JP7610368B2 - Scaffolding material for cell culture and cell culture vessel - Google Patents

Description

The present invention relates to a cell culture scaffold material used for culturing cells. The present invention also relates to a cell culture vessel using the above-mentioned cell culture scaffold material.

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

In particular, in industrial applications of regenerative medicine, it is necessary to handle large quantities of stem cells, and therefore it is necessary to support the proliferation of pluripotent stem cells using natural polymer materials, synthetic polymer materials, or feeder cells. For this reason, various culture methods using scaffold materials composed of natural polymer materials, synthetic polymer materials, etc. are being investigated.

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

The following Patent Document 2 discloses a composition including a first fibrous polymer scaffold material, in which the fibers of the first fibrous polymer scaffold material are aligned. It is described that the fibers constituting the first fibrous polymer scaffold material are composed of an aliphatic polyester such as polyglycolic acid or polylactic acid.

In addition, the following Patent Document 3 discloses a cell culture method for maintaining the undifferentiated state of pluripotent stem cells, which includes a step of culturing the pluripotent stem cells on a culture vessel having a surface coated with a polyrotaxane block copolymer.

JP 2006-314285 A Special Publication No. 2009-524507 JP 2017-23008 A

When the scaffold is made of a natural polymer material, the adhesion of cells after seeding can be improved. In particular, it is known that the adhesion of cells after seeding is very high when adhesive proteins such as laminin and vitronectin or Matrigel derived from mouse sarcoma are used as natural polymer materials. On the other hand, natural polymer materials are expensive, have large lot-to-lot variations due to being naturally derived substances, and have safety concerns due to animal-derived ingredients.

In contrast, scaffold materials using synthetic resins are easier to handle, less expensive, have less variation between lots, and are safer than scaffold materials using natural polymer materials. However, in scaffold materials using synthetic resins such as those described in Patent Documents 1 to 3, the synthetic resins used are highly hydrophilic, which can lead to excessive swelling of the synthetic resins. In addition, synthetic resins have a lower affinity for cells than natural polymer materials, which can lead to cell masses peeling off during culture. Thus, scaffold materials using synthetic resins have poor adhesion after seeding of cells, and cells may not grow sufficiently.

The object of the present invention is to provide a scaffold material for cell culture and a cell culture vessel that have excellent adhesion properties after cell seeding and can increase the cell proliferation rate.

The cell culture scaffold material of the present invention contains a synthetic resin, has a zeta potential of -60 mV or more at pH 7, and has multiple pores with an average diameter of 10 nm or more and 5 μm or less.

In a particular aspect of the cell culture scaffold material of the present invention, the zeta potential at pH 7 is +30 mV or less.

In another specific aspect of the cell culture scaffold material of the present invention, the elastic modulus at 25°C measured by the nanoindentation method is 0.01 GPa or more.

In yet another specific aspect of the cell culture scaffold material according to the present invention, the synthetic resin contains 0.2 mol % or more and 20 mol % or less of Bronsted basic groups in its constituent units.

In yet another specific aspect of the cell culture scaffold material of the present invention, the water swelling rate is 100% or less.

In yet another specific aspect of the cell culture scaffold material of the present invention, it is substantially free of animal-derived raw materials.

In yet another specific aspect of the cell culture scaffold material of the present invention, the synthetic resin includes a vinyl polymer.

In yet another specific aspect of the cell culture scaffold material according to the present invention, the synthetic resin contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin.

In yet another specific aspect of the cell culture scaffold material of the present invention, the synthetic resin includes a polyvinyl acetal resin.

In still another specific aspect of the cell culture scaffold material according to the present invention, the number of the pores per unit area is 0.01 pores/μm ² or more and 1000 pores/μm ² or less.

The cell culture vessel according to the present invention is provided with a cell culture scaffold material constructed according to the present invention in at least a portion of the cell culture area.

The present invention provides a scaffold material for cell culture and a cell culture vessel that have excellent adhesion after cell seeding and can increase the cell proliferation rate.

