Composite comprising substrate with immobilized porphyrin-containing polyamine, cell culture substrate containing the composite, methods for their production, method for cell stimulation, and method for producing antigen-presenting cells

Abstract

To provide a composite that enables cell culture, a cell culture substrate capable of applying oxidative stress injury to cells through light irradiation during cell culture, methods for their production, and a method for cell stimulation.SOLUTION: A composite comprises a substrate and a copolymer immobilized thereto, the copolymer comprising a repeating unit represented by formula (I), a repeating unit represented by formula (II) and a repeating unit represented by formula (III).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a complex comprising a substrate having a porphyrin-containing polyamine immobilized thereon, a cell culture substrate comprising the complex, a method for producing the same, a method for stimulating cells, and a method for producing antigen-presenting cells. [Background technology]

[0002] Living organisms take in oxygen through breathing and produce energy. Some of this oxygen becomes highly reactive reactive oxygen species, such as superoxide radicals, hydroxyl radicals, hydrogen peroxide, and alkoxy radicals, within the organism. Normally, the active oxygen is quickly eliminated in the organism because of antioxidants such as vitamin E and antioxidant enzymes such as superoxide peroxidase and catalase. However, excessive active oxygen cannot be eliminated by antioxidants and diffuses into the cells, oxidizing and damaging various cellular components such as nucleic acids, proteins, and lipids in the cells. This is called oxidative stress. Oxidative stress is known to be involved in various diseases such as aging, infectious diseases, cancer, myocardial infarction, arteriosclerosis, renal dysfunction, dementia, Parkinson's disease, autism, and depression.

[0003] In recent years, it has been reported that among these reactive oxygen species, lipid radicals and their metabolites act as causative molecules for various diseases and inflammation. For example, it has been reported that intracellular lipid radicals and oxidized lipids are involved in iron-dependent cell death (phoroptosis), and research on lipid radicals and oxidized lipids has been actively conducted. Therefore, there is a need for a method to selectively generate intracellular lipid radicals.

[0004] A conventional research method for oxidative stress is to add hydrogen peroxide to a cell culture solution to induce intracellular oxidative stress (see, for example, Non-Patent Document 1). However, hydrogen peroxide has cell membrane permeability and oxidizes various components in cells, so it cannot selectively generate lipid radicals in cells. On the other hand, a research method for lipid radicals is to add oxidation reaction initiators such as 2,2'-azobis(2-amidinopropane)dihydrochloride and 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) to a cell culture solution (see, for example, Non-Patent Document 2). However, when these oxidants are added to a cell culture solution, they diffuse into the cell and not only generate lipid radicals, but also generate various reactive oxygen species in the cytoplasm and nucleus. Therefore, there has been no method to generate only lipid radicals in cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kazuhiko Igarashi and Yoshihiro Doi, “Oxidative Stress Responsiveness in Cellular Senescence: Directed by Bach1,” Seikagaku Vol. 81 No. 6 (2009): 502-510 [Non-Patent Document 2] Yuta Matsuoka, "Development and application of fluorescent probes for highly sensitive and selective detection of redox substances in vivo", YAKUGAKU ZASSHI 141 (2021):1297-1304 Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above, one of the objects of the present invention is to provide a complex that enables cell culture (e.g., a glass substrate on which a specific copolymer is immobilized), and a cell culture substrate (e.g., a cell culture dish) that can cause oxidative stress damage to cells while culturing them by light irradiation, a method for producing these, and a method for stimulating cells. Another object of the present invention is to provide a complex, a cell culture substrate, a method for producing these, and a method for stimulating cells that selectively induce lipid radical production in cells and do not produce excessive reactive oxygen species in the cytoplasm. Another object of the present invention is to provide a method for producing antigen-presenting cells capable of producing anti-inflammatory cytokines. [Means for solving the problem]

[0007] The present inventors have discovered that a substrate (e.g., a glass substrate) on which a copolymer including a structural unit having a polyamine having a primary amino group and a structural unit having a porphyrin derivative or a salt thereof is immobilized allows cells to be electrostatically attached stably to the substrate surface and cultured, and that irradiation with light capable of exciting porphyrin selectively induces the production of lipid radicals without producing excessive reactive oxygen species in the cytoplasm, thereby completing the present invention.

[0008] The composite of the present invention comprises a substrate and a copolymer immobilized on the substrate, the copolymer comprising a repeating unit represented by the following formula (I), a repeating unit represented by the following formula (II), and a repeating unit represented by the following formula (III), thereby solving the above-mentioned problems. [ka] (In formula (I), (II) or (III), R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L 1 is O or NH, L 2 is a divalent group, X is a halogen atom, and Y represents a bonding mode for bonding to the substrate via a silane coupling agent, -C(=O)-NH-R 2-*, -C(=S)-NH-R 2 -*, -C(=O)-R 2 -* and -C(-OH)-R 2 -*, where * represents a bonding position with the substrate, and R 2 is selected from the group consisting of a linear or branched alkyl group having 1 to 5 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, and an aralkyl group having 7 to 10 carbon atoms, and Z is a group obtained by removing one arbitrary hydrogen atom from a porphyrin derivative or a salt thereof. Said L 2 is a divalent linear aliphatic hydrocarbon group having 1 to 5 carbon atoms, a divalent branched aliphatic hydrocarbon group having 3 to 5 carbon atoms, a divalent cyclic aliphatic hydrocarbon group having 5 to 10 carbon atoms, a divalent aromatic hydrocarbon group having 5 to 10 carbon atoms, -O-, -S-, -N(R 3 )-(wherein, R 3 represents a hydrogen atom, or a straight or branched alkyl group having 1 to 12 carbon atoms), -C(=O)-, -C(=O)-O-, -OC(=O)-O-, -C(=O)-NH-, -OC(=O)-NH-, -S(=O)-, -S(=O)-O-, -S(=O)2-, -S(=O)2-O-, -CH=N-, and a combination thereof. The ratio of the number of repeating units represented by formula (II) to the total number of repeating units in the copolymer may be 1 to 50%, and the ratio of the number of repeating units represented by formula (III) may be 1 to 50%. The porphyrin derivative may be selected from the group consisting of mesoporphyrin, deuteroporphyrin, hematoporphyrin, protoporphyrin, uroporphyrin, coproporphyrin, cytoporphyrin, rhodoporphyrin, pyroporphyrin, phyloporphyrin, heptacarboxyporphyrin, hexacarboxyporphyrin, pentacarboxyporphyrin, and salts of these porphyrins. R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms; L 2 may represent a linear or branched alkylene group having 1 to 5 carbon atoms. R 1represents -CH3, L 1 represents -NH-, L 2 represents -CH2-CH2-CH2-, and Z may be hematoporphyrin dihydrochloride. The molecular weight (Mw) of the copolymer may be 300 to 10,000,000. The substrate may be a glass substrate. The glass substrate may have a thickness of 0.04 mm to 0.60 mm and a transmittance of light with a wavelength of 300 nm to 1000 nm of 80% or more. The method for producing the above-mentioned composite of the present invention includes polymerizing a monomer represented by formula (IA) to synthesize a polyamine having a primary amino group, modifying the base material with a silane coupling agent, modifying the base material with the polyamine, and reacting the polyamine with a porphyrin derivative or a salt thereof to produce a porphyrin-containing polyamine, thereby solving the above-mentioned problem. [ka] (In formula (IA), R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L 1 is O or NH, L 2 is a divalent group, and X is a halogen atom. The method for producing the above-mentioned composite according to the present invention comprises polymerizing a monomer represented by formula (IA) to synthesize a polyamine having a primary amino group, reacting the polyamine with a porphyrin derivative or a salt thereof to produce a porphyrin-containing polyamine, modifying the base material with a silane coupling agent, and modifying the base material with the porphyrin-containing polyamine, thereby solving the above-mentioned problem. [ka] (In formula (IA), R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L 1is O or NH, L 2 is a divalent group, and X is a halogen atom. The cell culture substrate of the present invention comprises the above-described complex of the present invention. The cell culture substrate of the present invention may be a cell culture dish. The cell culture dish according to the present invention comprises a container and the composite of the present invention attached to the container, thereby solving the above-mentioned problems. The porphyrin contained in the porphyrin derivative or a salt thereof may be excited by irradiation with light having a wavelength of 300 nm to 650 nm to generate singlet oxygen. The method for producing a cell culture dish according to the present invention comprises polymerizing a monomer represented by formula (IA) to synthesize a polyamine having a primary amino group, modifying a glass substrate with a silane coupling agent, attaching the glass substrate to a container, modifying the glass substrate with the polyamine, and reacting the polyamine with a porphyrin derivative or a salt thereof to produce a porphyrin-containing polyamine, thereby solving the above-mentioned problems. [ka] (In formula (IA), R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L 1 is O or NH, L 2 is a divalent group, and X is a halogen atom. The method for manufacturing the above-mentioned cell culture dish according to the present invention comprises polymerizing a monomer represented by formula (IA) to synthesize a polyamine having a primary amino group, reacting the polyamine with a porphyrin derivative or a salt thereof to produce a porphyrin-containing polyamine, modifying a glass substrate with a silane coupling agent, modifying the glass substrate with the porphyrin-containing polyamine, and attaching the glass substrate to a container, thereby solving the above-mentioned problem. [ka] (In formula (IA), R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L 1 is O or NH, L 2 is a divalent group, and X is a halogen atom. The method for stimulating cells according to the present invention comprises contacting the above-mentioned complex with cells, and irradiating the complex in contact with the cells with light having a wavelength of 300 nm to 650 nm, thereby solving the above-mentioned problem. The porphyrin derivative or a salt thereof may generate singlet oxygen upon irradiation with light. The singlet oxygen may be brought into contact with the cells. Furthermore, the cells may be oxidized by the singlet oxygen. The energy density of the light irradiated onto the complex may be adjusted. The cells to be stimulated may be immature dendritic cells. The method for producing antigen-presenting cells capable of producing anti-inflammatory cytokines of the present invention includes contacting the above-mentioned complex of the present invention with immature dendritic cells, and irradiating the complex in contact with the immature dendritic cells with light having a wavelength of 300 nm to 650 nm. The method of the present invention for producing antigen-presenting cells capable of producing anti-inflammatory cytokines comprises stimulating immature dendritic cells with a cell culture substrate that generates singlet oxygen. The anti-inflammatory cytokine may be interleukin-10. Effect of the Invention

