Peptide, culture container, and method for producing culture container

Abstract

Provided is a peptide comprising an amino acid sequence represented by Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7-Cys-Gly-Gly-Za (SEQ ID NO: 1). In SEQ ID NO: 1, Xa1, Xa2, Xa3, Xa4, Xa5, Xa6, and Xa7 are arbitrary amino acids, and Za is an amino acid sequence including a cell adhesion sequence.

Description

Peptides, culture vessels, and methods for manufacturing culture vessels

[0001] The present invention relates to peptides, culture vessels, and methods for producing culture vessels.

[0002] Conventionally, as a technique for stably culturing cells in an in vitro environment, research has been conducted on coating the surface of artificial substrates such as culture vessels with molecules that have cell adhesion properties when culturing cells on the surface of the artificial substrate. These molecules require high adhesion to the surface of artificial substrates, especially to resins which are often used on the surface of culture vessels. For example, Patent Document 1 discloses an oligopeptide that has adhesion to norbornene-based polymers and contains the amino acid sequence Thr-Val-Asp-Ser-Cys-Leu-Thr.

[0003] International Publication No. 2018 / 117242

[0004] However, the conventional oligopeptides described above require the inclusion of the specific amino acid sequence mentioned above, and there was room for improvement in terms of further enhancing their adhesion to resins. Therefore, the present invention aims to provide a peptide with excellent adhesion to resins.

[0005] The inventors diligently conducted research with the aim of solving the above problems. As a result, the inventors newly discovered that a peptide containing the amino acid sequence Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7-Cys-Gly-Gly-Za has excellent adhesion to resins, and thus completed the present invention.

[0006] In other words, the present invention aims to advantageously solve the above problems, and the present invention is a peptide containing an amino acid sequence, represented as [1]Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7-Cys-Gly-Gly-Za (SEQ ID NO: 1), wherein Xa1, Xa2, Xa3, Xa4, Xa5, Xa6, and Xa7 are arbitrary amino acids, and Za is an amino acid sequence containing a cell adhesion sequence. The peptide containing the above amino acid sequence has excellent adhesion to resins.

[0007] [2] Here, it is preferable that the peptide in [1] above consists of the amino acid sequence Thr-Val-Asp-Xa4-Cys-Leu-Thr (SEQ ID NO: 2) or an amino acid sequence homologous thereto. The peptide having the above amino acid sequence exhibits even better adhesion to the resin.

[0008] [3] In addition, in the peptide of [1] or [2] above, it is preferable that Xa4 is an amino acid selected from the group consisting of Val, Ala, Gly, Thr, Phe, Tyr, Ser, and Leu. When Xa4 is any of the above amino acids, the adhesion of the peptide of the present invention to the resin is further improved.

[0009] [4] In any of the peptides [1] to [3] above, it is preferable that the cell adhesion sequence contained in Za is Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 3) or Ile-Lys-Val-Ala-Val (SEQ ID NO: 4), or an amino acid sequence homologous to the sequence shown in SEQ ID NO: 3 or 4. If the cell adhesion sequence is the above sequence, the adhesion between the peptide of the present invention and cells can be further enhanced.

[0010] [5] Preferably, any of the peptides in [1] to [4] above has adhesive properties to norbornene polymers.

[0011] [6] It is preferable that any of the peptides in [1] to [5] above contain one or more amino acid sequences selected from the following group. Ala-Cys-Thr-Val-Asp-Ser-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 7) Ala-Cys-Thr-Val-Asp-Val-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) Ala-Cys-Thr-Val-Asp-Ala-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 9) Ala-Cys-Thr-Val-Asp-Gly-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 10) Ala-Cys-Thr-Val-Asp-Leu-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 11) Ala-Cys-Thr-Val-Asp-Thr-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 12) Ala-Cys-Thr-Val-Asp-Phe-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 13)Ala-Cys-Thr-Val-Asp-Tyr-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-Tyr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 14) Ala-Cys-Thr-Val-Asp-Ser-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 15) Ala-Cys-Thr-Val-Asp-Val-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 16) Ala-Cys-Thr-Val-Asp-Ala-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 17) Ala-Cys-Thr-Val-Asp-Gly-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 18) Ala-Cys-Thr-Val-Asp-Leu-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 19) Ala-Cys-Thr-Val-Asp-Thr-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 20) Ala-Cys-Thr-Val-Asp-Phe-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 21)Ala-Cys-Thr-Val-Asp-Tyr-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 22)

[0012] [7] The present invention also relates to a culture vessel having a coating layer containing any of the peptides described in [1] to [6] above.

[0013] [8] The culture vessel in [7] above is preferably a sterilized culture vessel.

[0014] [9] Furthermore, the present invention is a method for manufacturing a culture vessel, comprising a coating layer formation step of forming a coating layer on the culture surface of a culture vessel, the coating layer being made of a coating agent containing any of the peptides in [1] to [6] above, and a sterilization step of sterilizing the culture vessel having the coating layer. According to the above manufacturing method, by forming a coating layer on the culture surface of the culture vessel and then performing sterilization, the sterility of the culture vessel can be easily ensured.

[0015] According to the present invention, a peptide with excellent adhesion to resins can be provided.

[0016] Embodiments of the present invention will be described in detail below.