FIG. 1 is a schematic front cross-sectional view showing a cell culture vessel according to an embodiment of the present invention. 1 is a SEM image at a magnification of 1000 times of the scaffold for cell culture obtained in Example 1. 1 is a SEM image of the scaffold for cell culture obtained in Example 1 at a magnification of 10,000 times.

The present invention will be clarified below by explaining specific embodiments of the present invention with reference to the drawings.

The cell culture scaffold of the present invention is a scaffold containing a synthetic resin. The cell culture scaffold has a zeta potential of -60 mV or more at pH 7. The cell culture scaffold has a plurality of pores with an average diameter of 10 nm or more and 5 μm or less.

The cell culture scaffold material of the present invention has the above-mentioned configuration, and therefore has excellent adhesion after cell seeding, and can increase the cell proliferation rate.

Conventional cell culture scaffolds made from natural polymer materials can increase the adhesion of cells after seeding, but they are expensive, have large lot-to-lot variations because they are made from natural substances, and have safety concerns due to animal-derived components. On the other hand, scaffolds made from synthetic resins tend to swell excessively or have low affinity for cells, which can cause cell masses to detach during culture. Therefore, scaffolds made from synthetic resins tend to have low adhesion after seeding, and cells may not grow sufficiently.

In contrast, the cell culture scaffold material of the present invention has a zeta potential of -60 mV or more at pH 7.

Furthermore, the inventors have focused on the structure of such a scaffold for cell culture and discovered that by having multiple pores with an average diameter of 10 nm or more and 5 μm or less, the cell proliferation rate can be increased.

The reason for this is unclear, but by forming a structure with multiple pores with average diameters in a specific range as described above, it is possible to form both cell adhesion areas, which are areas where no pores are formed, and cell non-adhesion areas, which are areas where pores are formed. This allows cells to move freely while maintaining their fixation in the cell adhesion areas. It is therefore believed that this can enhance the migration ability of cells and enhance their proliferation ability.

Therefore, the cell culture scaffold material of the present invention can increase adhesion to cells after seeding, thereby increasing the cell proliferation rate.

In addition, since the present invention can use synthetic resins as described above, it is easier to handle, less expensive, has less variation between lots, and is safer than scaffolding materials made from natural polymer materials.

It is preferable that the cell culture scaffold of the present invention is substantially free of animal-derived raw materials. In this case, the cell culture scaffold can be made even safer. Note that "substantially free of animal-derived raw materials" means that the animal-derived raw materials in the cell culture scaffold are 3% by weight or less. However, it is more preferable that the cell culture scaffold of the present invention is completely free of animal-derived raw materials.

In the cell culture scaffold of the present invention, the average pore size is preferably 50 nm or more, more preferably 200 nm or more, even more preferably 500 nm or more, and preferably 4.5 μm or less, more preferably 4 μm or less, even more preferably 3.5 μm or less. When the average pore size is within the above range, the adhesion of cells after seeding can be further improved, and the cell proliferation rate can be further increased.

In the cell culture scaffold of the present invention, the number of pores per unit area is preferably 0.01/μm ² or more, more preferably 0.1/μm ² or more, even more preferably 1/μm ² or more, particularly preferably 10/μm ² or more, preferably 1000/μm ² or less, more preferably 500/μm ² or less, and even more preferably 100/μm ² or less. When the number of pores per unit area is within the above range, the fixation of cells after seeding can be further improved, and the proliferation rate of cells can be further improved.

In this specification, the average diameter and number of pores can be measured by a scanning electron microscope (SEM), a transmission electron microscope (TEM), or the like. In this case, the average value of the pore diameters of any 100 pores confirmed by microscopic observation such as a scanning electron microscope (SEM), a transmission electron microscope (TEM), or the like can be regarded as the average diameter of the pores. The number of pores per unit area can be determined by dividing the number of pores in an observation area (5 μm×5 μm) by the area of the observation area. The number of pores can be, for example, the average value of the number of pores obtained by repeating the same operation 10 times. In addition, when measuring the average diameter and number of pores, pores with a pore diameter of 10 nm or more and 5 μm or less are judged to be pores.