[0009] Among the copolymers immobilized on the substrate (e.g., glass substrate), the portion having a positive charge has the property of promoting the adhesion of cells having a negative charge. In addition, among the copolymers immobilized on the substrate, the portion having porphyrin has the property of generating singlet oxygen by light irradiation. When cells are cultured on the complex (e.g., copolymer-immobilized glass substrate) and light irradiation is performed, singlet oxygen is generated from the vicinity of the surface of the complex, and the cells can be stimulated (e.g., oxidative stress damage can be given). The amount of singlet oxygen generated can be controlled by the light irradiation conditions. When strong light is irradiated, the entire cell is subjected to oxidative stress, but by irradiating with light of an appropriate intensity, there is no excess of reactive oxygen species in the cells, and only the lipids in the cells adhering to the complex can be selectively oxidized to generate lipid radicals. In this way, the substrate (i.e., the complex) and the cell culture substrate (e.g., cell culture dish) on which the copolymer of the present invention is immobilized can provide an experimental method capable of controlling the oxidative stress state of cells by controlling the light irradiation conditions. In addition, in the method for producing antigen-presenting cells of the present invention, by using a cell culture substrate that generates singlet oxygen, immature dendritic cells can be stimulated to promote maturation and produce anti-inflammatory cytokines. [Brief description of the drawings]

[0010] [Figure 1] Schematic diagram showing the complex of the present invention (glass substrate on which porphyrin-containing polyamine is immobilized) [Diagram 2] Schematic diagram showing the cell culture substrate (cell culture dish) of the present invention. [Diagram 3] Flowchart showing the process of stimulating (oxidizing) cells using cell culture dishes. [Figure 4] Chromatogram of poly(N-(3-aminopropyl) methacrylamide) [Diagram 5] 1H NMR spectrum of poly(N-(3-aminopropyl)methacrylamide) [Figure 6] FIG. 1 shows a procedure for producing a cell culture dish according to Example 1. [Figure 7]FIG. 1 shows the results of cell viability and cell membrane permeability tests using cell culture dishes of Example 1, Comparative Example 1, and Comparative Example 2. [Figure 8] FIG. 1 shows the results of singlet oxygen generation using cell culture dishes of Example 1 and Comparative Example 3. [Figure 9] FIG. 1 shows the results of measuring the temperature rise caused by laser irradiation in cell culture dishes in Example 1 and Comparative Example 3. [Figure 10] FIG. 1 shows the results of detection of intracellular reactive oxygen species by laser irradiation using the cell culture dish of Example 1. [Figure 11] FIG. 1 shows the results of detecting intracellular oxidized lipids by laser irradiation using the cell culture dish of Example 1. [Figure 12] Fluorescence micrographs of dendritic cells cultured using the cell culture dishes of Example 1 and Comparative Example 3 [Figure 13] Antigen presentation amount when ovalbumin OVA257-264 peptide was added to dendritic cells cultured using the cell culture dishes of Example 1 and Comparative Example 3 [Figure 14] Cytokine production amount when ovalbumin OVA257-264 peptide was added to dendritic cells cultured using the cell culture dishes of Example 1 and Comparative Example 3 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments. In this specification, a numerical range expressed using "~" means a range including the numerical values ​​before and after "~" as the lower and upper limits. For example, a numerical range of "1 to 100" includes both the upper limit "1" and the lower limit "100". The same applies to other numerical ranges.

[0012] [First embodiment] As a first embodiment of the present invention, a composite including a substrate and a copolymer (porphyrin-containing polyamine) having a specific structure immobilized on the substrate will be described. The substrate of the present invention is not particularly limited as long as it does not have cytotoxicity, but in this embodiment, a glass substrate 110 is used as the substrate (see FIG. 1).

[0013] <The composite of the present invention (glass substrate having copolymer immobilized thereon) and its manufacturing method> FIG. 1 is a schematic diagram showing the complex of the present invention.

[0014] The composite 100 of the present invention comprises a glass substrate 110 and a porphyrin-containing polyamine copolymer 120 (hereinafter sometimes simply referred to as a "copolymer") fixed thereon, the porphyrin-containing polyamine copolymer 120 including at least a repeating unit represented by the following formula (I), a repeating unit represented by the following formula (II), and a repeating unit represented by the following formula (III):

[0015] [ka] (In the formula (I), (II) or (III), R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, L 1 is O or NH, L 2 is a divalent group, X is a halogen atom, and examples of the halogen atom represented by X include fluorine, chlorine, bromine, and iodine. Y represents a bonding mode for bonding to the glass substrate 110 via a silane coupling agent, and is -C(=O)-NH-R 2 -*, -C(=S)-NH-R 2 -*, -C(=O)-R 2 -* and -C(-OH)-R 2 -*, where * represents a bonding position with the glass substrate 110, R 2is selected from the group consisting of linear or branched alkyl groups having 1 to 5 carbon atoms, cycloalkyl groups having 5 to 10 carbon atoms, and aralkyl groups having 7 to 10 carbon atoms; Z is a group in which any one hydrogen atom has been removed from a porphyrin derivative or a salt thereof.

[0016] R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms, and more preferably represents a hydrogen atom or a linear alkyl group having 1 to 3 carbon atoms.

[0017] L 1 is preferably NH. This prevents hydrolysis even during long-term culture, making it possible to prevent the porphyrin derivative from being released.

[0018] L 2 is preferably a divalent linear aliphatic hydrocarbon group having 1 to 5 carbon atoms, a divalent branched aliphatic hydrocarbon group having 3 to 5 carbon atoms, a divalent cyclic aliphatic hydrocarbon group having 5 to 10 carbon atoms, a divalent aromatic hydrocarbon group having 5 to 10 carbon atoms, -O-, -S-, -N(R 3 )-(wherein, R 3 represents a hydrogen atom or a straight or branched alkyl group having 1 to 12 carbon atoms), -C(=O)-, -C(=O)-O-, -OC(=O)-O-, -C(=O)-NH-, -OC(=O)-NH-, -S(=O)-, -S(=O)-O-, -S(=O)2-, -S(=O)2-O-, -CH=N-, or a combination thereof. R 3 More preferably, represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms.

[0019] L 2 is more preferably a divalent linear aliphatic hydrocarbon group having 1 to 5 carbon atoms or a divalent branched aliphatic hydrocarbon group having 3 to 5 carbon atoms, and particularly preferably a linear or branched alkylene group having 1 to 5 carbon atoms.

[0020] Some of the primary amino groups in formula (I) are protonated in water and in cell culture media, as in the structure of formula (IA). In that case, X- Examples of the monovalent anion represented by the formula (I) include inorganic anions and organic anions. Examples of the inorganic anion include a fluorine anion, a chlorine anion, a bromine anion, an iodine anion, and a hydroxy anion. Examples of the organic anion include an alkoxy anion having 1 to 4 carbon atoms, a carboxy anion, an alkyl carboxy anion having 2 to 5 carbon atoms, and a hydrogen carbonate anion. [ka]

[0021] Z is a group in which any one hydrogen atom has been removed from a porphyrin derivative or a salt thereof.

[0022] Porphyrin derivatives are organic compounds having a cyclic structure formed by combining four pyrroles called porphins, and examples thereof include alkylcarboxyporphyrins such as mesoporphyrin, deuteroporphyrin, hematoporphyrin, protoporphyrin, uroporphyrin, coproporphyrin, cytoporphyrin, rhodoporphyrin, pyroporphyrin, phyloporphyrin, heptacarboxyporphyrin, hexacarboxyporphyrin, and pentacarboxyporphyrin (preferably C1-10 alkylcarboxyporphyrin, more preferably C3-8 alkylcarboxyporphyrin).

[0023] The salts of the porphyrin derivatives include the acid addition salts and base addition salts of the above-mentioned porphyrins. The acid addition salts include inorganic acid salts and organic acid salts. The inorganic acid salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, and the organic acid salts include salts derived from organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.

[0024] The base addition salts include inorganic base salts and organic base salts. Examples of inorganic base salts include salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Examples of organic base salts include salts derived from organic bases such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, trifluoroacetyl, and ethanolamine.

[0025] The porphyrin in the copolymer 120 of the present invention is preferably a salt, and the salt is preferably an acid addition salt, more preferably an inorganic acid salt such as hydrochloride, which suppresses π-π stacking between the porphyrins and enables dissolution in a cell culture medium.

[0026] For example, specific copolymers 120 include those represented by the following formula: 1 is -CH3, L 1 is -NH-, L 2 is -CH2-CH2-CH2- and Z is hematoporphyrin dihydrochloride acid. [ka]

[0027] The number of repeating units represented by formula (II) in copolymer 120 immobilized on the surface of glass substrate 110 is preferably within a predetermined range since copolymer 120 needs to be covalently immobilized on the surface of glass substrate 110. Specifically, the ratio of the number of repeating units represented by formula (II) to the total number of all repeating units of copolymer 120 is preferably 0.001 to 20%, more preferably 0.01 to 10%, even more preferably 0.5 to 5%, and particularly preferably 1 to 3%.

