Scaffold material for cell culture and cell culture vessel

Abstract

The present application provides a scaffold material for cell culture, which is excellent in cell adhesion. The scaffold material for cell culture of the present application contains a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion, the peptide portion having a cyclic peptide backbone.

Description

Technical Field

[0001] This invention relates to scaffold materials for cell culture. Furthermore, this invention relates to cell culture containers using said scaffold materials. Background Technology

[0002] In research and development in academic, pharmaceutical, and regenerative medicine fields, animal cells from humans, mice, rats, pigs, cattle, and monkeys have been used. As scaffold materials for culturing animal cells, natural polymers such as laminin and vitrin, as well as matrix gel derived from mouse sarcoma, are used. By using natural polymers as scaffold materials, the domains of amino acid sequences (Arg-Gly-Asp, etc.) possessing cell-adhesive properties, such as those found in laminin and vitrin, bind to integrins on the cell surface, resulting in good cell adhesion to the scaffold material and efficient cell proliferation.

[0003] Furthermore, Patent Document 1 discloses a protein comprising a cell adhesion sequence including a repeating structure and an RGD sequence. This protein has a repeating structure formed by the linkage of specific Ala-rich sites with specific Ala-non-rich sites. Furthermore, Patent Document 1 describes the use of materials containing this protein as cell scaffold materials.

[0004] Furthermore, patent documents 2 to 5 below describe scaffold materials that use synthetic resins.

[0005] Patent Document 2 below discloses a cell culture carrier containing a molded article comprising a polyvinyl acetal compound or a molded article comprising the polyvinyl acetal compound and a water-soluble polysaccharide, wherein the degree of acetalization of the polyvinyl acetal compound is 20 to 60 mol%.

[0006] Furthermore, Patent Document 3 disclosed below discloses a composition (scaffold material) comprising a first fiber polymer scaffold material, wherein the fibers of the first fiber polymer scaffold material are arranged. As the material of the fiber polymer, an aliphatic polyester or the like is used.

[0007] Furthermore, Patent Document 4 below discloses a cell culture method for maintaining the undifferentiated nature of pluripotent stem cells, which includes a process of culturing the pluripotent stem cells on a culture vessel having a surface coated with a polyrotaxane block copolymer.

[0008] Furthermore, Patent Document 5 discloses a cell culture article comprising a substrate having a surface, a hydrophilic copolymer layer disposed on the surface of the substrate, and multiple peptide chains respectively bonded to the surface of the hydrophilic copolymer layer. The hydrophilic copolymer layer is a layer copolymerized from multiple polyvinyl alcohol units, multiple polyvinyl alcohol derivative units, and multiple carboxylic acid-containing units.

[0009] Existing technical documents

[0010] Patent documents

[0011] Patent Document 1: Japanese Patent Application Publication No. 2018-064542

[0012] Patent Document 2: Japanese Patent Application Publication No. 2006-314285

[0013] Patent Document 3: WO2007 / 090102A1

[0014] Patent Document 4: Japanese Patent Application Publication No. 2017-023008

[0015] Patent Document 5: Japanese Patent Application Publication No. 2015-070832 Summary of the Invention

[0016] The technical problem solved by the invention

[0017] By using natural polymer materials as scaffold materials, inoculated cells can proliferate well and pseudopodia can extend effectively. However, natural polymer materials are expensive, and due to their natural origin, there can be significant batch-to-batch inconsistencies, or safety risks due to animal-derived components. Furthermore, when using polypeptides with cell adhesion sequences such as RGD sequences as described in Patent Document 1 as scaffold materials, cell adhesion and proliferation can sometimes be uneven due to amino acid sequences other than the cell adhesion sequences.

[0018] On the other hand, the scaffold materials using synthetic resins described in Patent Documents 2-5 are less expensive, have less batch-to-batch inconsistency, and offer superior safety compared to scaffold materials using natural polymers. However, the scaffold materials using conventional synthetic resins described in Patent Documents 2-4 suffer from lower cell adhesion. Furthermore, while the scaffold material using synthetic resins with peptide chains described in Patent Document 5 improves cell adhesion to some extent, it is still insufficient.

[0019] The object of this invention is to provide a cell culture scaffold material with excellent cell adhesion. Furthermore, the object of this invention is to provide a cell culture container using the aforementioned cell culture scaffold material.

[0020] Technical means to solve the problem

[0021] According to a broad aspect of the present invention, a scaffold material for cell culture is provided, comprising a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion, wherein the peptide portion has a cyclic peptide backbone.

[0022] In one particular embodiment of the cell culture scaffold material of the present invention, the peptide portion has a cell-adhesive amino acid sequence.

[0023] In another specific embodiment of the cell culture scaffold material of the present invention, the cell adhesion amino acid sequence has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence.

[0024] In another specific embodiment of the cell culture scaffold material of the present invention, the cell-adhesive amino acid sequence has at least the RGD sequence represented by the following formula (1).

[0025] Arg-Gly-Asp-X···Equation (1)

[0026] In equation (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0027] In another specific embodiment of the cell culture scaffold material of the present invention, the cyclic peptide backbone is composed of 4 to 10 amino acids.

[0028] In another specific embodiment of the cell culture scaffold material of the present invention, the polyvinyl alcohol derivative portion and the peptide portion are combined via a connector portion.

[0029] In another specific embodiment of the cell culture scaffold material of the present invention, the cell culture scaffold material has an island structure.

[0030] In another specific embodiment of the cell culture scaffold material of the present invention, the peptide-containing polyvinyl alcohol derivative is a peptide-containing polyvinyl alcohol acetal resin having a polyvinyl alcohol acetal resin portion and the peptide portion.

[0031] According to a broad aspect of the present invention, a cell culture container is provided, comprising: a container body and a cell culture scaffold material disposed on the surface of the container body.

[0032] The effects of the invention

[0033] According to the present invention, cell culture scaffold materials and cell culture containers with excellent cell adhesion can be provided. Attached Figure Description

[0034] [Figure 1 ] Figure 1 This is a schematic cross-sectional view of a cell culture container according to one embodiment of the present invention.

[0035] [ Figure 2 ] Figure 2 (a) and (b) are images showing what it looks like with or without island structures.

[0036] [ Figure 3 ] Figure 3 (a), (b), and (c) are diagrams showing the relationship between SF and the planar shape of the cell.

[0037] Specific embodiments of the present invention

[0038] The present invention will now be described in detail.

[0039] The cell culture scaffold material of the present invention comprises a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. In the cell culture scaffold material of the present invention, the peptide portion has a cyclic peptide backbone.

[0040] The cell culture scaffold material of the present invention exhibits excellent cell adhesion due to the aforementioned configuration. Furthermore, the cell culture scaffold material of the present invention exhibits excellent pseudopodia extensibility and excellent cell proliferation.

