Coating solution for forming cell scaffold and production method thereof

Abstract

Provided is a coating solution for forming a cell scaffold that can easily form a thick cell scaffold and increase cell proliferation.　This coating solution for forming a cell scaffold, according to the present invention, includes an an alcohol solvent and peptide-containing resin that has a synthetic resin part and a peptide part. The content of the peptide-containing resin is 0.1 wt% or more.

Description

Coating liquid for forming cell scaffold material and its manufacturing method

[0001] The present invention relates to a coating liquid for forming a cell scaffold and a method for producing the same.

[0002] Animal cells from humans, mice, rats, pigs, cattle, monkeys, etc. are used in research and development in academic fields, drug discovery, regenerative medicine, etc. Scaffold materials used to culture animal cells include adhesive proteins such as laminin and vitronectin, and natural polymeric materials such as Matrigel derived from mouse sarcoma.

[0003] Also known are scaffold materials using synthetic resins and scaffold materials using synthetic resins to which peptides are bound. For example, Patent Documents 1 and 2 listed below disclose scaffold materials for cell culture that contain a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion.

[0004] WO2020 / 230884A1 WO2020 / 230885A1

[0005] Cells can be cultured in a liquid medium using a scaffold material (cell scaffold) that has been processed or molded into a predetermined shape. Conventionally, cell scaffolds containing synthetic resins bound to peptides have been prepared by the solid-phase method. However, this method tends to make the preparation process of the cell scaffold complicated.

[0006] Furthermore, the present inventors have found that the use of a thicker cell scaffold can enhance cell proliferation compared to the use of a thinner cell scaffold, but the process of producing a thicker cell scaffold tends to be more complicated in the solid-phase method.

[0007] An object of the present invention is to provide a coating liquid for forming a cell scaffold that can easily form a thick cell scaffold and enhance cell proliferation, and also to provide a method for producing the coating liquid for forming the cell scaffold.

[0008] According to a broad aspect of the present invention, there is provided a coating liquid for forming a cell scaffold (in this specification, "coating liquid for forming a cell scaffold" may be abbreviated as "coating liquid") comprising a peptide-containing resin having a synthetic resin portion and a peptide portion, and an alcohol solvent, wherein the content of the peptide-containing resin is 0.1 wt % or more.

[0009] In a specific aspect of the coating liquid according to the present invention, the amount of amino acids detected by amino acid composition analysis after hydrolysis is 75 μmol / L or more.

[0010] In a specific aspect of the coating liquid according to the present invention, the synthetic resin portion includes a (meth)acrylic copolymer portion or a polyvinyl alcohol derivative portion.

[0011] In a specific aspect of the coating liquid according to the present invention, the peptide-containing resin is a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion, and the (meth)acrylic copolymer portion has a structural unit derived from a (meth)acrylate compound (A) represented by the following formula (A1) or the following formula (A2):

[0012]

[0013] In the formula (A1), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0014]

[0015] In the formula (A2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0016] In a specific aspect of the coating liquid according to the present invention, the (meth)acrylic copolymer portion has a structural unit derived from a (meth)acrylate compound (B) having a functional group capable of reacting with an amino group or a carboxyl group, and in the peptide-containing (meth)acrylic copolymer, the peptide portion is bonded to the functional group capable of reacting with an amino group or a carboxyl group.

[0017] In a specific aspect of the coating liquid according to the present invention, the peptide-containing resin is a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion.

[0018] In a specific aspect of the coating liquid according to the present invention, the peptide-containing polyvinyl alcohol derivative further has a structural unit derived from a (meth)acrylate compound (C) represented by the following formula (C1) or the following formula (C2):

[0019]

[0020] In the formula (C1), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0021]

[0022] In the formula (C2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0023] In a specific aspect of the coating liquid according to the present invention, the peptide-containing polyvinyl alcohol derivative further has a structural unit derived from a (meth)acrylate compound (D) having a functional group capable of reacting with an amino group or a carboxyl group, and in the peptide-containing polyvinyl alcohol derivative, the peptide portion is bonded to the functional group capable of reacting with the amino group or the carboxyl group.

[0024] In a specific aspect of the coating liquid according to the present invention, the peptide-containing polyvinyl alcohol derivative is a peptide-containing polyvinyl acetal resin having a polyvinyl acetal portion and a peptide portion.

[0025] In a specific aspect of the coating liquid according to the present invention, the peptide portion has an RGD sequence.

[0026] According to a broad aspect of the present invention, there is provided a method for producing the above-mentioned coating liquid for forming a cell scaffold, which comprises a preparation step of preparing a solution containing a synthetic resin having a functional group capable of reacting with an amino group or a carboxyl group, a peptide, and a condensing agent, and a reaction step of reacting the synthetic resin with the peptide.

[0027] In a specific aspect of the method for producing a coating liquid according to the present invention, the method further includes a purification step.

[0028] Also provided herein are the following methods for producing peptide-containing resins.

[0029] According to a broad aspect of the present invention, there is provided a method for producing a peptide-containing resin, the method comprising the steps of: preparing a first solution containing a first solvent, a synthetic resin having a functional group capable of reacting with an amino group or a carboxyl group, and a peptide; preparing a second solution containing a second solvent and a condensing agent; and mixing the first solution with the second solution.

[0030] In a specific aspect of the method for producing a peptide-containing resin according to the present invention, the method further comprises a reaction step of reacting the peptide with a synthetic resin having a functional group capable of reacting with the amino group or carboxyl group.

[0031] In a specific aspect of the method for producing a peptide-containing resin according to the present invention, the method further comprises a purification step.

[0032] In a specific aspect of the method for producing a peptide-containing resin according to the present invention, the synthetic resin having a functional group capable of reacting with an amino group or a carboxyl group is a (meth)acrylic copolymer or a polyvinyl alcohol derivative.

[0033] In a specific aspect of the method for producing a peptide-containing resin according to the present invention, the peptide has an RGD sequence.

[0034] The coating solution for forming a cell scaffold according to the present invention comprises a peptide-containing resin having a synthetic resin portion and a peptide portion, and an alcohol solvent, and the content of the peptide-containing resin is 0.1 wt % or more. Because the coating solution for forming a cell scaffold according to the present invention has the above-mentioned configuration, a thick cell scaffold can be easily formed, and cell proliferation can be enhanced.

[0035] The present invention will be described in detail below.

[0036] [Coating liquid for forming a cell scaffold] The coating liquid for forming a cell scaffold according to the present invention (hereinafter sometimes abbreviated as "coating liquid") contains a peptide-containing resin having a synthetic resin portion and a peptide portion, and an alcohol solvent, and the content of the peptide-containing resin is 0.1% by weight or more.

[0037] The coating liquid according to the present invention has the above-described configuration, and therefore can easily form a thick cell scaffold, thereby enhancing cell proliferation. The coating liquid according to the present invention can be easily applied to the surface of a container or the like. Furthermore, since the coating liquid according to the present invention contains a relatively large amount of peptide-containing resin, a thick cell scaffold can be easily formed by drying the applied coating liquid. Furthermore, the coating liquid according to the present invention can easily form a thick cell scaffold, thereby enhancing cell proliferation.

[0038] Furthermore, the coating solution according to the present invention does not require the use of natural polymeric materials such as extracellular matrix (ECM) as materials, and is therefore inexpensive, has little variation between lots, and is excellent in safety.

[0039] (Peptide-Containing Resin) The coating liquid contains a peptide-conjugated resin. The peptide-conjugated resin is a synthetic resin to which a peptide is bound. The peptide-conjugated resin has a synthetic resin portion and a peptide portion. Only one type of peptide-conjugated resin may be used, or two or more types may be used in combination.

[0040] In order to achieve the effects of the present invention, the content of the peptide-containing resin in 100% by weight of the coating liquid is 0.1% by weight or more.

[0041] The content of the peptide-containing resin in 100% by weight of the coating liquid is preferably 0.3% by weight or more, more preferably 1% by weight or more, even more preferably 3% by weight or more, preferably 15% by weight or less, more preferably 10% by weight or less, and even more preferably 8% by weight or less. When the content of the peptide-containing resin is above the lower limit, a thick cell scaffold can be more easily formed, and cell proliferation can be further improved. When the content of the peptide-containing resin is below the upper limit, the solubility of the peptide-containing resin in alcohol solvents can be further improved, and coatability can be improved.

[0042] In the coating solution, the amount of amino acids detected by hydrolysis amino acid composition analysis is preferably 75 μmol / L or more, more preferably 250 μmol / L or more, even more preferably 750 μmol / L or more, and preferably 10 mmol / L or less, more preferably 5 mmol / L or less, and even more preferably 3 mmol / L or less. The amount of amino acids corresponds to the amount of amino acids (μmol) detected in 1 L of coating solution. When the amount of amino acids is above the lower limit, cell proliferation can be further improved. When the amount of amino acids is below the upper limit, the solubility of the peptide-containing resin in alcohol solvents can be further improved, and coatability can also be improved.