[0028] The number of repeating units represented by formula (III) in copolymer 120 immobilized on the surface of glass substrate 110 is preferably within a predetermined range in order to reduce π-π stacking of porphyrin between copolymers 120. Specifically, the ratio of the number of repeating units represented by formula (III) to the total number of all repeating units of copolymer 120 is preferably 0.01 to 60%, more preferably 0.1 to 45%, even more preferably 0.5 to 40%, and particularly preferably 1 to 35%.

[0029] The number of repeating units represented by formula (I) in copolymer 120 immobilized on the surface of glass substrate 110 is not particularly limited, and may be the remainder of the ratio of the numbers of repeating units of formulas (II) and (III).

[0030] Copolymer 120 preferably has 1 to 15 repeating units represented by formula (III), more preferably has 1 to 10 repeating units, and even more preferably has 1 to 7 repeating units represented by formula (III) in copolymer 120. When the number of porphyrin sites in copolymer 120 is increased, porphyrins in copolymer 120 undergo π-π stacking, which reduces the rate of singlet oxygen generation upon irradiation with light and generates heat.

[0031] The molecular weight (Mw) of the copolymer 120 is not particularly limited, and may be, for example, 300 to 10,000,000. However, since the molecular weight of the copolymer 120 is one of the factors that can affect properties such as water solubility and viscosity, it is preferably 1,000 to 100,000, and more preferably 3,000 to 70,000.

[0032] As used herein, unless otherwise specified, "molecular weight" refers to weight average molecular weight (Mw). In addition, both weight average molecular weight (Mw) and number average molecular weight (Mn) refer to values ​​measured by gel filtration chromatography under the following conditions. Measuring device: Shimadzu 20AD Columns: TSK gel G3000PW and G5000PW columns Eluent: Methanol Standard material: Polyethylene glycol Flow rate: 0.5mL / min

[0033] Although the copolymer 120 is immobilized only on one surface of the glass substrate 110 in FIG. 1, it may also be immobilized on the other surface.

[0034] The glass substrate 110 is not particularly limited, but may be a quartz glass substrate, a soda glass substrate, an aluminosilicate glass substrate, a borosilicate glass substrate, an alkali-containing glass substrate, an alkali-free glass substrate, etc. The thickness of the glass substrate 110 is not limited, but is preferably 0.04 mm to 0.60 mm. This range is advantageous for super-resolution confocal microscope observation.

[0035] The glass substrate 110 only needs to be transparent, but preferably has a transmittance of 80% or more for light with a wavelength of 300 nm to 1000 nm. This allows fluorescence observation using a microscope. The above-mentioned materials have this transmittance.

[0036] Such a composite 100 is suitable for cell culture, microscopic observation of cells, and light irradiation. A part of the copolymer 120 immobilized on the glass substrate 110 is protonated in water and cell culture medium as shown in formula (IV). Such protonated parts with increased positive charges can promote adhesion of negatively charged cells, thus promoting cell culture.

[0037] Next, a method for producing the composite 100 of the present invention will be described. Step S110: The monomer represented by formula (IA) is polymerized to synthesize a polyamine having a primary amino group. Step S120: The base material (glass substrate) is modified with a silane coupling agent. Step S130: The base material (glass substrate) is modified with polyamine. Step S140: A porphyrin-containing polyamine is produced by reacting a polyamine with a porphyrin derivative or a salt thereof.

[0038] [ka]

[0039] In the formula (IA), R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, L 1 is O or NH, L 2 is a divalent group, X is a halogen atom. 1 , L 1 , L 2 and X may be the same as in formula (I).

[0040] Each step will be explained in detail. Step S110: Synthesizing a polyamine having a primary amino group The polymerization reaction of the monomer of formula (IA) can be carried out, for example, by reacting a mixture of the monomer of formula (IA), a thermal polymerization initiator, and a solvent. This polymerization reaction can be carried out, for example, at 50°C to 100°C in an inert atmosphere such as nitrogen for 1 hour to 48 hours. In view of the required properties such as cell adhesiveness, the molecular weight is preferably in the range of 300 to 10,000,000. The polymer solution after the reaction may be dialyzed with a dialysis membrane, purified, and dried. This results in a powder.

[0041] The thermal polymerization initiator may be a water-soluble radical generator such as 4,4'-azobis(4-cyanovaleric acid), etc. The solvent may be water, ethanol, or a mixture of water and ethanol.

[0042] Monomers of formula (IA) are commercially available compounds and are readily available, but may also be prepared.

[0043] Step S120: A process of modifying the surface of the glass substrate with a silane coupling agent Y having a reactive group The method of modifying the surface of the glass substrate with a silane coupling agent having a reactive group can be a method well known in the art. For example, the surface of the glass substrate is etched by washing with a strong base such as potassium hydroxide or sodium hydroxide. Thereafter, the surface of the glass substrate is immersed in a solution of silane coupling agent Y and an alcohol such as ethanol, washed several times with alcohol, and dried at 80°C to 150°C for 15 minutes to 5 hours, thereby modifying the glass substrate with the silane coupling agent.

[0044] The silane coupling agent Y may have any reactive group capable of bonding with the primary amino group in the polyamine. Specifically, when bonded to the glass substrate, the silane coupling agent Y may have a reactive group capable of bonding with the primary amino group in the polyamine. 2 -*, isocyanate group -R 2 -*, carboxy group -R 2 -* and epoxy group -R 2 -*, where * represents a bonding position with the glass substrate, and R 2 has a group selected from the group consisting of a linear or branched alkyl group having 1 to 5 carbon atoms, a cycloalkyl group having 5 to 10 carbon atoms, and an aralkyl group having 7 to 10 carbon atoms. Silane coupling agents having an isocyanate group or an epoxy group are preferred.

[0045] Step S130: A process of modifying the glass substrate modified with the silane coupling agent with the polyamine synthesized in step S110 The reaction proceeds by impregnating a glass substrate modified with a silane coupling agent into a phosphate buffer solution (pH 7.4) in which a polyamine is dissolved, and the above formula (II) is introduced into the copolymer.

[0046] In step S130, a copolymer including a repeating unit represented by formula (I) and a repeating unit represented by formula (II) is immobilized on the surface of the glass substrate via a silane coupling agent.

[0047] Step S140: A porphyrin derivative or a salt thereof Z is reacted with a polyamine to produce a porphyrin-containing polyamine. The introduction of the porphyrin derivative or its salt Z into the polyamine can be carried out, for example, by reacting a mixture of the polyamine (here, a copolymer containing the repeating unit represented by formula (I) and the repeating unit represented by formula (II) immobilized on the surface of a glass substrate), the porphyrin derivative or its salt, and a condensing agent in a buffer solution of pH 6 to 8, methanol, or ethanol for 1 to 24 hours. As a result, a copolymer containing the repeating unit represented by formula (I), the repeating unit represented by formula (II), and the repeating unit represented by formula (III) is immobilized on the surface of the glass substrate via the silane coupling agent, and the complex 100 of the present invention (FIG. 1) is produced.

[0048] Here again, the porphyrin derivative is an organic compound having a cyclic structure formed by combining four pyrroles called porphins, and examples thereof include alkylcarboxyporphyrins such as mesoporphyrin, deuteroporphyrin, hematoporphyrin, protoporphyrin, uroporphyrin, coproporphyrin, cytoporphyrin, rhodoporphyrin, pyroporphyrin, phyloporphyrin, heptacarboxyporphyrin, hexacarboxyporphyrin, and pentacarboxyporphyrin (preferably C1-10 alkylcarboxyporphyrin, more preferably C3-8 alkylcarboxyporphyrin).

[0049] Salts of the porphyrin derivatives include acid addition salts and base addition salts of the above-mentioned porphyrins.

[0050] The mixing ratio of the polyamine and the porphyrin derivative or its salt is preferably adjusted according to the amount of the porphyrin derivative or its salt introduced, and may be 1 to 20 times the molar amount of the porphyrin derivative or its salt relative to the primary amino group in the polyamine to be reacted. From the viewpoint of production cost, it is preferable to use an amount of 1 to 5 times the molar amount. Examples of the condensation agent include N-hydroxysuccinimide and water-soluble carbodiimide hydrochloride.

[0051] Another method for producing the composite 100 of the present invention will now be described. In the above-mentioned steps S110 to S140, the porphyrin-containing polyamine is synthesized in a state where it is immobilized on the glass substrate. However, as shown in the following steps S210 to S240, the porphyrin-containing polyamine may be synthesized first and then immobilized on the glass substrate.

[0052] Step S210: The monomer represented by formula (IA) is polymerized to synthesize a polyamine having a primary amino group. Step S220: A porphyrin-containing polyamine is produced by reacting a polyamine with a porphyrin derivative or a salt thereof. Step S230: The base material (glass substrate) is modified with a silane coupling agent. Step S240: The base material (glass substrate) is modified with the porphyrin-containing polyamine.

[0053] [ka]

[0054] In the formula (IA), R 1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, L 1 is O or NH, L 2 is a divalent group, X is a halogen atom. 1 , L 1 , L2 and X may be the same as in formula (I).

[0055] Each step will be explained in detail. Step S210: Synthesizing a polyamine having a primary amino group Step S210 is similar to step S110 described above, and therefore a description thereof will be omitted.

[0056] Step S220: A step of producing a porphyrin-containing polyamine by reacting a polyamine with a porphyrin derivative or a salt thereof.

[0057] Step S220 is the same as step S140, except that the polyamine is not immobilized on the glass substrate. Introduction of porphyrin into polyamine can be carried out, for example, by reacting a mixture of polyamine (here, a copolymer containing a repeating unit represented by formula (I)), a porphyrin derivative or a salt thereof, and a condensing agent in a buffer solution of pH 6 to 8, methanol, or ethanol for 1 to 24 hours. A porphyrin-containing polyamine, which is a copolymer containing a repeating unit represented by formula (I) and a repeating unit represented by formula (III), is produced.

