Chemically modified silk fibroin and use thereof in cell and organoid culture

Abstract

Provided are chemically modified silk fibroin and a use thereof in cell and organoid culture. The chemically modified silk fibroin is a product obtained by sequentially carboxylating silk fibroin and modifying same by an organic amine having a phenol group. Also provided is a hydrogel based on the chemically modified silk fibroin. A hydrogel system has a hierarchical structure and mechanical properties similar to those of an extracellular matrix, and exhibits characteristics such as definite composition, controllable physicochemical properties, low cytotoxicity, good biocompatibility, and biodegradability, can support the growth and differentiation of cells and organoids, and is suitable as a matrigel for cell and organoid culture.

Description

Chemically modified silk fibroin and its use for cell and organoid culture TECHNICAL FIELD

[0001] The present application belongs to the field of tissue engineering and biomaterials, relates to the field of high polymer materials, hydrogel materials and biomaterials, and in particular relates to a chemically modified silk fibroin, a preparation method thereof and a use thereof for cell and organoid culture. BACKGROUND

[0002] Matrigel, which is widely used for cell and organoid culture at present, is a basement membrane matrix extracted from mouse sarcoma, which has problems such as tumor origin, complex and unclear composition, large batch difference, low yield and high price, which limits its wide application in drug development and regenerative medicine. In recent years, the development of hydrogel systems with clear composition, good repeatability, customizable component performance and the ability to simulate extracellular matrix (ECM) for three-dimensional culture of cells and organoids has become a hot spot in the field of biomedical research, and has a broad application and market prospect in the future.

[0003] Silk fibroin (simplified as silk protein) is a macromolecular protein derived from natural mulberry silk, which has excellent biocompatibility, mechanical properties and biodegradability. At present, silk fibroin-based materials have been widely used in many fields such as tissue engineering and biomedical engineering, and related medical devices have been approved for use in clinical practice.

[0004] Based on the many advantages of silk protein, it has great potential to develop silk protein-based hydrogel systems for three-dimensional culture of cells and organoids. In the past few years, various methods have been developed for preparing silk protein-based hydrogels, which can be divided into physical crosslinking and chemical crosslinking. Physical crosslinking is to promote the self-assembly of silk protein by applying external stimuli such as pH change, electric field, shear force, surfactant and organic solvent, etc., to promote the transition from random coil structure to beta-sheet structure, and finally form hydrogel. Chemical crosslinking of silk protein usually requires the introduction of chemical crosslinking agents to form a crosslinked network. At present, whether based on physical crosslinking or chemical crosslinking, the silk protein-based hydrogels developed cannot simultaneously have the properties of rapid and mild gelation, mechanical properties matching cells, good cell compatibility, stable gelation, etc., and inevitably bring certain cytotoxicity. Not suitable for three-dimensional cell culture.

[0005] Therefore, it is a current research hotspot to develop a new type of silk protein-based hydrogel material to meet the needs of cell and organoid culture. SUMMARY

[0006] OBJECTIVE

[0007] One technical objective of the present application is to provide a chemically modified silk fibroin and a preparation method thereof.

[0008] Another technical purpose of the present application is to provide a series of methods for forming hydrogels from the above-mentioned chemically modified silk proteins and chemically modified silk protein hydrogels prepared by these methods.

[0009] Another technical purpose of the present application is to provide the use of the above-mentioned chemically modified silk protein hydrogel materials in biomedical materials, particularly in cell or organoid culture.

[0010] SUMMARY

[0011] In one aspect, the present application provides a method for preparing an organic amine-modified carboxylated silk protein, the method comprising: subjecting a carboxylated silk protein to an amidation reaction with an organic amine to obtain an organic amine-modified carboxylated silk protein, wherein,

[0012] The carboxylated silk protein is a silk protein having a side chain containing the following structure:

[0013] wherein R represents a C2-C6 alkylene group, represents the connection to the side chain therefrom;

[0014] The organic amine carries a -NH2 group and a phenol group, and the amidation reaction is carried out between the -NH2 group and the carboxyl group of the carboxylated silk protein.

[0015] In a specific embodiment, the carboxylated silk protein is a silk protein having a serine side chain containing the above-mentioned structure.

[0016] Serine is also known as beta-hydroxyalanine, and has the structure In the carboxylated silk protein, the structure is connected to the hydroxyl group on the serine side chain.