[0043] The amount of the amino acid is measured, for example, as follows.

[0044] First, the coating solution is vacuum-dried to completely remove the solvent and obtain a dried product. 5 mg of the resulting dried product and 500 μL of 6 mol / L hydrochloric acid are placed in a vial, degassed, sealed, and heated at 110°C for 22 hours to hydrolyze the peptide. After the hydrolysis reaction, the hydrochloric acid is removed by N2 purging. Next, 500 μL of pH 2.2 sodium citrate buffer is added to redissolve the product. The resulting solution is placed in a centrifugal filter unit (Ultrafree-MC UFC30 series, 0.1 μm pore size, Merck) and centrifuged at 13,400 rpm for 3 minutes. The filtrate is analyzed by post-column derivatization using o-phthalaldehyde (hereinafter sometimes referred to as OPA) under the following analytical and detection conditions. The filtrate is diluted 1 to 125 times with pH 2.2 sodium citrate buffer depending on the spectral intensity before use.

[0045] <Analysis conditions> Column: Shim-pack Amino-Na (100 mmL x 6.0 mmI.D.) Ammonia trap column: ISC-30 / S0504 Na (50 mmL x 4.0 mmI.D.) Mobile phase: Amino acid mobile phase kit Na type (Shimadzu Corporation) Solution A: Sodium citrate buffer Solution B: Sodium citrate buffer Solution C: Sodium hydroxide solution Gradient elution (high separation mode) Flow rate: 0.4 mL / min Temperature: 60°C Injection volume: 10 μL

[0046] <Detection conditions> Reaction reagent: Amino acid analysis kit OPA reagent (Shimadzu Corporation) Solution A: Alkaline solution containing sodium hypochlorite Solution B: Alkaline solution containing OPA and N-acetyl-L-cysteine ​​Flow rate: 0.2 mL / min Temperature: 60°C Detection: Spectrofluorometric detector RF-20Axs Response: 1.5 sec Excitation wavelength: 350 nm Fluorescence wavelength: 450 nm Gain: ×1 Sensitivity: Low

[0047] Next, a calibration curve is prepared using an amino acid mixed standard solution type H for automatic amino acid analysis (manufactured by Wako Pure Chemical Industries, Ltd., each amino acid concentration 2.5 μmol / mL) as a quantitative standard reagent, and the obtained peak area value of each amino acid is quantified. The amount of amino acid (μmol / L) in the coating solution is calculated from the amount of coating solution used in the measurement, the amount of dried matter obtained, and the amino acid concentration.

[0048] The synthetic resin portion preferably has a (meth)acrylic copolymer portion or a polyvinyl alcohol derivative portion, more preferably a (meth)acrylic copolymer portion or a polyvinyl alcohol derivative portion. The peptide-containing resin is preferably a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion, or a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. In this case, cell proliferation can be further enhanced. The synthetic resin portion may have both a (meth)acrylic copolymer portion and a polyvinyl alcohol derivative portion.

[0049] In this specification, "(meth)acrylic" means one or both of "acrylic" and "methacrylic", and "(meth)acrylate" means one or both of "acrylate" and "methacrylate".

[0050] <Peptide-containing (meth)acrylic copolymer> From the viewpoint of further enhancing cell proliferation, the peptide-containing resin is preferably a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion. The peptide-containing (meth)acrylic copolymer is a (meth)acrylic copolymer to which a peptide is bound. The peptide-containing (meth)acrylic copolymer may be used alone or in combination of two or more types.

[0051] The (meth)acrylic copolymer portion preferably has a structural unit derived from a (meth)acrylate compound (A) represented by the following formula (A1) or (A2). In this case, the hydrophobicity of the peptide-containing resin (peptide-containing (meth)acrylic copolymer) can be increased, thereby making the cell scaffold less likely to dissolve into a liquid medium during cell culture and less likely to peel off from a container or the like during cell culture. As a result, the cell proliferation rate is less likely to decrease even when cells are cultured for a long period of time. The (meth)acrylate compound (A) may contain a (meth)acrylate compound represented by the following formula (A1), may contain a (meth)acrylate compound represented by the following formula (A2), or may contain both a (meth)acrylate compound represented by the following formula (A1) and a (meth)acrylate compound represented by the following formula (A2). When the (meth)acrylate compound (A) contains both a (meth)acrylate compound represented by the following formula (A1) and a (meth)acrylate compound represented by the following formula (A2), R in the following formula (A1) and R in the following formula (A2) may be the same or different. The (meth)acrylate compound (A) may be used alone or in combination of two or more. Furthermore, the (meth)acrylate compound represented by the following formula (A1) and the (meth)acrylate compound represented by the following formula (A2) may each be used alone or in combination of two or more.

[0052]

[0053] In the above formula (A1), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0054]

[0055] In the above formula (A2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0056] R in the above formula (A1) and R in the above formula (A2) may each be an aliphatic hydrocarbon group or an aromatic hydrocarbon group. From the viewpoint of improving the solubility of the peptide-containing (meth)acrylic copolymer, it is preferable that R in the above formula (A1) and R in the above formula (A2) each be an aliphatic hydrocarbon group. The aliphatic hydrocarbon group may be linear or may have a branched structure, and may or may not have a double bond. R in the above formula (A1) and R in the above formula (A2) may each be an alkyl group or an alkylene group.

[0057] The number of carbon atoms in R in the formula (A1) and the number of carbon atoms in R in the formula (A2) are each preferably 4 or more, more preferably 6 or more, even more preferably 8 or more, particularly preferably 10 or more, preferably 16 or less, more preferably 14 or less, and most preferably 12. When the carbon number is equal to or greater than the lower limit, the hydrophobicity of the peptide-containing (meth)acrylic copolymer can be increased, thereby making it difficult for the cell scaffold to dissolve into the liquid medium during cell culture and to peel off from the container during cell culture. When the carbon number is equal to or less than the upper limit, the solubility in alcohol solvents can be further improved, thereby further improving coatability and processability. In particular, when the carbon number is 12, the effects of the present invention can be even more effectively exerted and coatability and processability can be even further improved.

[0058] In 100 mol% of all structural units in the (meth)acrylic copolymer part, the content of structural units derived from the (meth)acrylate compound (A) is preferably 25 mol% or more, more preferably 30 mol% or more, even more preferably 40 mol% or more, particularly preferably 50 mol% or more, preferably 98 mol% or less, more preferably 95 mol% or less, even more preferably 90 mol% or less, even more preferably 80 mol% or less, particularly preferably 75 mol% or less. In 100 mol% of all structural units in the (meth)acrylic copolymer part, the content of structural units derived from the (meth)acrylate compound (A) is preferably 25 mol% or more, 98 mol% or less, more preferably 30 mol% or more, 95 mol% or less, even more preferably 40 mol% or more, 90 mol% or less, even more preferably 50 mol% or more, 80 mol% or less, particularly preferably 50 mol% or more, 75 mol% or less. When the content is equal to or higher than the lower limit, the hydrophobicity of the peptide-containing (meth)acrylic copolymer can be increased, and therefore the cell scaffold is less likely to be dissolved into the liquid medium during cell culture and is less likely to peel off from the container, etc. When the content is equal to or lower than the upper limit, the solubility in alcohol solvents can be further improved, thereby further improving the coatability and processability.

[0059] The (meth)acrylic copolymer portion preferably has a structural unit derived from a (meth)acrylate compound (B) having a functional group reactive with an amino group or a carboxyl group. The (meth)acrylate compound (B) may have a functional group reactive with an amino group, a functional group reactive with a carboxyl group, or a functional group reactive with an amino group and a functional group reactive with a carboxyl group. The (meth)acrylate compound (B) may be used alone or in combination of two or more.

[0060] Examples of the functional group capable of reacting with the amino group or carboxyl group include a carboxyl group, a thiol group, an amino group, and a cyano group.

[0061] From the viewpoint of effectively achieving the effects of the present invention, it is preferred that in the peptide-containing (meth)acrylic copolymer, the peptide moiety is bonded to a functional group capable of reacting with the amino group or carboxyl group. More specifically, it is preferred that the carboxyl group or amino group of the amino acid constituting the peptide moiety is bonded to a functional group capable of reacting with the amino group or carboxyl group.

[0062] The functional group capable of reacting with an amino group or a carboxyl group is preferably a carboxyl group or an amino group. The (meth)acrylate compound (B) preferably has a carboxyl group or an amino group.