[0058] Here too, the mixing ratio of the polyamine to the porphyrin derivative or its salt can be the same as that in step S 130. Examples of the condensing agent include N-hydroxysuccinimide and water-soluble carbodiimide hydrochloride.

[0059] Step S230: A process of modifying the surface of the glass substrate with a silane coupling agent having a reactive group Step S230 is similar to step S120 described above, and therefore a description thereof will be omitted.

[0060] Step S240: A step of modifying the glass substrate modified with the silane coupling agent with the porphyrin-containing polyamine synthesized in step S220 Step S240 is the same as step S130, except that the polyamine is a porphyrin-containing polyamine. The reaction proceeds by impregnating a glass substrate modified with a silane coupling agent with a phosphate buffer solution (pH 7.4) in which the porphyrin-containing polyamine is dissolved, and a copolymer including a repeating unit represented by formula (I), a repeating unit represented by formula (II), and a repeating unit represented by formula (III) is immobilized on the surface of the glass substrate via the silane coupling agent, thereby producing the composite 100 of the present invention (FIG. 1).

[0061] <Cell culture dish and method for producing same of the present invention> The composite 100 of the present invention functions as a scaffold (scaffold material) for cultured cells. A cell culture dish provided with the composite 100 will be described below.

[0062] FIG. 2 is a schematic diagram showing a cell culture dish of the present invention.

[0063] The cell culture dish 200 of the present invention includes a container 210 and a composite 100 (a glass substrate to which a copolymer is immobilized) attached to the container 210. Here, the composite 100 is similar to the composite 100 of the present invention described with reference to FIG. 1, and therefore a description thereof will be omitted.

[0064] The container 210 may be formed from a synthetic resin material, a glass material, etc. The glass material may be quartz glass, soda glass, aluminosilicate glass, borosilicate glass, alkali-containing glass, alkali-free glass, etc. The synthetic resin may be a styrene-based resin such as polystyrene or SBS, polycarbonate, acrylonitrile-based resin, polyolefin such as polypropylene or polyethylene, or a polyester resin such as polyvinyl chloride, PMMA (polymethyl methacrylate), polyethylene terephthalate, or polybutylene terephthalate.

[0065] The container 210 only needs to be transparent, but preferably has a transmittance of 80% or more for light with wavelengths of 300 nm to 1000 nm, which allows the complex 100 and the cells to be efficiently oxidatively damaged.

[0066] 2 shows a state in which the composite 100 is attached to a part of the bottom surface of the container 210, but the entire bottom surface of the container 210 may be the composite 100. The container 210 may also have a lid (not shown). In this case, the lid may also have the same material and optical properties as the container 210.

[0067] The cell culture dish 200 of the present invention includes the composite 100, so that cells can be easily attached to the composite 100 and can be used for cell experiments. There are no particular limitations on the type of cells, and examples of the cells include cancer cells, immune cells, nerve cells, digestive tract cells, pancreatic cells, mammary gland cells, skin cells, lung cells, kidney cells, bladder cells, liver cells, brain cells, vascular endothelial cells, adipocytes, germ cells, plant cells, and microorganisms.

[0068] The cell culture dish 200 of the present invention can generate singlet oxygen by exciting the porphyrin contained in the porphyrin derivative or its salt in the copolymer of the complex 100 when irradiated with light having a wavelength of 300 nm to 650 nm. Due to such characteristics, by using the cell culture dish 200 of the present invention, it is possible to induce a reaction of active oxygen in the cell components near the surface while culturing cells by irradiating the complex 100 with light such as an LED light or a semiconductor laser. That is, the cell culture dish 200 of the present invention can be used in an oxidative stress experiment. In addition, since it is possible to selectively oxidize lipids in the cell membrane closest to the surface of the complex 100, it can be used in an experiment on oxidized lipids.

[0069] Next, a method for producing the cell culture dish 200 of the present invention will be described. Step S310: The monomer represented by formula (IA) is polymerized to synthesize a polyamine having a primary amino group. Step S320: A porphyrin-containing polyamine is produced by reacting a polyamine with a porphyrin derivative or a salt thereof. Step S330: The glass substrate is modified with a silane coupling agent. Step S340: The glass substrate is modified with a porphyrin-containing polyamine. Step S350: The glass substrate is attached to the container.

[0070] Steps S310 to S340 are the same as steps S210 to S240 for producing the composite 100 of the present invention. That is, the composite 100 of the present invention may be simply attached to the cell culture dish 200 of the present invention. There are no particular limitations on the method of attachment, but for example, attachment can be performed as follows.

[0071] A hole capable of accommodating the composite 100 is formed in the container using a laser cutting machine or the like, the composite 100 is placed in the hole, and the composite 100 and the hole are bonded together with an adhesive such as hotmel. The shape of the hole may be rectangular or circular. The adhesive is preferably one that does not affect cell culture. A recess may be formed instead of the hole.

[0072] Another method for producing the cell culture dish 200 of the present invention will now be described. In the method for producing the composite in the above steps S110 to S140, a step of attaching the glass substrate modified with a silane coupling agent to a container may be inserted between steps S120 and S130.

[0073] Step S410: The monomer represented by formula (IA) is polymerized to synthesize a polyamine having a primary amino group. Step S420: The glass substrate is modified with a silane coupling agent. Step S430: The glass substrate is attached to the container. Step S440: The glass substrate is modified with polyamine. Step S450: A porphyrin-containing polyamine is produced by reacting a polyamine with a porphyrin derivative or a salt thereof.

[0074] That is, steps S410, S420, S440 and S450 are respectively the same as steps S110, S120, S130 and S140, and step S430 may be the same as step S350 described above.

[0075] It should be noted that, as in steps S410 to S450, if the glass substrate is attached to the container and then modified with polyamine, the amount of polyamine used can be advantageously reduced.

[0076] 2, the cell culture dish 200 of the present invention is shown in a state in which the composite 100 is attached to the container 210, but the cell culture dish 200 of the present invention may have a porphyrin-containing polyamine copolymer including the repeating unit represented by the above formula (I), the repeating unit represented by the formula (II), and the repeating unit represented by the formula (III) immobilized on the surface of the container 210. Such a modification would be easily conceived by a person skilled in the art.

[0077] <How to stimulate cells> Next, a method for stimulating cells will be described using the complex 100. In this embodiment, a method for stimulating cells using the cell culture dish 200 described above will be described. FIG. 3 is a flow chart showing the steps of stimulating cells using a cell culture dish.

[0078] Step S301: The complex is brought into contact with cells (specifically, the cells are seeded on a cell culture dish). Step S302: Light having a wavelength of 300 nm to 650 nm is irradiated onto the complex in contact with the cells (the complex on the cell culture dish).

[0079] According to step S301, when cells are seeded on the cell culture dish 200 of the present invention, the amino groups of the copolymer immobilized on the surface of the composite 100 of the present invention of the cell culture dish 200 are protonated by the cell culture medium, so that the cells easily adhere and culture is promoted.

[0080] In step S302, when the complex 100 on the cell culture dish 200 is irradiated with light having a wavelength of 300 nm to 650 nm, the porphyrin contained in the porphyrin or a salt thereof in the copolymer immobilized on the surface of the complex 100 is excited. The inventors of the present application have found that the excited porphyrin generates singlet oxygen, and the generated singlet oxygen comes into contact with the cells being cultured and stimulates the cells (for example, causes oxidative stress, i.e., oxidizes the cells).

[0081] In step S302, the wavelength of the light irradiated to the complex 100 is preferably 630 nm to 640 nm. Light in this range can suppress phototoxicity. Such light may be a semiconductor laser or a light emitting diode.

[0082] The light irradiation conditions are not particularly limited, but the light energy density, i.e., the light intensity and / or the irradiation time, may be adjusted. This allows the amount of singlet oxygen generated to be controlled. For example, the higher the light energy density, the greater the amount of singlet oxygen generated.

[0083] Furthermore, by using the cell culture dish of the present invention, it is possible to selectively induce lipid radical production within the cells while culturing the cells by simply irradiating them with light such as a laser, thereby suppressing the production of excessive reactive oxygen species in the cytoplasm.

[0084] [Variations] In the above-described first embodiment, a glass substrate was used as the base material 110 of the composite 100 (see Figs. 1 and 2), but the present invention is not limited thereto. The material of the base material 110 is not particularly limited as long as it is a material that does not have cytotoxicity, and examples of the material include synthetic resins, metals and oxides of these metals, ceramics, and the like in addition to the various glasses described above.

[0085] Examples of synthetic resins include styrene-based resins such as polystyrene and SBS, polycarbonate, acrylonitrile-based resins, polyolefins such as polypropylene and polyethylene, polyester resins such as polyvinyl chloride, PMMA (polymethyl methacrylate), polyethylene terephthalate, and polybutylene terephthalate, copolymers thereof, and polymer alloys thereof. Among them, polystyrene is preferable from the viewpoints of transparency, moldability, and cost. Examples of metals and oxides of metals include gold (Au), silver (Ag), copper (Cu), platinum (Pt), iron (Fe), chromium (Cr), cobalt (Co), alloys thereof, and oxides thereof, and magnetic materials such as various ferrites may also be used. Examples of ceramics include alumina, zirconia, reinforced alumina (zirconia-added alumina), silicon nitride (SiN), silicon carbide (SiC), and aluminum nitride (AlN).

[0086] In the above-described first embodiment, the substrate 110 is a plate-like body (glass substrate) (see FIG. 1), but the present invention is not limited to this and can have various shapes. In the above-described first embodiment, the cell culture dish 200 in which the composite 100 is attached to the container 210 functions as the cell culture substrate (see FIG. 2), but the present invention is not limited to this. For example, the composite 100 itself may be a cell culture substrate, and the shape (form) of the cell culture substrate can also be various shapes (forms) other than a dish shape.