[0017] In the carboxylated silk protein, the structure has a modification rate of serine of 20-90%. The modification rate refers to the molar percentage of serine connected to relative to the total amount of serine in the silk protein.

[0018] The source of the carboxylated silk protein is not particularly limited and can be a commercially available product or can be synthesized according to the methods of the prior art, for example, the methods disclosed in CN115433369A (the entire disclosure of which is incorporated herein by reference). For example, the carboxylated silk protein can be prepared by reacting a diacid anhydride (such as succinic anhydride) with a silk protein.

[0019] In an embodiment, R represents an ethylene group or a propylene group.

[0020] In embodiments, the organic amine can be represented by the following structure:

[0021] wherein R1represents a C1-C6 alkylene group.

[0022] In specific embodiments, R1represents a methylene, ethylene, propylene or butylene group.

[0023] In specific embodiments, in the organic amine, R1is attached to the para or meta position of the hydroxyl group, particularly the para position.

[0024] In specific embodiments, the organic amine is tyramine, i.e. p-hydroxyphenylethylamine, having the structure

[0025] In specific embodiments, the organic amine can be used as a free amine or as a salt (e.g. hydrochloride salt).

[0026] In specific embodiments, the molar ratio of carboxylated silk fibroin to organic amine can be 0.5-10:1, for example 1 :2, 1 : 1, 2: 1, 5: 1, 10: 1, in terms of the molar ratio of the carboxyl groups contained in the carboxylated silk fibroin to the amino groups contained in the organic amine; preferably 1 : 1.

[0027] In the specific embodiment, the amidation reaction can be performed in the presence of a condensing agent. The condensing agent can be, for example, an acyl chloride-based condensing agent such as thionyl chloride, phosgene, phosphorus trichloride, phosphorus oxychloride, phosphorus pentachloride; an acid anhydride-based condensing agent such as ethyl chloroformate, isobutyl chloroformate, N,N-carbonyldiimidazole (CDI), dimethyl triflate (CBMIT), methane sulfonyl chloride (MsCl), p-toluenesulfonyl chloride (TsCl), p-nitrobenzenesulfonyl chloride (NsCl), Boc anhydride; a carbodiimide-based condensing agent such as dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC); an onium salt-based condensing agent including a carbonium salt-based condensing agent such as 2-(7-oxabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate quaternary ammonium salt (TNTU), and a phosphonium salt-based condensing agent such as benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxyl)tris-1-pyrrolidinylphosphonium hexafluorophosphate (PyAOP); an organophosphorus-based condensing agent such as diphenylphosphoryl chloride (DPP-Cl), diethyl cyanophosphonate (DECP), diphenyl phosphorazide (DPPA), thiodimethylphosphoryl azide (MPTA), bis(2-oxo-3-oxazolidinyl)phosphoryl chloride (BOP-Cl), but the present application is not limited thereto.

[0028] In the case of using a carbodiimide-based condensing agent, a condensation activator is generally required to be added, and the condensation activator commonly used includes 4-N,N-dimethylpyridine (DMAP), 4-pyrrolidinylpyridine (4-PPy), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azobenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (NHPI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), pentafluorophenol (PFPOH), and the like, but the present application is not limited thereto.

[0029] In the specific embodiment, the method is performed as follows, but is not limited thereto,

[0030] S1: preparing a carboxylated silk fibroin solution;

[0031] S2: adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the carboxylated silk protein solution obtained from S1 to activate the carboxylated silk protein;

[0032] S3: adding tyramine hydrochloride to the activated carboxylated silk protein in S2 to react, obtaining tyramine-modified carboxylated silk protein.

[0033] Figure 1 exemplarily shows a synthesis schematic diagram of tyramine-modified carboxylated silk protein according to one embodiment, wherein the silk protein is first reacted with succinic anhydride to obtain carboxylated silk protein, and then reacted with tyramine in the presence of EDC / NHS to obtain amine-modified carboxylated silk protein, but the present application is not limited thereto.

[0034] In a specific embodiment, the carboxylated silk protein solution is prepared by dissolving carboxylated silk protein powder in an aqueous solution of 2-(N-morpholino)ethanesulfonic acid, the concentration of carboxylated silk protein in the carboxylated silk protein solution being 1-100 mg / mL, and the concentration of 2-(N-morpholino)ethanesulfonic acid being 5-100 mM.