[0017] (Peptide) The peptide of the present invention is characterized by containing an amino acid sequence represented by Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7-Cys-Gly-Gly-Za (Sequence ID 1). Here, Xa1, Xa2, Xa3, Xa4, Xa5, Xa6, and Xa7 are arbitrary amino acids. Za is an amino acid sequence containing an arbitrary cell adhesion sequence. The peptide containing the above amino acid sequence has excellent adhesion to resin. In the sequence listing attached to this specification, the amino acid sequence of Sequence ID 1 is represented as "ACXXXXXXXXXCGGZ" using single-letter amino acid notation. In this sequence, each X corresponds to Xa1, Xa2, Xa3, Xa4, Xa5, Xa6, and Xa7 from left to right. Z corresponds to Za.

[0018] The peptide of the present invention may consist solely of the above amino acid sequence, or it may be a modified peptide containing the above amino acid sequence at its terminal or internally, insofar as the peptide is adhesive to the resin. In this specification, a modified peptide refers to one that can be obtained by adding, inserting, or substituting any peptide for some of the amino acids in the above amino acid sequence. Examples of the above-mentioned optional peptides include extracellular matrix molecules such as laminin, fibronectin, collagen, and vitronectin; cytokines such as interleukins, platelet-derived growth factor, hepatocyte growth factor, nerve growth factor, tumor necrosis factor, epidermal growth factor, fibroblast growth factor, transforming growth factor, and adiponectin; (poly)peptides with physiological activity such as cell membrane receptors such as integrins and PD-1, and cell surface antigens such as CD2 and CD60, as well as antibodies that can recognize hapten antigens and protein antigens; (poly)peptides with genetically useful functions such as histidine tags, thioredoxin tags, glutathione S-transferase tags, maltose-binding protein tags, and fluorescently labeled proteins such as GFP and luciferase; and (poly)peptides consisting of amino acid sequences of unknown function. Furthermore, linker sequences can be inserted to link the amino acid sequence of SEQ ID NO: 1 with the above-mentioned optional peptides or to adjust the peptide length.

[0019] Of the above amino acid sequence (SEQ ID NO: 1), "Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7" is a sequence that has adhesive properties to resin (hereinafter also referred to as the resin adhesion sequence), and "Za" is a sequence that includes a sequence that has adhesive properties to cells (hereinafter also referred to as the cell adhesion sequence). In this specification, "Cys-Gly-Gly" which links the above resin adhesion sequence and the sequence including the cell adhesion sequence is also referred to as the linker sequence. In this specification, the adhesive properties of the peptide of the present invention to resin refer to the combined adhesive properties of the above resin adhesion sequence to resin and the adhesive properties of the above cell adhesion sequence to cells (i.e., the adhesion of cells to resin via the peptide of the present invention).

[0020] <Resin Adhesion Sequence> The resin adhesion sequence (Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7) has adhesive properties to resin. Xa1 to Xa7 are not particularly limited, and any amino acids can be selected, but from the viewpoint of further enhancing the adhesion to resin, it is preferable that Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7 consists of the amino acid sequence Thr-Val-Asp-Xa4-Cys-Leu-Thr (Sequence ID 2) or an amino acid sequence homologous thereto. In the sequence listing attached to this specification, the amino acid sequence of Sequence ID 2 is represented as "TVDXCLT" using single-letter amino acid notation. In this sequence, X corresponds to Xa4 as described in this specification.

[0021] In this specification, an amino acid sequence homologous to the amino acid sequence shown in Sequence ID No. 2 refers to an amino acid sequence in which the sequence of amino acid residues, excluding the fourth amino acid residue from the N-terminus, is preferably identical to that of the amino acid sequence shown in Sequence ID No. 2 by three or more residues, more preferably identical to that of four or more residues, and even more preferably identical to that of five or more residues.

[0022] Furthermore, Xa4 is preferably an amino acid selected from the group consisting of Val, Ala, Gly, Thr, Phe, Tyr, Ser, and Leu, more preferably an amino acid selected from the group consisting of Val, Ala, Gly, Thr, Phe, Tyr, and Leu, and even more preferably an amino acid selected from the group consisting of Val, Ala, Gly, and Leu. If Xa4 is any of the above amino acids, the hydrophobicity of the resin adhesion sequence is increased, thereby further improving the adhesion of the peptide of the present invention to the resin.

[0023] <<Resin>> There are no particular limitations on the type of resin to which a resin-binding sequence can bind, that is, the type of resin to which the peptide of the present invention can bind, and the type can be arbitrarily selected according to the purpose. From the viewpoint of using the peptide of the present invention in a coating agent for a culture vessel described below, it is preferable that the peptide of the present invention has adhesiveness to highly transparent resins that can be used in culture vessels, such as cycloolefin polymers, polyethylene terephthalate (PET), acrylic resins, and polycarbonates.

[0024] Among these, the resin-binding sequence preferably has adhesiveness to cycloolefin polymers. A cycloolefin polymer is a polymer having an alicyclic structure in one or both of the main chain and side chains. Examples thereof include norbornene-based polymers, monocyclic cycloolefin-based polymers, cyclic conjugated diene-based polymers, vinyl alicyclic hydrocarbon-based polymers, and hydrides of these. Among these, norbornene-based polymers are preferable from the viewpoint of transparency and moldability, and norbornene-based polymers having no polar group in the side chain are more preferable.

[0025] Examples of the norbornene-based polymer include ring-opened polymers of monomers having a norbornene structure, ring-opened polymers of a monomer having a norbornene structure and an optional monomer, or hydrides thereof; addition polymers of monomers having a norbornene structure, addition polymers of a monomer having a norbornene structure and an optional monomer, or hydrides thereof; and the like.