[0087] The shape of the substrate 110, i.e., the shape of the composite 100 and the cell culture substrate, is not particularly limited, and may be, in addition to a plate-like body, for example, particles (powder) such as beads, fibers such as hollow fibers, fabrics (woven fabrics, nonwoven fabrics, knitted fabrics), films (thin films), etc. If the substrate 110 is a particle, a composite 100 with a large surface area is obtained, and it is easy to efficiently culture and stimulate (e.g., oxidize) cells in large quantities. As the substrate 110 for particles, for example, magnetic powder (magnetic particles) such as ferromagnetic material is preferable because suspension culture can be performed and the substrate 110 for particles and the cultured and stimulated (e.g., oxidized) cells can be easily separated.

[0088] In addition, the substrate 110 may also function as a container 210 (see FIG. 2) capable of holding a culture medium, etc. The shape of such substrate 110 is not particularly limited, and may be, for example, a petri dish (dish) type, a flask type, a plate type, a culture pack type, or the like.

[0089] The manufacturing method of the composite and cell culture substrate of various shapes in this modification is similar to the manufacturing method of the composite 100 or the manufacturing method of the cell culture dish 200 described in the first embodiment, except that a corresponding substrate is used instead of a glass substrate as the substrate, and the preferred embodiments are also the same. The composite and cell culture substrate of this modification can achieve the same effects as the composite 100 and cell culture dish 200 of the first embodiment, and can be used to culture cells and stimulate (e.g., oxidize) cells.

[0090] [Second embodiment] In this embodiment, a method for producing antigen-presenting cells capable of producing anti-inflammatory cytokines will be described.

[0091] Anti-inflammatory cytokines are produced by helper T (Th) cells, dendritic cells, macrophages, monocytes, B cells, etc., and act on immune cells such as T cells and macrophages to directly suppress cell activation and weaken the antigen-presenting ability of macrophages, thereby calming excessive immune responses. Therefore, anti-inflammatory cytokines play an extremely important role in the treatment and prevention of inflammatory and autoimmune conditions, as well as in the suppression of immune rejection after transplant surgery.

[0092] The present inventors have surprisingly discovered that when immature dendritic cells are stimulated using a cell culture substrate that generates singlet oxygen, the dendritic cells mature to become antigen-presenting cells, and that the mature dendritic cells selectively produce anti-inflammatory cytokines while suppressing the production of inflammatory cytokines such as TNF-α and interleukin 6 (IL-6), and have arrived at the present invention based on this discovery. The mechanism is unclear, but is speculated as follows. It is possible that a trace amount of singlet oxygen generated from the cell culture substrate reacts with lipids and proteins in the vicinity of the cell membrane, causing immature dendritic cells to receive cell membrane stimulation and mature, and further react to an oxidation reaction to selectively produce anti-inflammatory cytokines. Note that this mechanism is merely speculation and does not limit the technical scope of the present invention in any way.

[0093] The production method of this embodiment is a simple method of simply stimulating immature dendritic cells using a cell culture substrate that generates singlet oxygen, and can efficiently produce antigen-presenting cells that produce anti-inflammatory cytokines, and can also be mass-produced. When dendritic cells combine antigen presentation and secretion of anti-inflammatory cytokines, antigen-specific immune tolerance is possible, and it is known that when administered to a living body, they attenuate cytotoxic T cells and induce regulatory T cells that suppress immunity, and are expected to be excellent as a cell medicine for autoimmune diseases and the like. The antigen-presenting cells produced by the production method of this embodiment can be suitably used as a therapeutic or preventive drug for inflammatory and autoimmune pathologies, a drug for suppressing immune rejection after transplant surgery, and the like. The manufacturing method of this embodiment will be described in detail below.

[0094] The production method of this embodiment is a method for producing antigen-presenting cells capable of producing anti-inflammatory cytokines, and includes stimulating immature dendritic cells with a cell culture substrate that generates singlet oxygen.

[0095] Anti-inflammatory cytokines produced by the antigen-presenting cells (immunotolerant (immunosuppressive) dendritic cells) of this embodiment include interleukin 10 (IL-10), TGF-β, interleukin 27 (IL-27), etc., of which IL-10 is preferred.

[0096] The cell culture substrate that generates singlet oxygen is not particularly limited, but examples thereof include substrates having a cell adhesive surface (e.g., a surface having a cationic functional group) to which porphyrin or a salt thereof has been introduced (modified with porphyrin or a salt thereof). The material, shape, etc. of the substrate modified with porphyrin or a salt thereof are not particularly limited, and are, for example, similar to the substrate 110 (see FIG. 1) of the complex 100 listed in the first embodiment and its modified examples, and the preferred embodiments are also the same. For example, when a substrate made of magnetic particles is used, mass production of antigen-presenting cells and separation of the produced antigen-presenting cells from the cell culture substrate can be easily performed.

[0097] As a cell culture substrate that generates singlet oxygen, for example, a cell culture substrate containing the complex of the first embodiment is preferable. Antigen-presenting cells that produce anti-inflammatory cytokines can be produced, for example, by stimulating (e.g., oxidizing) immature dendritic cells while culturing them, using the above-mentioned method of stimulating cells using the complex (see FIG. 3). The production method of this embodiment may include, for example, the following steps.

[0098] Step S501: The complex is contacted with immature dendritic cells. Step S502: The complex in contact with the immature dendritic cells is irradiated with light having a wavelength of 300 nm to 650 nm.

[0099] In step S501, when immature dendritic cells are brought into contact with complex 100, the amino groups of the copolymer immobilized on the surface of complex 100 are protonated by the cell culture medium, allowing the cells to adhere easily and promoting culture. The immature dendritic cells used in this embodiment may be commercially available products, or may be prepared by inducing differentiation of cultured cells, monocytes, or stem cells into immature dendritic cells.

[0100] In step S502, when the complex 100 is irradiated with light having a wavelength of 300 nm to 650 nm, the porphyrin contained in the porphyrin or its salt in the copolymer is excited, generating singlet oxygen. This singlet oxygen stimulates (e.g., oxidizes) immature dendritic cells and differentiates them into a mature state in which dendrites are extended. These mature dendritic cells selectively produce anti-inflammatory cytokines (e.g., IL-10).

[0101] In step S502, the wavelength of the light to be irradiated is preferably 630 nm to 640 nm. Light in this range can suppress phototoxicity. Such light may be a semiconductor laser or a light emitting diode.

[0102] The light irradiation conditions are not particularly limited, but the light energy density, i.e., the light intensity and / or the irradiation time, may be adjusted. This allows the amount of singlet oxygen generated to be controlled, and as a result, the amount of antigen-presenting cells (i.e., mature dendritic cells) that produce anti-inflammatory cytokines to be produced can be controlled. For example, the higher the light energy density, the greater the amount of singlet oxygen generated, and as a result, the greater the amount of antigen-presenting cells produced. EXAMPLES

[0103] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0104] [Synthesis of polyamine: Poly N-(3-aminopropyl) methacrylamide (PA)] Using N-(3-aminopropyl)methacrylamide (APAM) as the monomer represented by formula (IA), poly N-(3-aminopropyl)methacrylamide (PA) was synthesized by free radical polymerization (step S410 described above).

[0105] Specifically, as shown in the following formula, 143.7 mg of N-(3-aminopropyl)methacrylamide (APAM, monomer, manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 2 mL of ethanol. Then, 10 mg of 4,4'-azobis(4-cyanovaleric acid) (ACVA, free radical initiator, manufactured by Tokyo Chemical Industry Co., Ltd.) was added. Oxygen gas in the mixture was removed by bubbling with N2 gas. The reaction was carried out at 70 °C for 24 hours. The polymer solution was purified by dialysis against water using a dialysis membrane (MWCO, 3500) for 2 days. The resulting poly N-(3-aminopropyl)methacrylamide solution was then freeze-dried to obtain a white powder.

[0106] [ka]

[0107] The resulting white powder was analyzed by proton nuclear magnetic resonance ( 1 The product was characterized by 1 H NMR and gel filtration chromatography, and the results are shown in Figures 4 and 5.

[0108] FIG. 4 is a chromatogram of poly N-(3-aminopropyl) methacrylamide. FIG. 5 shows the structure of poly(N-(3-aminopropyl) methacrylamide). 1 FIG. 1 shows a H NMR spectrum.

[0109] The measurement conditions for gel filtration chromatography were as follows. Measuring device: Shimadzu 20AD Columns: TSK gel G3000PW and G5000PW columns Eluent: Methanol Standard material: Polyethylene glycol Flow rate: 0.5mL / min

[0110] 1 The measurement conditions for 1 H NMR were as follows. Measuring device: JNM-AL300 JEOL Deuterated solvent: Deuterium oxide Number of times accumulated: 64

[0111] The chromatogram in FIG. 4 is a chromatogram obtained by detecting the powder obtained by polymerization of APAM using a gel filtration chromatography column and a differential refractometer, and the horizontal axis indicates the elution time.

[0112] Figure 5 1 The H NMR spectrum showed that the powder was the desired poly(N-(3-aminopropyl) methacrylamide). Gel filtration chromatography showed that the number average molecular weight (Mn) of poly(N-(3-aminopropyl) methacrylamide) was about 3800 g / mol, the weight average molecular weight (Mw) was about 4200 g / mol, and the polydispersity (Mw / Mn) was about 1.1.

[0113] [Example 1] In Example 1, a glass substrate (composite) of the present invention having porphyrin-containing polyamine (PAHpD) immobilized thereon using PA, and a cell culture dish including the same were produced according to the procedure in FIG. FIG. 6 is a diagram showing a procedure for producing the cell culture dish of Example 1.