The multiple pores can be formed, for example, by applying a solution containing a synthetic resin and alcohol to the substrate, freezing it with liquid nitrogen or the like, and then freeze-drying it. In this case, it is believed that the pores are formed by the parts from which the alcohol has been removed. When using a synthetic resin that does not expand easily, such as polyvinyl acetal resin, the size of the pores can be made more uniform, making it even easier to form pores with a controlled average diameter.

The average diameter and number of pores can be controlled by adjusting the type and amount of alcohol added, or by adding small amounts of water to the alcohol.

In the present invention, a scaffold for cell culture may be formed by crushing a synthetic resin that has multiple pores formed in advance and placing it on a substrate. Also, a particulate or fibrous scaffold for cell culture having multiple pores may be used.

In the present invention, the zeta potential of the cell culture scaffold at pH 7 is preferably -40 mV or more, more preferably -35 mV or more, even more preferably -30 mV or more, and preferably +30 mV or less, more preferably less than +30 mV, even more preferably less than +25 mV. In this case, the adhesiveness of cells can be further improved.

The zeta potential of a cell culture scaffold is the potential difference between the surface of the cell culture scaffold, which is the interface when the cell culture scaffold is placed in a solution, and the bulk part in the solution that is sufficiently distant from the surface of the cell culture scaffold. The value of the zeta potential in this specification can be determined by appropriately selecting a method such as streaming potential method, electrophoresis method, or electroosmosis method depending on the shape of the cell culture scaffold.

For example, if the cell culture scaffold is a membrane, it can be measured by streaming potential method. If the cell culture scaffold is a fiber or particle, it can be measured by electroosmosis method.

The streaming potential method can be performed, for example, using 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. An example of the electrophoretic method is a method in which light scattering is performed in water using a zeta potential/particle size/molecular weight measuring system (manufactured by Otsuka Electronics Co., Ltd.).

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

The elastic modulus of the cell culture scaffold of the present invention at 25°C measured by the nanoindentation method is preferably 0.01 GPa or more, more preferably 0.05 GPa or more, even more preferably 0.1 GPa or more, particularly preferably 0.5 GPa or more, preferably 5 GPa or less, more preferably 3 GPa or less, and even more preferably 1 GPa or less. When the elastic modulus is within the above range, the adhesiveness of cells can be further improved.

The elastic modulus can be measured, for example, using a nanoindenter (Hysitron, Tryboindenter, manufactured by Bruker). In this measurement, a Berkovich (triangular pyramid-shaped) indenter with a tip diameter R of 100 nm is used, and a single indentation measurement is performed in air at room temperature, with the indentation depth being 50 nm.

The cell culture scaffold of the present invention preferably has a water swelling rate of 100% or less, more preferably 50% or less, and even more preferably 40% or less. In this case, the fixation of cells after seeding can be further improved. The lower limit of the water swelling rate is not particularly limited, but can be, for example, 0.5%. The water swelling rate can be measured, for example, by the following method.

First, the scaffold for cell culture is immersed in water at 25°C for 24 hours. Next, the scaffold for cell culture is removed from the water and the water remaining in the pores is drained by placing it on a water-absorbent material, for example. Next, the weight of the sample before and after immersion is measured, and the water swelling rate is calculated as follows: (sample weight after immersion - sample weight before immersion) / (sample weight before immersion) x 100 (%).

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

[Synthetic resin]
The cell culture scaffold of 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 constituting the synthetic resin. In addition, when the synthetic resin has a graft chain, it includes a repeating unit of a monomer constituting the graft chain.

Synthetic resin X preferably contains 0.2 mol% or more of Bronsted basic groups in its structural units, more preferably 2 mol% or more, and preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 15 mol% or less. In this case, the effects of the present invention can be more effectively achieved. Bronsted basic groups and the like will be described later.

(Vinyl polymer)
The synthetic resin X is preferably 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 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. Furthermore, the vinyl polymer is preferably a polyvinyl alcohol derivative or a poly(meth)acrylic acid ester from the viewpoint of easier enhancement of adhesion to cells.