[0026] Here, the norbornene-based polymer is a polymer containing monomer units having a norbornene skeleton in an amount of 50% by mass or more, preferably 60% by mass or more, relative to all monomer units constituting the norbornene-based polymer. More specifically, a norbornene-based polymer is obtained by polymerizing a norbornene-based monomer, which is a monomer having a norbornene skeleton, and is broadly classified into those obtained by ring-opening polymerization and those obtained by addition polymerization.

[0027] Examples of those obtained by ring-opening polymerization include ring-opening polymers of norbornene monomers, ring-opening polymers of a norbornene monomer and another monomer capable of ring-opening copolymerization therewith, and hydrides of the foregoing. Examples of those obtained by addition polymerization include addition polymers of norbornene monomers and addition polymers of a norbornene monomer and another monomer capable of copolymerization therewith. Norbornene polymers may be used alone or in combination of two or more types. Among these, hydrides of ring-opening polymers of norbornene monomers are preferred. Examples of norbornene monomers that can be used for synthesizing norbornene polymers include bicyclo[2.2.1]hept-2-ene (common name: norbornene), 5-methyl-bicyclo[2.2.1]hept-2-ene, 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene, 5-ethyl-bicyclo[2.2.1]hept-2-ene, 5-ethylidene-bicyclo[2.2.1]hept-2-ene, 5-vinyl-bicyclo[2.2.1]hept-2-ene, 5-propenylbicyclo[2.2.1]hept-2-ene, 5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene, 5-cyanobicyclo[2.2.1]hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene and other bicyclic monomers; tricyclo[4.3.0 １，６ .1 ２，５ deca-3,8-diene (common name: dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene and other tricyclic monomers; tetracyclo[4.4.0.1 ２，５ .1 ７，１０ -3-dodecene (tetracyclododecene), tetracyclo[4.4.0.1 ２，５ .1 ７，１０ -3-dodecene, 8-methyltetracyclo[4.4.0.1 ２，５ .1 ７，１０ -3-dodecene, 8-ethyltetracyclo[4.4.0.1 ２，５ .1 ７，１０ -3-dodecene, 8-ethylidenetetracyclo[4.4.0.1 ２，５ .1 ７，１０-3-dodecene, 8,9-dimethyltetracyclo[4.4.0.1 ２，５ .1 ７，１０ -3-dodecene, 8-ethyl-9-methyltetracyclo[4.4.0.1 ２，５ .1 ７，１０ -3-dodecene, 8-ethylidene-9-methyltetracyclo[4.4.0.1 ２，５ .1 ７，１０ -3-dodecene, 8-methyl-8-carboxymethyltetracyclo[4.4.0.1 ２，５ .1 ７，１０ -3-dodecene, 7,8-benzotricyclo[4.3.0.1 ２，５ deca-3-ene (common name: methanotetrahydrofluorene; also referred to as 1,4-methano-1,4,4a,9a-tetrahydrofluorene), 1,4-methano-8-methyl-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-bromo-1,4,4a,9a-tetrahydrofluorene and other tetracyclic monomers; etc. These norbornene-based monomers may have one or two or more types of substituents. Examples of the substituents include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, an alkylidene group and the like.

[0028] Examples of other monomers capable of undergoing ring-opening copolymerization with norbornene-based monomers include monocyclic cycloolefin monomers such as cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, 1,5,9-cyclododecatriene, and 1,5,9,13-cyclohexadecatetraene.

[0029] Examples of other monomers capable of undergoing addition copolymerization with norbornene-based monomers include α-olefin monomers having 2 to 20 carbon atoms such as ethylene, propylene, 1-butene, 1-pentene, and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetracyclo[9.2.1.0 ２，１０ .0 ３，８Examples include cycloolefin monomers such as tetradeca-3,5,7,12-tetraene (also known as 3a,5,6,7a-tetrahydro-4,7-methano-1H-indene); and non-conjugated diene monomers such as 1,4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene. Among these, α-olefin monomers are preferred as other monomers that can be addition copolymerized with norbornene monomers, and ethylene is more preferred. These other monomers may have one or more substituents. Examples of substituents include alkyl groups, alkylene groups, aryl groups, silyl groups, alkoxycarbonyl groups, alkylidene groups, etc.

[0030] Ring-opening polymers of norbornene monomers, or ring-opening polymers of norbornene monomers and other monomers copolymerizable thereto, can be obtained by polymerizing the monomer components in the presence of a known ring-opening polymerization catalyst. As the ring-opening polymerization catalyst, for example, a catalyst consisting of a metal halide such as ruthenium or osmium, a nitrate or acetylacetone compound, and a reducing agent, or a catalyst consisting of a metal halide such as titanium, zirconium, tungsten, or molybdenum or an acetylacetone compound and an organoaluminum compound can be used. Hydrogenated ring-opening polymers of norbornene monomers can usually be obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to the polymerization solution of the above-mentioned ring-opening polymer and hydrogenating the carbon-carbon unsaturated bond.

[0031] Addition polymers of norbornene monomers, or addition polymers of norbornene monomers with other monomers copolymerizable thereto, can be obtained by polymerizing the monomer components in the presence of a known addition polymerization catalyst. As the addition polymerization catalyst, for example, a catalyst consisting of a titanium, zirconium, or vanadium compound and an organoaluminum compound can be used.