[0063] Examples of the (meth)acrylate compound (B) include (meth)acrylic acid, 3-butenoic acid, 4-pentenoic acid, 5-hexenoic acid, 6-heptenoic acid, 7-octenoic acid, benzeneacrylic acid, (meth)acryloyloxyethyl succinic acid, (meth)acryloyloxyethyl phthalic acid, (meth)acryloyloxypropyl succinic acid, (meth)acryloyloxypropyl phthalic acid, (meth)acryloyloxyethyl hexahydrosuccinic acid, (meth)acryloyloxyethyl hexahydrophthalic acid, (meth)acryloyloxypropyl hexahydrosuccinic acid, and (meth)acryloyloxypropyl hexahydrophthalic acid.

[0064] The (meth)acrylate compound (B) is preferably (meth)acrylic acid, (meth)acryloyloxyethyl succinic acid, (meth)acryloyloxypropyl succinic acid, (meth)acryloyloxyethyl hexahydrosuccinic acid, (meth)acryloyloxypropyl hexahydrosuccinic acid, or butenoic acid, and more preferably (meth)acrylic acid. In this case, the effects of the present invention can be more effectively exhibited.

[0065] Of the total structural units (100 mol%) of the (meth)acrylic copolymer portion, the content of structural units derived from the (meth)acrylate compound (B) is preferably 2 mol% or more, more preferably 5 mol% or more, even more preferably 10 mol% or more, even more preferably 20 mol% or more, particularly preferably 25 mol% or more, preferably 75 mol% or less, more preferably 70 mol% or less, even more preferably 60 mol% or less, and particularly preferably 50 mol% or less. When the content is above the lower limit, the solubility in alcohol solvents can be increased. When the content is below the upper limit, cell culture stability can be easily maintained over a long period of time.

[0066] In the total structural units (100 mol%) of the (meth)acrylic copolymer part, the total content of the structural units derived from the (meth)acrylate compound (A) and the structural units derived from the (meth)acrylate compound (B) is preferably 50 mol% or more, more preferably 65 mol% or more, even more preferably 80 mol% or more, even more preferably 90 mol% or more, particularly preferably 95 mol% or more, and most preferably 100 mol%.When the total content is above the lower limit, the effects of the present invention can be more effectively exhibited.In addition, the total content may be 100 mol% or less, or may be 90 mol% or less.

[0067] The (meth)acrylic copolymer portion may contain structural units derived from a (meth)acrylate compound different from both the (meth)acrylate compound (A) and the (meth)acrylate compound (B), provided that the object of the present invention is not violated. Furthermore, the (meth)acrylic copolymer portion may contain structural units derived from a vinyl compound copolymerizable with the (meth)acrylate compound, provided that the object of the present invention is not violated.

[0068] The content of the structural unit derived from the (meth)acrylate compound (A) in the (meth)acrylic copolymer portion, the content of the structural unit derived from the (meth)acrylate compound (B), and the content of the structural unit derived from the (meth)acrylate compound (C) in the peptide-containing polyvinyl alcohol derivative described below can be measured, for example, by NMR (nuclear magnetic resonance).

[0069] <Peptide-Containing Polyvinyl Alcohol Derivative> From the viewpoint of further enhancing cell proliferation, the peptide-containing resin is preferably a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. The polyvinyl alcohol derivative portion is a portion derived from a polyvinyl alcohol derivative. The polyvinyl alcohol derivative is a compound derived from polyvinyl alcohol. From the viewpoint of further enhancing the adhesiveness between the cell scaffold and cells, the polyvinyl alcohol derivative is preferably a polyvinyl acetal resin, and the polyvinyl alcohol derivative portion is preferably a polyvinyl acetal portion. That is, the peptide-containing polyvinyl alcohol derivative is more preferably a peptide-containing polyvinyl acetal resin having a polyvinyl acetal portion and a peptide portion. Note that the polyvinyl alcohol derivative and the polyvinyl acetal resin may each be used alone, or two or more types may be used in combination.

[0070] The polyvinyl alcohol derivative portion and the polyvinyl acetal portion preferably have an acetal group, a hydroxyl group, and an acetyl group in their side chains. However, the polyvinyl alcohol derivative portion and the polyvinyl acetal portion may not have an acetyl group, for example. For example, by bonding all of the acetyl groups in the polyvinyl alcohol derivative portion and the polyvinyl acetal portion to a linker, the polyvinyl alcohol derivative portion and the polyvinyl acetal portion may not have an acetyl group.

[0071] The polyvinyl acetal resin can be synthesized by acetalizing polyvinyl alcohol with an aldehyde.

[0072] The aldehyde used for acetalization of polyvinyl alcohol is not particularly limited. Examples of the aldehyde include aldehydes having 1 to 10 carbon atoms. The aldehyde may or may not have a chain aliphatic group, a cyclic aliphatic group, or an aromatic group. The aldehyde may be a chain aldehyde or a cyclic aldehyde. Only one type of the aldehyde may be used, or two or more types may be used in combination.

[0073] From the viewpoint of further enhancing the adhesiveness between the cell scaffold and cells, the aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, or pentanal, and more preferably butyraldehyde. Therefore, the polyvinyl acetal resin is more preferably a polyvinyl butyral resin, and the polyvinyl acetal moiety is more preferably a polyvinyl butyral moiety. From the viewpoint of further enhancing the adhesiveness between the cell scaffold and cells, the peptide-containing resin is preferably a peptide-containing polyvinyl butyral resin having a polyvinyl butyral moiety and a peptide moiety.

[0074] In the peptide-containing polyvinyl alcohol derivative, the degree of acetalization of the polyvinyl alcohol derivative portion and the polyvinyl acetal portion (the degree of butyralization in the case of the polyvinyl butyral portion) is preferably 40 mol% or more, more preferably 50 mol% or more, preferably 90 mol% or less, more preferably 85 mol% or less. When the degree of acetalization is above the lower limit, the cell fixation can be further improved, and the cells can grow efficiently. When the degree of acetalization is below the upper limit, the solubility in solvents can be improved.

[0075] In the peptide-containing polyvinyl alcohol derivative, the hydroxyl group content (hydroxyl group amount) of the polyvinyl alcohol derivative portion and the polyvinyl acetal portion is preferably 15 mol % or more, more preferably 20 mol % or more, and preferably 45 mol % or less, more preferably 30 mol % or less, and even more preferably 25 mol % or less.

[0076] In the peptide-containing polyvinyl alcohol derivative, the degree of acetylation (amount of acetyl groups) of the polyvinyl alcohol derivative portion and the polyvinyl acetal portion is preferably 1 mol % or more, more preferably 2 mol % or more, and preferably 5 mol % or less, more preferably 4 mol % or less. When the degree of acetylation is equal to or greater than the above lower limit and equal to or less than the above upper limit, the reaction efficiency between the polyvinyl acetal resin and the linker can be increased.

[0077] The degree of acetalization, the degree of acetylation and the amount of hydroxyl groups in the polyvinyl alcohol derivative portion and the polyvinyl acetal portion are １ It can be measured by H-NMR (nuclear magnetic resonance spectroscopy).

[0078] The peptide-containing polyvinyl alcohol derivative preferably further has a structural unit derived from a (meth)acrylate compound (C) represented by the following formula (C1) or (C2). In this case, the formation of hydrogen bonds between peptides (particularly intermolecular hydrogen bonds) can be suppressed, thereby further improving solubility in alcohol solvents. The (meth)acrylate compound (C) may contain a (meth)acrylate compound represented by the following formula (C1), may contain a (meth)acrylate compound represented by the following formula (C2), or may contain both a (meth)acrylate compound represented by the following formula (C1) and a (meth)acrylate compound represented by the following formula (C2). When the (meth)acrylate compound (C) contains both a (meth)acrylate compound represented by the following formula (C1) and a (meth)acrylate compound represented by the following formula (C2), R in the following formula (C1) and R in the following formula (C2) may be the same or different. The (meth)acrylate compound (C) may be used alone or in combination of two or more thereof. Furthermore, the (meth)acrylate compound represented by the following formula (C1) and the (meth)acrylate compound represented by the following formula (C2) may each be used alone or in combination of two or more thereof.

[0079]

[0080] In the formula (C1), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0081]

[0082] In the above formula (C2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0083] R in the above formula (C1) and R in the above formula (C2) may each be an aliphatic hydrocarbon group or an aromatic hydrocarbon group. From the viewpoint of improving the solubility of the peptide-containing polyvinyl alcohol derivative in an alcohol solvent, it is preferable that R in the above formula (C1) and R in the above formula (C2) are each an aliphatic hydrocarbon group. In this case, the aliphatic hydrocarbon group may be linear, may have a branched structure, may have a double bond, or may not have a double bond. R in the above formula (C1) and R in the above formula (C2) may each be an alkyl group or an alkylene group.