(Synthetic resin having a polyvinyl acetal skeleton)
The cell culture scaffold preferably contains a synthetic resin having a polyvinyl acetal skeleton. The synthetic resin X preferably contains a synthetic resin having a polyvinyl acetal skeleton.

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

It is preferable that the polyvinyl acetal resin X has an acetal group, an acetyl group, and a hydroxyl group in the side chain. However, the polyvinyl acetal resin X may not have an acetyl group, for example. For example, the polyvinyl acetal resin X may not have an acetyl group by bonding all of the acetyl groups of the polyvinyl acetal resin X to a linker described later.

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

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

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 above aldehydes may be used alone or in combination of two or more.

The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, or pentanal, and more preferably butyraldehyde. Therefore, the polyvinyl acetal skeleton is preferably a polyvinyl butyral skeleton. The polyvinyl acetal resin X is preferably a synthetic resin having a polyvinyl butyral skeleton, and more preferably a polyvinyl butyral resin.

The polyvinyl acetal resin X may be copolymerized with a vinyl compound. That is, the polyvinyl acetal resin X may be a copolymer of the 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.

The vinyl compound is a compound having a vinyl group (H ₂ C═CH—). The vinyl compound may be a polymer having a structural unit having a vinyl group.

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.

The above copolymer can be synthesized, for example, by the following methods (1) to (3). (1) A method of synthesizing a polyvinyl acetal resin using polyvinyl alcohol copolymerized with a vinyl compound. (2) A method of synthesizing a polyvinyl acetal resin using polyvinyl alcohol and polyvinyl alcohol copolymerized with a vinyl compound. (3) A method of graft copolymerizing a vinyl compound onto a polyvinyl acetal resin before graft copolymerization.

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

From the viewpoint of further enhancing the adhesiveness of cells, it is preferable that the polyvinyl acetal resin X has a Brønsted basic group or a Brønsted acidic group, and it is more preferable that the polyvinyl acetal resin X has a Brønsted basic group. In other words, 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 it is more preferable that a part of the polyvinyl acetal resin X is modified with a Brønsted basic group.

The polyvinyl acetal resin X preferably has a Brønsted basic group or a Brønsted acidic group in a portion thereof, and more preferably has a Brønsted basic group. In that case, the adhesiveness of cells can be further increased. The Brønsted basic group or the Brønsted acidic group may be present in a portion 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 in 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 adhesiveness of cells can be further increased.

The Bronsted basic group is a general term for a functional group that can receive a hydrogen ion H+ from another substance. Examples of the Bronsted basic group include amine-based basic groups such as a substituent having an imine structure, a substituent having an imide structure, a substituent having an amine structure, and a substituent having an amide structure. Examples of Bronsted basic groups include, but are not limited to, hydroxyamino groups, urea groups, conjugated amine functional groups such as guanidine and biguanide, 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, melamine, and other heterocyclic amino functional groups, porphyrin, chlorine, choline, and other cyclic pyrrole functional groups and derivatives thereof.

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.

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. In this case, it may have only one type of these structural units, or two or more types.

The polyvinyl acetal resin X may have a structural unit having an imine structure. An imine structure refers to a structure having a C=N bond. In particular, it is preferable that the polyvinyl acetal resin X has an imine structure in the side chain.

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).

It is preferable that the polyvinyl acetal resin X has an imino group in the 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.

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.

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

It is preferable that the polyvinyl acetal resin X has an amine structure or an amide structure in the side chain. In this case, the amine structure or the amide structure may be directly bonded to a carbon atom constituting the main chain of the polyvinyl acetal resin X, or may be bonded to the main chain via a linking group such as an alkylene group.

The content of structural units having an imine structure, the content of structural units having an imide structure, the content of structural units having an amine structure, and the content of structural units having an amide structure can be measured by ¹ H-NMR (nuclear magnetic resonance spectroscopy).

(Synthetic resin having a poly(meth)acrylic acid ester skeleton)
The cell culture scaffold of the present invention preferably contains a synthetic resin 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.