[0035] In a specific embodiment, in S2, the amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide added is 172.5-1725 mg, preferably 345 mg, and the amount of N-hydroxysuccinimide added is 103.5-1035 mg, preferably 207 mg, per 1 g of carboxylated silk protein.

[0036] In a specific embodiment, in S3, the amount of tyramine hydrochloride added is 31.4-314 mg per 1 g of carboxylated silk protein. In some embodiments, the reaction temperature is 18-25°C, the reaction time is 1-24 hours, preferably 2 hours, the pH of the reaction system is 6, and preferably, the reaction is carried out under shaking at a rotation speed of 25-100 rpm.

[0037] In a specific embodiment, after step S3, dialysis, lyophilization, and powdering operations can also be included to prepare the tyramine-modified carboxylated silk protein in powder form.

[0038] In another aspect, the present application provides a carboxylated silk protein modified with an organic amine, which contains

[0039] wherein R represents C2-C6 alkylene, preferably ethylene or propylene;

[0040] R1 represents C1-C6 alkylene, preferably methylene, ethylene, propylene, or butylene;

[0041] denotes the attachment to the side chain.

[0042] In particular embodiments, R1is attached to the para or meta position of the hydroxyl group, in particular the para position.

[0043] In particular embodiments, For wherein R is defined as above.

[0044] In particular embodiments, the total content of phenol groups in the organic amine-modified carboxylated silk protein is 4.6-10 mol%, for example 7.1 mol%, based on the total number of moles of amino acid units in the silk protein.

[0045] In particular embodiments, the organic amine-modified carboxylated silk protein can be obtained by the method for preparing an organic amine-modified carboxylated silk protein according to the present application.

[0046] In another aspect, the present application provides a method for preparing an organic amine-modified carboxylated silk protein hydrogel, the method comprising:

[0047] S1-1: dissolving the above organic amine-modified carboxylated silk protein in water to obtain an organic amine-modified carboxylated silk protein aqueous solution;

[0048] S1-2: dividing the solution obtained in S1-1 into two equal parts, one part is added with hydrogen peroxide to prepare solution A, and the other part is added with horseradish peroxidase (HRP) to prepare solution B;

[0049] S1-3: mixing solutions A and B in equal volumes to obtain an organic amine-modified carboxylated silk protein hydrogel.

[0050] In particular embodiments, in S1-1, the concentration of the organic amine-modified carboxylated silk protein in the organic amine-modified carboxylated silk protein solution is 25-50 mg / mL.

[0051] In particular embodiments, in S1-2, the concentration of hydrogen peroxide in solution A is 0.01-0.16 wt%, for example 0.06 wt%; and the concentration of horseradish peroxidase in solution B is 1-20 U / mL. It should be noted that the concentration of hydrogen peroxide will affect the mechanical properties of the hydrogel formed, for example, when the concentration of hydrogen peroxide in the finally prepared hydrogel is 0.03 wt%, the hydrogel shows excellent mechanical properties.

[0052] In particular embodiments, in S1-3, the mixing temperature of solutions A and B is 20-55°C, for example, mixing at 37°C.

[0053] In the above hydrogel formation process, HRP catalyzes H2O2 to generate oxygen free radicals, so as to generate active phenol groups in the organic amine modified carboxylated silk protein, and coupling between the ortho-phenolic hydroxyl groups of tyrosine residues or grafted tyramines to form di-tyrosines.

[0054] In another aspect, the present application provides a carboxylated silk protein hydrogel prepared by the above method.

[0055] In another aspect, the present application provides a method for preparing a carboxylated silk protein hydrogel, which comprises:

[0056] The carboxylated silk protein hydrogel is prepared by standing the aqueous solution of carboxylated silk protein at room temperature or under heating.

[0057] In a specific embodiment, the concentration of carboxylated silk protein in the aqueous solution of carboxylated silk protein is 1-100 mg / mL.

[0058] In a specific embodiment, the standing temperature of the solution is preferably 20-80°C, and the standing time is 0.5-240 hours.

[0059] In another aspect, the present application provides a carboxylated silk protein hydrogel prepared by the above method.

[0060] In another aspect, the present application provides a Matrigel for cell or organoid culture, which comprises at least the above organic amine modified carboxylated silk protein hydrogel or the above carboxylated silk protein hydrogel.