[0114] A cover glass (24 mm x 24 mm, thickness 0.12-0.17 mm, manufactured by Matsunami Glass, made of borosilicate glass) was used as the glass substrate. The cover glass was immersed in ethanol containing 5% potassium hydroxide, ultrasonicated for 20 minutes, and washed. Next, the surface of the washed cover glass was modified with a silane coupling agent (step S420 described above). Specifically, the cover glass was immersed in water, ultrasonicated for 10 minutes, and then placed in ethanol containing 1% 3-(triethoxysilyl)propyl isocyanate as a silane coupling agent for 5 minutes. Next, the surface of the cover glass was rinsed with ethanol and dried at 120°C for 1 hour. In this way, an isocyanate group was introduced into the surface of the cover glass, as shown in FIG. 6(A).

[0115] A carbon dioxide laser cutting machine (SC300; SMART DIYs, Japan) was used to create a square hole measuring 18 mm × 18 mm in the center of a petri dish (35 mm diameter × 10 mm height, made of polystyrene). A cover glass modified with a silane coupling agent was attached to the square hole of the petri dish using a glue gun-type hot melt adhesive (step S430 described above).

[0116] Next, the surface of the cover glass modified with the silane coupling agent was modified with PA (step S440 described above). -1 , pH 7.4) was added to the surface of a cover glass attached to a petri dish and left to stand at room temperature for 1 hour. The surface of the cover glass modified with PA was then washed with phosphate-buffered saline (PBS). In this way, some of the primary amino groups in the polyamine (PA) were reacted with isocyanate groups, and the polyamine was introduced onto the glass substrate, as shown in Figure 6(B).

[0117] Next, a porphyrin-containing polyamine was produced by reacting PA with hematoporphyrin dihydrochloride as a porphyrin derivative or its salt (step S450 described above). Specifically, a mixture (200 μL) of hematoporphyrin dihydrochloride (0.1 mg / mL, HpD, MedChem Express), water-soluble carbodiimide hydrochloride (0.16 mg / mL, WSCD·HCl, Peptide Institute, Inc.), and N-hydroxysuccinimide (0.15 mg / mL, NHS, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the surface of the cover glass modified with PA and reacted at room temperature for 2 hours. The surface of the cover glass was washed three times with 70% ethanol and dried overnight in a light-shielded clean bench.

[0118] In this way, as shown in Fig. 6(C), hematoporphyrin (HpD) was introduced into some of the primary amino groups in the polyamine (PA) on the surface of the cover glass to obtain the cell culture dish of Example 1. The obtained cell culture dish was covered with aluminum foil and stored in a dark place at room temperature.

[0119] As shown in FIG. 6, it is clear that after the porphyrin-containing polyamine is immobilized on a cover glass, the cover glass may be attached to the container with an adhesive or the like.

[0120] Here, the PAHpD obtained by the above-mentioned operation was a copolymer containing a repeating unit represented by formula (Ia), a repeating unit represented by formula (IIa), and a repeating unit represented by formula (IIIa). 1 is -CH3, L 1 is -NH-, L 2 is the divalent group -CH2-CH2-CH2-, X is chlorine, Y is -C(=O)-NH-C3H6-*, * represents the bonding position with the glass substrate, and Z is a porphyrin derivative in which one hydrogen atom has been removed from hematoporphyrin dihydrochloride.

[0121] [ka]

[0122] Here, the molecular weight (Mw) of PAHpD was measured by gel filtration chromatography to be about 4200 g / mol. In addition, the number of repeating units represented by formula (IIIa) was found to be 31% of the total number of all repeating units of PAHpD using an X-ray photoelectron spectrometer (Ulvac PHI Quantera SXM).

[0123] A cell membrane permeability test was carried out using the cell culture dish of Example 1. 5 × 10 per 18 mm × 18 mm cover glass area. 4Mouse colon cancer cells (Colon26) were seeded on a cover glass at a cell density of 100 μg / ml and cultured at 37°C for 48 hours. Live cells were then stained green and dead cells and cells with damaged cell membranes were stained red using calcein AM and propidium iodide. When the cell membrane becomes more permeable, propidium iodide, a red staining reagent, is taken up into the cell and intercalates into the nucleic acids in the nucleus, emitting strong red fluorescence. The results are shown in Figure 7.

[0124] Singlet oxygen was generated using the cell culture dish of Example 1. To confirm the presence or absence of singlet oxygen generation, singlet oxygen sensor green reagent (SOSG, Thermo Fisher Scientific) was added to the surface of the cover glass of the cell culture dish of Example 1 at a concentration of 5 μM using methanol as a solvent. Then, 12 J / cm 2 The cover glass was irradiated with a 635 nm laser with an energy density of 100 Hz from the side without SOSG solution. The fluorescence signal of SOSG was measured at an excitation wavelength of 488 nm and an emission wavelength of 535 nm using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co.). The results are shown in Figure 8.

[0125] The temperature change due to laser irradiation in the cell culture dish of Example 1 was investigated. 200 μL of phosphate buffer solution was added to the glass substrate of the cell culture dish of Example 1, and a laser (wavelength: 635 nm) with different light energy densities was irradiated, and the temperature change of the solution was measured using a thermal camera (FLIR, C5). The results are shown in Figure 9.

[0126] Using the cell culture dish of Example 1, intracellular active oxygen was detected by laser irradiation. 4 Mouse-derived dendritic cells, JAWII cells (American Type Culture Collection), were seeded onto the cover glass at a cell density of 100 μg / ml and cultured at 37° C. for 48 hours.

[0127] The reactive oxygen species-sensitive dyes used in this experiment were DCFH-DA (Invitrogen, Thermo Fisher Scientific) for detecting reactive oxygen species in the cytoplasm, siDMA (Dojindo Laboratories, Ltd.) for detecting mitochondrial singlet oxygen, and HPF and APF (Goryo Chemical Co., Ltd.) for detecting highly reactive reactive oxygen species in the cytoplasm.

[0128] DCFH-DA reacts with various reactive oxygen species such as singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals, and emits green fluorescence. siDMA accumulates in mitochondria, reacts with singlet oxygen produced near mitochondria, and emits green fluorescence. HPF reacts with intracellular hydroxyl radicals and peroxynitrite, and emits green fluorescence. APF reacts with intracellular hydroxyl radicals, peroxynitrite, and hypochlorite ions, and emits green fluorescence.

[0129] After adding these staining reagents to the cell culture medium, the cells were incubated at 37°C for 30 minutes, and the staining reagents outside the cells were removed with PBS before irradiation. Then, the cells were irradiated with a laser (635 nm, 12 J / cm 2 The cells were then observed under a fluorescence microscope (Keysense, BZ-X710).

[0130] The final concentrations of DCFH-DA, siDMA, HPF, and APF were 5 μM, 50 nM, 5 μM, and 5 μM, respectively. All dye solutions were prepared in PBS and used immediately. The intracellular fluorescence intensity was analyzed using ImageJ software. The results are shown in Figure 10.

[0131] Detection of intracellular oxidized lipids by laser irradiation was carried out using the cell culture dish of Example 1. Mouse-derived dendritic cells JAWII cells (American Type Culture Collection) were cultured at 5 × 10 cells per 18 mm × 18 mm cover glass area. 4 Cells were seeded onto glass substrates at a density of 100x and incubated at 37°C for 48 hours.

[0132] The reactive oxygen species-sensitive dye used in this experiment was LipiRADICAL Green (Funakoshi Co., Ltd.), which is used to detect lipid oxidation. Dendritic cells attached to a cover glass were irradiated with a laser (635 nm, 12 J / cm 2 After incubation at 37°C for 10 min, LipiRADICAL Green was added to the cells at a final concentration of 1 μM, and the cells were incubated at 37°C for 15 min. After washing with PBS, the cells were observed under a fluorescence microscope (Keysense, BZ-X710). The intracellular fluorescence intensity was analyzed using ImageJ software. The results are shown in Figure 11.

[0133] [Comparative Example 1] In Comparative Example 1, a cell culture dish on which porphyrin-containing polyethyleneimine was immobilized was produced. Comparative Example 1 is similar to Example 1 except that polyethyleneimine was used instead of PA, and therefore a description thereof will be omitted. The cell culture dish thus obtained is called the cell culture dish of Comparative Example 1. Using the cell culture dish of Comparative Example 1, a cell membrane permeability test was performed in the same manner as in Example 1. The results are shown in FIG. 7.

[0134] [Comparative Example 2] In Comparative Example 2, a cell culture dish on which porphyrin was immobilized was produced. In Comparative Example 2, 1% (3-aminopropyl)triethoxysilane was used as a silane coupling agent in Example 1, and hematoporphyrin dihydrochloride was directly immobilized without modifying PA.

[0135] Specifically, the surface of the cleaned cover glass was modified with a silane coupling agent in the same manner as in Example 1, and then the cover glass was immersed in water and ultrasonically treated for 10 minutes, and then placed in ethanol containing 1% (3-aminopropyl)triethoxysilane as a silane coupling agent for 5 minutes. Next, the surface of the cover glass was rinsed with ethanol and dried at 120°C for 1 hour.

[0136] A cover glass modified with (3-aminopropyl)triethoxysilane was attached using a glue gun type hot melt adhesive to a petri dish in which holes had been formed in the same manner as in Example 1. The subsequent procedures were the same as in Example 1, and a cell culture dish on which hematoporphyrin was immobilized was obtained using hematoporphyrin dihydrochloride.

[0137] The cell culture dish obtained in this manner is referred to as the cell culture dish of Comparative Example 2. Using the cell culture dish of Comparative Example 2, a cell membrane permeability test was carried out in the same manner as in Example 1. The results are shown in FIG.