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

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

Poly(meth)acrylic acid ester resin X is obtained by polymerization of a (meth)acrylic acid ester or by copolymerization of a (meth)acrylic acid ester with another monomer.

(Meth)acrylic acid esters 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.

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.

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 esters include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

Examples of (meth)acrylic acid cyclic alkyl esters include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

Examples of (meth)acrylic acid aryl esters include phenyl (meth)acrylate and benzyl (meth)acrylate.

(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.

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.

Examples of (meth)acrylate phosphorylcholine include 2-(meth)acryloyloxyethyl phosphorylcholine.

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.

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

The poly(meth)acrylic acid ester resin X preferably has a Brønsted basic group or a Brønsted acidic group in part, as in the polyvinyl acetal resin X. In that case, the extensibility can be further improved. The Brønsted 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 Brønsted basic group may be copolymerized or grafted. The degree of modification in 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 adhesiveness of the cells can be further improved.

(Synthetic resin having a peptide portion)
The cell culture scaffold of the present invention preferably contains a synthetic resin having a peptide portion. The synthetic resin X preferably contains a synthetic resin having a peptide portion. By using a synthetic resin having a peptide portion as a material for the cell culture scaffold, the cell adhesion after seeding can be further improved, and the cell proliferation rate can be further increased.

The synthetic resin having a peptide portion can be obtained by reacting a synthetic resin, a linker, and a peptide. The synthetic resin having a peptide portion is preferably a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion, a linker portion, and a peptide portion, and more preferably a peptide-containing polyvinyl butyral resin having a polyvinyl butyral resin portion, a linker portion, and a peptide portion. Therefore, the synthetic resin having the polyvinyl acetal skeleton (polyvinyl acetal resin X) is preferably a peptide-containing polyvinyl acetal resin, and more preferably a peptide-containing polyvinyl butyral resin. Only one type of synthetic resin having a peptide portion may be used, or two or more types may be used in combination.

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

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

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

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

Arg-Gly-Asp-X...Formula (1)

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

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

In the synthetic resin having a peptide portion, the content of the peptide portion is preferably 0.1 mol% or more, more preferably 1 mol% or more, even more preferably 5 mol% or more, and particularly preferably 10 mol% or more. In the synthetic resin having a peptide portion, the content of the peptide portion is preferably 60 mol% or less, more preferably 50 mol% or less, even more preferably 35 mol% or less, and particularly preferably 25 mol% or less. When the content of the peptide portion is within the above range, the adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased. In addition, when the content of the peptide portion is equal to or less than the above upper limit, the manufacturing cost can be reduced. The content of the peptide portion (mol%) is the amount of substance of the peptide portion relative to the sum of the amounts of substance of each structural unit constituting the synthetic resin having a peptide portion.

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

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

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

Examples of the linker having a carboxyl group include (meth)acrylic acid and acrylamide containing a carboxyl group. By using a carboxylic acid (carboxylic acid monomer) having a polymerizable unsaturated group as the linker having a carboxyl group, the carboxylic acid monomer can be polymerized by graft polymerization when reacting the linker with a synthetic resin, so that the number of carboxyl groups that can be reacted with a peptide can be increased.

(Other resins)
The cell culture scaffold may contain a polymer other than the above-mentioned synthetic resins, such as polyvinylpyrrolidone, polystyrene, ethylene-vinyl acetate copolymer, polyolefin resin, polyether resin, polyvinyl alcohol resin, polyester, epoxy resin, polyamide resin, polyimide resin, polyurethane resin, polycarbonate resin, etc.

[Other details of the cell culture scaffold]
The cell culture scaffold material according to 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.

The cell culture scaffold material may contain components other than the synthetic resin X. Examples of components other than the synthetic resin X include polysaccharides, cellulose, synthetic peptides, etc.

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.

The cell culture scaffold material according to the present invention preferably does not contain any raw materials derived from animals. By not containing raw materials derived from animals, it is possible to provide a cell culture scaffold material that is highly safe and has little variation in quality during production.