[0061] In a specific embodiment, the hydrogel further comprises natural materials used in the art to promote the interaction between cells and hydrogels, improve cell adhesion, and promote cell differentiation. The types of these natural materials are not limited, and can be selected from, for example, laminin, collagen, fibronectin, gelatin, serum protein, milk protein, hyaluronic acid, sodium alginate, chitosan, dextran, chondroitin sulfate, carboxymethyl cellulose.

[0062] In another aspect, the present application provides the use of the above organic amine modified carboxylated silk protein, the above organic amine modified carboxylated silk protein hydrogel, or the above carboxylated silk protein hydrogel in the preparation of a biomedical material.

[0063] In a specific embodiment, the biomedical material can be used for organoid culture, cell delivery, biological 3D printing, tissue filling, tissue regeneration, drug release, and the like.

[0064] In another aspect, the present application provides a kit for cell and organoid culture, which comprises:

[0065] the carboxylated silk protein modified by the organic amine described above; a hydrogen peroxide solution and a horseradish peroxidase; and optionally, a natural material for promoting the interaction between cells and the material, improving cell adhesion or promoting cell differentiation; or

[0066] the carboxylated silk protein described above; and optionally, a natural material for promoting the interaction between cells and the material, improving cell adhesion or promoting cell differentiation.

[0067] In specific embodiments, the natural material can be selected from the group consisting of laminin, collagen, fibronectin, gelatin, serum protein, milk protein, hyaluronic acid, sodium alginate, chitosan, dextran, chondroitin sulfate, carboxymethyl cellulose.

[0068] In specific embodiments, the concentration of the hydrogen peroxide solution is 1 wt%.

[0069] In specific embodiments, the kit can further comprise a culture medium for cell and organoid culture. For example, the culture medium can be a DMEM medium used in three-dimensional culture of cells, or an EB formation medium, an induction medium, an expansion medium and a maturation medium used in culture of organoids. For the EB formation medium, the induction medium, the expansion medium and the maturation medium, for example, reference can be made to STEMdiff TM A brain-like organ kit.

[0070] In another aspect, the present application provides a method for three-dimensional culture of cells, the method comprising:

[0071] S2-1: Dissolve the carboxylated silk protein modified by the organic amine described above in a DMEM medium, and divide the solution into two parts, one part is added with hydrogen peroxide to obtain solution A', and the other part is added with horseradish peroxidase to obtain solution B';

[0072] S2-2: Digest, collect, centrifuge and resuspend the adherent cells to prepare a cell suspension;

[0073] S2-3: Add the cell suspension to solution B' of step S2-1, and then mix solutions A' and B' in equal volumes, and incubate in a 37°C incubator for 1 minute;

[0074] S2-4: After the gel of S2-3 is completely formed into a hydrogel, add a culture medium to cover the hydrogel, and place in a 37°C incubator for three-dimensional culture of cells.

[0075] In yet another aspect, the present application provides a method for three-dimensional culture of organoids, the method being one of the following methods:

[0076] Method (i), using the carboxylated silk protein modified by the organic amine described above as a Matrigel for organoid culture, the method comprising:

[0077] The carboxylated silk fibroin modified by the organic amine is dissolved in the expansion culture medium, mixed with the natural material, and the solution is divided into two parts, one part is added with hydrogen peroxide to obtain solution A", and the other part is added with horseradish peroxidase to obtain solution B";

[0078] The same volume of solution A" and B" is added dropwise on the human induced pluripotent stem cell induced culture to form embryoid bodies (EBs), which are incubated in a 37°C incubator for 1-20 minutes to obtain EBs encapsulated by the hydrogel, and then the expansion culture medium is added and placed in a 37°C shaker for shaking culture; starting from the 11th day, the medium is replaced with an induced brain-like organ maturation culture medium for subsequent culture.

[0079] Method (ii), using the carboxylated silk fibroin hydrogel described above as Matrigel for organoid culture, the method comprising:

[0080] The carboxylated silk fibroin is dissolved in the expansion culture medium, mixed with the natural material, and placed in a 37°C incubator for incubation to form a carboxylated silk fibroin hydrogel;

[0081] The carboxylated silk fibroin hydrogel is added dropwise on the human induced pluripotent stem cell induced culture to form embryoid bodies (EBs), which are incubated in a 37°C incubator for 60-180 minutes to obtain EBs encapsulated by the hydrogel, and then the expansion culture medium is added and placed in a 37°C shaker for shaking culture; starting from the 11th day, the medium is replaced with an induced brain-like organ maturation culture medium for subsequent culture.

