzyl (meth)acrylate.

[0096] 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.

[0097] 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.

[0098] Examples of the (meth)acrylate phosphorylcholine include 2-(meth)acryloyloxyethyl phosphorylcholine.

[0099] 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.

[0100] In this specification, "(meth)acrylic" means "acrylic" or "methacrylic", and "(meth)acrylate" means "acrylate" or "methacrylate".

[0101] The poly(meth)acrylic acid resin X may have a Brønsted basic group or a Brønsted acidic group in part, similar to the synthetic resin X having a polyvinyl acetal skeleton. The Brønsted basic group and the Brønsted acidic group are as described above.

[0102] (Resin other than synthetic resin X) The scaffold material for cell culture may contain a polymer other than the synthetic resin X. Examples of the polymer include polyolefin resin, polyether resin, polyvinyl alcohol resin, polyester, epoxy resin, polyamide resin, polyimide resin, polyurethane resin, and polycarbonate resin.

[0103] [Scaffolding materials for cell culture] The cell culture scaffold material used in the present invention 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 cell culture scaffold material 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 cell culture scaffold material 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] (Cell culture using resin membrane made of scaffold material 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 the cells when culturing the cells.

[0108] 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.

[0109] 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.

[0110] Examples of the mature cells include nerve cells, cardiomyocytes, retinal cells, and hepatic cells.

[0111] (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.

[0112] 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.

[0113] The film containing the scaffold material for cell culture is preferably used for flat surface 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.

[0114] (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 schematic front cross-sectional view showing a cell culture vessel according to one embodiment of the present invention.

[0115] 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.

[0116] 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, a resin film may be disposed on the surface of the second container body.

[0117] The container body may be a conventionally known container body (container). The shape and size of the container body are not particularly limited.

[0118] 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.

[0119] 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

[0120] The present invention will now be described in more detail with reference to specific examples and comparative examples, although the present invention is not limited to the following examples. The following Example 6 is a reference example.

[0121] The following synthetic resins were synthesized as raw materials for cell culture scaffold materials.

[0122] Example 1 In a reactor equipped with a stirrer, 2700 mL of ion-exchanged water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700, a degree of saponification of 98 mol%, and a degree of acetyl group of 2 mol% were charged, 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 was added while stirring. Next, 148 parts by weight of n-butyl aldehyde was 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. 99.7 parts by weight of the obtained polyvinyl butyral resin was dissolved in tetrahydrofuran to give a 20% by weight solution, and 5 parts by weight of Irgacure 184 as an initiator and 0.3 parts by weight of dimethylaminoacrylate were added thereto, and graft polymerization was performed to obtain a synthetic resin.

[0123] The synthetic resin thus obtained had a butyralization degree of 70.7 mol%, an acetylation degree of 1 mol%, a hydroxyl group content of 28 mol%, and a cation modification degree of 0.3 mol%. The content of structural units in the synthetic resin thus obtained was measured by dissolving the synthetic resin in DMSO-d6 (dimethyl sulfoxide) and then 1 The measurement was performed by H-NMR (nuclear magnetic resonance spectroscopy). The same measurement was performed in the following examples and comparative examples.

[0124] Example 2 98 parts by weight of polyvinyl butyral resin obtained in the same manner as in Example 1 was dissolved in tetrahydrofuran to give a 20% by weight solution, and 5 parts by weight of Irgacure 184 as an initiator and 2 parts by weight of dimethylaminoacrylate were added thereto, followed by graft polymerization to obtain a synthetic resin.

[0125] Example 3 90 parts by weight of the polyvinyl butyral resin obtained in the same manner as in Example 1 was dissolved in tetrahydrofuran to give a 20% by weight solution, and 5 parts by weight of Irgacure 184 as an initiator and 10 parts by weight of dimethylaminoacrylate were added thereto, and graft polymerization was performed to obtain a synthetic resin.