(Cell culture using scaffolds for cell culture)
The scaffold for cell culture according to the present invention is used for culturing cells. The scaffold for cell culture according to the present invention is used as a scaffold for cells when culturing the cells.

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.

The above stem cells include mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

The above-mentioned mature cells include nerve cells, cardiomyocytes, retinal cells, and hepatic cells.

(Shape of scaffold for cell culture)
The shape of the scaffold for cell culture of the present invention is not particularly limited, and may be a particle, a fiber, a porous body, or a film. The particle, fiber, porous body, or film may contain components other than the scaffold for cell culture.

When the scaffold material for cell culture has the shape of a film, it is preferably used for planar culture (two-dimensional culture). When the scaffold material for cell culture has the shape of particles, fibers, or a porous body, it is preferably used for three-dimensional culture of cells.

(Cell culture vessel)
The present invention also relates to a cell culture vessel comprising the above-mentioned cell culture scaffold 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.

The cell culture vessel 1 comprises a vessel body 2 and a cell culture scaffold 3. The cell culture scaffold 3 is disposed on the surface 2a of the vessel body 2. The cell culture scaffold 3 is disposed on the 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 cell culture scaffold 3, the cells can be cultured on a surface or in three dimensions.

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 cell culture scaffold material may be disposed on the surface of the second container body.

A conventionally known container body (container) can be used as the container body. There are no particular limitations on the shape and size of the container body.

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 well bottom may be, but is not particularly limited to, a flat bottom, a round bottom, an uneven bottom, etc.

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.

Next, the present invention will be clarified by presenting specific examples and comparative examples. Note that the present invention is not limited to the following examples.

The following synthetic resin X1 was synthesized as a raw material for cell culture scaffolding.

2700 mL of ion-exchanged water and 300 parts by weight of amine-modified polyvinyl alcohol with an average degree of polymerization of 800, an amine modification degree of 1 mol%, and a saponification degree of 97.5 mol% were added to a reactor equipped with a stirring device, and the mixture was heated and dissolved while stirring to obtain a solution. 35% by weight hydrochloric acid was added as a catalyst to the obtained solution 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 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 synthetic resin X1, which is a polyvinyl butyral resin.

The obtained polyvinyl butyral resin (synthetic resin X1) had an average degree of polymerization of 800, a hydroxyl group content of 20 mol%, an amine group content of 1 mol%, an acetylation degree of 1 mol%, and a butyralization degree of 78 mol%.

The content of the structural units in the obtained synthetic resin was measured by dissolving the synthetic resin in DMSO-D6 (dimethyl sulfoxide) and then using ¹ H-NMR (nuclear magnetic resonance spectrum).

(Examples 1 to 10 and Comparative Examples 2 and 3)
In Examples 1 and 2, the synthetic resin X1 was used, and in Examples 3 to 7, 9 and 10, the synthetic resins X2 to X8 were used, respectively.

Synthetic resin X2 was obtained by blending 5 mol% of dimethylaminoacrylamide as a monomer with polyvinyl butyral resin having a butyralization degree of 68 mol%, an acetylation degree of 1 mol%, and a hydroxyl group content of 26 mol%. The synthesis conditions were as follows: the polyvinyl butyral resin was dissolved in THF at a concentration of 20% by weight, 5 parts by weight of dimethylaminoacrylamide was added, and then 3 parts by weight of Irgacure 184 (manufactured by IGM Resins B.V.) was dissolved as an initiator, and UV irradiation was performed for 20 minutes using a UV polymerization tester.

Synthetic resin X3 was obtained in the same manner as synthetic resin X2, except that the content of dimethylaminoacrylamide was changed from 5 mol% to 20 mol%, as shown in Table 1.

Synthetic resin X4 was obtained in the same manner as synthetic resin X2, except that diethylaminoacrylamide was used as the monomer and its content was 28 mol%.

Synthetic resin X5 was obtained in the same manner as synthetic resin X2, except that vinyl imidazole was used as the monomer and its content was 13 mol%.