[0084] The number of carbon atoms in R in the above formula (C1) and the number of carbon atoms in R in the above formula (C2) are each preferably 4 or more, more preferably 6 or more, even more preferably 8 or more, particularly preferably 10 or more, preferably 16 or less, more preferably 14 or less, and most preferably 12. When the number of carbon atoms is equal to or more than the above lower limit and equal to or less than the above upper limit, the formation of hydrogen bonds between peptides can be suppressed, and therefore, the solubility in alcohol solvents can be further improved.

[0085] In the peptide-containing polyvinyl alcohol derivative, the content of the structural unit derived from the (meth)acrylate compound (C) is preferably 5 mol % or more, more preferably 10 mol % or more, and preferably 50 mol % or less, more preferably 30 mol % or less. When the content is equal to or more than the lower limit and equal to or less than the upper limit, the formation of hydrogen bonds between peptides can be suppressed, and therefore the solubility in alcohol solvents can be further improved.

[0086] The peptide-containing polyvinyl alcohol derivative preferably further comprises a structural unit derived from a (meth)acrylate compound (D) having a functional group capable of reacting with an amino group or a carboxyl group. The (meth)acrylate compound (D) may have a functional group capable of reacting with an amino group, a functional group capable of reacting with a carboxyl group, or a functional group capable of reacting with an amino group and a functional group capable of reacting with a carboxyl group. The (meth)acrylate compound (D) may be used alone or in combination of two or more.

[0087] Examples of the functional group capable of reacting with the amino group or carboxyl group include a carboxyl group, a thiol group, an amino group, a hydroxyl group, and a cyano group.

[0088] To effectively achieve the effects of the present invention, the peptide portion of the peptide-containing polyvinyl alcohol derivative is preferably bound to a functional group capable of reacting with the amino or carboxyl group. More specifically, the carboxyl or amino group of the amino acid constituting the peptide portion is preferably bound to a functional group capable of reacting with the amino or carboxyl group.

[0089] The functional group capable of reacting with an amino group or a carboxyl group is preferably a carboxyl group or an amino group. The (meth)acrylate compound (D) preferably has a carboxyl group or an amino group.

[0090] Examples of the (meth)acrylate compound (D) include the compounds listed above as the (meth)acrylate compound (B).

[0091] The (meth)acrylate compound (D) is preferably (meth)acrylic acid, (meth)acryloyloxyethyl succinic acid, (meth)acryloyloxypropyl succinic acid, (meth)acryloyloxyethyl hexahydrosuccinic acid, (meth)acryloyloxypropyl hexahydrosuccinic acid, or butenoic acid, and more preferably (meth)acrylic acid. In this case, the effects of the present invention can be more effectively exhibited.

[0092] <Peptide portion> The peptide portion is a structural portion derived from a peptide. The peptide portion has an amino acid sequence. The peptide constituting the peptide portion may be an oligopeptide or a polypeptide. Only one type of the peptide may be used, or two or more types may be used in combination.

[0093] The number of amino acid residues in the peptide portion is preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, and preferably 10 or less, more preferably 8 or less, and even more preferably 6 or less. When the number of amino acid residues is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased. However, the number of amino acid residues in the peptide portion may be more than 10 or more than 15.

[0094] 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 phage display, Sepharose bead, or plate coating. The phage display method can be, for example, the method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995, pp. 1189-1196." The Sepharose bead method can be, for example, the method described in "Protein, Nucleic Acid, Enzyme, Vol. 45, No. 15 (2000) 2477." The plate coating method can be, for example, the method described in "Protein, Nucleic Acid, Enzyme, Vol. 45, No. 15 (2000) 2477."

[0095] Examples of the cell adhesive amino acid sequence 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 those described in "Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Medical Center Journal, Vol. 8, No. 1, pp. 58-66, 1992." The peptide portion may have only one or more of the cell-adhesive amino acid sequences.

[0096] The cell adhesive amino acid sequence preferably has at least one of the above-mentioned cell adhesive amino acid sequences, more preferably has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, even more preferably has an RGD sequence, and particularly preferably has at least an RGD sequence represented by the following formula (1): In this case, adhesiveness to cells after seeding can be further increased, and the cell proliferation rate can be further increased.

[0097] Arg-Gly-Asp-X...Formula (1)

[0098] In the above formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0099] The peptide portion may be linear or may have a cyclic peptide backbone. The cyclic peptide backbone is a cyclic backbone composed of a plurality of amino acids. From the viewpoint of more effectively exerting the effects of the present invention, the cyclic peptide backbone is preferably composed of four or more amino acids, more preferably five or more amino acids, and preferably ten or less amino acids.

[0100] In the peptide-containing resin, the content of the peptide moiety is preferably 0.5 mol% or more, more preferably 1 mol% or more, even more preferably 2 mol% or more, particularly preferably 5 mol% or more, preferably 25 mol% or less, more preferably 20 mol% or less, even more preferably 15 mol% or less, and particularly preferably 10 mol% or less. When the content of the peptide moiety is above the lower limit, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased. Furthermore, when the content of the peptide moiety is below the upper limit, production costs can be reduced. The content of the peptide moiety (mol%) is the amount of the peptide moiety relative to the sum of the amounts of the substances of each structural unit constituting the peptide-containing resin.

[0101] The content of the peptide moiety can be measured, for example, by NMR (nuclear magnetic resonance).

[0102] <Other Details of Peptide-Containing Resin> The number-average molecular weight of the peptide-containing (meth)acrylic copolymer is preferably 5,000 or more, more preferably 10,000 or more, even more preferably 50,000 or more, and preferably 5,000,000 or less, more preferably 2,500,000 or less, and even more preferably 1,000,000 or less. When the number-average molecular weight is equal to or greater than the lower limit, the hydrophobicity of the peptide-containing (meth)acrylic copolymer can be increased, thereby making it difficult for the cell scaffold to dissolve into a liquid medium during cell culture and for the cell scaffold to peel off from a container or the like during cell culture. When the number-average molecular weight is equal to or less than the upper limit, the solubility in alcohol solvents can be increased.

[0103] The number-average molecular weight of the peptide-containing polyvinyl alcohol derivative is preferably 10,000 or more, more preferably 50,000 or more, even more preferably 100,000 or more, preferably 5,000,000 or less, more preferably 2,500,000 or less, and even more preferably 1,000,000 or less. If the number-average molecular weight is above the lower limit, the cell scaffold material is less likely to dissolve into the liquid medium during cell culture, and the cell scaffold material is less likely to peel off from the container or the like during cell culture. If the number-average molecular weight is below the upper limit, the solubility in alcohol solvents can be increased.

[0104] The number average molecular weight of the peptide-containing resin (peptide-containing (meth)acrylic copolymer, peptide-containing polyvinyl alcohol derivative) can be measured, for example, by the following method. The peptide-containing resin is dissolved in tetrahydrofuran (THF) to prepare a 0.2 wt % solution of the peptide-containing resin. Next, the molecular weight is evaluated using a gel permeation chromatography (GPC) measuring device (APC system, manufactured by Waters) under the following measurement conditions.

[0105] Column: HSPgel HR MB-M 6.0 x 150 mm Flow rate: 0.5 mL / min Column temperature: 40°C Injection volume: 10 μL Detector: RI, PDA Standard sample: polystyrene

[0106] (Alcohol Solvent) The coating liquid contains an alcohol solvent. The alcohol solvent may be used alone or in combination of two or more.

[0107] Examples of the alcohol solvent include methanol, ethanol, butanol, isobutanol, sec-butanol, tert-butanol, isopropanol, propanol, benzyl alcohol, octyl alcohol, lauryl alcohol, and ethylene glycol.

[0108] From the viewpoint of successfully forming a resin film (cell scaffolding material) having a uniform thickness, the alcohol solvent is preferably a lower alcohol having 2 to 4 carbon atoms, more preferably ethanol, propanol or butanol, and even more preferably butanol.

[0109] The content of the alcohol solvent in 100% by weight of the coating liquid is preferably 80% by weight or more, more preferably 85% by weight or more, even more preferably 90% by weight or more, and preferably 99.9% by weight or less, more preferably 99% by weight or less, and even more preferably 98% by weight or less. When the content of the alcohol solvent is equal to or more than the above lower limit and equal to or less than the above upper limit, the solubility of the peptide-containing resin in the alcohol solvent can be further increased, and the coatability can also be improved.

[0110] The total content of the peptide-containing resin and the alcohol solvent in the coating solution (100% by weight) is preferably 80% by weight or more, more preferably 85% by weight or more, and even more preferably 90% by weight or more. The total content may be 100% by weight or less, or may be less than 100% by weight.

[0111] (pH Adjusting Agent) The coating liquid preferably contains a pH adjusting agent. By using a pH adjusting agent, the solubility of the peptide-containing resin in an alcohol solvent can be adjusted. Examples of the pH adjusting agent include organic acids, inorganic acids, organic bases, and inorganic bases. Examples of the pH adjusting agent include acetic acid, trifluoroacetic acid, hydrochloric acid, triethylamine, and N,N-diisopropylethylamine. The pH adjusting agent may be used alone or in combination of two or more.

