Method for producing peptide-containing polyvinyl alcohol derivative and scaffold material for cell culture

Abstract

To provide a cell culture scaffold material having excellent cell adhesion.SOLUTION: A cell culture scaffold material according to the present invention contains a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion, and the peptide portion has a cyclic peptide skeleton.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a scaffold material for cell culture used for culturing cells. The present invention also relates to a cell culture vessel using the scaffold material for cell culture. [Background technology]

[0002] In research and development in academic fields, drug discovery, regenerative medicine, and the like, animal cells such as human, mouse, rat, pig, cow, and monkey are used. As scaffold materials used for culturing animal cells, adhesive proteins such as laminin and vitronectin, and natural polymer materials such as Matrigel derived from mouse sarcoma are used. By using natural polymer materials as scaffold materials, domains having cell adhesive amino acid sequences (Arg-Gly-Asp, etc.) contained in laminin, vitronectin, etc. bind to integrins on the cell surface, allowing cells to adhere well to the scaffold material and proliferate well.

[0003] In addition, the following Patent Document 1 discloses a protein containing a repeat structure and a cell adhesion sequence such as an RGD sequence. This protein has a structure in which a specific Ala-rich site and a specific Ala-non-rich site are linked as the repeat structure. Patent Document 1 also describes that a material containing this protein can be used as a cell scaffold material.

[0004] Furthermore, as shown in the following Patent Documents 2 to 5, scaffold materials using synthetic resins are also known.

[0005] Patent Document 2 listed below discloses a carrier for cell culture which comprises a molded product made of a polyvinyl acetal compound or a molded product made of the polyvinyl acetal compound and a water-soluble polysaccharide, and in which the degree of acetalization of the polyvinyl acetal compound is 20 to 60 mol %.

[0006] In addition, the following Patent Document 3 discloses a composition (scaffold material) including a first fibrous polymer scaffold material, in which the fibers of the first fibrous polymer scaffold material are aligned. The material of this fibrous polymer is an aliphatic polyester or the like.

[0007] Furthermore, the following Patent Document 4 discloses a cell culture method for maintaining the undifferentiated state of pluripotent stem cells, the method comprising a step of culturing the pluripotent stem cells on a culture vessel having a surface coated with a polyrotaxane block copolymer.

[0008] Furthermore, the following Patent Document 5 discloses a cell culture product comprising a substrate having a surface, a hydrophilic copolymer layer provided on the surface of the substrate, and a plurality of peptide chains each bound to the surface of the hydrophilic copolymer layer. The hydrophilic copolymer layer is a layer copolymerized with a plurality of polyvinyl alcohol units, a plurality of polyvinyl alcohol derivative units, and a plurality of carboxylic acid group-containing units. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2018-064542 A [Patent Document 2] JP 2006-314285 A [Patent Document 3] WO2007 / 090102A1 [Patent Document 4] JP 2017-023008 A [Patent Document 5] JP 2015-070832 A Summary of the Invention [Problem to be solved by the invention]

[0010] By using a natural polymer material as a scaffold material, cells can proliferate well after seeding, and pseudopodia can extend well. However, natural polymer materials are expensive, have large lot-to-lot variations because they are naturally derived substances, and have safety concerns due to animal-derived components. Even when a polypeptide having a cell adhesion sequence such as the RGD sequence described in Patent Document 1 is used as a scaffold material, the adhesion and proliferation of cells can vary depending on the amino acid sequence other than the cell adhesion sequence.

[0011] On the other hand, scaffold materials using synthetic resins as described in Patent Documents 2 to 5 are less expensive, have smaller lot-to-lot variation, and are safer than scaffold materials using natural polymer materials. However, the conventional scaffold materials using synthetic resins as described in Patent Documents 2 to 4 have a problem of low cell adhesion. In addition, the scaffold material using synthetic resins having peptide chains as described in Patent Document 5 can increase cell adhesion to some extent, but may not be sufficient.

[0012] An object of the present invention is to provide a scaffold material for cell culture having excellent cell adhesiveness. Another object of the present invention is to provide a cell culture vessel using the scaffold material for cell culture. [Means for solving the problem]

[0013] According to a broad aspect of the present invention, there is provided a scaffold material for cell culture, comprising a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion, the peptide portion having a cyclic peptide backbone.

[0014] In a particular aspect of the cell culture scaffold material according to the present invention, the peptide portion has a cell adhesive amino acid sequence.

[0015] In another specific aspect of the scaffold material for cell culture according to the present invention, the cell adhesive amino acid sequence has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence.

[0016] In still another specific aspect of the scaffold material for cell culture according to the present invention, the cell adhesive amino acid sequence has at least an RGD sequence represented by the following formula (1):

[0017] Arg-Gly-Asp-X...Formula (1)

[0018] In the formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0019] In still another specific aspect of the scaffold material for cell culture according to the present invention, the cyclic peptide backbone is constituted of 4 to 10 amino acids.

[0020] In still another specific aspect of the scaffold material for cell culture according to the present invention, the polyvinyl alcohol derivative portion and the peptide portion are bonded via a linker portion.

[0021] In still another specific aspect of the scaffold material for cell culture according to the present invention, the scaffold material for cell culture has an islands-in-a-sea structure.

[0022] In still another specific aspect of the scaffold material for cell culture according to the present invention, the peptide-containing polyvinyl alcohol derivative is a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion and the peptide portion.

[0023] According to a broad aspect of the present invention, there is provided a cell culture vessel comprising a vessel body and the above-mentioned cell culture scaffold material, the cell culture scaffold material being disposed on a surface of the vessel body. Effect of the Invention

[0024] According to the present invention, it is possible to provide a scaffold material for cell culture and a vessel for cell culture which have excellent cell adhesiveness. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 is a cross-sectional view that illustrates a cell culture vessel according to one embodiment of the present invention. [Diagram 2] Figures 2(a) and (b) are images showing the presence or absence of an island structure. [Diagram 3] 3(a), (b) and (c) are diagrams showing the relationship between SF and the planar shape of a cell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention will be described in detail below.

[0027] The cell culture scaffold material according to the present invention includes a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. In the cell culture scaffold material according to the present invention, the peptide portion has a cyclic peptide backbone.

[0028] The scaffold material for cell culture according to the present invention has the above-mentioned configuration, and therefore has excellent cell adhesiveness. In addition, the scaffold material for cell culture according to the present invention has excellent pseudopod extensibility and excellent cell proliferation properties.