[0041] In the case of cell culture scaffold materials comprising peptide-containing polyvinyl alcohol derivatives having a polyvinyl alcohol derivative portion and a peptide portion, cell adhesion can be improved to a certain extent. However, when the peptide portion does not have a cyclic peptide backbone, it is sometimes difficult to improve cell adhesion when using cells with poor adhesion. In contrast, in the case of the cell culture scaffold material of the present invention, the peptide portion has a cyclic peptide backbone, therefore, even with cells with poor adhesion, cell adhesion can be improved.

[0042] For example, in cell culture scaffold materials where the peptide portion does not have a cyclic peptide backbone, it can be difficult for aging mesenchymal stem cells that have undergone long passages, or certain iPS cell lines with low adhesion, to adhere to the scaffold material. In contrast, in the case of the cell culture scaffold material of the present invention, the peptide portion has a cyclic peptide backbone, thus improving cell adhesion even with cells exhibiting poor adhesion.

[0043] The mechanism by which the cell culture scaffold material of the present invention improves cell adhesion can be presumed to be based on the following mechanisms (1) and (2), but is not limited thereto.

[0044] (1) The three-dimensional structure of the cyclic peptide backbone allows specific amino acid sequences (e.g., cell-adhesive amino acid sequences) of the peptide to be effectively exposed. Therefore, specific amino acid sequences are easily recognized by cells, and the peptide can bind stably to cells.

[0045] (2) Peptides are not easily broken down by enzymes produced by cells, and peptides have high stability.

[0046] In the case of the cell culture scaffold material of the present invention, similar to the case where natural polymer materials such as matrix gel are used, the extension of filamentous pseudopodia-like pseudopodia is observed in the inoculated cells. This extension of pseudopodia is almost never observed in scaffold materials using conventional synthetic resin materials.

[0047] The cell culture scaffold material of the present invention allows cells to adhere well to the scaffold material even at low cell seeding densities, and the cells proliferate well.

[0048] Furthermore, the cell culture scaffold material of the present invention is less expensive, exhibits less batch-to-batch heterogeneity, and demonstrates superior safety compared to conventional cell scaffold materials using natural polymers. Using the cell scaffold material of the present invention can reduce the burden of cell quality management.

[0049] (Scaffold materials for cell culture)

[0050] The cell culture scaffold material of the present invention comprises 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.

[0051] The peptide-containing polyvinyl alcohol derivative has a polyvinyl alcohol derivative portion and a peptide portion. Preferably, the polyvinyl alcohol derivative portion and the peptide portion are bonded together via a connector portion. Therefore, the peptide-containing polyvinyl alcohol derivative preferably has a polyvinyl alcohol derivative portion, a peptide portion, and a connector portion.

[0052] The peptide-containing polyvinyl alcohol derivative, as described below, can be obtained, for example, by reacting a polyvinyl alcohol derivative, a linker, and a peptide. It should be noted that the peptide-containing polyvinyl alcohol derivative can be obtained, for example, by reacting polyvinyl alcohol, a linker, and a peptide.

[0053] <Polyvinyl alcohol derivatives section>

[0054] The polyvinyl alcohol derivative portion is the structural part of the peptide-containing polyvinyl alcohol derivative derived from the polyvinyl alcohol derivative. The polyvinyl alcohol derivative is a compound obtained by derivatizing polyvinyl alcohol. The polyvinyl alcohol derivative is preferably a polyvinyl alcohol acetal resin, and the polyvinyl alcohol derivative portion is preferably a polyvinyl alcohol acetal resin portion. That is, the peptide-containing polyvinyl alcohol derivative is preferably a peptide-containing polyvinyl alcohol acetal resin having both a polyvinyl alcohol acetal resin portion and the peptide portion. Only one of the polyvinyl alcohol derivative and the polyvinyl alcohol acetal resin may be used, or two or more may be used in combination.

[0055] The polyvinyl alcohol derivative portion and the polyvinyl alcohol acetal resin portion preferably have acetal, hydroxyl, and acetyl groups on their side chains. However, the polyvinyl alcohol derivative portion and the polyvinyl alcohol acetal resin portion may, for example, lack acetyl groups. For instance, all the acetyl groups in the polyvinyl alcohol derivative portion and the polyvinyl alcohol acetal resin portion may be bonded to the connector, thus rendering the polyvinyl alcohol derivative portion and the polyvinyl alcohol acetal resin portion acetic-free.

[0056] Polyvinyl alcohol acetal resin can be synthesized by acetalizing polyvinyl alcohol with aldehydes.

[0057] The aldehyde used in the acetalization of polyvinyl alcohol is not particularly limited. Examples of such aldehydes include those with 1 to 10 carbon atoms. The aldehyde may or may not have chain aliphatic groups, cyclic aliphatic groups, or aromatic groups. The aldehyde may be a chain aldehyde or a cyclic aldehyde.

[0058] Examples of aldehydes include: formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, pentanaldehyde, hexanal, heptanaldehyde, octanaldehyde, nonanaldehyde, decanaldehyde, acrolein, benzaldehyde, cinnamaldehyde, perillaldehyde, formylpyridine, formylimidazolium, formylpyrrole, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formylisoindole, formylpurine, formylbenzimidazole, formylbenzotriazole, formylquinoline, formylisoquinoline, formylquinoxaline, formylcinnamoline, formylpteridine, formylfuran, formyloxacyclopentane, formyloxane, formylthiophene, formyltetrahydrothiophene, formylcyclopentane sulfide, formyladenine, formylguanine, formylcytosine, formylthymidine, and formyluracil, etc. The aldehyde can be used alone or in combination with two or more.

[0059] The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, or valeraldehyde, and more preferably butyraldehyde. Therefore, the polyvinyl alcohol acetal resin is more preferably polyvinyl alcohol butyral resin, the polyvinyl alcohol acetal resin portion is more preferably polyvinyl alcohol butyral resin portion, and the peptide-containing polyvinyl alcohol derivative is more preferably peptide-containing polyvinyl alcohol butyral resin.

[0060] It should be noted that the mixing amount of the aldehyde can be appropriately set according to the desired amount of acetal group. From the viewpoint of improving the efficiency of the acetalization reaction and easily removing unreacted aldehyde, the amount of aldehyde added relative to 100 mol% of polyvinyl alcohol is preferably 60 mol% or more, more preferably 65 mol% or more, more preferably 95 mol% or less, and more preferably 90 mol% or less.

[0061] The average degree of polymerization of the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol acetal resin is preferably 100 or more, more preferably 200 or more, further preferably 500 or more, particularly preferably 1500 or more, preferably 6000 or less, more preferably 3000 or less, and further preferably 2500 or less. When the average degree of polymerization is above the lower limit, swelling caused by liquid culture medium can be effectively suppressed, thus maintaining the strength of the scaffold material for cell culture well. Therefore, cell proliferation can be improved. In addition, when the average degree of polymerization is below the upper limit, operability and formability of the scaffold material for cell culture can be improved. It should be noted that the average degree of polymerization of the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol acetal resin is usually the same as the average degree of polymerization of the polyvinyl alcohol used as raw material, and can be determined based on the average degree of polymerization of polyvinyl alcohol.