[0082] In a specific embodiment, in the above-mentioned cell three-dimensional culture method or the above-mentioned organoid three-dimensional culture method, the natural material is as described above. The amount of the natural material added can be determined by a person skilled in the art according to his common sense or routine operation, and is not limited.

[0083] In a specific embodiment, in the above-mentioned cell three-dimensional culture method or the above-mentioned organoid three-dimensional culture method, the selection of the culture medium depends on the cultured cells, and a person skilled in the art can reasonably select it according to his common knowledge, for example, using DMEM culture medium in cell three-dimensional culture, and using STEMdiff TM A brain-like organ kit, the kit comprising an EB formation culture medium, an induction culture medium, an expansion culture medium and a maturation culture medium, specifically, during the organoid culture process, using the EB formation culture medium for 0-5 days, using the induction culture medium for 5-7 days, using the expansion culture medium for 7-10 days, and using the maturation culture medium for 10-40 days or more, the use of each culture medium can be referred to STEMdiff TM Instructions for using the brain-like organ kit.

[0084] In specific embodiments, in the above-mentioned cell three-dimensional culture method or the above-mentioned organoid three-dimensional culture method, the type of cells for culture includes but is not limited to mouse fibroblasts, human umbilical vein vascular endothelial cells, human induced pluripotent stem cells, and human embryonic stem cells, and the final concentration of the cells in the hydrogel is 3 x 10 5 -1 x 10 6

[0085] In specific embodiments, in the above-mentioned cell culture method or the above-mentioned organoid culture method, the concentration of the organic amine-modified carboxylated silk protein in the culture medium is 25-50 mg / mL; and the concentration of the carboxylated silk protein in the culture medium is 1-100 mg / mL.

[0086] In specific embodiments, the natural material is selected from one or all of laminin, collagen, and fibronectin, and preferably, the concentration of the natural material in the culture medium is: laminin 2-200 μg / mL; collagen 10-1000 μg / mL; fibronectin 4-400 μg / mL; and hyaluronic acid 0.02-2 mg / mL.

[0087] In specific embodiments, the concentration of horseradish peroxidase in the finally formed organic amine-modified carboxylated silk protein hydrogel is 5-10 U / mL; and the concentration of hydrogen peroxide in the finally formed organic amine-modified carboxylated silk protein hydrogel is 0.005wt%-0.03wt%.

[0088] In specific embodiments, human induced pluripotent stem cells are inoculated in an ultra-low adsorption 96-well plate at 9000 cells / well, and after 7 days of induction culture, embryoid bodies (EBs) are formed, and the diameter of the formed embryoid bodies is 400-600 μm. Advantages

[0089] The present application further uses an organic amine containing a phenol group (e.g., tyramine) to modify the carboxylated silk protein, thereby increasing the phenol group in the silk protein and accelerating the formation of the silk protein hydrogel.

[0090] Further, according to the present application, the hydrogel system obtained from the carboxylated silk protein or the tyramine-modified carboxylated silk protein has a multi-level structure and mechanical properties similar to those of the extracellular matrix, and has the characteristics of clear composition, controllable physical and chemical properties, low cytotoxicity, good biocompatibility, and biodegradability, and can support the growth and differentiation of cells and organoids. At the same time, the chemical modification silk protein hydrogel has a simple preparation process, can be prepared in large quantities, and the cost is controllable, which helps to realize the batch and high-throughput culture of cells and organoids, and solves the pain points of traditional Matrigel, such as tumor origin, complex composition, and large batch differences.

[0091] ​Further, the present application combines chemical modification and physical complexation of silk fibroin to avoid the use of chemical cross-linking agents, further improving the biocompatibility of silk fibroin-based hydrogel materials. In addition, by combining chemical modification with active substance compounding, not only the physical and chemical properties of the hydrogel material can be precisely adjusted, but also the biological activity of the hydrogel material is improved, and the interaction between cells and the hydrogel material is enhanced, which is suitable for three-dimensional culture of cells and organoids.

[0092] Further, the carboxylated silk fibroin or tyramine-modified carboxylated silk fibroin hydrogel developed by the present application also has injectability, printability and biodegradability, and has broad application prospects in the fields of organoid culture, cell delivery, biological 3D printing, tissue filling, tissue regeneration, drug release and other biomedical fields. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 is a schematic diagram of the synthesis of tyramine-modified carboxylated silk fibroin.