[0112] The content of the pH adjuster is preferably 0.1 parts by weight or more, more preferably 0.5 parts by weight or more, and preferably 20 parts by weight or less, more preferably 15 parts by weight or less, based on 100 parts by weight of the alcohol solvent.

[0113] (Other Components) The coating liquid may contain other components different from the above-described components (the peptide-containing resin, the alcohol solvent, and the pH adjuster) within a range that does not impair the effects of the present invention. Examples of the other components include solvents other than alcohol solvents, polysaccharides, etc. One type of the other components may be used alone, or two or more types may be used in combination.

[0114] Examples of solvents other than the alcohol solvents include water, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, dichloromethane, and acetonitrile.

[0115] The coating liquid preferably contains substantially no animal-derived raw materials. By not containing any animal-derived raw materials, a cell scaffold can be provided that is highly safe and has little variation in quality during production. Note that "substantially free of animal-derived raw materials" means that the content of animal-derived raw materials in the coating liquid is 3% by weight or less. In the coating liquid, the content of animal-derived raw materials in the coating liquid is preferably 1% by weight or less, and most preferably 0% by weight. In other words, it is most preferable that the coating liquid does not contain any animal-derived raw materials at all.

[0116] (Method for Producing Coating Liquid) The method for producing a coating liquid according to the present invention is the method for producing the coating liquid described above. The method for producing a coating liquid according to the present invention includes a preparation step of preparing a solution containing a synthetic resin having a functional group reactive with an amino group or a carboxyl group, a peptide, and a condensing agent, and a reaction step of reacting the synthetic resin with the peptide.

[0117] In this specification, a "synthetic resin having a functional group capable of reacting with an amino group or a carboxyl group" may be referred to as a "synthetic resin X."

[0118] Therefore, the method for producing a coating liquid according to the present invention comprises a preparation step of preparing a solution containing synthetic resin X, a peptide, and a condensing agent, and a reaction step of reacting the synthetic resin X with the peptide.

[0119] The synthetic resin X may have a functional group reactive with an amino group, or may have a functional group reactive with a carboxyl group, or may have a functional group reactive with an amino group and a functional group reactive with a carboxyl group.

[0120] Examples of the synthetic resin X include a (meth)acrylate copolymer and a polyvinyl acetal derivative. More specifically, examples of the synthetic resin X include a (meth)acrylate copolymer obtained by polymerizing a monomer mixture containing the (meth)acrylate compound (A) and the (meth)acrylate compound (B), and a polyvinyl acetal derivative having a structural unit derived from the (meth)acrylate compound (D). Only one type of the synthetic resin X may be used, or two or more types may be used in combination.

[0121] Examples of the peptide include peptides having the number of amino acid residues and amino acid sequence described in the above-mentioned column for the peptide portion. Only one type of the peptide may be used, or two or more types may be used in combination.

[0122] As the condensing agent, a conventionally known condensing agent can be used, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, diphenylphosphoric azide, hexamethylphosphoric triamide, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, or 2-chloro-4,6-dimethoxy-1,3,5-triazine. , 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 4,6-dimethoxy-1,3,5-triazin-2-yl)-(2-octoxy-2-oxoethyl)dimethylammonium trifluoromethanesulfonate, 2,2,6,6-tetramethylpiperidine, and (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate. The above condensing agents may be used alone or in combination of two or more.

[0123] In the preparation step, a solution containing synthetic resin X, a peptide, a condensing agent, and a solvent is prepared. The solution containing synthetic resin X, a peptide, a condensing agent, and a solvent may be a dispersion. In the preparation step, the solution may be obtained by mixing synthetic resin X, a peptide, and a condensing agent in the same solvent. For example, the solution may be obtained by mixing solution A containing at least one component selected from synthetic resin X, a peptide, and a condensing agent with solution B containing at least one component selected from synthetic resin X, a peptide, and a condensing agent.

[0124] The preparation step preferably includes the steps of preparing a first solution containing a first solvent, synthetic resin X, and a peptide, preparing a second solution containing a second solvent and a condensing agent, and mixing the first solution with the second solution. In this case, the condensation reaction can be easily controlled. The first solvent and the second solvent may be the same type of solvent or different types of solvent. The first solution may be prepared before the second solution, or the second solution may be prepared before the first solution. The first solution and the second solution may be prepared simultaneously.

[0125] The solvent, the first solvent, and the second solvent may each be an alcohol solvent or a solvent other than an alcohol solvent. When the method for producing the coating liquid does not include a purification step described below, the solvent, the first solvent, and the second solvent are each preferably an alcohol solvent.

[0126] Examples of the solvent, the first solvent, and the second solvent include methanol, ethanol, butanol, dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, acetone, methyl ethyl ketone (MEK), dimethyl sulfoxide (DMSO), and water.

[0127] In the reaction step, the synthetic resin X is reacted with the peptide. It is preferable to bond a functional group of the synthetic resin X that can react with the amino group or carboxyl group to a carboxyl group or amino group of an amino acid that constitutes the peptide. This allows the peptide-containing resin to be obtained. When an alcohol solvent is used as the solvent, a liquid containing the peptide-containing resin and the alcohol solvent can be obtained in the reaction step. The reaction conditions in the reaction step are not particularly limited.

[0128] The method for producing the coating liquid preferably further comprises a purification step. The purification method in the purification step is not particularly limited, but examples include (1) a method in which a liquid phase containing a peptide-containing resin is recovered after liquid-liquid phase separation, (2) a method in which a peptide-containing resin is reprecipitated and then recovered, and (3) a method in which ion exchange is performed using an ion exchange resin. When the method for producing the coating liquid comprises the purification step, a coating liquid with fewer impurities can be obtained by removing unreacted condensing agent and peptide. The purification step may involve a combination of multiple purification methods. Furthermore, the solution obtained after purification may be used as is as the coating liquid, or the solvent may be evaporated and then redissolved in an alcohol solvent, and the resulting solution may be used as the coating liquid.

[0129] (Other Details of Coating Liquid) The coating liquid can be applied to the surface of a container or the like and then dried to form a cell scaffold. Examples of the coating method include spin coating, gravure coating, flexo coating, spray coating, cast coating, dip coating, jet dispensing, and inkjet methods.

[0130] The thickness of the cell scaffold is not particularly limited. The average thickness of the cell scaffold is preferably 10 nm or more, more preferably 20 nm or more, even more preferably 30 nm or more, preferably 1 μm or less, more preferably 500 nm or less, and even more preferably 200 nm or less. When the average thickness is equal to or greater than the lower limit, cell proliferation can be enhanced. The coating liquid according to the present invention can easily form a cell scaffold having an average thickness equal to or greater than the lower limit. When the average thickness is equal to or less than the upper limit, a cell scaffold with less surface irregularities and a good appearance can be easily obtained. The average thickness of the cell scaffold may be 50 nm or more, 100 nm or more, 1000 μm or less, or 500 μm or less.

[0131] The surface roughness Ra of the cell scaffold is preferably 10 nm or less, more preferably 5 nm or less. When the surface roughness Ra of the cell scaffold is equal to or less than the upper limit, a cell scaffold with less variation in cell proliferation can be obtained. The surface roughness Ra of the cell scaffold can be adjusted to equal to or less than the upper limit by appropriately selecting the coating method and drying conditions of the coating liquid.

[0132] The cell scaffold material is used as a scaffold for cells when the cells are cultured.

[0133] The cells include animal cells from humans, mice, rats, pigs, cows, monkeys, etc. The cells also include somatic cells, such as stem cells, progenitor cells, and mature cells. The somatic cells may be cancer cells.

[0134] Examples of the stem cells include somatic stem cells and embryonic stem cells, such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0135] Examples of the mature cells include nerve cells, cardiomyocytes, retinal cells, and hepatocytes.

[0136] The above-mentioned cell scaffold material is preferably used for two-dimensional culture (plate culture), three-dimensional culture or suspension culture of cells, more preferably for two-dimensional culture (plate culture) or three-dimensional culture, and even more preferably for two-dimensional culture.

[0137] The cell scaffold is preferably used for serum-free culture, and since the cell scaffold contains the peptide-containing resin, it can enhance cell adhesion, particularly the initial cell adhesion rate after cell seeding, even in serum-free culture without feeder cells or adhesive proteins.

[0138] (Cell culture substrate) A cell culture substrate can be produced by applying the coating liquid to the surface of a substrate and drying the applied coating liquid. The cell culture substrate has a substrate and the cell scaffold material formed on the surface of the substrate. In the cell culture substrate, the cell scaffold material is a dried layer of the coating liquid. In the cell culture substrate, the cell scaffold material is preferably a resin film.