[0062] The number-average molecular weight (Mn) of the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol 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 alcohol acetal resin portion, and the polyvinyl alcohol acetal resin is preferably 2,000 or more, and preferably 1,200,000 or less. Furthermore, the ratio (Mw / Mn) of the weight-average molecular weight (Mw) to the number-average molecular weight (Mn) in the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol acetal resin is preferably 2.0 or more, and preferably 40 or less. When Mn, Mw, and Mw / Mn are above the lower limit and below the upper limit, the strength of the scaffold material for cell culture can be improved.

[0063] It should be noted that the number-average molecular weight (Mn) and weight-average molecular weight (Mw) of the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol acetal resin can be obtained, for example, as polystyrene conversion values ​​by gel permeation chromatography (GPC) analysis using tetrahydrofuran (THF) as a solvent.

[0064] The degree of acetalization (or butyralization in the case of polyvinyl butyral resin) of the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol acetal resin is preferably 40 mol% or more, more preferably 50 mol% or more, more preferably 90 mol% or less, and more preferably 85 mol% or less. When the degree of acetalization is above or below the lower limit, cell fixation can be further improved, resulting in efficient cell proliferation. When the degree of acetalization is below the upper limit, solubility in solvents can be improved.

[0065] The content (hydroxyl content) of hydroxyl groups in the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol 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.

[0066] The degree of acetylation (acetyl group content) of the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol acetal resin is preferably 1 mol% or more, more preferably 2 mol% or more, more preferably 5 mol% or less, and more preferably 4 mol% or less. When the degree of acetylation is above the lower limit and below the upper limit, the reaction efficiency between the polyvinyl alcohol acetal resin and the connector can be improved.

[0067] The degree of acetalization, degree of acetylation, and hydroxyl content of the polyvinyl alcohol derivative portion, the polyvinyl alcohol acetal resin portion, and the polyvinyl alcohol acetal resin can be determined by... 1 The determination was performed using H-NMR (nuclear magnetic resonance spectroscopy).

[0068] <Peptide Section>

[0069] The peptide portion is a structural part 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 can be an oligopeptide or a polypeptide. Only one peptide may be used, or two or more may be used in combination.

[0070] The peptide portion has a cyclic peptide backbone. The cyclic peptide backbone refers to a cyclic backbone composed of multiple amino acids. The peptide portion may have only a cyclic peptide backbone, or it may have a cyclic peptide backbone and a backbone other than the cyclic peptide backbone (such as a chain peptide backbone).

[0071] The peptide portion preferably consists of 4 or more amino acids, more preferably 5 or more amino acids, more preferably 15 or fewer amino acids, and even more preferably 10 or fewer amino acids. When the number of amino acids constituting the peptide portion is above the lower limit and below the upper limit, the recognition of specific amino acid sequences by cells can be improved, and cell adhesion and proliferation can be further enhanced. In addition, when the number of amino acids constituting the peptide portion is above the lower limit and below the upper limit, the extensibility of pseudopodia can be further improved.

[0072] The cyclic peptide backbone is preferably composed of 4 or more amino acids, more preferably 5 or more amino acids, more preferably 10 or fewer amino acids, and even more preferably 7 or fewer amino acids. When the number of amino acids constituting the cyclic peptide backbone is above the lower limit, the recognition of specific amino acid sequences by cells can be improved, and cell adhesion and proliferation can be further enhanced. Furthermore, when the number of amino acids constituting the cyclic peptide backbone is above the lower limit and below the upper limit, the extensibility of 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.

[0073] The peptide portion preferably has an amino acid sequence with cell-adhesive properties. It should be noted that the amino acid sequence with cell-adhesive properties refers to an amino acid sequence whose cell-adhesive activity has been confirmed by phage display, agarose bead method, or plate coating method. For example, the method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995 1189-1196" can be used as the phage display method. For example, the method described in "Protein Nuclease Vol. 45 No. 15 (2000) 2477" can be used as the plate coating method.

[0074] Examples of amino acid sequences that contribute to cell adhesion include, for example: 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). Furthermore, examples of the amino acid sequences that contribute to cell adhesion include those described in "Pathological Physiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Health Center Journal, Vol. 8, No. 1, pp. 58-66, 1992". The peptide may contain only one such amino acid sequence contributing to cell adhesion, or it may contain two or more.

[0075] The amino acid sequence for cell adhesion preferably has at least one of the above-mentioned amino acid sequences for cell adhesion, 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, cell adhesion and proliferation can be further improved, and pseudopodia extension can be further improved.

[0076] Arg-Gly-Asp-X···Equation (1)

[0077] In equation (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0078] 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 improving cell recognition of the cell-adhesive amino acid sequence and further enhancing cell adhesion and proliferation, the cyclic peptide backbone preferably has the cell-adhesive amino acid sequence.

[0079] When the peptide portion has the cell-adhesive amino acid sequence, the amino acid at the N-terminus or C-terminus of the cell-adhesive amino acid sequence may bind to the linker, or the amino acid sequence constituting a portion different from the cell-adhesive amino acid sequence may bind to the linker.

[0080] When the peptide portion has the amino acid sequence that promotes cell adhesion, it is more preferable that an amino acid sequence constituting a portion different from the amino acid sequence that promotes cell adhesion binds to the linker portion. In this case, cell adhesion and proliferation can be further improved, and pseudopodia extension can be further enhanced.

[0081] In the 100% by weight of the peptide-containing polyvinyl alcohol derivative, the content of the peptide portion is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, further preferably 1% by weight or more, and particularly preferably 5% by weight or more. In the 100% by weight of the peptide-containing polyvinyl alcohol derivative, the content of the peptide portion is preferably 30% by weight or less, more preferably 25% by weight or less, further preferably 20% by weight or less, and particularly preferably 15% by weight or less. When the content of the peptide portion is above the lower limit, it can further improve cell adhesion and proliferation, and can further improve the extensibility of pseudopodia.

[0082] 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, further preferably 1 mol% or more, 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, further preferably 35 mol% or less, and particularly preferably 25 mol% or less. When the content of the peptide portion is above the lower limit, it can further improve cell adhesion and proliferation, and further improve the extensibility of pseudopodia. When the content of the peptide portion is below the upper limit, it can suppress manufacturing costs. It should be noted that the content of the peptide portion (mol%) is the ratio of the mass of the peptide portion to the sum of the masses of each structural unit constituting the peptide-containing polyvinyl alcohol derivative.