[0029] A scaffold material for cell culture containing a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion can increase the adhesiveness of cells to a certain extent. However, if the peptide portion does not have a cyclic peptide backbone, it may be difficult to increase the adhesiveness of cells when poorly adhesive cells are used. In contrast, in the scaffold material for cell culture according to the present invention, the peptide portion has a cyclic peptide backbone, so that the adhesiveness of cells can be increased even for poorly adhesive cells.

[0030] For example, in a cell culture scaffold material in which the peptide portion does not have a cyclic peptide skeleton, when mesenchymal stem cells that have been passaged for a long period of time and aged, or some iPS cell lines with low adhesiveness, etc. are used, the cells may not adhere well to the scaffold material. In contrast, in the cell culture scaffold material according to the present invention, the peptide portion has a cyclic peptide skeleton, so that the adhesiveness of the cells can be increased even for such poorly adhesive cells.

[0031] The mechanism by which the cell adhesiveness is enhanced by the scaffold material for cell culture according to the present invention is presumed to be the following mechanisms (1) and (2), but is not limited thereto. (1) The three-dimensional structure of the cyclic peptide backbone effectively exposes a specific amino acid sequence in the peptide portion (e.g., a cell-adhesive amino acid sequence), making it easier for cells to recognize the specific amino acid sequence and enabling the peptide portion to stably bind to cells. (2) The peptide portion is not easily degraded by degradative enzymes produced by cells, and is therefore highly stable.

[0032] In the case of the scaffold material for cell culture according to the present invention, the extension of filopodia-like pseudopods is observed in the cells after seeding, as in the case of using natural polymer materials such as Matrigel, whereas this extension of pseudopods is hardly observed in the case of the scaffold material using the conventional synthetic resin material.

[0033] The scaffold material for cell culture according to the present invention allows cells to adhere to the scaffold material for cell culture well and to proliferate well, even when the seeding density of the cells is low.

[0034] Furthermore, the cell culture scaffold material according to the present invention is less expensive, has less variation between lots, and is safer than conventional cell scaffold materials using natural polymers. By using the cell scaffold material according to the present invention, the burden of quality control of cells can be reduced.

[0035] (Scaffolding material for cell culture) The cell culture scaffold material according to the present invention includes a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. The peptide-containing polyvinyl alcohol derivative may be used alone or in combination of two or more kinds.

[0036] The peptide-containing polyvinyl alcohol derivative has a polyvinyl alcohol derivative portion and a peptide portion. In the peptide-containing polyvinyl alcohol derivative, the polyvinyl alcohol derivative portion and the peptide portion are preferably bonded via a linker portion. Therefore, the peptide-containing polyvinyl alcohol derivative preferably has a polyvinyl alcohol derivative portion, a peptide portion, and a linker portion.

[0037] The peptide-containing polyvinyl alcohol derivative can be obtained, for example, by reacting a polyvinyl alcohol derivative, a linker, and a peptide, as described below. The peptide-containing polyvinyl alcohol derivative may also be obtained, for example, by reacting a polyvinyl alcohol, a linker, and a peptide.

[0038] <Polyvinyl alcohol derivative part> The polyvinyl alcohol derivative portion is a structural portion derived from the polyvinyl alcohol derivative in the peptide-containing polyvinyl alcohol derivative. The polyvinyl alcohol derivative is a compound derived from polyvinyl alcohol. The polyvinyl alcohol derivative is preferably a polyvinyl acetal resin, and the polyvinyl alcohol derivative portion is preferably a polyvinyl acetal resin portion. That is, the peptide-containing polyvinyl alcohol derivative is preferably a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion and the peptide portion. The polyvinyl alcohol derivative and the polyvinyl acetal resin may each be used alone or in combination of two or more.

[0039] The polyvinyl alcohol derivative part and the polyvinyl acetal resin part preferably have an acetal group, a hydroxyl group, and an acetyl group in the side chain. However, the polyvinyl alcohol derivative part and the polyvinyl acetal resin part may not have an acetyl group, for example. For example, the polyvinyl alcohol derivative part and the polyvinyl acetal resin part may not have an acetyl group by bonding all of the acetyl groups of the polyvinyl alcohol derivative part and the polyvinyl acetal resin to the linker.

[0040] The polyvinyl acetal resin can be synthesized by acetalizing polyvinyl alcohol with an aldehyde.

[0041] The aldehyde used for acetalization of polyvinyl alcohol is not particularly limited. The aldehyde may be, for example, an aldehyde 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.

[0042] Examples of the aldehyde include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal, acrolein, benzaldehyde, cinnamaldehyde, perillaldehyde, formylpyridine, formylimidazole, formylpyrrole, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formylisoindole, formylpurine, formylbenzimidazole, formylbenzotriazole, formylquinoline, formylisoquinoline, formylquinoxaline, formylcinnoline, formylpteridine, formylfuran, formyloxolane, formyloxane, formylthiophene, formylthiolane, formylthiane, formyladenine, formylguanine, formylcytosine, formylthymine, and formyluracil. The aldehydes may be used alone or in combination of two or more.

[0043] The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, or pentanal, and more preferably butylaldehyde. Therefore, the polyvinyl acetal resin is more preferably a polyvinyl butyral resin, the polyvinyl acetal resin portion is more preferably a polyvinyl butyral resin portion, and the peptide-containing polyvinyl alcohol derivative is more preferably a peptide-containing polyvinyl butyral resin.

[0044] The amount of the aldehyde to be added can be appropriately set according to the amount of the desired acetal group. From the viewpoint of increasing the efficiency of the acetalization reaction and easily removing the unreacted aldehyde, the amount of the aldehyde to be added is preferably 60 mol% or more, more preferably 65 mol% or more, and preferably 95 mol% or less, more preferably 90 mol% or less, based on 100 mol% of polyvinyl alcohol.

[0045] The average polymerization degree of the polyvinyl alcohol derivative part, the polyvinyl acetal resin part, and the polyvinyl acetal resin is preferably 100 or more, more preferably 200 or more, even more preferably 500 or more, particularly preferably 1500 or more, preferably 6000 or less, more preferably 3000 or less, and even more preferably 2500 or less. When the average polymerization degree is equal to or more than the lower limit, swelling due to a liquid medium can be effectively suppressed, and the strength of the scaffold material for cell culture can be well maintained. Therefore, the cell proliferation can be improved. When the average polymerization degree is equal to or less than the upper limit, the handling property can be improved, and the moldability of the scaffold material for cell culture can be improved. The average polymerization degree of the polyvinyl alcohol derivative part, the polyvinyl acetal resin part, and the polyvinyl acetal resin is usually the same as the average polymerization degree of the polyvinyl alcohol used as a raw material, and can be determined by the average polymerization degree of polyvinyl alcohol.