[0139] The shape and size of the substrate are not particularly limited, and examples of the substrate that can be used include fibers, nonwoven fabrics, hollow fibers, particles, films, and porous membranes.

[0140] The fibers may have an average length of 1 μm to 10 mm and an average diameter of 100 nm to 300 μm. Examples of the material for the fibers include synthetic resin, cellulose, and glass. Examples of the synthetic resin include styrene copolymer, polyester, polyamide, (meth)acrylic resin, and polyvinyl alcohol.

[0141] The nonwoven fabric may have, for example, an average fiber diameter of 0.01 μm to 10 μm and an apparent density of 1 kg / m ３ ~100 kg / m ３ A nonwoven fabric having an average thickness of 10 μm to 1000 μm can be used. Examples of materials for the nonwoven fabric include synthetic resins and cellulose. Examples of the synthetic resins include polypropylene, polyester, polyamide, and polyphenylene sulfide.

[0142] The hollow fibers may have an inner diameter of 50 μm to 1000 μm, a membrane thickness of 10 μm to 400 μm, and a pore size of 0.001 μm to 0.5 μm. Examples of materials for the hollow fibers include polyethylene, polyamide, polyester, polyethersulfone, polysulfone, polyvinylidene fluoride, polytetrafluoroethylene, polypropylene, cellulose, polyacrylonitrile copolymer, polyvinyl chloride, polyethylene vinyl alcohol, and ceramic.

[0143] The particles may have an average particle diameter of 10 μm to 1000 μm and a specific gravity of 0.5 to 5.0. Examples of the material for the particles include synthetic resins, polysaccharides, and silica. Examples of the synthetic resins include polystyrene, (meth)acrylic resins, and divinylbenzene.

[0144] The film may have a thickness of, for example, 1 μm to 1000 μm. The film may be a single layer or a multilayer. Examples of materials for the film include polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, polyamide, silicone elastomer, polystyrene elastomer, and ionomer resin.

[0145] The porous membrane may have an average pore size of 0.01 μm to 10 μm, a porosity of 1 vol % to 95 vol %, and an average thickness of 1 μm to 100 μm. Examples of materials for the porous membrane include polyester, polyethylene, polyimide, and fluororesin.

[0146] The coating method and conditions for applying the coating liquid to the surface of the substrate can be appropriately selected to be optimal depending on the size, shape, etc. of the substrate.

[0147] For example, in the case of a film-like substrate, the coating liquid can be applied to the substrate using a roll coating method. Examples of roll coating methods include gravure coating, flexo coating, reverse coating, slot die coating, lip coating, and knife coating. The coating liquid may be applied to the entire surface of the film-like substrate, or may be applied to form a pattern such as a dot pattern or a stripe pattern.

[0148] Furthermore, for example, in the case of a porous substrate or a fibrous substrate, the coating liquid can be applied to the substrate by a dip coating method.

[0149] (Cell culture vessel) A cell culture vessel can be produced by applying the coating liquid to the surface of a container body and drying the applied coating liquid. The cell culture vessel has a container body and the cell scaffold material formed on the surface of the container body. In the cell culture vessel, the cell scaffold material is a dried layer of the coating liquid described above. In the cell culture vessel, the cell scaffold material is preferably a resin film.

[0150] As the container body, a conventionally known container body (container) can be used. The shape and size of the container body are not particularly limited. Examples of the container body that can be used include 2- to 384-well plates, single-layer flasks, multi-layer flasks, multi-sided flasks, dishes, roller bottles, bags, insert cups, and microchannel chips.

[0151] Examples of materials for the container body include synthetic resins, metals, glass, etc. Examples of synthetic resins include polystyrene, polyethylene, polypropylene, polyethersulfone, polycarbonate, polyester, polyisoprene, cycloolefin polymers, polyimides, polyamides, polyamideimides, (meth)acrylic resins, epoxy resins, and silicones.

[0152] The coating method and conditions for applying the coating liquid to the surface of the container body can be appropriately selected to be optimal depending on the size, shape, etc. of the container body.

[0153] For example, the coating liquid can be applied to the surface of the container body using a cast coating method, a spray coating method, a spin coating method, etc. The coating liquid may also be applied to the surface of the container body so as to form a pattern such as dots or stripes. Methods for applying the coating liquid so as to form a pattern include, for example, an inkjet method, a screen printing method, and a microcontact printing method.

[0154] (Method for Producing Peptide-Containing Resin) The present specification also discloses a method for producing a peptide-containing resin having the following configuration.

[0155] A method for producing a peptide-containing resin, comprising: (1) preparing a first solution containing a first solvent, a synthetic resin (synthetic resin X) having a functional group capable of reacting with an amino group or a carboxyl group, and a peptide; (2) preparing a second solution containing a second solvent and a condensing agent; and (3) mixing the first solution with the second solution.

[0156] The method for producing the peptide-containing resin is a method for producing a peptide-containing resin having a synthetic resin portion and a peptide portion. The method for producing the peptide-containing resin allows for the suitable production of a coating liquid, as described above. The peptide-containing resin obtained by the method for producing the peptide-containing resin is preferably used as a coating liquid containing the peptide-containing resin and an alcohol solvent, but does not necessarily have to be used as a coating liquid.

[0157] In the method for producing the peptide-containing resin, the first solution may be prepared before the second solution, or the second solution may be prepared before the first solution, or the first solution and the second solution may be prepared simultaneously.

[0158] The details of the synthetic resin X (synthetic resin having a functional group capable of reacting with an amino group or a carboxyl group) in the method for producing the peptide-containing resin are the same as the details of the synthetic resin X described in the section on the method for producing the coating liquid mentioned above.

[0159] In the method for producing a peptide-containing resin, the synthetic resin X is preferably a (meth)acrylate copolymer or a polyvinyl acetal derivative. In the method for producing a peptide-containing resin, the synthetic resin X is more preferably a (meth)acrylate copolymer obtained by polymerizing a monomer mixture containing the (meth)acrylate compound (A) and the (meth)acrylate compound (B), or a polyvinyl acetal derivative having a structural unit derived from the (meth)acrylate compound (D). Only one type of synthetic resin X may be used, or two or more types may be used in combination.

[0160] The details of the peptide in the method for producing the peptide-containing resin are the same as those described in the section on the method for producing the coating liquid. That is, the peptide includes a peptide having the number of amino acid residues and amino acid sequence described in the section on the peptide portion. Only one type of the peptide may be used, or two or more types may be used in combination.

[0161] The number of amino acid residues in the peptide is preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, and preferably 10 or less, more preferably 8 or less, and even more preferably 6 or less. When the number of amino acid residues is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased. However, the number of amino acid residues in the peptide may be more than 10 or more than 15.

[0162] The peptide preferably has a cell-adhesive amino acid sequence, more preferably has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, even more preferably has an RGD sequence, and particularly preferably has at least the RGD sequence represented by formula (1) above.

[0163] The peptide may be linear or have a cyclic peptide backbone. The cyclic peptide backbone is a cyclic backbone composed of a plurality of amino acids. The cyclic peptide backbone is preferably composed of four or more amino acids, more preferably five or more amino acids, and preferably ten or fewer amino acids.

[0164] The details of the condensing agent in the method for producing the peptide-containing resin are the same as those described in the section on the method for producing the coating liquid. The condensing agents may be used alone or in combination of two or more.

[0165] The details of the first solvent and the second solvent in the method for producing the peptide-containing resin are the same as those of the first solvent and the second solvent described in the section on the method for producing the coating liquid. Only one type of the first solvent and the second solvent may be used, or two or more types may be used in combination.

[0166] In the method for producing a peptide-containing resin, the first solvent is preferably an alcohol solvent, and the second solvent is preferably an alcohol solvent.

[0167] The method for producing the peptide-containing resin preferably further comprises a reaction step of reacting the synthetic resin X with the peptide.

[0168] In the reaction step, the synthetic resin X is reacted with the peptide. It is preferable to bond a functional group of the synthetic resin X that can react with the amino group or carboxyl group to a carboxyl group or amino group of an amino acid that constitutes the peptide. This allows the peptide-containing resin to be obtained. When an alcohol solvent is used as the solvent, a liquid containing the peptide-containing resin and the alcohol solvent can be obtained in the reaction step. The reaction conditions in the reaction step are not particularly limited.

[0169] The method for producing the peptide-containing resin preferably further comprises a purification step. The purification method in the purification step is not particularly limited, but examples include (1) a method in which the liquid phase containing the peptide-containing resin is recovered after liquid-liquid phase separation, (2) a method in which the peptide-containing resin is reprecipitated and then recovered, and (3) a method in which ion exchange is performed using an ion exchange resin. When the method for producing the peptide-containing resin comprises the purification step, the unreacted condensing agent and peptide are removed, thereby obtaining a peptide-containing resin with few impurities. A plurality of purification methods may be combined in the purification step.