[0083] In the peptide-containing polyvinyl alcohol derivative, the molar ratio of the peptide content to the total content of the acetal, hydroxyl, and acetyl groups (the content of the peptide / the total content of the acetal, hydroxyl, and acetyl groups) is preferably 0.0001 or more, more preferably 0.001 or more. When the molar ratio (the content of the peptide / the total content of the acetal, hydroxyl, and acetyl groups) is at or above the lower limit, cell adhesion and proliferation can be further improved, and pseudopodia extensibility can be further improved. The upper limit of the molar ratio of the peptide content to the total content of the acetal, hydroxyl, and acetyl groups (the content of the peptide / the total content of the acetal, hydroxyl, and acetyl groups) in the peptide-containing polyvinyl alcohol derivative is not particularly limited. From the viewpoint of manufacturing cost, the molar ratio (the content of the peptide / the total content of the acetal, hydroxyl, and acetyl groups) is preferably 0.2 or less.

[0084] The content of the peptide can be determined by FT-IR or LC-MS.

[0085] <Joint section>

[0086] The connector portion is a structural part of the peptide-containing polyvinyl alcohol derivative derived from the connector. The connector portion is located between the polyvinyl alcohol derivative portion and the peptide portion. The polyvinyl alcohol derivative portion and the peptide portion are bonded together through the connector portion. The connector portion is formed by a connector (crosslinking agent). Only one connector may be used, or two or more may be used in combination.

[0087] The linker is preferably a compound having a functional group capable of condensing with the carboxyl or amino group of the peptide. Examples of functional groups capable of condensing with the carboxyl or amino group of the peptide include carboxyl, thiol, and amino groups. From the viewpoint of good reactivity with the peptide, the linker is preferably a compound having a carboxyl group.

[0088] Examples of carboxyl-containing linkers include (meth)acrylic acid and carboxyl-containing acrylamide. By using a carboxylic acid (carboxylic acid monomer) with a polymerizable unsaturated group as the carboxyl-containing linker, the carboxylic acid monomer can be polymerized by graft polymerization during linker introduction, thereby increasing the number of carboxyl groups that can react with the peptide.

[0089] From the viewpoint of enabling good binding between polyvinyl alcohol derivatives and peptides, the connector is preferably (meth)acrylic acid, more preferably acrylic acid.

[0090] The peptide-containing polyvinyl alcohol derivative can be synthesized, for example, in the manner described below.

[0091] (1) React a polyvinyl alcohol derivative (e.g., polyvinyl alcohol acetal resin) with a linker to obtain a reactant formed by the combination of polyvinyl alcohol acetal resin and the linker. (2) React the obtained reactant with a peptide to obtain a peptide-containing polyvinyl alcohol derivative (peptide-containing polyvinyl alcohol acetal resin).

[0092] In (1), as a method for obtaining the reactant formed by combining the polyvinyl acetal resin and the connector, examples include: acetalizing a copolymer of polyvinyl alcohol and a carboxylic acid having polymerizable unsaturated groups; and graft copolymerizing the polyvinyl acetal resin and the connector (e.g., a carboxylic acid monomer) under ultraviolet light. The graft copolymerization method is preferred for obtaining the reactant. In this case, the carboxylic acid monomer can be polymerized by graft polymerization, thus increasing the number of carboxyl groups that can react with the peptide.

[0093] In step (2), the carboxyl group originating from the linker in the reactant can undergo dehydration condensation with the amino group of the peptide to obtain a peptide-containing polyvinyl alcohol derivative (peptide-containing polyvinyl alcohol acetal resin) having a polyvinyl alcohol acetal resin part, a peptide part, and a linker part.

[0094] The peptide-containing polyvinyl alcohol derivative may or may not contain carboxyl groups derived from the linker. The content of the carboxyl groups 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, and more preferably 1.5 mol% or less. When the content of the carboxyl groups is above the lower limit and below the upper limit, it can further improve cell adhesion and proliferation, and further improve the extensibility of pseudopodia. It should be noted that the content of the carboxyl groups (mol%) is the ratio of the mass of the carboxyl groups to the sum of the masses of each structural unit constituting the peptide-containing polyvinyl alcohol derivative.

[0095] From the viewpoint of further improving cell adhesion and proliferation, the cell culture scaffold material preferably has a phase-separated structure. The phase-separated structure has at least a first phase and a second phase.

[0096] Examples of phase-separation structures include microphase-separation structures such as island structures, cylindrical structures, gyroscopic structures, and layered structures. In an island structure, for example, the first phase can be designated as the sea portion, and the second phase as the island portion. In a cylindrical, gyroscopic, or layered structure, for example, the phase with the largest surface area can be designated as the first phase, and the phase with the second largest surface area as the second phase. The cell culture scaffold material has both continuous and discontinuous phases, thereby improving its affinity with cells and further enhancing cell adhesion and proliferation.

[0097] The phase separation structure is preferably an island structure. The cell culture scaffold material preferably has an island structure. In this case, cell adhesion and proliferation can be further improved.

[0098] When the cell culture scaffold material has an island structure, the surface integral number of the island (second phase) relative to the overall surface of the cell culture scaffold material is preferably 0.01 or more, more preferably 0.1 or more, even more preferably 0.2 or more, preferably 0.95 or less, more preferably 0.9 or less, and even more preferably 0.8 or less. When the surface integral number is above or below the lower limit and below the upper limit, the adhesion of cells can be further improved.

[0099] When the cell culture scaffold material has an island structure, the island portion preferably includes a peptide portion. That is, the cell culture scaffold material preferably has a sea portion and an island portion, with the island portion containing a peptide portion. In this case, the cell adhesion domains can accumulate in the island portion, thereby further improving cell adhesion.

[0100] The presence or absence of phase separation structures can be confirmed, for example, by atomic force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), etc. Furthermore, the surface integral can be obtained from microscopic images using image analysis software such as ImageJ.

[0101] The phase separation structure, for example, can increase the content of the peptide portion by forming a phase separation structure between or within molecules of the peptide-containing polyvinyl alcohol derivative.

[0102] From the viewpoint of effectively utilizing the effects of the present invention and improving productivity, the content of the peptide-containing polyvinyl alcohol derivative in 100% by weight of the cell culture scaffold material is preferably 90% by weight or more, more preferably 95% by weight or more, further preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total). Therefore, the cell culture scaffold material is most preferably the peptide-containing polyvinyl alcohol derivative. When the content of the peptide-containing polyvinyl alcohol derivative is at or above the lower limit, the effects of the present invention can be further effectively utilized.

[0103] The cell culture scaffold material may contain polymers other than the peptide-containing polyvinyl alcohol derivative. Examples of such polymers include: polyvinyl acetal resin, polyolefin resin, polyether resin, polyvinyl alcohol resin, polyester, epoxy resin, polyamide resin, polyimide resin, polyurethane resin, polycarbonate resin, cellulose, and polypeptides. Only one polymer may be used, or two or more may be used in combination.