[0046] The number average molecular weight (Mn) of the polyvinyl alcohol derivative portion, the polyvinyl acetal resin portion, and the polyvinyl acetal resin is preferably 10,000 or more, and preferably 600,000 or less. The weight average molecular weight (Mw) of the polyvinyl alcohol derivative portion, the polyvinyl acetal resin portion, and the polyvinyl acetal resin is preferably 2,000 or more, and preferably 1,200,000 or less. In addition, in the polyvinyl alcohol derivative portion, the polyvinyl acetal resin portion, and the polyvinyl acetal resin, the ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is preferably 2.0 or more, and preferably 40 or less. When the Mn, Mw, and Mw / Mn are equal to or more than the lower limit and equal to or less than the upper limit, the strength of the scaffold material for cell culture can be increased.

[0047] The number average molecular weight (Mn) and weight average molecular weight (Mw) of the polyvinyl alcohol derivative portion, the polyvinyl acetal resin portion, and the polyvinyl acetal resin can be determined, for example, as polystyrene-equivalent values ​​by gel permeation chromatography (GPC) analysis using tetrahydrofuran (THF) as a solvent.

[0048] The degree of acetalization of the polyvinyl alcohol derivative portion, the polyvinyl acetal resin portion, and the polyvinyl acetal resin (the degree of butyralization in the case of polyvinyl butyral resin) 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 equal to or more than the lower limit, the fixation property of cells can be improved, and cells can grow efficiently. When the degree of acetalization is equal to or less than the upper limit, the solubility in a solvent can be improved.

[0049] The hydroxyl group content (hydroxyl group amount) of the polyvinyl alcohol derivative portion, the polyvinyl acetal resin portion and the polyvinyl acetal resin is preferably 15 mol % or more, more preferably 20 mol % or more, preferably 45 mol % or less, more preferably 30 mol % or less, and even more preferably 25 mol % or less.

[0050] The degree of acetylation (amount of acetyl groups) of the polyvinyl alcohol derivative portion, the polyvinyl acetal resin portion, and the polyvinyl acetal resin 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 more than the lower limit and equal to or less than the upper limit, the reaction efficiency between the polyvinyl acetal resin and the linker can be increased.

[0051] The acetalization degree, the acetylation degree and the hydroxyl group amount of the polyvinyl alcohol derivative portion, the polyvinyl acetal resin portion and the polyvinyl acetal resin are 1 It can be measured by H-NMR (nuclear magnetic resonance spectroscopy).

[0052] <Peptide section> The peptide portion is a structural portion derived from the peptide in the peptide-containing polyvinyl alcohol derivative. 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.

[0053] The peptide portion has a cyclic peptide backbone. The cyclic peptide backbone is a cyclic backbone composed of a plurality of amino acids. The peptide portion may have only a cyclic peptide backbone, or may have a cyclic peptide backbone and a backbone other than a cyclic peptide backbone (e.g., a linear peptide backbone).

[0054] The peptide portion is preferably composed of 4 or more amino acids, more preferably 5 or more amino acids, preferably 15 or less amino acids, and more preferably 10 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 recognition of a specific amino acid sequence by cells can be improved, and the adhesiveness and proliferation of cells can be further improved. Furthermore, 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 extension of pseudopodia can be further improved.

[0055] The cyclic peptide backbone is preferably composed of 4 or more amino acids, more preferably composed of 5 or more amino acids, preferably composed of 10 or less amino acids, and more preferably composed of 7 or less amino acids. When the number of amino acids constituting the cyclic peptide backbone is equal to or greater than the lower limit, the recognition of a specific amino acid sequence by cells can be improved, and the adhesiveness and proliferation of cells can be further improved. When the number of amino acids constituting the cyclic peptide backbone is equal to or greater than the lower limit and equal to or less than the upper limit, the extension of the pseudopodia can be further improved. From the viewpoint of keeping costs low, the number of amino acids constituting the cyclic peptide backbone is preferably 5.

[0056] 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 pp. 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.

[0057] Examples of the cell adhesive amino acid sequences include the RGD sequence (Arg-Gly-Asp), YIGSR sequence (Tyr-Ile-Gly-Ser-Arg), PDSGR sequence (Pro-Asp-Ser-Gly-Arg), HAV sequence (His-Ala-Val), ADT sequence (Ala-Asp-Thr), QAV sequence (Gln-Ala-Val), LDV sequence (Leu-Asp-Val), IDS sequence (Ile-Asp-Ser), REDV sequence (Arg-Glu-Asp-Val), IDAPS sequence (Ile-Asp-Ala-Pro-Ser), KQAGDV sequence (Lys-Gln-Ala-Gly-Asp-Val), and TDE sequence (Thr-Asp-Glu). Further, examples of the cell adhesive amino acid sequence include the 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 two or more types.

[0058] The cell adhesive amino acid sequence preferably has at least any one of the above-mentioned cell adhesive amino acid sequences, more preferably has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, and even more preferably has at least an RGD sequence represented by the following formula (1). In this case, the adhesiveness and proliferation of cells can be further improved, and the extension of pseudopodia can be further improved.

[0059] Arg-Gly-Asp-X...Formula (1)

[0060] In the above formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0061] When the peptide portion has the cell adhesive amino acid sequence, the cyclic peptide backbone may or may not have the cell adhesive amino acid sequence. From the viewpoint of enhancing the recognition of the cell adhesive amino acid sequence by cells and further enhancing the adhesiveness and proliferation of cells, it is preferable that the cyclic peptide backbone has the cell adhesive amino acid sequence.

[0062] When the peptide portion has the cell-adhesive amino acid sequence, a linker may be bonded to an amino acid at the N-terminus or C-terminus of the cell-adhesive amino acid sequence, or a linker may be bonded to an amino acid constituting a portion of the amino acid sequence different from the cell-adhesive amino acid sequence.

[0063] When the peptide portion has the cell adhesive amino acid sequence, it is more preferable that the linker portion is bound to amino acids constituting a portion of the amino acid sequence different from the cell adhesive amino acid sequence, which can further enhance the adhesiveness and proliferation of cells and further improve the extension of pseudopods.

[0064] The content of the peptide moiety in 100% by weight of the peptide-containing polyvinyl alcohol derivative is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, even more preferably 1% by weight or more, and particularly preferably 5% by weight or more. The content of the peptide moiety in 100% by weight of the peptide-containing polyvinyl alcohol derivative is preferably 30% by weight or less, more preferably 25% by weight or less, even more preferably 20% by weight or less, and particularly preferably 15% by weight or less. When the content of the peptide moiety is equal to or more than the lower limit, the adhesiveness and proliferation of cells can be further improved, and the extension of pseudopods can be further improved.