[0032] There are no particular restrictions on the molecular weight of norbornene polymers, but the weight-average molecular weight in terms of polyisoprene, measured by gel permeation chromatography (GPC) in a cyclohexane solution (or toluene solution if the polymer does not dissolve), is usually 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and particularly preferably 10,000 to 100,000. When the weight-average molecular weight is within this range, a high balance between mechanical strength and moldability is achieved, making it preferable.

[0033] The glass transition temperature of norbornene-based polymers can be appropriately selected depending on the intended use, but is typically 50 to 300°C, preferably 100 to 280°C. A glass transition temperature within this range provides a high balance between heat resistance and moldability, making it ideal. The glass transition temperatures of the norbornene-based polymers mentioned above were measured according to JIS K 7121.

[0034] <Sequence containing cell adhesion sequence> The cell adhesion sequence contained in Za has adhesive properties to cells. The cell adhesion sequence contained in Za can be any sequence without particular limitation, as long as it is an amino acid sequence that has adhesive properties to cells. In particular, it is preferable that the cell adhesion sequence is Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 3) or Ile-Lys-Val-Ala-Val (SEQ ID NO: 4), or an amino acid sequence homologous to the sequence shown in SEQ ID NO: 3 or 4. If the cell adhesion sequence is one of the above sequences, the adhesion between the peptide of the present invention and cells can be further enhanced. In this specification, an amino acid sequence homologous to the sequence shown in SEQ ID NO: 3 or 4 refers to an amino acid sequence that is preferably identical to the sequence shown in SEQ ID NO: 3 or 4 by three or more residues, and more preferably identical to the sequence shown in SEQ ID NO: 3 or 4 by four or more residues.

[0035] The number of amino acid residues contained in Za is not particularly limited, but it is preferably 5 or more, more preferably 7 or more, even more preferably 10 or more, preferably 40 or less, more preferably 30 or less, and even more preferably 20 or less. If the number of amino acid residues contained in Za is above the lower limit, there is a sufficiently large amount of positive charge contained in Za, which can improve the adhesion between the peptide and the cell. If the number of amino acid residues contained in Za is below the upper limit, the higher-order structure of the peptide of the present invention is sufficiently maintained, and the positive charge derived from Za is located on the surface of the peptide structure, which can improve the adhesion between the peptide and the cell. Furthermore, if the number of amino acid residues contained in Za is below the upper limit, the cost of synthesizing the peptide of the present invention can be reduced, making it economical.

[0036] <<Cells>> The types of cells to which the cell adhesion array can adhere, that is, the types of cells to which the peptide of the present invention can adhere, include, for example, cancer cells (A549, MCF-7, PANC-1, HeLa, HepG2, U-2OS, SH-SY5Y, etc.), established cell lines (MRC5 cells, HEK293 cells, HEK293T cells, C2C12 cells, V79 cells, Vero cells, CHO cells, NIH / 3T3 cells, etc.), iPS-derived cells (e.g., iPS cells, iPS-derived cardiomyocytes, etc.), stem cells (e.g., mesenchymal stem cells), adipocytes, osteoblasts, skeletal muscle cells, etc., but are not particularly limited and can be arbitrarily selected depending on the purpose. Among these, cancer cells, established cell lines, iPS-derived cardiomyocytes, and mesenchymal stem cells are preferred, and iPS-derived cardiomyocytes and mesenchymal stem cells are even more preferred.

[0037] (Culture Vessel) The culture vessel of the present invention is characterized by having a coating layer containing the peptide of the present invention. Preferably, the culture vessel of the present invention has the above-mentioned coating layer on the culture surface (for example, if the culture vessel is a well plate, the bottom surface inside each well). The coating layer may cover the entire bottom surface, or it may exist as a pattern such as dots or lines.

[0038] As long as the culture vessel of the present invention is made of resin, any shape can be used. Examples of culture vessel shapes include dishes, plates, microfluidic tips, bags, tubes, scaffolds, cups, jars, and fermenters.

[0039] The culture vessel only needs to have a culture surface made of the resin described above in the "Resin" section of the "Peptide" section. Preferably, the culture surface of the culture vessel is made of a cycloolefin polymer, and more preferably, from the viewpoint of transparency, moldability, and adhesion to the peptide of the present invention, the culture surface is made of a norbornene polymer, and even more preferably, the culture surface is made of a norbornene polymer that does not have polar groups in its side chains. For example, if the culture vessel is a 96-well plate, it is preferable that the inner bottom surface of each well is made of the above polymer. If the culture vessel is a bag, for example, if it is made of a laminate of films made of different polymer materials, it is preferable that the innermost layer (inner surface of the bag) is made of the above polymer. Alternatively, the entire culture vessel may be made of the above polymer. For example, if it is a culture dish, flask, or plate with multiple wells, the entire container can be made of the above polymer by molding the entire container with the above polymer.

[0040] The following describes the case where the culture surface of the culture vessel is composed of norbornene polymers. Norbornene polymers can be used individually or in combination of two or more types. In addition to norbornene polymers, as components constituting the culture surface of the culture vessel, compounding agents commonly used in thermoplastic resin materials, such as soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near-infrared absorbers, mold release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, and fluorescent whitening agents, can be added in amounts commonly used. When using a mixture of norbornene polymers and soft polymers, the amount of soft polymer is usually 0.01 to 20 parts by mass, preferably 0.05 to 10 parts by mass, and more preferably 0.05 to 5 parts by mass, per 100 parts by mass of norbornene polymer.