[0170] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0171] The content of structural units and the content of peptide moieties in the resulting peptide-containing resin were measured by dissolving the peptide-containing resin in DMSO-d6 (dimethyl sulfoxide) and then using 1H-NMR (nuclear magnetic resonance spectroscopy).

[0172] The following peptides (A) and (B) were prepared.

[0173] Peptide (A): A linear peptide having the amino acid sequence Gly-Arg-Gly-Asp-Ser (5 amino acid residues, shown as GRGDS in the table). Peptide (B): A cyclic peptide having the amino acid sequence Arg-Gly-Asp-Phe-Lys (5 amino acid residues, Arg and Lys bond to form a cyclic skeleton, Phe is in the D-configuration, shown as c-RGDfK in the table).

[0174] Example 1 Preparation of Coating Liquid Preparation of Synthetic Resin (Synthetic Resin X) Having Functional Groups Reactive with Amino or Carboxyl Groups: A polyvinyl butyral resin having an acetalization degree (butyralization degree) of 65 mol%, a hydroxyl group content of 32 mol%, and an acetyl group content of 3 mol% was prepared as a polyvinyl acetal resin. 30 parts by weight of acrylic acid and 70 parts by weight of the polyvinyl acetal resin were dissolved in 255 parts by weight of tetrahydrofuran to obtain a polymer mixture solution. 0.015 parts by weight of Perbutyl O (manufactured by NOF Corporation) was dissolved in the obtained polymer mixture solution, and the mixture was reacted at 90°C for 6 hours. The reacted solution was then mixed with 30,000 parts by weight of water. The resulting precipitate was vacuum dried at 80°C for 3 hours to prepare a polyvinyl acetal resin X having structural units derived from acrylic acid (a polyvinyl butyral resin having structural units derived from acrylic acid) as the synthetic resin X.

[0175] Preparation step: DMF was prepared as the first solvent. DMF was prepared as the second solvent. Peptide (A) was prepared as the peptide. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was prepared as the condensing agent. 50 parts by weight of polyvinyl acetal resin X and 2 parts by weight of peptide were mixed with 1,000 parts by weight of the first solvent to prepare a first solution. 1 part by weight of the condensing agent was mixed with 1,000 parts by weight of the second solvent to prepare a second solution. The first solution and the second solution were mixed to prepare a solution containing polyvinyl acetal resin X, the peptide, and the condensing agent.

[0176] Reaction step: The obtained solution was reacted at 40°C for 2 hours to cause dehydration condensation between the carboxyl group in the structural unit derived from acrylic acid of polyvinyl acetal resin X and the amino group of Gly of the peptide, thereby obtaining a solution containing a peptide-containing polyvinyl acetal resin (referred to as resin X1 in the Tables).

[0177] Purification step: The resulting solution containing the peptide-containing polyvinyl acetal resin was diluted 100-fold with DMF and washed by adding dropwise at a rate of 0.3 mL / min to a column packed with ion exchange resin (manufactured by Organo Corporation). The washed solution was vacuum dried at 60°C for 3 hours to obtain a dried product, which was then dissolved in butanol (alcohol solvent). 5 parts by weight of acetic acid (pH adjuster) was added to 100 parts by weight of butanol (alcohol solvent) to obtain a coating solution containing the peptide-containing polyvinyl acetal resin (resin X1) and butanol (alcohol solvent). The content of the peptide-containing polyvinyl acetal resin in the coating solution was 0.1% by weight.

[0178] <Preparation of cell culture vessel> 20 μL of the obtained coating liquid was applied to each well of a 6-well plate by cast coating, and the alcohol solvent was then removed by vacuum drying for 3 hours at 60° C. In this way, a cell culture vessel was obtained in which a cell scaffold material (resin film), which was a dried layer of the coating liquid, was placed on the bottom surface of each well.

[0179] (Examples 2 and 4 and Comparative Example 2) Coating solutions and cell culture vessels were obtained in the same manner as in Example 1, except that the content of the peptide-containing polyvinyl acetal resin in the coating solution was set to the contents shown in Tables 3 and 4.

[0180] (Examples 3 and 5) The amount of peptide contained in the first solution was changed to 0.3 parts by weight (Example 3) and 4 parts by weight (Example 5), respectively, so that the content of the peptide moiety in the peptide-containing polyvinyl acetal resin was the content shown in Table 1. Furthermore, the content of the peptide-containing polyvinyl acetal resin in the coating liquid was the content shown in Table 3. Except for these, a coating liquid and a cell culture vessel were obtained in the same manner as in Example 1. In the tables, the peptide-containing polyvinyl acetal resin used in Example 3 is referred to as resin X2, and the peptide-containing polyvinyl acetal resin used in Example 5 is referred to as resin X3.

[0181] Example 6: Peptide (B) was prepared as the peptide, and the amount of peptide contained in the first solution was 2 parts by weight. A peptide-containing polyvinyl acetal resin (referred to as resin X4 in the table) was synthesized by dehydration condensation of a carboxyl group in a structural unit derived from acrylic acid of polyvinyl acetal resin X with an amino group of Lys in the peptide. A coating solution and a cell culture vessel were obtained in the same manner as in Example 1, except that this peptide-containing polyvinyl acetal resin was used and the content of the peptide-containing polyvinyl acetal resin in the coating solution was set to the content shown in Table 4.

[0182] Example 7 A polymer mixed solution was obtained by dissolving 15 parts by weight of acrylic acid, 15 parts by weight of dodecyl acrylate, and 70 parts by weight of polyvinyl acetal resin in 255 parts by weight of tetrahydrofuran. Except for this, a polyvinyl acetal resin X having structural units derived from acrylic acid and structural units derived from dodecyl acrylate (a polyvinyl butyral resin having structural units derived from acrylic acid and structural units derived from dodecyl acrylate) was prepared as the synthetic resin X in the same manner as in Example 1.

[0183] Furthermore, peptide (B) was prepared as the peptide, and the amount of peptide contained in the first solution was 4 parts by weight. A peptide-containing polyvinyl acetal resin (referred to as resin X5 in the table) was synthesized in the same manner as in Example 6. A coating solution and a cell culture vessel were obtained in the same manner as in Example 1, except that this peptide-containing polyvinyl acetal resin was used and the content of the peptide-containing polyvinyl acetal resin in the coating solution was set to the content shown in Table 4.

[0184] (Example 8) A coating solution and a cell culture vessel were obtained in the same manner as in Example 1, except that the alcohol solvent in the coating solution was changed to ethanol and the content of the peptide-containing polyvinyl acetal resin in the coating solution was set to the content shown in Table 4.

[0185] Comparative Example 1: The polyvinyl acetal resin X (the resin before reaction with the peptide, shown as resin Y1 in the table) prepared in Example 1 was used. In addition, the alcohol solvent in the coating liquid was changed to ethanol, and the content of resin Y1 in the coating liquid was set to the content shown in Table 4. Other than these, a coating liquid and a cell culture vessel were obtained in the same manner as in Example 1.

[0186] Example 9 Preparation of Coating Liquid Preparation of a synthetic resin (synthetic resin X) having a functional group capable of reacting with an amino group or a carboxyl group: 70 parts by weight of dodecyl acrylate and 30 parts by weight of acrylic acid were dissolved in 27 parts by weight of tetrahydrofuran to obtain an acrylic monomer solution. 0.0575 parts by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained acrylic monomer solution, and the obtained solution was applied to a PET film. The coated 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 (meth)acrylic copolymer solution was obtained by irradiating the (meth)acrylic copolymer solution with light at 80° C. for 3 hours, thereby obtaining a (meth)acrylic copolymer. In this way, (meth)acrylic copolymer X was prepared as the synthetic resin X.

[0187] Preparation step: DMF was prepared as the first solvent. DMF was prepared as the second solvent. Peptide (A) was prepared as the peptide. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was prepared as the condensing agent. 50 parts by weight of (meth)acrylic copolymer X and 2 parts by weight of peptide were mixed with 1000 parts by weight of the first solvent to prepare a first solution. Furthermore, 1 part by weight of the condensing agent was mixed with 1000 parts by weight of the second solvent to prepare a second solution. The first solution and the second solution were mixed to prepare a solution containing (meth)acrylic copolymer X, the peptide, and the condensing agent.

[0188] Reaction step: The obtained solution was reacted at 40°C for 2 hours to cause dehydration condensation between the carboxyl group in the structural unit derived from acrylic acid of (meth)acrylic copolymer X and the amino group of Gly of the peptide, thereby synthesizing a peptide-containing (meth)acrylic copolymer (referred to as resin X6 in the table).