[0065] In the peptide-containing polyvinyl alcohol derivative, the content of the peptide portion is preferably 0.01 mol% or more, more preferably 0.1 mol% or more, even more preferably 1 mol% or more, even more preferably 5 mol% or more, and particularly preferably 10 mol% or more. In the peptide-containing polyvinyl alcohol derivative, 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 equal to or more than the lower limit, the adhesiveness and proliferation of cells can be further improved, and the extension of pseudopodia can be further improved. When the content of the peptide portion is equal to or less than the upper limit, the production 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 peptide-containing polyvinyl alcohol derivative.

[0066] In the peptide-containing polyvinyl alcohol derivative, the molar ratio of the content of the peptide portion to the total content of the acetal group, the hydroxyl group, and the acetyl group (the content of the peptide portion / the total content of the acetal group, the hydroxyl group, and the acetyl group) is preferably 0.0001 or more, more preferably 0.001 or more. When the molar ratio (the content of the peptide portion / the total content of the acetal group, the hydroxyl group, and the acetyl group) is equal to or more than the lower limit, the adhesiveness and proliferation of the cells can be further improved, and the extension of the pseudopods can be further improved. In the peptide-containing polyvinyl alcohol derivative, the upper limit of the molar ratio of the content of the peptide portion to the total content of the acetal group, the hydroxyl group, and the acetyl group (the content of the peptide portion / the total content of the acetal group, the hydroxyl group, and the acetyl group) is not particularly limited. From the viewpoint of production costs, etc., the molar ratio (the content of the peptide portion / the total content of the acetal group, the hydroxyl group, and the acetyl group) is preferably 0.2 or less.

[0067] The content of the above peptide portion can be measured by FT-IR or LC-MS.

[0068] <Linker> The linker part is a structural part derived from the linker in the peptide-containing polyvinyl alcohol derivative. The linker part is located between the polyvinyl alcohol derivative part and the peptide part. The polyvinyl alcohol derivative part and the peptide part are bonded via the linker part. The linker part is formed by a linker (crosslinking agent). Only one type of the linker may be used, or two or more types may be used in combination.

[0069] The linker is preferably a compound having a functional group capable of condensing with a carboxyl group or an amino group of the peptide. Examples of the functional group capable of condensing with a carboxyl group or an 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.

[0070] Examples of the linker having a carboxyl group include (meth)acrylic acid and acrylamide containing a carboxyl group, etc. By using a carboxylic acid (carboxylic acid monomer) having a polymerizable unsaturated group as the linker having a carboxyl group, the carboxylic acid monomer can be polymerized by graft polymerization when the linker is introduced, so that the number of carboxyl groups that can be reacted with the peptide can be increased.

[0071] From the viewpoint of effectively binding the polyvinyl alcohol derivative and the peptide, the linker is preferably (meth)acrylic acid, and more preferably acrylic acid.

[0072] The above peptide-containing polyvinyl alcohol derivative can be synthesized, for example, as follows.

[0073] (1) A polyvinyl alcohol derivative (e.g., a polyvinyl acetal resin) is reacted with a linker to obtain a reaction product in which the polyvinyl acetal resin and the linker are bonded, and (2) the reaction product is reacted with a peptide to obtain a peptide-containing polyvinyl alcohol derivative (peptide-containing polyvinyl acetal resin).

[0074] In the above (1), examples of a method for obtaining a reaction product in which the polyvinyl acetal resin and the linker are bonded include a method for acetalizing a copolymer of polyvinyl alcohol and a carboxylic acid having a polymerizable unsaturated group, and a method for graft copolymerizing a polyvinyl acetal resin and a linker (e.g., a carboxylic acid monomer) under ultraviolet irradiation. The method for obtaining the reaction product is preferably the above graft copolymerization method. In this case, the carboxylic acid monomer can be polymerized by graft polymerization, so that the number of carboxyl groups that can react with peptides can be increased.

[0075] In the above (2), the carboxyl group derived from the linker in the obtained reaction product and the amino group of the peptide are dehydrated and condensed to obtain a peptide-containing polyvinyl alcohol derivative (peptide-containing polyvinyl acetal resin) having a polyvinyl acetal resin portion, a peptide portion, and a linker portion.

[0076] The peptide-containing polyvinyl alcohol derivative may or may not have a carboxyl group derived from the linker remaining. The content of the carboxyl group in the peptide-containing polyvinyl alcohol derivative is preferably 0.1 mol% or more, more preferably 0.5 mol% or more, preferably 2 mol% or less, more preferably 1.5 mol% or less. When the content of the carboxyl group is equal to or more than the lower limit and equal to or less than the upper limit, the adhesiveness and proliferation of cells can be further improved, and the extension of pseudopods can be further improved. The content of the carboxyl group (mol%) is the amount of the carboxyl group relative to the sum of the amounts of the respective structural units constituting the peptide-containing polyvinyl alcohol derivative.

[0077] From the viewpoint of further enhancing the adhesiveness and proliferation of cells, the cell culture scaffold material preferably has a phase-separated structure having at least a first phase and a second phase.

[0078] Examples of the phase separation structure include microphase separation structures such as a sea-island structure, a cylindrical structure, a gyroid structure, and a lamellar structure. In the sea-island structure, for example, the first phase can be the sea portion, and the second phase can be the island portion. In the cylindrical structure, the gyroid structure, or the lamellar structure, for example, the phase with the largest surface area can be the first phase, and the phase with the second largest surface area can be the second phase. The cell culture scaffold material has a continuous phase and a discontinuous phase, which can increase the affinity with cells and further increase the adhesiveness and proliferation of cells.

[0079] The phase-separated structure is preferably an island-in-a-sea structure. The scaffold material for cell culture preferably has an island-in-a-sea structure. In this case, the adhesiveness and proliferation of cells can be further improved.

[0080] When the scaffold material for cell culture has an island-in-a-sea structure, the surface area ratio of the islands (second phase) to the entire surface of the scaffold material for cell culture is preferably 0.01 or more, more preferably 0.1 or more, even more preferably 0.2 or more, and preferably 0.95 or less, more preferably 0.9 or less, even more preferably 0.8 or less. When the surface area ratio is equal to or more than the lower limit and equal to or less than the upper limit, the adhesiveness of cells can be further increased.

[0081] When the scaffold material for cell culture has a sea-island structure, it is preferable that the island portion contains a peptide portion. That is, it is preferable that the scaffold material for cell culture has a sea portion and an island portion, and the island portion contains a peptide portion. In this case, the adhesion domain of the cell accumulates in the island portion, thereby further enhancing the adhesiveness of the cell.