For synthetic resin X6, a synthetic resin that is a polyvinyl acetal resin was obtained in the same manner as in Example 1, except that polyvinyl alcohol with an average degree of polymerization of 2400 and a degree of saponification of 97 mol% was used, and acetaldehyde was used instead of n-butylaldehyde (n-BA). The obtained polyvinyl acetal resin was obtained by blending 5 mol% of dimethylaminoacrylamide as a monomer into the obtained polyvinyl acetal resin. The synthesis conditions were as follows. That is, the obtained polyvinyl acetal resin was dissolved in THF at a concentration of 20% by weight, 5 parts by weight of dimethylaminoacrylamide was added, and then 3 parts by weight of an initiator (Irgacure 184) was dissolved and irradiated with UV for 20 minutes using a UV polymerization tester.

Synthetic resin X7 was prepared as follows. First, 81.4 parts by weight of butyl methacrylate and 18.6 parts by weight of hydroxyethyl methacrylate were mixed to obtain a (meth)acrylic monomer solution. Next, 0.5 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 irradiated with light of a wavelength of 365 nm at an integrated light quantity of 2000 mJ/cm ² using a UV conveyor device "ECS301G1" manufactured by Eye Graphics Co., Ltd. at 25 ° C. to obtain a poly(meth)acrylic acid ester resin solution. The obtained poly(meth)acrylic acid ester resin solution was vacuum dried at 80 ° C. for 3 hours to obtain synthetic resin X7 as a poly(meth)acrylic acid ester resin.

For synthetic resin X8, a synthetic resin that is a polyvinyl acetal resin was obtained in the same manner as in Example 1, except that polyvinyl alcohol with an average degree of polymerization of 800 and a degree of saponification of 1 mol% was used. 95 parts by weight of the obtained polyvinyl acetal resin and 5 parts by weight of acrylic acid (linker) were dissolved in 300 parts by weight of THF, and reacted for 20 minutes under ultraviolet irradiation in the presence of a photoradical polymerization initiator, and the polyvinyl acetal resin and acrylic acid were graft-copolymerized to form a linker portion.

A linear peptide having the amino acid sequence Gly-Arg-Gly-Asp-Ser (5 amino acid residues, indicated as GRGDS in Table 1) was prepared as a peptide. Five parts by weight of this peptide and 5 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) were added to 90 parts by weight of methanol containing neither calcium nor magnesium to prepare a peptide-containing liquid. 100 parts by weight of this peptide-containing liquid and 97 parts by weight of polyvinyl acetal resin having a linker portion formed thereon were added to 300 parts by weight of methanol, and reacted to dehydrate and condense the carboxyl group of the linker portion and the amino group of Arg of the peptide. After the reaction was completed, the mixture was vacuum dried at 60°C for 1 hour to volatilize the methanol. In this way, synthetic resin X8, which is a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion, a linker portion, and a peptide portion, was prepared.

In addition, synthetic resin X1 was used in Comparative Example 2, and polyvinyl butyral resin with the composition shown in Table 1 was used in Example 8 and Comparative Example 3.

Fabrication of cell culture vessels;
(Examples 1 to 10, Comparative Example 3)
In Examples 1 to 10 and Comparative Example 3, the synthetic resin shown in Table 1 was first dissolved in a butanol/methanol mixed solvent (9:1) at a concentration of 5 wt %. 40 μL of the resulting solution was cast onto a polystyrene dish with a diameter of 22 mm, and then immersed in liquid nitrogen to freeze together with the solvent. Then, the dish was freeze-dried for two days using a freeze dryer to obtain a cell culture vessel equipped with a cell culture scaffold having fine pores.

The average pore size of the cell culture scaffold was determined using a scanning electron microscope (SEM). Figure 2 is an SEM image of the cell culture scaffold obtained in Example 1 at a magnification of 1000 times, and Figure 3 is an SEM image of the cell culture scaffold obtained in Example 1 at a magnification of 10000 times.

(Comparative Examples 1 and 2)
In Comparative Example 1, a polystyrene dish itself was used as the cell culture vessel.