[0104] From the viewpoint of effectively utilizing the effects of the present invention, the lower the content of the polymer other than the peptide-containing polyvinyl alcohol derivative, the better. In 100% by weight of the cell culture scaffold material, the content of this polymer is preferably 10% by weight or less, more preferably 5% by weight or less, further preferably 2.5% by weight or less, particularly preferably 1% by weight or less, and most preferably 0% by weight (excluding). Therefore, the cell culture scaffold material most preferably does not contain polymers other than the peptide-containing polyvinyl alcohol derivative.

[0105] The cell culture scaffold material of the present invention preferably does not contain animal-derived raw materials. By not containing animal-derived raw materials, batch-to-batch inhomogeneity can be reduced, providing a cell culture scaffold material with excellent cost and safety. It should be noted that "not containing animal-derived raw materials" means that the animal-derived raw materials in the cell culture scaffold material are 3% by weight or less. In the case of the cell culture scaffold material of the present invention, the animal-derived raw materials in the cell culture scaffold material are preferably 1% by weight or less, more preferably 0% by weight. That is, the cell culture scaffold material is more preferably free of animal-derived raw materials.

[0106] It should be noted that the scaffold material for cell culture can be prepared on the surface of the container body described later. For example, a synthetic resin having a polyvinyl alcohol derivative portion and a connector can be coated on the surface of the container body to form a resin film, and the synthetic resin can be reacted with a peptide on the surface of the resin film to obtain the peptide-containing polyvinyl alcohol derivative.

[0107] (Further details on scaffold materials for cell culture)

[0108] The cell culture scaffold material of the present invention is used for culturing cells. The cell culture scaffold material of the present invention serves as a scaffold for culturing cells.

[0109] Examples of such cells include animal cells from humans, mice, rats, pigs, cattle, and monkeys. Furthermore, examples of such cells include somatic cells, such as stem cells, progenitor cells, and mature cells. The somatic cells may be cancer cells.

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

[0111] Examples of stem cells include: mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germline stem cells, and mGS cells.

[0112] The shape of the scaffold material for cell culture is not particularly limited. The scaffold material for cell culture can be membrane-like, particle-like, fibrous, or porous. Membrane-like materials include thin films and sheets.

[0113] The cell culture scaffold material is preferably used in two-dimensional (planar) culture, three-dimensional culture, or floating culture of cells, and more preferably in two-dimensional (planar) culture.

[0114] Furthermore, the cell culture scaffold material can also be used as a cell culture carrier (medium) comprising the cell culture scaffold material and a polysaccharide. The polysaccharide is not particularly limited and can be any conventionally known polysaccharide. Preferably, the polysaccharide is water-soluble.

[0115] Furthermore, the cell culture scaffold material can be used as a cell culture fiber comprising a fibrous body and a scaffold material disposed on the surface of the fibrous body. In this case, the cell culture scaffold material is preferably coated on the surface of the fibrous body, and preferably a coating. In this cell culture fiber, the cell culture scaffold material can be present within the fibrous body. For example, the fibrous body can be impregnated or kneaded with liquid cell culture scaffold material, thereby making the cell culture scaffold material present within the fibrous body. Stem cells generally have the property of easily adhering to three-dimensional structures such as linear structures rather than planar structures, therefore, cell culture fibers are suitable for three-dimensional culture of stem cells. Among stem cells, they are particularly suitable for three-dimensional culture of adipose stem cells.

[0116] The synthetic resin in the cell culture scaffold material can be cross-linked. Cell culture scaffold materials containing cross-linked synthetic resin can effectively suppress water swelling and improve strength. The synthetic resin can be cross-linked by using a cross-linking agent.

[0117] (Containers for cell culture)

[0118] The cell culture container of the present invention comprises: a container body and a cell culture scaffold material disposed on the surface of the container body. At least a portion of the cell culture region of the cell culture container is provided with the cell culture scaffold material.

[0119] Figure 1 This is a schematic cross-sectional view of a cell culture container according to one embodiment of the present invention.

[0120] The cell culture container 1 comprises: a container body 2 and a cell culture scaffold material 3. The cell culture scaffold material 3 is disposed on the surface 2a of the container body 2. The cell culture scaffold material 3 is disposed on the bottom surface of the container body 2. Liquid culture medium can be added to the cell culture container 1, and cells such as cell blocks can be inoculated onto the surface of the cell culture scaffold material 3 to culture cells in a planar manner.

[0121] It should be noted that the container body may include: a first container body and a second container body, such as a coverslip on the bottom surface of the first container body. The first container body and the second container body may be separable. In this case, the cell culture scaffold material may be disposed on the surface of the second container body.

[0122] As the container body, conventionally known container bodies (containers) can be used. The shape and size of the container body are not particularly limited.

[0123] Examples of the main body of the container include cell culture plates and cell culture flasks having one or more wells (cavities). The number of wells in the plate is not particularly limited. Examples of the number of wells include 2, 4, 6, 12, 24, 48, 96, and 384. The shape of the well is not particularly limited and can include perfect circles, ellipses, triangles, squares, rectangles, and pentagons. The shape of the bottom surface of the well is not particularly limited and can include flat bottoms, round bottoms, and convex / concave bottoms.

[0124] The material of the container body is not particularly limited, and examples include resin, metal, and inorganic materials. Examples of resins include: polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymers, polyimide, polyamide, polyamide-imide, (meth)acrylic resins, epoxy resin, polysiloxane, etc. Examples of metals include: stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, platinum, etc. Examples of inorganic materials include: silicon dioxide (glass), aluminum oxide, titanium oxide, zirconium oxide, iron oxide, silicon nitride, etc.

[0125] The present invention will be described in detail below with examples and comparative examples. The present invention is not limited to these examples.

[0126] It should be noted that the content of structural units in the obtained synthetic resin can be determined by dissolving the synthetic resin in DMSO-d6 (dimethyl sulfoxide) and then... 1 The determination was performed using 1H-NMR (nuclear magnetic resonance spectroscopy). Furthermore, the peptide content in the peptide-containing polyvinyl alcohol derivatives could be determined by FT-IR or LC-MS. The degree of acetalization (degree of butyralization), hydroxyl content, degree of acetylation, carboxyl group content, and peptide content of the obtained synthetic resins are shown in Tables 1–3.

[0127] (Example 1)

[0128] Preparation of polyvinyl acetal resin (PVB1):

[0129] 2700 mL of deionized water and 300 parts by weight of polyvinyl alcohol (PVA) with an average degree of polymerization of 1700 and a saponification degree of 99 mol% were added to a reactor equipped with a stirrer. The mixture was heated and dissolved while stirring to obtain a solution. 35 wt% hydrochloric acid was added to the obtained solution as a catalyst to achieve a hydrochloric acid concentration of 0.2 wt%. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butyraldehyde were added while stirring. Then, 148 parts by weight of n-butyraldehyde were added, precipitating white granular polyvinyl acetal resin (polyvinyl butyral resin). After 15 minutes of precipitation, 35 wt% hydrochloric acid was added to achieve a hydrochloric acid concentration of 1.8 wt%, and the mixture was heated to 50°C and maintained at 50°C for 2 hours. Next, the solution was cooled, neutralized, washed with water, and dried to obtain polyvinyl butyral resin (polyvinyl butyral resin (PVB1), average degree of polymerization 1700, degree of acetalization (degree of butyralization) 70 mol%, hydroxyl content 27 mol%, degree of acetylation 3 mol%)).