[0082] The presence or absence of a phase-separated structure can be confirmed by, for example, an atomic force microscope (AFM), a transmission electron microscope (TEM), a scanning electron microscope (SEM), etc. The surface area ratio can be determined from a microscope observation image by using image analysis software such as ImageJ.

[0083] The phase-separated structure can be formed, for example, by increasing the content of peptide moieties and forming an inter- or intramolecular phase-separated structure in the peptide-containing polyvinyl alcohol derivative.

[0084] From the viewpoint of effectively exerting the effects of the present invention and from the viewpoint of increasing productivity, the content of the peptide-containing polyvinyl alcohol derivative in 100% by weight of the scaffold material for cell culture is preferably 90% by weight or more, more preferably 95% by weight or more, even more preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). Therefore, the scaffold material for cell culture is most preferably the peptide-containing polyvinyl alcohol derivative. When the content of the peptide-containing polyvinyl alcohol derivative is equal to or more than the lower limit, the effects of the present invention can be exerted more effectively.

[0085] The cell culture scaffold material may contain a polymer other than the peptide-containing polyvinyl alcohol derivative. Examples of the polymer include polyvinyl acetal resin, polyolefin resin, polyether resin, polyvinyl alcohol resin, polyester, epoxy resin, polyamide resin, polyimide resin, polyurethane resin, polycarbonate resin, cellulose, and polypeptide. The above polymers may be used alone or in combination of two or more.

[0086] From the viewpoint of effectively exerting the effects of the present invention, the content of polymers other than the peptide-containing polyvinyl alcohol derivative is preferably as small as possible. In 100% by weight of the scaffold material for cell culture, the content of the polymer 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 polymers other than the peptide-containing polyvinyl alcohol derivative.

[0087] The scaffold material for cell culture according to the present invention is preferably substantially free of animal-derived raw materials. By being substantially free of animal-derived raw materials, a scaffold material for cell culture can be provided that has less variation between lots and is excellent in cost and safety. Here, "substantially free of animal-derived raw materials" means that the amount of animal-derived raw materials in the scaffold material for cell culture is 3% by weight or less. The scaffold material for cell culture according to the present invention is preferably 1% by weight or less, more preferably 0% by weight, of animal-derived raw materials in the scaffold material for cell culture. In other words, it is more preferable that the scaffold material for cell culture does not contain any animal-derived raw materials in the scaffold material for cell culture.

[0088] The cell culture scaffold material may be prepared on the surface of the container body described below. For example, a synthetic resin having a polyvinyl alcohol derivative portion and a linker may be coated on the surface of the container body to form a resin film, and the synthetic resin may be reacted with a peptide on the surface of the resin film to obtain the peptide-containing polyvinyl alcohol derivative.

[0089] (Other details of cell culture scaffold materials) The scaffold material for cell culture according to the present invention is used for culturing cells. The scaffold material for cell culture according to the present invention is used as a scaffold for cells when culturing the cells.

[0090] 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.

[0091] Examples of the mature cells include nerve cells, cardiomyocytes, retinal cells, and hepatic cells.

[0092] Examples of the stem cells include mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0093] The shape of the scaffold material for cell culture is not particularly limited. The scaffold material for cell culture may be in the form of a membrane, a particle, a fiber, or a porous body. The membrane form includes a film form and a sheet form.

[0094] The above-mentioned scaffold material for cell culture is preferably used for two-dimensional culture (plate culture), three-dimensional culture or suspension culture of cells, and more preferably for two-dimensional culture (plate culture).

[0095] The scaffold material for cell culture can also be used as a cell culture carrier (medium) containing the scaffold material for cell culture and a polysaccharide. The polysaccharide is not particularly limited, and any conventionally known polysaccharide can be used. The polysaccharide is preferably a water-soluble polysaccharide.

[0096] The above-mentioned cell culture scaffold material can also be used as a cell culture fiber comprising a fiber body and a cell culture scaffold material arranged on the surface of the fiber body. In this case, the cell culture scaffold material is preferably applied to the surface of the fiber body, and is preferably a coating. In this cell culture fiber, the cell culture scaffold material may be present in the fiber body. For example, the cell culture scaffold material can be present in the fiber body by impregnating or kneading the fiber body with a liquid cell culture scaffold material. Stem cells generally have a property of being difficult to adhere to planar structures and easily adhere to three-dimensional structures such as fibrous structures, so that the cell culture fiber is preferably used for three-dimensional culture of stem cells. Among stem cells, the cell culture fiber is more preferably used for three-dimensional culture of adipose stem cells.

[0097] The synthetic resin in the above-mentioned cell culture scaffold material may be crosslinked. A cell culture scaffold material containing a crosslinked synthetic resin can effectively suppress water swelling and increase strength. The synthetic resin can be crosslinked by using a crosslinking agent.

[0098] (Cell culture container) The cell culture vessel according to the present invention comprises a vessel body and the above-mentioned scaffold material for cell culture, the scaffold material for cell culture being disposed on a surface of the vessel body. The cell culture vessel comprises the scaffold material for cell culture in at least a part of a cell culture region.

[0099] FIG. 1 is a cross-sectional view that illustrates a cell culture vessel according to one embodiment of the present invention.

[0100] The cell culture vessel 1 comprises a vessel body 2 and a cell culture scaffold material 3. The cell culture scaffold material 3 is disposed on a surface 2a of the vessel body 2. The cell culture scaffold material 3 is disposed on a bottom surface of the vessel body 2. By adding a liquid medium to the cell culture vessel 1 and seeding cells such as cell clumps on the surface of the cell culture scaffold material 3, cells can be cultured on a surface.

[0101] The container body may include a first container body and a second container body such as a cover glass on the bottom surface of the first container body. The first container body and the second container body may be separable. In this case, the scaffold material for cell culture may be disposed on the surface of the second container body.

[0102] The container body may be a conventionally known container body (container). The shape and size of the container body are not particularly limited.

[0103] The container body may be a cell culture plate or a cell culture flask having one or more wells (holes). The number of wells in the plate is not particularly limited. The number of wells may be, but is not particularly limited to, 2, 4, 6, 12, 24, 48, 96, 384, etc. The shape of the well may be, but is not particularly limited to, a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, etc. The shape of the bottom of the well may be, but is not particularly limited to, a flat bottom, a round bottom, an uneven bottom, etc.

[0104] 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.

[0105] 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.