In Comparative Example 2, synthetic resin X1 was first dissolved in a butanol/methanol mixed solvent (9:1) at a concentration of 5 wt %. 40 μL of the resulting solution was cast onto a φ22 mm polystyrene dish, and then dried in an oven at 45°C for 6 hours without freezing to form a flat membrane. In Comparative Examples 1 and 2, no pores were present whose average diameter could be measured.

Zeta potential measurement;
The zeta potential of the cell culture scaffolds of Examples 1 to 10 and Comparative Examples 1 to 3 at pH 7 was measured using a zeta potential measuring device (model number SURPASS 3, manufactured by Anton Paar). First, a 0.1 mM KCl solution was prepared. Next, the resin film of the scaffold was set in the 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.

In addition, in Comparative Example 3, the zeta potential was below the lower measurement limit and therefore could not be measured.

Water swelling rate:
When determining the water swelling rate of each scaffold, the scaffold was removed from the cell culture vessel and immersed in water at 25°C for 24 hours. After immersion, the sample was placed on Kimtowel for 1 minute to drain the water from the pores, and then the weight was measured. 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) x 100 (%).

Surface elasticity measurement;
A nanoindenter (Hysitron, Triboindenter, manufactured by Bruker) was used to determine the surface elastic modulus at 25° C. of the cell culture scaffold materials of Examples 1 to 10 and Comparative Examples 1 to 3. A Berkovich (triangular pyramid) indenter with a tip diameter R of 100 nm was used as the indenter, and a single indentation measurement was performed in air at room temperature, with the indentation depth at that time being 50 nm.

Average pore size:
The surface of the cell culture scaffold was observed using a scanning electron microscope (SEM), and the average pore size of 100 pores randomly selected within the observation area (5 μm×5 μm) was calculated.

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

TeSR E8 medium (manufactured by STEM CELL)
ROCK-Inhibitor (Y27632)

1 mL of phosphate buffered saline was added to the resulting cell culture vessel and 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.

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 clumps were obtained that were crushed to sizes of 50 μm to 200 μm by pipetting with 1 mL of TeSR E8 medium. The obtained cell clumps (cell count 0.2 x 105 cells) were clamp-seeded into the above cell culture vessel.

At the time of seeding, 1.5 mL of liquid medium and a ROCK-specific inhibitor were added to the cell culture vessel so that the final concentration was 10 μM, and the cells were cultured in an incubator at 37 ° C and a _CO2 concentration of 5%. After that, the liquid in the dish was replaced with 1.5 mL of fresh liquid medium every 24 hours, and the culture was continued for 5 days. After the culture, 1 mL of Triple Express (manufactured by Thermo-Fisher) was added to the dish and left to stand at 37 ° C for 2 minutes, and the cells were collected and the cell count was measured using an auto cell counter EVE.

The results are shown in Table 1 below.

1... Container for cell culture 2... Container body 2a... Surface 3... Scaffolding material for cell culture

Claims (8)

Contains synthetic resin,
the synthetic resin contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin;
The synthetic resin contains 0.2 mol % or more and 20 mol % or less of a Bronsted basic group in a constituent unit,
The zeta potential at pH 7 is −60 mV or more;
A scaffold for cell culture having a plurality of pores with an average diameter of 10 nm or more and 5 μm or less. The scaffold for cell culture according to claim 1, which has a zeta potential of +30 mV or less at pH 7. The scaffold for cell culture according to claim 1 or 2, which has an elastic modulus of 0.01 GPa or more at 25°C as measured by the nanoindentation method. The scaffold for cell culture according to any one of claims 1 to 3 , which has a water swelling rate of 100% or less. The scaffold for cell culture according to any one of claims 1 to 4 , which is substantially free of animal-derived raw materials. The cell culture scaffold according to any one of claims 1 to 5 , wherein the synthetic resin comprises a synthetic resin having a polyvinyl acetal skeleton . The scaffold for cell culture according to any one of claims 1 to 6 , wherein the number of pores per unit area is 0.01 pores/µm2 or ^more and 1000 pores/µm2 ^or less. A cell culture vessel comprising the scaffold for cell culture according to any one of claims 1 to 7 in at least a part of a cell culture region.

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