[0189] Purification step: The resulting solution containing the peptide-containing (meth)acrylic copolymer was diluted 100-fold with DMF and washed by adding dropwise at a rate of 0.3 mL / min to a column packed with ion exchange resin (manufactured by Organo Corporation). The washed solution was vacuum dried at 60°C for 3 hours to obtain a dried product, which was then dissolved in butanol (alcohol solvent). 5 parts by weight of acetic acid (pH adjuster) was added to 100 parts by weight of butanol (alcohol solvent) to obtain a coating solution containing the peptide-containing (meth)acrylic copolymer (resin X6) and butanol (alcohol solvent). The content of the peptide-containing (meth)acrylic copolymer in the coating solution was 0.1% by weight.

[0190] <Preparation of cell culture vessel> 20 μL of the obtained coating liquid was applied to each well of a 6-well plate by cast coating, and the alcohol solvent was then removed by vacuum drying for 3 hours at 60° C. In this way, a cell culture vessel was obtained in which a cell scaffold material (resin film), which was a dried layer of the coating liquid, was placed on the bottom surface of each well.

[0191] (Examples 10 and 11 and Comparative Example 3) Coating solutions and cell culture vessels were obtained in the same manner as in Example 9, except that the content of the peptide-containing (meth)acrylic copolymer in the coating solution was set to the content shown in Table 5.

[0192] Example 12: Peptide (B) was prepared as the peptide, and the amount of peptide contained in the first solution was 2 parts by weight. A peptide-containing (meth)acrylic copolymer (referred to as resin X7 in the table) was synthesized by dehydration condensation of a carboxyl group in a structural unit derived from acrylic acid in (meth)acrylic copolymer X with an amino group of Lys in the peptide. A coating solution and a cell culture vessel were obtained in the same manner as in Example 9, except that this peptide-containing (meth)acrylic copolymer was used and the content of the peptide-containing (meth)acrylic copolymer in the coating solution was the content shown in Table 5.

[0193] (Example 13) A coating liquid and a cell culture vessel were obtained in the same manner as in Example 9, except that 70 parts by weight of butyl acrylate was used instead of dodecyl acrylate and the content of the peptide-containing (meth)acrylic copolymer in the coating liquid was set to the content shown in Table 5. The peptide-containing (meth)acrylic copolymer prepared in Example 13 is referred to as Resin X8 in the table.

[0194] Example 14 A coating liquid and a cell culture vessel were obtained in the same manner as in Example 9, except that 70 parts by weight of octyl acrylate was used instead of dodecyl acrylate and the content of the peptide-containing (meth)acrylic copolymer in the coating liquid was set to the content shown in Table 5. The peptide-containing (meth)acrylic copolymer prepared in Example 14 is referred to as Resin X9 in the table.

[0195] (Evaluation) (1) Solubility in Ethanol (Maximum Dissolution Amount at 60° C.) The solubility in ethanol (maximum dissolution amount at 60° C.) of the obtained resins X1 to X9 and Y1 was evaluated according to the following criteria.

[0196] <Evaluation criteria for solubility in ethanol> ○○: Maximum solubility is 1% by weight or more ○: Maximum solubility is 0.5% by weight or more and less than 1% by weight △: Maximum solubility is 0.1% by weight or more and less than 0.5% by weight ×: Maximum solubility is less than 0.1% by weight

[0197] (2) Amount of Amino Acids Detected in Coating Solution The amount of amino acids detected in the resulting coating solution by the amino acid composition analysis method after hydrolysis was measured by the method described above.

[0198] (3) Average Thickness of Cell Scaffold Material The average thickness of the cell scaffold material (resin film) in the obtained cell culture vessel was measured using a reflection spectroscopic film thickness measuring instrument ("OPTELICS" manufactured by Lasertec).

[0199] (4) Cell Culture Evaluation (Cell Proliferation) The following liquid media were prepared.

[0200] R-STEM (Rohto Pharmaceutical Co., Ltd.)

[0201] 1 mL of phosphate buffered saline was added to the obtained cell culture vessel, and the vessel was allowed to stand in an incubator at 37° C. for 1 hour, after which the phosphate buffered saline was removed from the cell culture vessel.

[0202] 5 x 10 in 1.5 mL of liquid medium ４ A cell suspension containing 100 cells (Lonza, human adipose-derived mesenchymal stem cells, model number: PT-5006) was prepared. This cell suspension was seeded into each well of a 6-well plate. The 6-well plate was then shaken left and right five times and incubated at 37°C and CO ２ The mixture was cultured in an incubator containing 5% of the lysate.

[0203] After 5 days of culture, the number of cells was counted using a NucleoCounter NC-3000 (MS Techno Systems). The doubling time was calculated according to the following calculation method, and the proliferation of the cells was evaluated according to the following criteria.

[0204] <Method of calculating doubling time> X = log2 ÷ {log(N(5)) - log(N(0))} × T X: doubling time (hours) N(0): number of cells seeded (cells) N(5): number of cells 5 days after seeding (cells) T: culture time (hours) Note that T = 120 because the culture was for 5 days.

[0205] For example, if the number of cells 5 days after seeding is 65,000 cells and the number of cells seeded is 20,000 cells, the doubling time (X) is log2÷(log65,000−log20,000)×120≈70 hours.

[0206] <Criteria for cell culture evaluation (cell proliferation)> AA: Doubling time (X) is less than 24 hours. A: Doubling time (X) is 24 hours or more but less than 28 hours. B: Doubling time (X) is 28 hours or more but less than 36 hours. C: Doubling time (X) is 36 hours or more.

[0207] Details and results are shown in Tables 1 to 5 below.

[0208]

[0209]

[0210]

[0211]

[0212]

Claims

1. a peptide-containing resin having a synthetic resin portion and a peptide portion; an alcohol solvent; A coating solution for forming a cell scaffold, wherein the content of the peptide-containing resin is 0.1% by weight or more.

2. The coating solution for forming a cell scaffold according to claim 1, wherein the amount of amino acids detected by an amino acid composition analysis method after hydrolysis is 75 μmol / L or more.

3. The coating liquid for forming a cell scaffold according to claim 1 or 2, wherein the synthetic resin portion has a (meth)acrylic copolymer portion or a polyvinyl alcohol derivative portion.

4. the peptide-containing resin is a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion, The coating liquid for forming a cell scaffold material according to claim 1 or 2, wherein the (meth)acrylic copolymer portion has a structural unit derived from a (meth)acrylate compound (A) represented by the following formula (A1) or the following formula (A2): 【Chemistry 1】 In the formula (A1), R represents a hydrocarbon group having 2 to 18 carbon atoms. 【Chemistry 2】 In the formula (A2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

5. the (meth)acrylic copolymer portion has a structural unit derived from a (meth)acrylate compound (B) having a functional group capable of reacting with an amino group or a carboxyl group, The coating liquid for forming a cell scaffold according to claim 4 , wherein in the peptide-containing (meth)acrylic copolymer, the peptide portion is bonded to a functional group capable of reacting with the amino group or carboxyl group.

6. The coating liquid for forming a cell scaffold according to claim 1 or 2, wherein the peptide-containing resin is a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion.

7. The coating liquid for forming a cell scaffold material according to claim 6, wherein the peptide-containing polyvinyl alcohol derivative further has a structural unit derived from a (meth)acrylate compound (C) represented by the following formula (C1) or the following formula (C2): 【Transformation 3】 In the formula (C1), R represents a hydrocarbon group having 2 to 18 carbon atoms. 【Chemistry 4】 In the formula (C2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

8. the peptide-containing polyvinyl alcohol derivative further has a structural unit derived from a (meth)acrylate compound (D) having a functional group capable of reacting with an amino group or a carboxyl group, The coating liquid for forming a cell scaffold according to claim 6 , wherein in the peptide-containing polyvinyl alcohol derivative, the peptide portion is bonded to a functional group capable of reacting with the amino group or carboxyl group.

9. The coating liquid for forming a cell scaffold according to claim 6 , wherein the peptide-containing polyvinyl alcohol derivative is a peptide-containing polyvinyl acetal resin having a polyvinyl acetal portion and a peptide portion.

10. The coating liquid for forming a cell scaffold according to claim 1 or 2, wherein the peptide portion has an RGD sequence.

11. A coating liquid for forming a cell scaffold material as described in claim 1 or 2, wherein the alcohol solvent is ethanol, propanol or butanol.

12. A coating liquid for forming a cell scaffold material as described in claim 1 or 2, wherein the content of the alcohol solvent is 80% by weight or more.

13. A coating liquid for forming a cell scaffold material as described in claim 1 or 2, wherein the total content of the peptide-containing resin and the alcohol solvent is 90% by weight or more.

14. A method for producing the coating solution for forming a cell scaffold according to claim 1 or 2, a preparation step of preparing a solution containing a synthetic resin having a functional group capable of reacting with an amino group or a carboxyl group, a peptide, and a condensing agent; a reaction step of reacting the synthetic resin with the peptide.

15. The method for producing a coating solution for forming a cell scaffold according to claim 14, further comprising a purification step.

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