[0041] Furthermore, in addition to norbornene polymers and the soft polymer which is one of the compounding agents mentioned above, other polymers (hereinafter simply referred to as "other polymers") may be mixed as components constituting the culture surface. The amount of other polymers mixed with norbornene polymer is usually 200 parts by mass or less, preferably 150 parts by mass or less, and more preferably 100 parts by mass or less, per 100 parts by mass of norbornene polymer. If the proportion of various compounding agents and other polymers added to the norbornene polymer is too high, the cells will have difficulty floating, so it is preferable to add them in a range that does not impair the properties of the norbornene polymer.

[0042] The method of mixing norbornene polymers with compounding agents or other polymers is not particularly limited, as long as the compounding agent is sufficiently dispersed within the polymer. Furthermore, there are no particular restrictions on the order of compounding. Examples of compounding methods include: mixing the resin in a molten state using a mixer, single-screw kneader, twin-screw kneader, rolls, brabender, extruder, etc.; dissolving and dispersing in a suitable solvent, followed by removal of the solvent by solidification, casting, or direct drying; and so on. When using a twin-screw kneader, after mixing, the mixture is usually extruded into a rod shape in a molten state, cut to an appropriate length with a strand cutter, and then pelletized for use.

[0043] The method for molding the culture vessel can be arbitrarily selected according to the desired shape of the culture vessel. Examples of molding methods include injection molding, extrusion molding, cast molding, inflation molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, calendering, rolling, cutting, and spinning. These molding methods can be combined, and post-processing such as stretching can be performed as needed after molding.

[0044] <Coating Layer> The coating layer on the culture surface of the culture vessel contains the peptide of the present invention. The coating layer can be formed by bringing a coating agent containing the peptide of the present invention into contact with the culture surface of the culture vessel.

[0045] The specific method for forming the coating layer is not particularly limited, but may be similar to the method for coating a culture vessel with a general cell substrate. Typically, the above-mentioned coating agent is placed in the culture vessel, left to stand for 10 minutes to 5 hours, preferably 30 minutes to 2 hours, to allow the coating agent to come into contact with the culture surface, and then the coating agent is removed. If the contact time is above the lower limit, the peptide contained in the coating agent can be sufficiently adhered to the culture surface, and sufficient coating can be achieved. The temperature at which the coating agent comes into contact is preferably near the culture temperature, specifically, usually 15°C to 65°C, preferably 20°C to 60°C. If the temperature is above the lower limit, sufficient adhesion of the peptide of the present invention to the resin constituting the culture surface can be obtained. Also, if the temperature is below the upper limit, the progress of evaporation of the coating agent can be suppressed. After removing the coating agent, the culture vessel can be dried by air drying or the like to form the coating layer on the culture surface. Therefore, the formed coating layer is a dried coating agent and may contain the peptide of the present invention, and optionally pH adjusters, osmotic pressure adjusters, and other additives described later.

[0046] In culture vessels where the culture surface is made of cycloolefin polymer, the cycloolefin polymer tends to repel aqueous solutions, but adsorption occurs rapidly. Therefore, the amount of coating agent added to the culture vessel can be the same as the amount added to a typical polystyrene cell culture vessel. Specifically, the culture surface 1 cm ２ It is preferable to add 0.10 mL to 0.50 mL of the solution.

[0047] <<Coating Agent>> The coating agent is not particularly limited as long as it contains the peptide of the present invention, and may be a solution containing the peptide of the present invention and a solvent. The solvent can be any solvent that can dissolve the peptide, such as water, organic solvents, buffer solutions, etc. Examples of organic solvents include acetonitrile, ethanol, isopropanol, N-methyl-2-pyrrolidone (NMP), and dimethyl sulfoxide (DMSO).

[0048] From the viewpoint of culturing cells on a coating layer, it is preferable that the coating agent has pH buffering properties, an osmotic pressure suitable for cells, and low toxicity to cells. Examples of components that impart pH buffering properties to the coating agent include Tris hydrochloride, various phosphates, and various carbonates. From the viewpoint of making the osmotic pressure of the coating agent suitable for cells, it is preferable that the coating agent is a solution containing potassium ions, sodium ions, calcium ions, glucose, etc. The osmotic pressure of the coating agent is usually adjusted to be approximately the same as the osmotic pressure of cells. Specifically, it is preferable to adjust the osmotic pressure of the coating agent by adding, for example, physiological salines such as phosphate-buffered saline, Tris-buffered saline, and HEPES-buffered saline; Ringer's solutions such as lactated Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution.

[0049] Additives can also be added to the coating agent. Examples of additives include nucleic acids; amino acids; peptides; minerals; metals; vitamin components; ligands, agonists, and antagonists that act on cell surface receptors; ligands, agonists, and antagonists for nuclear receptors; extracellular matrix such as collagen and Fibunectin; a portion of the extracellular matrix or compounds that mimic the extracellular matrix; components that act on proteins involved in intracellular signaling pathways; components that act on enzymes involved in primary or secondary metabolism within cells; and components that affect the expression of genes in the nucleus or mitochondria within cells. These additives can be used individually or in combination of two or more.