[0126] Example 4 90 parts by weight of the polyvinyl butyral resin obtained in the same manner as in Example 1 was dissolved in tetrahydrofuran to give a 20% by weight solution, and 5 parts by weight of Irgacure 184 as an initiator, 5 parts by weight of vinylpyrrolidone, and 5 parts by weight of lauryl methacrylate were added thereto, and graft polymerization was performed to obtain a synthetic resin.

[0127] Example 5 90 parts by weight of the polyvinyl butyral resin obtained in the same manner as in Example 1 was dissolved in tetrahydrofuran to give a 20% by weight solution, and 5 parts by weight of Irgacure 184 as an initiator and 10 parts by weight of vinylimidazole were added thereto, followed by graft polymerization to obtain a synthetic resin.

[0128] Example 6 5 parts by weight of vinylpyrrolidone, 71 parts by weight of butyl methacrylate, and 24 parts by weight of ethyl acrylate were mixed to obtain a (meth)acrylic monomer solution. 0.1 parts by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained (meth)acrylic monomer solution and applied onto a PET film. The applied material was exposed to light of 365 nm wavelength at an integrated light intensity of 2000 mJ / cm using a UV conveyor device "ECS301G1" manufactured by iGraphics Co., Ltd. at 25°C. 2 A poly(meth)acrylic acid ester resin solution was obtained by irradiating the solution with light at 400 nm with 1000 V of UV light. The poly(meth)acrylic acid ester resin solution was dried in a vacuum at 80°C for 3 hours to obtain a synthetic resin as a poly(meth)acrylic acid ester resin.

[0129] Comparative Example 1 As the synthetic resin, polystyrene resin was used as it is.

[0130] Comparative Example 2 As the synthetic resin, polyethylene terephthalate resin was used as it is.

[0131] Comparative Example 3 As the synthetic resin, polystyrene resin was used as it is.

[0132] Comparative Example 4 As the synthetic resin, polymethyl methacrylate resin was used as it is.

[0133] Comparative Example 5 As the synthetic resin, polytetrafluoroethylene resin was used as it is.

[0134] Comparative Example 6 90 parts by weight of polyvinyl butyral resin A obtained in the same manner as in Example 1 was dissolved in tetrahydrofuran to give a 20% by weight solution, and 5 parts by weight of Irgacure 184 as an initiator and 10 parts by weight of acrylic acid were added thereto to carry out graft polymerization to obtain a synthetic resin.

[0135] Comparative Example 7 90 parts by weight of polyvinyl butyral resin A obtained in the same manner as in Example 1 was dissolved in tetrahydrofuran to give a 20% by weight solution, and 5 parts by weight of Irgacure 184 as an initiator and 10 parts by weight of diethylaminoacrylate were added thereto, followed by graft polymerization to obtain a synthetic resin.

[0136] [evaluation] (isoelectric point) Fabrication of cell culture vessels; 0.1 parts by weight of each of the synthetic resins obtained in Examples 1 to 6 and Comparative Examples 1 to 7 was dissolved in 19.9 parts by weight of a butanol / methanol mixed solvent (9:1). 200 μL of the resulting solution was cast into a polystyrene dish and heated at 60° C. for 120 minutes to obtain a cell culture vessel on which a resin film (a resin film made of a scaffold material for cell culture) with a smooth surface was formed. This resin film was used in the following measurements.

[0137] Determination of isoelectric point; The zeta potential of the resin films obtained in Examples 1 to 6 and Comparative Examples 1 to 7 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. Using HCl and NaOH solutions, the pH was titrated from pH 3.0 to 9.0 to measure the pH (isoelectric point) at which the zeta potential became 0.

[0138] (Storage modulus) The storage modulus at 25°C and 100°C of the resin films obtained in Examples 1 to 6 and Comparative Examples 1 to 7 was measured under tensile conditions with a dynamic viscoelasticity measuring device (IT Measurement and Control, DVA-200) at a frequency of 10Hz and a temperature range of -150°C to 150°C at a heating rate of 5°C / min. The storage modulus at 25°C and 100°C was obtained from the graph of the tensile storage modulus obtained, and the 25°C storage modulus / 100°C storage modulus was calculated. The measurement was performed using a measurement sample with a length of 50mm, a width of 5mm to 20mm, and a thickness of 0.1mm to 1.0mm under the conditions of 10Hz, strain of 0.1%, temperature of -150°C to 150°C, and a heating rate of 5°C / min. 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.