[0130] Connector insertion:

[0131] 99 parts by weight of the obtained polyvinyl acetal resin and 1 part by weight of acrylic acid (joint) were dissolved in 300 parts by weight of THF. The mixture was reacted for 20 minutes under ultraviolet irradiation in the presence of a photoradical polymerization initiator to allow the polyvinyl acetal resin and acrylic acid to undergo graft copolymerization, thereby introducing the joint. 1 part by weight of the jointed polyvinyl acetal resin was dissolved in 19 parts by weight of butanol. 150 μL of the resulting solution was poured onto the surface of a φ22 mm coverslip (Matsunami Corporation "22-piece No. 1") that had been dusted using a dust collector. The coverslip was then spin-coated at 2000 rpm for 20 seconds, followed by heating at 60°C for 60 minutes to obtain a smooth resin film.

[0132] Peptide formation:

[0133] A cyclic peptide (5 amino acid residues, forming a cyclic backbone through Arg-Gly-Asp-Phe-Lys binding, with Phe as the D-form, listed as c-RGDf K in the table) with the amino acid sequence Arg-Gly-Asp-Phe-Lys was prepared. One part by weight of this peptide and one part by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) were added to phosphate-buffered saline (excluding calcium and magnesium) to achieve a final peptide concentration of 1 mM, thus preparing a peptide-containing solution. One part by weight of this peptide-containing solution was added to a spin-coated resin film (forming a linker-connected polyvinyl acetal resin) and reacted to allow dehydration condensation of the carboxyl group of the linker and the amino group of the Lys group of the peptide. This yielded a peptide-containing polyvinyl acetal resin with a polyvinyl acetal resin portion, a linker portion, and a peptide portion.

[0134] The obtained peptide-containing polyvinyl alcohol acetal resin has the following characteristics: degree of acetalization (degree of butyralization) 69.3 mol%, hydroxyl content 26.7 mol%, degree of acetylation 3.0 mol%, carboxyl content 0.9 mol%, and peptide content 0.1 mol%.

[0135] Preparation of containers for cell culture:

[0136] The obtained peptide-containing polyvinyl acetal resin and coverslip were stacked on a φ22mm polystyrene dish to obtain a cell culture container.

[0137] (Example 2)

[0138] Preparation of polyvinyl acetal resin (PVB2):

[0139] 2700 mL of deionized water and 300 parts by weight of polyvinyl alcohol (PVA) with an average degree of polymerization of 1700 and a saponification degree of 99 mol% were added to a reactor equipped with a stirrer. The mixture was heated and dissolved while stirring to obtain a solution. 35 wt% hydrochloric acid was added to the obtained solution as a catalyst to achieve a hydrochloric acid concentration of 0.2 wt%. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butyraldehyde were added while stirring. Then, 143 parts by weight of n-butyraldehyde were added, precipitating white granular polyvinyl acetal resin (polyvinyl butyral resin). After 15 minutes of precipitation, 35 wt% hydrochloric acid was added to achieve a hydrochloric acid concentration of 1.8 wt%, and the mixture was heated to 50°C and maintained at 50°C for 2 hours. Next, the solution was cooled, neutralized, washed with water, and dried to obtain polyvinyl butyral resin (polyvinyl butyral resin (PVB2), average degree of polymerization 1700, degree of acetalization (degree of butyralization) 69 mol%, hydroxyl content 28 mol%, degree of acetylation 3 mol%)).

[0140] Connector insertion:

[0141] 70 parts by weight of the obtained polyvinyl acetal resin and 30 parts by weight of acrylic acid (joint) were dissolved in 300 parts by weight of THF. The mixture was reacted for 20 minutes under ultraviolet irradiation in the presence of a photoradical polymerization initiator to allow the polyvinyl acetal resin and acrylic acid to undergo graft copolymerization, thereby introducing the joint. 1 part by weight of the jointed polyvinyl acetal resin was dissolved in 19 parts by weight of butanol. 150 μL of the resulting solution was poured onto the surface of a φ22 mm coverslip (Matsunami Corporation "22-piece No. 1") that had been dusted using a dust collector. The coverslip was then spin-coated at 2000 rpm for 20 seconds, followed by heating at 60°C for 60 minutes to obtain a smooth resin film.

[0142] Peptide formation:

[0143] Using the obtained resin membrane (introduced with a connectored polyvinyl acetal resin), the amount of peptide added was set to 30 parts by weight. Otherwise, the peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 1.

[0144] Preparation of containers for cell culture:

[0145] The cell culture container was obtained in the same manner as in Example 1.

[0146] (Example 3)

[0147] Preparation of polyvinyl acetal resin (PVB3):

[0148] 2700 mL of deionized water and 300 parts by weight of polyvinyl alcohol (PVA) with an average degree of polymerization of 1700 and a saponification degree of 99 mol% were added to a reactor equipped with a stirrer. The mixture was heated and dissolved while stirring to obtain a solution. 35 wt% hydrochloric acid was added to the obtained solution as a catalyst to achieve a hydrochloric acid concentration of 0.2 wt%. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butyraldehyde were added while stirring. Then, 133 parts by weight of n-butyraldehyde were added, precipitating white granular polyvinyl acetal resin (polyvinyl butyral resin). After 15 minutes of precipitation, 35 wt% hydrochloric acid was added to achieve a hydrochloric acid concentration of 1.8 wt%, and the mixture was heated to 50°C and maintained at 50°C for 2 hours. Next, the solution was cooled, neutralized, washed with water, and dried to obtain polyvinyl butyral resin (polyvinyl butyral resin (PVB3), average degree of polymerization 1700, degree of acetalization (degree of butyralization) 63 mol%, hydroxyl content 34 mol%, degree of acetylation 3 mol%)).

[0149] Connector insertion:

[0150] The obtained polyvinyl acetal resin was used, except that the connector was introduced in the same manner as in Example 2. Furthermore, a resin film with a smooth surface was obtained in the same manner as in Example 2.

[0151] Peptide formation:

[0152] Using the obtained resin membrane (introduced with a jointed polyvinyl acetal resin), a peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 2.

[0153] Preparation of containers for cell culture:

[0154] The cell culture container was obtained in the same manner as in Example 1.