[0106] The content of structural units in the obtained synthetic resin was measured by dissolving the synthetic resin in DMSO-d6 (dimethyl sulfoxide) and then 1The measurement was performed by H-NMR (nuclear magnetic resonance spectrum). The peptide content in the peptide-containing polyvinyl alcohol derivative was measured by FT-IR or LC-MS. The acetalization degree (butyralization degree), hydroxyl group amount, acetylation degree, carboxyl group content, and peptide content of the obtained synthetic resin are shown in Tables 1 to 3.

[0107] Example 1 Preparation of polyvinyl acetal resin (PVB1): In a reactor equipped with a stirrer, 2700 mL of ion-exchanged water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 99 mol% were added, and the mixture was heated and dissolved while stirring to obtain a solution. To the obtained solution, 35% by weight hydrochloric acid was added as a catalyst so that the hydrochloric acid concentration was 0.2% by weight. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butyl aldehyde was added while stirring. Next, 148 parts by weight of n-butyl aldehyde was added to precipitate a white particulate polyvinyl acetal resin (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 the mixture was heated to 50°C and held at 50°C for 2 hours. Next, the solution was cooled and neutralized, and then the polyvinyl butyral resin was washed with water and dried to obtain a polyvinyl acetal resin (polyvinyl butyral resin (PVB1), average polymerization degree 1700, acetalization degree (butyralization degree) 70 mol%, hydroxyl group amount 27 mol%, acetylation degree 3 mol%).

[0108] Introducing the linker: 99 parts by weight of the obtained polyvinyl acetal resin and 1 part 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 to introduce the linker by graft copolymerization of the polyvinyl acetal resin and acrylic acid. 1 part by weight of the polyvinyl acetal resin to which the linker was introduced was dissolved in 19 parts by weight of butanol. 150 μL of the obtained solution was discharged onto the surface of a φ22 mm cover glass (Matsunami Co., Ltd. "22 Maru No. 1") that had been dusted with an air duster, and rotated at 2000 rpm for 20 seconds using a spin coater, and then heated at 60 ° C for 60 minutes to obtain a resin film with a smooth surface.

[0109] Formation of the peptide part: A cyclic peptide having an amino acid sequence of Arg-Gly-Asp-Phe-Lys (5 amino acid residues, Arg and Lys are bonded to form a cyclic skeleton, Phe is D-form, and is indicated as c-RGDfK in the table) was prepared. 1 part by weight of this peptide and 1 part by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) were added to phosphate buffered saline containing neither calcium nor magnesium so that the final concentration of the peptide was 1 mM to prepare a peptide-containing liquid. 1 part by weight of this peptide-containing liquid was added to a spin-coated resin film (polyvinyl acetal resin with a linker formed) and reacted to dehydrate and condense the carboxyl group of the linker and the amino group of Lys of the peptide. In this way, a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion, a linker portion, and a peptide portion was prepared.

[0110] The obtained peptide-containing polyvinyl acetal resin had a degree of acetalization (degree of butyralization) of 69.3 mol%, a hydroxyl group amount of 26.7 mol%, a degree of acetylation of 3.0 mol%, a carboxyl group content of 0.9 mol%, and a peptide moiety content of 0.1 mol%.

[0111] Preparation of cell culture vessels: The resulting laminate of the peptide-containing polyvinyl acetal resin and cover glass was placed in a polystyrene dish having a diameter of 22 mm to obtain a cell culture vessel.

[0112] Example 2 Preparation of polyvinyl acetal resin (PVB2): In a reactor equipped with a stirrer, 2700 mL of ion-exchanged water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 99 mol% were added, and the mixture was heated and dissolved while stirring to obtain a solution. To the obtained solution, 35% by weight hydrochloric acid was added as a catalyst so that the hydrochloric acid concentration was 0.2% by weight. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butylaldehyde were added while stirring. Next, 143 parts by weight of n-butylaldehyde were added to precipitate a white particulate polyvinyl acetal resin (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 the mixture was heated to 50°C and held at 50°C for 2 hours. Next, the solution was cooled and neutralized, and then the polyvinyl butyral resin was washed with water and dried to obtain a polyvinyl acetal resin (polyvinyl butyral resin (PVB2), average polymerization degree 1700, acetalization degree (butyralization degree) 69 mol%, hydroxyl group amount 28 mol%, acetylation degree 3 mol%).

[0113] Introducing the linker: 70 parts by weight of the obtained polyvinyl acetal resin and 30 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 to introduce the linker by graft copolymerization of the polyvinyl acetal resin and acrylic acid. 1 part by weight of the polyvinyl acetal resin to which the linker was introduced was dissolved in 19 parts by weight of butanol. 150 μL of the obtained solution was discharged onto the surface of a φ22 mm cover glass (Matsunami Co., Ltd. "22 Maru No. 1") that had been dusted with an air duster, and rotated at 2000 rpm for 20 seconds using a spin coater, and then heated at 60 ° C for 60 minutes to obtain a resin film with a smooth surface.

[0114] Formation of the peptide part: A peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 1, except that the obtained resin film (polyvinyl acetal resin with a linker introduced therein) was used and the amount of peptide added was 30 parts by weight.

[0115] Preparation of cell culture vessels: In the same manner as in Example 1, a cell culture vessel was obtained.

[0116] Example 3 Preparation of polyvinyl acetal resin (PVB3): In a reactor equipped with a stirrer, 2700 mL of ion-exchanged water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 99 mol% were added, and the mixture was heated and dissolved while stirring to obtain a solution. To the obtained solution, 35% by weight hydrochloric acid was added as a catalyst so that the hydrochloric acid concentration was 0.2% by weight. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butylaldehyde was added while stirring. Next, 133 parts by weight of n-butylaldehyde was added to precipitate a white particulate polyvinyl acetal resin (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 the mixture was heated to 50°C and held at 50°C for 2 hours. Next, the solution was cooled and neutralized, and then the polyvinyl butyral resin was washed with water and dried to obtain a polyvinyl acetal resin (polyvinyl butyral resin (PVB3), average degree of polymerization 1700, degree of acetalization (degree of butyralization) 63 mol%, amount of hydroxyl groups 34 mol%, degree of acetylation 3 mol%).

[0117] Linker installation: Except for using the obtained polyvinyl acetal resin, a linker was introduced in the same manner as in Example 2. Moreover, a resin film having a smooth surface was obtained in the same manner as in Example 2.

[0118] Formation of the peptide part: A peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 2, except that the obtained resin film (polyvinyl acetal resin into which a linker had been introduced) was used.

[0119] Preparation of cell culture vessels: In the same manner as in Example 1, a cell culture vessel was obtained.