[0139] (Water swelling ratio) The resin films (measurement samples) obtained in Examples 1 to 6 and Comparative Examples 1 to 7 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 rate was calculated as follows: (sample weight after immersion−sample weight before immersion) / (sample weight before immersion)×100(%).

[0140] (Surface Free Energy) The surface free energy of the resin films obtained in Examples 1 to 6 and Comparative Examples 1 to 7 was measured using a contact angle meter (Kyowa Interface Science Co., Ltd., DMo-701). 1 μL of pure water was dropped onto the resin film, and an image of the droplet was taken after 30 seconds to obtain the contact angle of pure water. In addition, 1 μL of diiodomethane was dropped onto the resin film, and an image of the droplet was taken after 30 seconds to obtain the contact angle of diiodomethane. From the obtained contact angles, the dispersion term component of the surface free energy γ d (dSFE) and dipole component γ p (pSFE) was calculated.

[0141] (Adhesiveness) 1 mL of phosphate buffered saline was added to the obtained cell culture vessel, which was then left to stand in an incubator at 37°C for 1 hour, after which the phosphate buffered saline in the culture vessel was removed. A colony of h-iPS cells 253G1 that had reached a confluent state was added to a 35 mm dish, and then 1 mL of 0.5 mM ethylenediamine / phosphate buffer solution was added and left to stand at room temperature for 2 minutes. Thereafter, the ethylenediamine / phosphate buffer solution was removed, and the cell clumps, which had been crushed to 50 μm to 200 μm by pipetting, were mixed with 1.5 mL of TeSRE8 medium, and 0.5 x 10 5 The cells were seeded in a cell culture vessel and cultured in the presence of 1 mL of TeSR E8 medium (STEM CELL) and 10 μM ROCK-Inhibitor (Y27632) in an incubator at 37°C and a CO2 concentration of 5%. Every 24 hours, 1000 μL of the medium was removed and 1000 μL of new TeSR E8 was added to replace the medium.

[0142] In the cell cluster culture test, the time that the cell clusters could maintain adhesion before they were detached was measured and evaluated according to the following criteria.

[0143] [Evaluation Criteria] XX: Adhesion was maintained for more than 72 hours after medium change. ○: Adhesion was maintained for more than 48 hours after medium change, but all cells detached within 72 hours. ×: All cells were detached within 24 hours after medium change.

[0144] The results are shown in Table 1 below.

[0145] [Table 1] [Explanation of symbols]

[0146] 1...Cell culture container 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 degree of cation modification of the synthetic resin is 0.2 mol% or more and 10 mol% or less, The cationic modification degree is the content of cationic functional groups contained in the structural units of the synthetic resin, The isoelectric point of the resin film is 2.8 or more and 5.4 or less; The dispersion term component of the surface free energy is 30.0 mJ / m ２ Above, 38.0mJ / m ２ or less, and the polar term component is 1.5 mJ / m ２ Above, 7.5mJ / m ２ is as follows: the synthetic resin includes a synthetic resin having a polyvinyl acetal skeleton, 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. The resin film according to claim 1 , which has a water swelling ratio of 50% or less.

3. Storage modulus at 100°C is 1.0 x 10 ４ Pa or more, 1.0×10 ８ Pa or less, The ratio of the storage modulus at 25° C. to the storage modulus at 100° C. ((storage modulus at 25° C.) / (storage modulus at 100° C.)) is 1.0×10 １ That's it, 1.0 x 10 ５ The resin film according to claim 1 or 2, wherein:

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 container body; The resin film according to any one of claims 1 to 4, A cell culture vessel, comprising the resin film disposed on the surface of the vessel body.

Images