[0050] The method for preparing the coating agent is not particularly limited. For example, the peptide of the present invention can be dissolved in a solvent such as water, an organic solvent, or a buffer solution as described above, and then optionally a solution containing the above-mentioned pH buffering component, osmotic pressure regulating component, and additives, or some or all of these components, can be added to prepare the coating agent.

[0051] The culture vessel of the present invention is preferably sterilized. In this specification, "sterilized culture vessel" means a vessel with a Sterility Assurance Level (SAL) of 10. －６ The following refers to the culture vessel. SAL is an index that indicates the sterilization standard defined in ISO, and it shows the probability that a product will contain viable bacteria after sterilization. The SAL of the culture vessel is 10 -７ Preferably, the following: 10 -８ The following is more preferable:

[0052] There are no particular limitations on the sterilization method for the culture vessel of the present invention. A suitable method can be selected from among those commonly used in the medical field, depending on the cells used, such as heating methods including high-pressure steam or dry heat; radiation methods including irradiation with gamma rays or electron beams or high-frequency irradiation; gas methods including contact with gas such as ethylene oxide gas (EOG); and filtration methods using sterilization filters. Among these, ethylene oxide gas (EOG) sterilization is particularly preferred because it results in less deterioration of the coating layer due to sterilization, excellent adhesion of cells to the culture vessel after sterilization, and less discoloration of norbornene polymers.

[0053] The type of cells cultured using the culture vessel of the present invention is not particularly limited, but for example, the cells described above can be cultured in the "Cells" section of the "Peptides" section. The cell culture conditions are not particularly limited and can be appropriately determined depending on the cells used and the purpose. For example, cells can be cultured using a humidified incubator with a carbon dioxide concentration of about 5% and a temperature maintained at a constant range of 20°C to 37°C.

[0054] The culture medium for culturing the above-mentioned cells is not particularly limited as long as it can culture and maintain the cells; commercially available cell culture media can be used. Additives may also be added to the culture medium. Examples of additives include minerals, metals, and vitamins. These additives can be used individually or in combination of two or more.

[0055] There are no particular restrictions on the method of seeding the above cells into the culture vessel. After forming a coating layer on the culture surface of the culture vessel using the method described above, seed the cells suspended in the culture medium into the culture vessel using a pipette or the like, shake the vessel as needed to evenly distribute the cells within the culture vessel, and then leave it to stand in the incubator.

[0056] (Method for manufacturing a culture vessel) The method for manufacturing a culture vessel of the present invention is characterized by comprising a coating layer formation step of forming a coating layer made of a coating agent containing the peptide of the present invention on the culture surface of a culture vessel, and a sterilization step of sterilizing the culture vessel having the coating layer.

[0057] <Coating Layer Formation Process> The coating layer formation process is not particularly limited, as long as a coating layer can be formed on the culture surface of the culture vessel. Specifically, for example, a coating agent containing the peptide of the present invention can be prepared by the method and conditions described above in the "Culture Vessel" section, the coating agent can be brought into contact with the culture surface of the culture vessel and then removed, and the culture vessel can be dried to form a coating layer.

[0058] <Sterilization Process> After the coating layer formation process, a sterilization process is performed to sterilize the culture vessel having the coating layer. As for the sterilization method, the method described above in the "Culture Vessel" section can be appropriately selected. Among these, ethylene oxide gas (EOG) sterilization is particularly preferred because it causes less deterioration of the coating layer due to sterilization, has excellent adhesion of cells to the culture vessel after sterilization, and does not easily cause discoloration of norbornene polymers. The specific conditions of the sterilization process can be appropriately set according to the purpose, but the SAL of the culture vessel after sterilization should be 10 －６ The following conditions shall be met: The SAL of the culture vessel after sterilization shall be 10 -７ Preferably, the following: 10 -８ The following is more preferable.

[0059] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. In the following description, "%" representing quantities refers to mass unless otherwise specified. In the examples and comparative examples, various operations and evaluations were performed in the following manner under the conditions shown in Tables 1 and 2.

[0060] <Preparation of 96-well plates with a coating layer> The following norbornene-based ring-opening polymer hydrides were molded to produce 96-well plates. COP1: ZEONEX® 690R manufactured by Nippon Zeon Co., Ltd., Tg: 136°C COP2: ZEONEX® 790R manufactured by Nippon Zeon Co., Ltd., Tg: 163°C COP3: ZEONOR® 1060R manufactured by Nippon Zeon Co., Ltd., Tg: 100°C COC: TOPAS® 6013M07 manufactured by Polyplastics Co., Ltd., Tg: 142°C Synthesized peptides, each consisting of the amino acid sequence described later, were dissolved in DMSO (Biotechnology Grade Preparation Solution #09659-85 manufactured by Nacalai Tesque Co., Ltd.) in the above 96-well plates, and then diluted with D-PBS(-) to prepare 10 μg / mL solutions (for evaluation 1) and 0.625 μg / mL solutions (for evaluation 2), respectively. These solutions were dispensed at 100 μL / well and refrigerated at 37°C and 5% CO2. ２ The samples were left to stand in an incubator for 30 minutes. However, for Comparative Example 1, a sequence lacking Za from the amino acid sequence described later was used instead of the synthetic peptide consisting of the amino acid sequence described later. For Comparative Examples 2 and 3, sequences consisting only of Za, described later, and not including the sequence that exhibits adhesion to the resin, were used instead of the synthetic peptide consisting of the amino acid sequence described later. After that, the liquid was aspirated to remove the peptide coating agent, and 200 μL / well of sterile water was added to remove the liquid in the same manner. After air-drying in a safety cabinet, the samples were sterilized and used in the experiment.