[0138] [Comparative Example 3] In Comparative Example 3, a cell culture dish with immobilized PA was produced. Specifically, the procedure in Example 1 was completed without reacting hematoporphyrin dihydrochloride, to obtain the cell culture dish of Comparative Example 3. Using the cell culture dish of Comparative Example 3, the generation of singlet oxygen and the temperature change due to laser irradiation were examined in the same manner as in Example 1. The results are shown in Figs. 8 and 9.

[0139] For simplicity, the cell culture dishes of Example 1 and Comparative Examples 1 to 3 are shown in a table, and the results are summarized and explained.

[0140] [Table 1]

[0141] FIG. 7 shows the results of cell survival and cell membrane permeability tests using the cell culture dishes of Example 1, Comparative Example 1, and Comparative Example 2.

[0142] Fig. 7 shows the results of culturing mouse colon cancer cells (Colon26) in the cell culture dishes of Example 1, Comparative Example 1, and Comparative Example 2, and staining them with calcein AM and propidium iodide. Fig. 7 shows the results in grayscale, but in reality, the cells stained with calcein AM emit green light, and the cells stained with propidium iodide emit red light, and the areas shown brightly in the grayscale indicate the emission of fluorescence.

[0143] According to FIG. 7, when the cell culture dishes of Comparative Example 1 and Comparative Example 2 were used, most of the cells survived, but strong red fluorescence was observed in some of the cells. This suggests that the cell membrane may have been damaged in the process of electrostatically adhering the cells to the cover glass (glass substrate) surface. On the other hand, when the cell culture dish of Example 1 was used, all the cells survived, and no red fluorescence was observed from the cell nuclei. This result indicates that cells can be cultured on the cover glass (glass substrate) of the cell culture dish of Example 1 without damage to the cell membrane. This shows that the complex of the present invention and the cell culture dish attached thereto are advantageous for cell culture.

[0144] FIG. 8 shows the results of singlet oxygen generation using the cell culture dishes of Example 1 and Comparative Example 3.

[0145] 8, no increase in the fluorescent signal of SOSG was observed on the surface of the cover glass of the cell culture dish in Comparative Example 1 due to laser irradiation, but the fluorescent signal of SOSG increased on the surface of the cover glass of the cell culture dish in Example 1. This result indicates that the amount of singlet oxygen was generated due to the presence of porphyrin bound to PAHpD on the surface of the cover glass.

[0146] FIG. 9 shows the results of measuring the temperature rise caused by laser irradiation in the cell culture dishes of Example 1 and Comparative Example 3.

[0147] 9, a temperature rise of about 2° C. occurred in both the cell culture dishes of Example 1 and Comparative Example 3. This shows that no temperature rise was observed depending on the presence or absence of porphyrin.

[0148] FIG. 10 shows the results of detection of intracellular active oxygen by laser irradiation using the cell culture dish of Example 1.

[0149] FIG. 10(A) shows the result of staining cells with the reagent DCFH-DA, which reacts with intracellular reactive oxygen species and shows fluorescence, FIG. 10(B) shows the result of staining cells with the reagent siDMA, which reacts with intramitochondrial singlet oxygen and shows fluorescence, FIG. 10(C) shows the result of staining cells with APF, which reacts with intracellular reactive oxygen species and shows fluorescence, and FIG. 10(D) shows the result of staining cells with HPF, which reacts with intracellular reactive oxygen species and shows fluorescence. In each case, cell images and intracellular fluorescent signal intensity are shown. The scale bar in FIG. 10 indicates 50 μm.

[0150] As shown in Figure 10, with these four reactive oxygen species detection reagents, no increase in intracellular fluorescent signal was observed upon laser irradiation of dendritic cells cultured on the cover glass of the cell culture dish in Example 1. This result indicates that the amount of reactive oxygen species released into the cells is extremely small.

[0151] FIG. 11 shows the results of detection of intracellular oxidized lipids by laser irradiation using the cell culture dish of Example 1.

[0152] Figure 11 shows cell images stained with LipiRADICAL Green, which specifically reacts with lipid radicals, and the intracellular fluorescent signal intensity. The scale bar in Figure 11 indicates 50 μm.

[0153] As shown in FIG. 11, a significant increase in the fluorescent signal of the LipiRADICAL Green reagent was confirmed by irradiating the dendritic cells cultured on the cover glass of the cell culture dish of Example 1 with a laser. This result indicates that lipid radicals are produced in the cells. This shows that by using the cell culture dish of the present invention, singlet oxygen can be generated by irradiating light such as a laser while culturing the cells, thereby oxidizing the cells and inducing oxidative stress in the cells.

[0154] [Production of antigen-presenting cells] (1) Fluorescence microscopy Using the cell culture dishes of Example 1 and Comparative Example 3, immature dendritic cells were cultured and observed under a fluorescent microscope according to the method described below.

[0155] 8×10 3 Mouse-derived dendritic cells JAWII cells (American Type Culture Collection) were seeded on the glass substrate of a cell culture dish, and the dendritic cells attached to the glass substrate were irradiated with laser light (635 nm, 12 J / cm 2 , for 10 minutes) and then cultured for 24 hours at 37° C. The medium used was MEM-α medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 5 ng / mL GM-CSF.

[0156] The cultured cells were washed twice with PBS and fixed with 4% formaldehyde at room temperature for 15 min. Next, the cells were permeabilized with 0.1% Triton X-100 at room temperature for 15 min. After that, the cells were washed twice with PBS and actin was stained with Alexa FluoreTM 488 phalloidin (Invitrogen, Thermo Fisher Scientific, Inc., USA) at room temperature for 45 min. After washing twice with PBS, the cell nuclei were stained with Hoechst 33258 (Invitrogen, Thermo Fisher Scientific, Inc., USA) at room temperature for 5 min. After staining, the cells were washed twice with PBS and observed under a Keyence BZ-X710 microscope (Keyence, Japan). The results are shown in Figure 12.

[0157] For comparison, immature dendritic cells were cultured using the cell culture dishes of Example 1 and Comparative Example 3 in the same manner, except that the dendritic cells were not irradiated with laser light, and observed under a fluorescent microscope. The results are shown in Figure 12.

[0158] The top four photographs in Figure 12 are shown in grayscale, but in reality, the stained cell nuclei are blue and the stained actin is green, and the areas shown bright in grayscale are fluorescent. The bottom four photographs in Figure 12 are bright-field images.

[0159] Generally, immature dendritic cells have a spherical shape, but when they mature, they form dendrites. As shown in Fig. 12, no morphological changes were observed with or without laser irradiation in the dendritic cells cultured in the cell culture dish of Comparative Example 3, and the cells maintained their spherical cell shape. On the other hand, in the dendritic cells cultured in the cell culture dish of Example 1, although they had a similar spherical cell shape in the absence of laser irradiation, it was confirmed that they formed dendrites when cultured for 24 hours after laser irradiation.

[0160] (2) Measurement of antigen presentation amount Using the cell culture dishes of Example 1 and Comparative Example 3, immature dendritic cells were cultured by the method described below, and the amount of antigen presentation when ovalbumin OVA257-264 peptide was added was measured.

[0161] 5×10 4 JAWII cells derived from mice were seeded on the glass substrate of a cell culture dish, and the dendritic cells attached to the glass substrate were irradiated with laser light (635 nm, 12 J / cm 2 After the addition of ovalbumin OVA257-264 peptide (Sigma-Aldrich, USA), the cells were cultured for 16 hours at 37° C. The medium used was the same as that used in “(1) Fluorescence microscopy observation” above.

[0162] To evaluate antigen presentation, the cultured cells were harvested and stained with APC-labeled anti-OVA257-264 peptide antibody and FITC-labeled anti-mouse CD80 antibody. The cells were washed twice with PBS, and the amount of fluorescence (relative amount of antigen presentation) of the cells was quantified using a flow cytometer (BD AccuriTM C6 Plus System, BD Biosciences, USA). The evaluation results are shown in Figure 13 as "1a" and "1b". In Figure 13, the relative values ​​are shown when the value of 3b is set to 1.0.

[0163] For comparison, immature dendritic cells were cultured using the cell culture dishes of Example 1 and Comparative Example 3 and the amount of antigen presentation was measured using the same method, except that the dendritic cells were not irradiated with laser light. Also, instead of irradiating with laser light, lipopolysaccharide (LPS), which is known to promote the maturation of immature dendritic cells, was added to the immature dendritic cells, and the amount of antigen presentation was measured using the cell culture dish of Comparative Example 3 using the same method, except that the immature dendritic cells were cultured using the cell culture dish of Comparative Example 3 and the amount of antigen presentation was measured. The respective evaluation results are shown in Figure 13 as "3a", "3b", and "3c".

[0164] As shown in FIG. 13, when the cell culture dish of Comparative Example 3 (PA having no porphyrin structure) was used and LPS was not used, there was no difference in the amount of antigen presented with or without laser light irradiation (evaluation results 3a and 3b). Below, the evaluation results 3a and 3b will be compared with the other evaluation results 3c, 1a, and 1b. First, when LPS, which promotes the maturation of immature dendritic cells, was used (evaluation result 3c), the amount of antigen presented tended to increase compared to when LPS was not used (evaluation results 3a and 3b), but the difference was not significant. Furthermore, when the cell culture dish of Example 1 was used without irradiation with laser light (evaluation result 1a), the amount of antigen presentation tended to increase compared with evaluation results 3a and 3b, but there was no significant difference. The reason why evaluation result 1a showed a tendency to increase the amount of antigen presentation is presumed to be that the porphyrin structure contained in the copolymer (PAHpD) had some effect on the maturation of immature dendritic cells. On the other hand, when the cell culture dish of Example 1 was used and laser light was irradiated (evaluation result 1b), the amount of antigen presentation was significantly increased compared to evaluation results 3a and 3b. It was confirmed that the combination of a copolymer having a porphyrin structure and laser light irradiation greatly contributes to the maturation of immature dendritic cells.