[0155] (Example 4)

[0156] Preparation of polyvinyl acetal resin (PVB4):

[0157] 2700 mL of deionized water and 300 parts by weight of polyvinyl alcohol (PVA) with an average degree of polymerization of 1700 and a saponification degree of 99 mol% were added to a reactor equipped with a stirrer. The mixture was heated and dissolved while stirring to obtain a solution. 35 wt% hydrochloric acid was added to the obtained solution as a catalyst to achieve a hydrochloric acid concentration of 0.2 wt%. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butyraldehyde were added while stirring. Then, 133 parts by weight of n-butyraldehyde were added, precipitating white granular polyvinyl acetal resin (polyvinyl butyral resin). After 15 minutes of precipitation, 35 wt% hydrochloric acid was added to achieve a hydrochloric acid concentration of 1.8 wt%, and the mixture was heated to 50°C and maintained at 50°C for 2 hours. Next, the solution was cooled, neutralized, washed with water, and dried to obtain polyvinyl butyral resin (polyvinyl butyral resin (PVB4), average degree of polymerization 1700, degree of acetalization (degree of butyralization) 50 mol%, hydroxyl content 47 mol%, degree of acetylation 3 mol%)).

[0158] Connector insertion:

[0159] The obtained polyvinyl acetal resin was used, except that the connector was introduced in the same manner as in Example 2. Furthermore, a resin film with a smooth surface was obtained in the same manner as in Example 2.

[0160] Peptide formation:

[0161] Using the obtained resin membrane (introduced with a jointed polyvinyl acetal resin), a peptide-containing polyvinyl acetal resin was prepared in the same manner as in Example 2.

[0162] Preparation of containers for cell culture:

[0163] The cell culture container was obtained in the same manner as in Example 1.

[0164] (Comparative Example 1)

[0165] A linear peptide (4 amino acid residues, referred to as RGDS in the table) with the amino acid sequence Arg-Gly-Asp-Ser was used to dehydrate and condense the carboxyl group of the linker with the amino group of Arg in the peptide. Otherwise, the peptide-containing polyvinyl acetal resin and cell culture container were prepared in the same manner as in Example 1.

[0166] (Comparative Example 2)

[0167] A linear peptide (5 amino acid residues, referred to as GRGDS in the table) with the amino acid sequence Gly-Arg-Gly-Asp-Ser was used to dehydrate and condense the carboxyl group of the linker with the amino group of the peptide's Gly group. Otherwise, the peptide-containing polyvinyl acetal resin and cell culture containers were prepared in the same manner as in Example 2.

[0168] (Comparative Example 3)

[0169] A linear peptide (5 amino acid residues, referred to as GRGDS in the table) with the amino acid sequence Gly-Arg-Gly-Asp-Ser was used to dehydrate and condense the carboxyl group of the linker with the amino group of the peptide's Gly group. Otherwise, the peptide-containing polyvinyl acetal resin and cell culture containers were prepared in the same manner as in Example 3.

[0170] (Comparative Example 4)

[0171] A linear peptide (5 amino acid residues, referred to as GRGDS in the table) with the amino acid sequence Gly-Arg-Gly-Asp-Ser was used to dehydrate and condense the carboxyl group of the linker with the amino group of the peptide's Gly group. Otherwise, the peptide-containing polyvinyl acetal resin and cell culture containers were prepared in the same manner as in Example 4.

[0172] (Comparative Example 5)

[0173] No peptide portion was formed. Otherwise, the cell culture container was prepared in the same manner as in Example 1.

[0174] (Example 5)

[0175] 99 parts by weight of polyvinyl alcohol (PVA) with an average degree of polymerization of 1700 and a saponification degree of 90 mol%, and 1 part by weight of acrylic acid (linker) were dissolved in 300 parts by weight of ethanol. The mixture was reacted for 20 minutes under ultraviolet light in the presence of a photoradical polymerization initiator to allow the PVA and acrylic acid to undergo graft copolymerization, thereby introducing the linker. In addition, peptide-containing PVA derivatives and cell culture containers were prepared in the same manner as in Example 1.

[0176] (Comparative Example 6)

[0177] 99 parts by weight of polyvinyl alcohol (PVA) with an average degree of polymerization of 1700 and a saponification degree of 90 mol%, and 1 part by weight of acrylic acid (linker) were dissolved in 300 parts by weight of ethanol. The mixture was reacted for 20 minutes under UV irradiation in the presence of a photoradical polymerization initiator to graft copolymerize PVA and acrylic acid, thereby introducing the linker. A linear peptide (5 amino acid residues, listed as GRGDS in the table) with the amino group of Gly-Arg-Gly-Asp-Ser was used to dehydrate and condense the carboxyl group of the linker with the amino group of the Gly group of the peptide. Otherwise, peptide-containing PVA derivatives and cell culture containers were prepared in the same manner as in Example 1.

[0178] (Refer to Example A)

[0179] Preparation of scaffold materials derived from natural sources:

[0180] 1 ml of a 5 μg / ml Vitronectin (Corning) solution was added to a φ35 mm dish. A φ22 mm coverslip (Matsunami Corporation "22 pellets No.1") was then immersed in the dish and incubated at 37°C for 1 hour to obtain a natural scaffold material (referred to as VTN in the table) in which Vitronectin is smoothly adsorbed on the surface.

[0181] Preparation of containers for cell culture:

[0182] Cell culture containers were obtained in the same manner as in Example 1. It should be noted that Vitron ectin denatures when dried, which significantly reduces its adhesive properties; therefore, the cell culture containers were immediately immersed in PBS solution after preparation.

[0183] (evaluate)

[0184] (1) Whether or not there is an island structure

[0185] The obtained peptide-containing polyvinyl acetal resin membrane was immersed in PBS solution for 30 minutes. The immersed resin membrane was observed using an atomic force microscope (AFM, Bruker "Dimension XR"). Under QNM mode with the peak set point set to 2nN, a range of 1μm × 1μm was observed. The obtained height mapping image and elasticity mapping image were compared to determine the presence or absence of island structures. It should be noted that in the table, cases where island structures were observed are recorded as "A", and cases where no island structures were observed are recorded as "B".

[0186] Figure 2 (a) is an instance of an image that is determined to show observed island structures. Figure 2 (b) is an example of an image that was determined to have no observed island structure.

[0187] (3) Cell Culture Evaluation

[0188] (3-1) Seeding and culture of iPS cells (201B7)

[0189] The following steps were performed using cell culture containers obtained in Examples 1-4, Comparative Examples 1-5, and Reference Example A.

[0190] Prepare the following liquid culture medium and ROCK (Rho-binding kinase) specific inhibitor.

[0191] TeSR E8 medium (manufactured by STEM CELL)

[0192] ROCK-Inhibitor (Y27632)

[0193] Add 1 mL of phosphate-buffered saline to the obtained cell culture container and let it stand in an incubator at 37°C for 1 hour, then remove the phosphate-buffered saline from the cell culture container.