[0061] [Amino Acid Sequence] Ala-Cys-Thr-Val-Asp-Xa4-Cys-Leu-Thr-Cys-Gly-Gly-Za Xa4 in each example is as shown in Tables 1 and 2. For the cell adhesion sequence Za, the sequence shown in Sequence ID No. 5 below was used for Examples 1-12 and Comparative Example 2, and the sequence shown in Sequence ID No. 6 below was used for Examples 13-24 and Comparative Example 3. Gly-Gly-Gly-Tyr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (Sequence ID No. 5) Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (Sequence ID No. 6)

[0062] <Sterilization> The above sterilization method involved ethylene oxide gas (EOG) sterilization under the following conditions: Temperature: 45°C, Humidity: 50%, Pressure: -0.053 MPa to 0.049 MPa, Time: 360 minutes

[0063] <Culture of Cardiomyocytes> iPS cell-derived cardiomyocytes (iCell® Cardiomyocytes (#C1016, FUJIFILM Cellular Dynamics) (hereinafter referred to as cardiomyocytes)) were thawed in a 37°C incubator for 3 minutes, and then seeded into the above 96-well plates at a density of 75,000 cells / well using iCell Cardiomyocytes Plating Medium (#M1001, FUJIFILM Cellular Dynamics). (Cells for Evaluation 1 were seeded on 96-well plates coated with a 10 μg / mL coating agent, and cells for Evaluation 2 were seeded on 96-well plates coated with a 0.625 μg / mL coating agent.) Subsequently, cultured at 37°C and 5% CO2. ２ The cells were left to stand in an incubator for 4 hours. After 4 hours, the entire culture medium was replaced with iCell Cardiomytocytes Maintenance Medium (#M1003, FUJIFILM Cellular Dynamics). Half of the culture medium was replaced every other day at 37°C and 5% CO2. ２ The cells were cultured in an incubator for three days.

[0064] <Evaluation of cell coverage (Evaluation 1)> Cells cultured on a 96-well plate coated with a 10 μg / mL coating agent were subjected to the addition of Cal-520 (registered trademark) AM (#21130, AAT Bioquest) to each well on day 3 of culture, resulting in a final concentration of 1 μg / mL. The cells were then cultured at 37°C and 5% CO2. ２ Cardiomyocytes were stained after being allowed to stand in the environment for 60 minutes. The stained cells were observed using a confocal quantitative image cytometer CellVoyager CQ1 (Yokogawa Electric Corporation) with an excitation wavelength of 488 nm, emission filter BP525 / 50, and objective lens 4X, and images were captured. The captured images were analyzed using the image analysis software ImageJ, and the coverage rate, which is the percentage of cells in the bottom area of ​​the well, was calculated. The calculated cell coverage rate was evaluated according to the following criteria. The evaluation results are shown in Tables 1 and 2. Cell coverage rate of 80% or more: A Cell coverage rate of 60% or more but less than 80%: B Cell coverage rate less than 60%: C

[0065] <Evaluation of cell detachment at low coating concentration (0.625 μg / mL) (Evaluation 2)> Cells cultured on a 96-well plate coated with a 0.625 μg / mL coating agent were cultured, and on day 3 of culture, Cal-520 (registered trademark) AM (#21130, AAT Bioquest) was added to each well to a final concentration of 1 μg / mL, and the cells were cultured at 37°C and 5% CO2. ２ The cardiomyocytes were allowed to stand in the environment for 60 minutes and then stained. The stained cells were observed using a confocal quantitative image cytometer CellVoyager CQ1 (Yokogawa Electric Corporation) with an excitation wavelength of 488 nm, emission filter BP525 / 50, and objective lens 4X to check for the presence or absence of cardiomyocyte detachment from the well edges. Tables 1 and 2 show the results, with "Present" indicating detachment and "Absent" indicating no detachment.

[0066] <Evaluation of cell coverage of non-cardiac cells (Evaluation 3)> Under the conditions described below, various cells were cultured on a 96-well plate coated with a 10 μg / mL coating agent, and calcein AM (#19177-14, Nacalai Tesque) was added to each well to a concentration of 2.38 μM. At 37°C and 5% CO2, the cells were cultured. ２Adherent live cells were stained after standing for 5 minutes in the environment. [Cell type and culture conditions] Cells: C2C12 (myoblast cell line derived from mouse striated muscle) Catalog number: #91031101, ECACC (European Collection of Authenticated Cell Cultures) Culture medium: DMEM (#08458-45, Nacalai Tesque) + 10% FBS + 1% P / S (FBS (fetal bovine serum) #172012-500 mL (Thermo Fisher Scientific), P / S (Penicilin-streptomycin) #26253-84 (Nacalai Tesque Co., Ltd.) was used) Culture environment: 37°C, 5% CO ２ Seeding conditions: 10,000 cells / well, culture days: 3 days, Cells: PANC-1 (human pancreatic cancer cells), Catalog number: #87092802, ECACC (European Collection of Authenticated Cell Cultures), Medium: DMEM (#08458-45, Nacalai Tesque) + 10% FBS + 1% P / S (FBS (fetal bovine serum) #172012-500mL (Thermo Fisher Scientific), P / S (Penicilin-streptomycin) #26253-84 (Nacalai Tesque Co., Ltd.) used), Culture environment: 37°C, 5% CO2 ２ Seeding conditions: 10,000 cells / well Culture days: 7th day ・Cells: hMSC-AT (human mesenchymal stem cells) Product number: #C-12977, manufactured by Promocell Medium: Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use) (#C-28009, manufactured by Promocell) Culture environment: 37°C, 5% CO ２ Seeding conditions: 10,000 cells / well, culture duration: 7 days