[0165] (3) Evaluation of cytokine production Using the cell culture dishes of Example 1 and Comparative Example 3, immature dendritic cells were cultured by the method described below, and cytokine production when ovalbumin OVA257-264 peptide was added was evaluated.

[0166] Immature dendritic cells were cultured in the same manner as in "(2) Measurement of antigen presentation amount" above, and the medium after cell culture was collected, and the cytokine concentration in the medium was measured using an ELISA kit (Biolegend, USA). The concentrations of interleukin-6 (IL-6) and TNF-α (Tumor Necrosis Factor-α) were measured as inflammatory cytokines, and the concentration of IL-10 was measured as an anti-inflammatory cytokine. The results are shown in Figures 14(A) to (C).

[0167] First, the evaluation results using the cell culture dish of Comparative Example 3 will be considered. As shown in Figures 14(A) and (B), regardless of the presence or absence of laser irradiation, immature dendritic cells cultured in the cell culture dish of Comparative Example 3 produced very low amounts of inflammatory cytokines (IL-6 and TNF-α) even when the OVA257-264 peptide was added. However, when LPS, which promotes the maturation of immature dendritic cells, was added to the cells, the production of inflammatory cytokines increased significantly. On the other hand, as shown in Figure 14(C), the addition of LPS did not significantly increase the anti-inflammatory cytokine (IL-10).

[0168] Next, the evaluation results using the cell culture dish of Example 1 will be considered. As shown in Figures 14(A) to (C), when the cell culture dish of Example 1 was used and not irradiated with laser light, the production amounts of all cytokines (IL-6, TNF-α, IL-10) increased slightly compared to when the cell dish of Example 3 was used. On the other hand, as shown in Figure 14(C), when the cell culture dish of Example 1 was used and irradiated with laser light, only the production amount of the anti-inflammatory cytokine (IL-10) increased significantly. [Industrial Applicability]

[0169] The complex of the present invention has excellent cell adhesion due to the immobilization of the porphyrin-containing polyamine copolymer, and is advantageous for cell culture. Furthermore, the cell culture substrate provided with the complex of the present invention can generate singlet oxygen by irradiating light such as laser light, thereby causing oxidative stress damage to cells. [Explanation of symbols]

[0170] 100 Composite (glass substrate with copolymer) 110 Substrate (glass substrate) 120 Porphyrin-containing polyamine copolymer 200 cell culture dishes 210 Container

Claims

1. A substrate; A complex comprising a copolymer including a repeating unit represented by the following formula (I), a repeating unit represented by the following formula (II), and a repeating unit represented by the following formula (III), which is immobilized on the substrate. 【Chemistry 1】 (In the formula (I), (II) or (III), R １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L １ is O or NH, L ２ is a divalent group, X is a halogen atom; Y represents a bonding mode for bonding to the substrate via a silane coupling agent, and is represented by -C(=O)-NH-R ２ -*, -C(=S)-NH-R ２ -*, -C(=O)-R ２ -* and -C(-OH)-R ２ -*, where * represents a bonding position with the substrate, R ２ is selected from the group consisting of linear or branched alkyl groups having 1 to 5 carbon atoms, cycloalkyl groups having 5 to 10 carbon atoms, and aralkyl groups having 7 to 10 carbon atoms; Z is a group obtained by removing one arbitrary hydrogen atom from a porphyrin derivative or a salt thereof.

2. Said L ２ represents a divalent linear aliphatic hydrocarbon group having 1 to 5 carbon atoms, a divalent branched aliphatic hydrocarbon group having 3 to 5 carbon atoms, a divalent cyclic aliphatic hydrocarbon group having 5 to 10 carbon atoms, a divalent aromatic hydrocarbon group having 5 to 10 carbon atoms, -O-, -S-, -N(R ３ )-(wherein, R ３ represents a hydrogen atom or a straight or branched alkyl group having 1 to 12 carbon atoms), -C(=O)-, -C(=O)-O-, -O-C(=O)-O-, -C(=O)-NH-, -O-C(=O)-NH-, -S(=O)-, -S(=O)-O-, -S(=O) ２ -, -S(=O) ２ The complex according to claim 1, wherein the group is selected from the group consisting of -O-, -CH=N-, and combinations thereof.

3. The composite according to claim 1, wherein the ratio of the number of repeating units represented by formula (II) to the total number of repeating units of the copolymer is 1 to 50%, and the ratio of the number of repeating units represented by formula (III) to the total number of repeating units of the copolymer is 1 to 50%.

4. 2. The complex of claim 1, wherein the porphyrin derivative is selected from the group consisting of mesoporphyrin, deuteroporphyrin, hematoporphyrin, protoporphyrin, uroporphyrin, coproporphyrin, cytoporphyrin, rhodoporphyrin, pyroporphyrin, phyloporphyrin, heptacarboxyporphyrin, hexacarboxyporphyrin, pentacarboxyporphyrin, and salts of these porphyrins.

5. R １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms; L ２ The complex according to claim 1, wherein represents a linear or branched alkylene having 1 to 5 carbon atoms.

6. R １ is -CH ３ represents L １ represents -NH-; L ２ is -CH ２ -CH ２ -CH ２ - represents 2. The conjugate of claim 1, wherein Z is hematoporphyrin dihydrochloride.

7. The composite according to claim 1, wherein the molecular weight (Mw) of the copolymer is 300 to 10,000,000.

8. The composite of claim 1 , wherein the substrate is a glass substrate.

9. The thickness of the glass substrate is 0.04 mm to 0.60 mm; The composite according to claim 8, having a transmittance of 80% or more for light having a wavelength of 300 nm to 1000 nm.

10. polymerizing a monomer represented by formula (IA) to synthesize a polyamine having a primary amino group; modifying the substrate with a silane coupling agent; modifying the substrate with the polyamine; reacting the polyamine with a porphyrin derivative or a salt thereof to produce a porphyrin-containing polyamine; The method for producing the complex according to any one of claims 1 to 9, comprising: 【Chemistry 2】 (In formula (IA), R １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L １ is O or NH, L ２ is a divalent group, X is a halogen atom.

11. polymerizing a monomer represented by formula (IA) to synthesize a polyamine having a primary amino group; reacting the polyamine with a porphyrin derivative or a salt thereof to produce a porphyrin-containing polyamine; modifying the substrate with a silane coupling agent; modifying the substrate with the porphyrin-containing polyamine; The method for producing the complex according to any one of claims 1 to 9, comprising: 【Chemistry 3】 (In formula (IA), R １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L １ is O or NH, L ２ is a divalent group, X is a halogen atom.

12. A cell culture substrate comprising the complex according to any one of claims 1 to 9.

13. The cell culture substrate according to claim 12 , which is a cell culture dish.

14. The cell culture substrate according to claim 12, wherein the porphyrin contained in the porphyrin derivative or a salt thereof is excited by irradiation with light having a wavelength of 300 nm to 650 nm to generate singlet oxygen.

15. A method for producing a cell culture dish according to claim 13, comprising the steps of: polymerizing a monomer represented by formula (IA) to synthesize a polyamine having a primary amino group; Modifying a glass substrate with a silane coupling agent; attaching the glass substrate to a container; modifying the glass substrate with the polyamine; reacting the polyamine with a porphyrin derivative or a salt thereof to produce a porphyrin-containing polyamine; The method for producing a cell culture dish includes the steps of: 【Chemistry 4】 (In formula (IA), R １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L １ is O or NH, L ２ is a divalent group, X is a halogen atom.

16. A method for producing a cell culture dish according to claim 13, comprising the steps of: polymerizing a monomer represented by formula (IA) to synthesize a polyamine having a primary amino group; reacting the polyamine with a porphyrin derivative or a salt thereof to produce a porphyrin-containing polyamine; Modifying a glass substrate with a silane coupling agent; modifying the glass substrate with the porphyrin-containing polyamine; Attaching the glass substrate to a container; The method for producing a cell culture dish includes the steps of: 【Chemistry 5】 (In formula (IA), R １ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; L １ is O or NH, L ２ is a divalent group, X is a halogen atom.

17. Contacting a cell with the complex according to any one of claims 1 to 9; irradiating the complex in contact with the cell with light having a wavelength of 300 nm to 650 nm; The present invention relates to a method for stimulating a cell.

18. The method for stimulating a cell according to claim 17 , wherein the porphyrin derivative or a salt thereof generates singlet oxygen upon irradiation with light.

19. The method of stimulating a cell according to claim 18 , wherein the singlet oxygen is contacted with the cell.

20. The method of stimulating a cell according to claim 18, wherein the cell is oxidized by the singlet oxygen.

21. The method for stimulating cells according to claim 17 , further comprising adjusting the energy density of light irradiated to the complex.

22. The method of stimulating cells according to claim 17, wherein the cells are immature dendritic cells.

23. A method for producing an antigen-presenting cell capable of producing an anti-inflammatory cytokine, comprising the steps of: Contacting immature dendritic cells with the complex according to any one of claims 1 to 9; and irradiating the complex in contact with the immature dendritic cells with light having a wavelength of 300 nm to 650 nm.

24. The method for producing an antigen-presenting cell according to claim 23, wherein the anti-inflammatory cytokine is interleukin-10.

25. A method for producing antigen-presenting cells capable of producing anti-inflammatory cytokines, comprising stimulating immature dendritic cells with a cell culture substrate that generates singlet oxygen.

26. The method for producing an antigen-presenting cell according to claim 25, wherein the anti-inflammatory cytokine is interleukin-10.

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