[0120] Example 4 Preparation of polyvinyl acetal resin (PVB4): In a reactor equipped with a stirrer, 2700 mL of ion-exchanged water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 99 mol% were added, and the mixture was heated and dissolved while stirring to obtain a solution. To the obtained solution, 35% by weight hydrochloric acid was added as a catalyst so that the hydrochloric acid concentration was 0.2% by weight. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butylaldehyde was added while stirring. Next, 133 parts by weight of n-butylaldehyde was added to precipitate a white particulate polyvinyl acetal resin (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 the mixture was heated to 50°C and held at 50°C for 2 hours. Next, the solution was cooled and neutralized, and then the polyvinyl butyral resin was washed with water and dried to obtain a polyvinyl acetal resin (polyvinyl butyral resin (PVB4), average polymerization degree 1700, acetalization degree (butyralization degree) 50 mol%, hydroxyl group amount 47 mol%, acetylation degree 3 mol%).

[0121] Introducing the linker: Except for using the obtained polyvinyl acetal resin, a linker was introduced in the same manner as in Example 2. Moreover, a resin film having a smooth surface was obtained in the same manner as in Example 2.

[0122] Formation of the peptide part: A peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 2, except that the obtained resin film (polyvinyl acetal resin into which a linker had been introduced) was used.

[0123] Preparation of cell culture vessels: In the same manner as in Example 1, a cell culture vessel was obtained.

[0124] Comparative Example 1 A peptide-containing polyvinyl acetal resin and a cell culture vessel were prepared in the same manner as in Example 1, except that a linear peptide having the amino acid sequence Arg-Gly-Asp-Ser (four amino acid residues, indicated as RGDS in the table) was used and the carboxyl group of the linker and the amino group of Arg of the peptide were subjected to dehydration condensation.

[0125] Comparative Example 2 A peptide-containing polyvinyl acetal resin and a cell culture vessel were prepared in the same manner as in Example 2, except that a linear peptide having the amino acid sequence Gly-Arg-Gly-Asp-Ser (five amino acid residues, indicated as GRGDS in the table) was used and the carboxyl group of the linker and the amino group of Gly of the peptide were subjected to dehydration condensation.

[0126] Comparative Example 3 A peptide-containing polyvinyl acetal resin and a cell culture vessel were prepared in the same manner as in Example 3, except that a linear peptide having the amino acid sequence Gly-Arg-Gly-Asp-Ser (five amino acid residues, indicated as GRGDS in the table) was used and the carboxyl group of the linker and the amino group of Gly of the peptide were subjected to dehydration condensation.

[0127] Comparative Example 4 A peptide-containing polyvinyl acetal resin and a cell culture vessel were prepared in the same manner as in Example 4, except that a linear peptide having the amino acid sequence Gly-Arg-Gly-Asp-Ser (five amino acid residues, indicated as GRGDS in the table) was used and the carboxyl group of the linker and the amino group of Gly of the peptide were subjected to dehydration condensation.

[0128] Comparative Example 5 A cell culture vessel was produced in the same manner as in Example 1, except that the peptide portion was not formed.

[0129] Example 5 99 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 90 mol% and 1 part by weight of acrylic acid (linker) were dissolved in 300 parts by weight of ethanol, and reacted for 20 minutes under ultraviolet irradiation in the presence of a photoradical polymerization initiator to graft-copolymerize the polyvinyl alcohol and the acrylic acid, thereby introducing the linker. A peptide-containing polyvinyl alcohol derivative and a cell culture vessel were prepared in the same manner as in Example 1, except for the above.

[0130] Comparative Example 6 99 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 90 mol% and 1 part by weight of acrylic acid (linker) were dissolved in 300 parts by weight of ethanol, and reacted for 20 minutes under ultraviolet irradiation in the presence of a photoradical polymerization initiator to graft copolymerize the polyvinyl alcohol and the acrylic acid, thereby introducing the linker. A peptide-containing polyvinyl alcohol derivative and a cell culture vessel were prepared in the same manner as in Example 1, except that a linear peptide (5 amino acid residues, indicated as GRGDS in the table) having an amino acid sequence of Gly-Arg-Gly-Asp-Ser was used and the carboxyl group of the linker and the amino group of Gly of the peptide were dehydrated and condensed.

[0131] (Reference example A) Fabrication of scaffolds from natural products: 1 ml of a solution of Vitronectin (manufactured by Corning) adjusted to 5 μg / ml in phosphate buffer (PBS) was added to a φ35 mm dish. A φ22 mm cover glass (Matsunami Co., Ltd. "22 Maru No. 1") was immersed in the solution and cured at 37°C for 1 hour to obtain a scaffold material derived from natural products (referred to as VTN in the table) with Vitronectin smoothly adsorbed on the surface.

[0132] Preparation of cell culture vessels: A cell culture vessel was obtained in the same manner as in Example 1. Note that since Vitronectin is denatured when dried and its adhesive performance is significantly reduced, the cell culture vessel was immersed in a PBS solution immediately after production.

[0133] (evaluation) (1) Presence or absence of an island structure The resulting resin film of the peptide-containing polyvinyl acetal resin was immersed in a PBS solution for 30 minutes. The resin film after immersion was observed using an atomic force microscope (AFM, Bruker's "Dimension XR"). The measurement conditions were set to a peak set point of 2 nN in QNM mode, and an area of ​​1 μm x 1 μm was observed. The obtained height mapping image and elastic modulus mapping image were compared to determine whether or not a sea-island structure was present. In the table, "A" is indicated when a sea-island structure was observed, and "B" is indicated when a sea-island structure was not observed.

[0134] FIG. 2(a) is an example of an image in which it is determined that a sea-island structure is observed, and FIG. 2(b) is an example of an image in which it is determined that a sea-island structure is not observed.

[0135] (3) Cell culture evaluation (3-1) Seeding and culturing of iPS cells (201B7) The cell culture vessels obtained in Examples 1 to 4, Comparative Examples 1 to 5, and Reference Example A were used in the following experiments.

[0136] The following liquid medium and ROCK (Rho-associated kinase) specific inhibitor were prepared.

[0137] TeSR E8 medium (STEM CELL) ROCK Inhibitor (Y27632)

[0138] 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.

[0139] 1 mL of 0.5 mM ethylenediaminetetraacetic acid / phosphate buffer solution was added to h-iPS cells 201B7 that had become confluent in a φ35 mm dish, and the cells were left to stand at room temperature for 2 minutes. After removing the ethylenediaminetetraacetic acid / phosphate buffer solution, cell clusters were obtained that were broken into pieces of 50 μm to 200 μm in size by pipetting with 1 mL of liquid medium. The obtained cell clusters (cell number 1.0 × 10 5 The cells were clamp-seeded in the above cell culture vessel.