[0067] For the various cell types described above, stained cells were observed using a confocal quantitative image cytometer CellVoyager CQ1 (manufactured by Yokogawa Electric Corporation) with an excitation wavelength of 488 nm, an emission filter EM B525 / 50, and a 4X objective lens. The images were then captured using the image analysis software ImageJ, and the cell coverage rate, which is the percentage of cells in the bottom area of ​​the well, was calculated. The calculated cell coverage rate was evaluated according to the following criteria. The evaluation results are shown in Tables 1 and 2. In all examples where evaluation 3 was performed (Examples 2-3, 10-12, 14-15, and 22-24), an evaluation of A was obtained for each of the various cell types described above. Cell coverage rate of 80% or more: A Cell coverage rate of 60% or more and less than 80%: B Cell coverage rate of less than 60%: C

[0068]

[0069]

[0070] A comparison of Examples 1-24 and Comparative Examples 1-3 shows that the examples prepared using a peptide containing the amino acid sequence represented by Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7-Cys-Gly-Gly-Za (SEQ ID NO: 1) yielded a coating layer with excellent cell coverage evaluation (Evaluation 1) and superior adhesion to the resin. Furthermore, a comparison of Examples 1 and 2-8, Examples 9 and 10, Examples 13 and 14-20, and Examples 21 and 22 shows that when Xa4 in the peptide's resin adhesion sequence (Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7) is an amino acid selected from the group consisting of Val, Ala, Gly, Thr, Phe, Tyr, and Leu, cell detachment is reduced. Therefore, it is clear that if Xa4 is an amino acid selected from the above, the adhesion of the peptide to the resin is further improved. Furthermore, from Examples 2-3, 10-12, 14-15, and 22-24, it can be seen that when the peptide of the present invention is used, the evaluation of cell coverage is excellent even when cells other than cardiomyocytes are cultured.

[0071] According to the present invention, a peptide with excellent adhesion to resins can be provided.

Claims

1. A peptide containing an amino acid sequence, represented as Ala-Cys-Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7-Cys-Gly-Gly-Za (SEQ ID NO: 1), where Xa1, Xa2, Xa3, Xa4, Xa5, Xa6, and Xa7 are arbitrary amino acids, and Za is an amino acid sequence containing a cell adhesion sequence.

2. The peptide according to claim 1, wherein Xa1-Xa2-Xa3-Xa4-Xa5-Xa6-Xa7 consists of the amino acid sequence Thr-Val-Asp-Xa4-Cys-Leu-Thr (SEQ ID NO: 2) or an amino acid sequence homologous thereto.

3. The peptide according to claim 1, wherein Xa4 is an amino acid selected from the group consisting of Val, Ala, Gly, Thr, Phe, Tyr, Ser, and Leu.

4. The peptide according to claim 1, wherein the cell adhesion sequence contained in Za is Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 3) or Ile-Lys-Val-Ala-Val (SEQ ID NO: 4), or an amino acid sequence homologous to the sequence shown in SEQ ID NO: 3 or 4.

5. The peptide according to claim 1, which has adhesive properties to norbornene polymers.

6. The peptide according to claim 1, comprising one or more amino acid sequences selected from the following group. Ala-Cys-Thr-Val-Asp-Ser-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 7) Ala-Cys-Thr-Val-Asp-Val-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) Ala-Cys-Thr-Val-Asp-Ala-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 9) Ala-Cys-Thr-Val-Asp-Gly-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 10) Ala-Cys-Thr-Val-Asp-Leu-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 11) Ala-Cys-Thr-Val-Asp-Thr-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 12) Ala-Cys-Thr-Val-Asp-Phe-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-T yr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 13)Ala-Cys-Thr-Val-Asp-Tyr-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Gly-Gly-Tyr-Ile-Gly-Ser-Arg-Cys-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 14) Ala-Cys-Thr-Val-Asp-Ser-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 15) Ala-Cys-Thr-Val-Asp-Val-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 16) Ala-Cys-Thr-Val-Asp-Ala-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 17) Ala-Cys-Thr-Val-Asp-Gly-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 18) Ala-Cys-Thr-Val-Asp-Leu-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 19) Ala-Cys-Thr-Val-Asp-Thr-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 20) Ala-Cys-Thr-Val-Asp-Phe-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Ala-Val-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 21)Alpha-Cys-Thr-Val-Asp-Tyr-Cys-Leu-Thr-Cys-Gly-Gly-Gly-Ser-Ile-Lys-Val-Al-Val-Trp-Ser-His-Pro-Gln-Phe-Gly-Lys (sequence number 22) 7. A culture vessel having a coating layer containing the peptide described in any one of claims 1 to 6.

8. A sterilized culture vessel according to claim 7.

9. A method for manufacturing a culture vessel, comprising: a coating layer formation step of forming a coating layer made of a coating agent containing the peptide described in claim 1 on the culture surface of a culture vessel; and a sterilization step of sterilizing the culture vessel having the coating layer.

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