[0194] For h-iPS cells 201B7 that have reached a confluence state in a φ35 mm dish, add 1 mL of 0.5 mM ethylenediaminetetraacetic acid / phosphate buffer and incubate at room temperature for 2 minutes. After removing the ethylenediaminetetraacetic acid / phosphate buffer, pipette 1 mL of liquid culture medium to obtain cell fragments with a size of 50 μm to 200 μm. Analyze the resulting cell fragments (cell count 1.0 × 10⁻⁶) 5 Cells are inoculated into the cell culture container using forceps.

[0195] Add 1 mL of liquid culture medium to the cell culture vessel and add the ROCK-specific inhibitor to achieve a final concentration of 10 μM. Incubate in an incubator at 37°C and 5% CO2. Remove 1 mL of liquid culture medium every 24 hours and add 1 mL of fresh liquid culture medium for medium exchange.

[0196] (3-2) Seeding and culture of iPS cells (253G1)

[0197] The cell culture containers obtained in Example 5, Comparative Example 6 and Reference Example A were used, and h-iPS cells 253G1 were used. Otherwise, the cells were seeded and cultured in the same manner as in “(3-1) Seeding and Culture of iPS Cells (201B7)”.

[0198] (3-3) Extensibility of pseudopodia

[0199] Twenty-four hours after cell seeding, cells were observed using a phase-contrast microscope (OLYMPUS IX73, 10×20x). During observation, images representing the most uniform adhesion morphology within the cell culture vessel were acquired. Based on the obtained images, the shape factor (SF) was calculated to evaluate the extensibility of pseudopodia. It should be noted that the shape factor (SF) is the shape evaluation coefficient of a region in a top-view view of the cells after culture, and can be calculated using the following formula.

[0200] Formula: SF = 4 × π × (area of ​​the cell) / (length of the cell's outer perimeter) 2

[0201] Figure 3 Figures (a), (b), and (c) are diagrams illustrating the relationship between SF and the planar shape of the cell. For example... Figure 3 (a) indicates that when SF is 1, the cell's planar shape is circular. The smaller the SF, the further away from a circle it is from a circle, meaning that the cell's pseudopodia are well extended. Figure 3 (b) is a photograph showing the planar shape of a cell when SF≒0.3. Figure 3 (c) is a photograph showing the planar shape of a cell in the case of SF≒1.

[0202] <Criteria for determining the extensibility of pseudopodia>

[0203] ○: SF is 0.1 or higher and 0.6 or lower

[0204] ×: SF is greater than 0.6 and less than 1

[0205] (3-4) Cell proliferation

[0206] Five days after cell seeding, the fixed cell blocks were peeled off using 1.0 mL of TryPLE Express peeling solution, and the cell count was determined using a cell counter (Chemometec NucleoCounter NC-3000). Next, the cell proliferation rate relative to Reference Example A was calculated using the following formula. It should be noted that the cell proliferation rate relative to Reference Example A was compared between experiments using the same cell species (iPS cells (201B7) or iPS cells (253G1)).

[0207] Cell proliferation rate (%) relative to Reference Example A = (Number of cells in Example / Comparative Example) / (Number of cells in Reference Example A) × 100

[0208] <Criteria for determining cell adhesion>

[0209] A: The cell proliferation rate is more than 50% compared to reference example A.

[0210] B: The cell proliferation rate compared to reference example A is 10% or more but less than 50%.

[0211] C: Cell proliferation rate less than 10% compared to reference example A.

[0212] Details and results are shown in Tables 1-3 below.

[0213] [Table 1]

[0214]

[0215] [Table 2]

[0216]

[0217] [Table 3]

[0218]

[0219] The cell culture scaffold materials obtained in the examples exhibit superior cell adhesion compared to those obtained in the comparative examples, as shown in Tables 1-3, as well as superior pseudopodia extensibility and cell proliferation.

[0220] Explanation of symbols

[0221] 1…Cell culture containers

[0222] 2… Container body

[0223] 2a…Surface

[0224] 3… Scaffold materials for cell culture sequence list <110> Sekisui Chemicals Co., Ltd. <120> Scaffold materials and containers for cell culture <130> F2891PCT <150> JP 2019-092083 <151> 2019-05-15 <150> JP 2019-119079 <151> 2019-06-26 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 1 Arg Gly Asp Gly 1 <210> 2 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 2 Arg Gly Asp Ala 1 <210> 3 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 3 Arg Gly Asp Val 1 <210> 4 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 4 Arg Gly Asp Ser 1 <210> 5 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 5 Arg Gly Asp Thr 1 <210> 6 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 6 Arg Gly Asp Phe 1 <210> 7 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 7 Arg Gly Asp Met 1 <210> 8 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 8 Arg Gly Asp Pro 1 <210> 9 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 9 Arg Gly Asp Asn 1 <210> 10 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 10 Tyr Ile Gly Ser Arg 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 11 Pro Asp Ser Gly Arg 1 5 <210> 12 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 12 Arg Glu Asp Val 1 <210> 13 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 13 Ile Asp Ala Pro Ser 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 14 Lys Gln Ala Gly Asp Val 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Cell adhesion peptides <400> 15 Gly Arg Gly Asp Ser 1 5

Claims

1. 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-containing polyvinyl alcohol derivative is a peptide-containing polyvinyl alcohol acetal resin having a polyvinyl alcohol acetal resin portion and the peptide portion. The peptide portion has a cyclic peptide backbone and a cell-adhesive amino acid sequence. The cell culture scaffold material has an island structure. The island portion of the island structure contains the peptide portion. In the peptide-containing polyvinyl alcohol derivative, the content of the peptide is more than 0.01 mol% and less than 25 mol%. The polyvinyl acetal resin portion and the peptide portion are joined by a connector portion, wherein a carboxylic acid having a polymerizable unsaturated group is used as the connector portion. In the peptide-containing polyvinyl alcohol derivative, the content of carboxyl groups from the connector portion is more than 1.0 mol% and less than 2 mol%. Of the 100% by weight of the cell culture scaffold material, the content of the peptide-containing polyvinyl alcohol derivative is 90% or more.

2. The cell culture scaffold material according to claim 1, wherein, The amino acid sequence for cell adhesion has at least the RGD sequence, YIGSR sequence, or PDSGR sequence.

3. The cell culture scaffold material according to claim 1 or 2, wherein, The amino acid sequence for cell adhesion has at least the RGD sequence represented by the following formula (1). Arg-Gly-Asp-X ···Equation (1) In equation (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

4. The cell culture scaffold material according to claim 1 or 2, wherein, The cyclic peptide backbone is composed of 4 to 10 amino acids.

5. The cell culture scaffold material according to claim 1 or 2, wherein, The carboxylic acid with polymerizable unsaturated groups is (meth)acrylic acid.

6. A cell culture container, comprising: Container body, and The cell culture scaffold material according to any one of claims 1 to 5, The cell culture scaffold material is disposed on the surface of the container body.

Images