[0140] 1 mL of liquid medium and a ROCK-specific inhibitor were added to a cell culture vessel to a final concentration of 10 μM, and the cells were cultured in an incubator at 37°C and 5% CO2 concentration. Every 24 hours, 1 mL of the liquid medium was removed and 1 mL of new liquid medium was added to replace the medium.

[0141] (3-2) Seeding and culturing of iPS cells (253G1) Cell seeding and culture were performed in the same manner as in “(3-1) Seeding and culture of iPS cells (201B7)”, except that the cell culture vessels obtained in Example 5, Comparative Example 6, and Reference Example A were used, and h-iPS cells 253G1 were used.

[0142] (3-3) Pseudopod extension The cells were observed 24 hours after seeding using a phase contrast microscope (Olympus IX73, 10x20x magnification). During the observation, an image of the field of view showing the most average adhesion form in the cell culture vessel was obtained. From the obtained image, the shape factor (SF) was calculated to evaluate the extensibility of the pseudopods. The shape factor (SF) is an evaluation coefficient of the shape of the area of ​​the cells in a planar view after the cells have been cultured, and is calculated using the following formula.

[0143] Formula: SF = 4 x π x (cell area) / (cell perimeter length) 2

[0144] Figures 3(a), (b), and (c) are diagrams showing the relationship between SF and the planar shape of a cell. As shown in Figure 3(a), if the SF is 1, the planar shape of the cell is circular. The smaller the SF, the more it is away from a circular shape, which means that the cell's pseudopodia are well extended. Figure 3(b) is a photograph showing the planar shape of a cell when SF is 0.3, and Figure 3(c) is a photograph showing the planar shape of a cell when SF is 1.

[0145] <Criteria for determining pseudopod extensibility> ○: SF is 0.1 or more and 0.6 or less ×: SF is greater than 0.6 and less than or equal to 1

[0146] (3-4) Cell proliferation The fixed cell mass 5 days after cell seeding was detached using 1.0 mL of TryPLE Express detachment solution, and the cell number was determined using a cell counter (Chemometec's "NucleoCounter NC-3000"). Next, the cell proliferation rate relative to Reference Example A was calculated using the following formula. The cell proliferation rate relative to Reference Example A was compared between experiments using the same cell type (iPS cells (201B7) or iPS cells (253G1)).

[0147] Cell proliferation rate (%) relative to Reference Example A=(cell number in Examples and Comparative Examples) / (cell number in Reference Example A)×100

[0148] <Criteria for determining cell adhesion> A: Cell proliferation rate is 50% or higher compared to Reference Example A B: Cell proliferation rate is 10% or more but less than 50% compared to Reference Example A C: Cell proliferation rate is less than 10% compared to Reference Example A

[0149] Details and results are shown in Tables 1 to 3 below.

[0150] [Table 1]

[0151] [Table 2]

[0152] [Table 3]

[0153] The scaffold materials for cell culture obtained in the Examples had better cell adhesion than the scaffold materials for cell culture obtained in the Comparative Examples, and as shown in Tables 1 to 3, had better pseudopod extension and cell proliferation. [Explanation of symbols]

[0154] 1...Cell culture container 2…Container body 2a…Surface 3. Scaffolding for cell culture

Claims

1. A method for producing a polyvinyl alcohol derivative by reacting a polyvinyl alcohol derivative with a linker, comprising the steps of: (1) obtaining a reaction product in which the polyvinyl alcohol derivative and the linker are bonded; and (2) reacting the resulting reaction product with a peptide to obtain a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion, a linker portion, and a peptide portion. the linker is a compound having a carboxyl group, a thiol group, or an amino group, the peptide portion has a cyclic peptide backbone, A method for producing a peptide-containing polyvinyl alcohol derivative, wherein the cyclic peptide backbone has a cell-adhesive amino acid sequence.

2. A method for producing the peptide-containing polyvinyl alcohol derivative described in claim 1, wherein the cell adhesive amino acid sequence has at least an RGD sequence represented by the following formula (1): Arg-Gly-Asp-X...Formula (1) In the formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

3. The peptide portion is composed of 15 or less amino acids, The method for producing a peptide-containing polyvinyl alcohol derivative according to claim 1 or 2, wherein the cyclic peptide backbone is composed of 10 or less amino acids.

4. A method for producing a peptide-containing polyvinyl alcohol derivative described in any one of claims 1 to 3, wherein the cyclic peptide backbone is composed of 4 to 10 amino acids.

5. The linker is a compound having a carboxyl group, The method for producing a peptide-containing polyvinyl alcohol derivative according to any one of claims 1 to 4, wherein the content of the carboxyl group in the peptide-containing polyvinyl alcohol derivative is 0.1 mol % or more and 2 mol % or less.

6. The linker comprises a carboxylic acid having a polymerizable unsaturated group, The method for producing a peptide-containing polyvinyl alcohol derivative according to any one of claims 1 to 5, wherein in the step (1), the polyvinyl alcohol derivative and the linker are graft-copolymerized under ultraviolet irradiation.

7. The polyvinyl alcohol derivative is a polyvinyl acetal resin, the polyvinyl alcohol derivative portion is a polyvinyl acetal resin portion, The method for producing a peptide-containing polyvinyl alcohol derivative according to any one of claims 1 to 6, wherein the peptide-containing polyvinyl alcohol derivative is a peptide-containing polyvinyl acetal resin.

8. The peptide-containing polyvinyl alcohol derivative has a polyvinyl alcohol derivative portion, a peptide portion, and a linker portion, In the peptide-containing polyvinyl alcohol derivative, the polyvinyl alcohol derivative portion and the peptide portion are bonded via the linker portion, In the peptide-containing polyvinyl alcohol derivative, the linker portion is a structural portion derived from a linker, the linker is a compound having a carboxyl group, a thiol group, or an amino group, the peptide portion has a cyclic peptide backbone, A scaffold material for cell culture, wherein the cyclic peptide backbone has a cell adhesive amino acid sequence.

9. The linker is a compound having a carboxyl group, 9. The scaffold material for cell culture according to claim 8, wherein the content of the carboxyl groups in the peptide-containing polyvinyl alcohol derivative is 0.1 mol % or more and 2 mol % or less.

10. The polyvinyl alcohol derivative portion is a polyvinyl acetal resin portion, The scaffold material for cell culture according to claim 8 or 9, wherein the peptide-containing polyvinyl alcohol derivative is a peptide-containing polyvinyl acetal resin.

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