[0094] Figure 2 is the nuclear magnetic hydrogen spectrum of the tyramine-modified carboxylated silk fibroin obtained in Preparation Example 1.

[0095] Figure 3 is a photograph of the sample before and after the preparation of the tyramine-modified carboxylated silk fibroin hydrogel.

[0096] Figure 4 is the change of storage modulus during the gelation of the tyramine-modified carboxylated silk fibroin hydrogel in Example 4.

[0097] Figure 5 is the ultraviolet spectrum of the tyramine-modified carboxylated silk fibroin hydrogel before and after gelation in Example 4.

[0098] Figure 6 is the change of ultraviolet absorption at 335 nm during the gelation of the tyramine-modified carboxylated silk fibroin hydrogel in Example 4.

[0099] Figure 7 is the storage modulus of the tyramine-modified carboxylated silk fibroin hydrogel (prepared in Examples 1-5, respectively) cross-linked under different hydrogen peroxide concentrations. Note: The hydrogen peroxide concentration in the figure is the concentration in the hydrogel.

[0100] Figure 8 is the compression modulus of the tyramine-modified carboxylated silk fibroin hydrogel (prepared in Examples 1-5, respectively) cross-linked under different hydrogen peroxide concentrations. Note: The hydrogen peroxide concentration in the figure is the concentration in the hydrogel.

[0101] Figure 9 is the compression modulus of the tyramine-modified carboxylated silk fibroin hydrogel prepared in Example 4 at different time points during 28 days of storage at 37°C.

[0102] Figure 10 is the β-sheet structure content of the tyramine-modified carboxylated silk fibroin hydrogel prepared in Example 4 at different time points during 28 days of storage at 37°C.

[0103] Figure 11: Cell viability of NIH / 3T3 cells cultured on tyramine-modified carboxylated silk fibroin hydrogel surface at day 1 and day 3 in Example 10.

[0104] Figure 12: Cell survival rate of NIH / 3T3 cells cultured inside tyramine-modified carboxylated silk fibroin hydrogel for 48 hours in Example 11. The scale bar is 200 pm in size.

[0105] Figure 13: Schematic diagram of the process of encapsulating embryoid bodies inside tyramine-modified carboxylated silk fibroin hydrogel / carboxylated silk fibroin hydrogel in Examples 12 and 13.

[0106] Figure 14: Process of growing brain organoids inside tyramine-modified carboxylated silk fibroin hydrogel in Examples 12 and Comparative Examples. The scale bar is 500 pm in size in bright field images.

[0107] Figure 15: Immunofluorescence staining results of neural differentiation specific markers of brain organoids inside tyramine-modified carboxylated silk fibroin hydrogel at day 15 in Example 12. The scale bar is 200 pm in size in fluorescence images.

[0108] Figure 16: Immunofluorescence staining results of neural differentiation specific markers of brain organoids inside tyramine-modified carboxylated silk fibroin hydrogel at day 40 in Example 12. The scale bar is 50 pm in size in fluorescence images.

[0109] Figure 17: Process of growing brain organoids inside carboxylated silk fibroin hydrogel in Example 13. The scale bar is 500 pm in size in bright field images.

[0110] Figure 18: Immunofluorescence staining results of neural differentiation specific markers of brain organoids inside carboxylated silk fibroin hydrogel at day 15 in Example 13.

[0111] Figure 19: Immunofluorescence staining results of neural differentiation specific markers of brain organoids inside carboxylated silk fibroin hydrogel at day 40 in Example 13.

[0112] Description: SA in each of the above figures represents carboxylated silk fibroin; SA-TA represents tyramine-modified carboxylated silk fibroin. SF represents silk fibroin. DETAILED DESCRIPTION

[0113] Reagents and cell lines:

[0114] 2-(N-morpholino)ethanesulfonic acid (Aladdin), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (TCI), N-hydroxysuccinimide (TCI), hydrogen peroxide (Aladdin), horseradish peroxidase (ThermoFisher), mouse fibroblast NIH / 3T3 cell line (BeNa Bio), human induced pluripotent stem cells, hiPSCs (Frontier Innovation Center of Basic Medicine, Fudan University), Alamar Blue kit (Thermo Fisher), Calcein AM / PI Live-Dead Staining Kit (Lanboer), Gibco TM DMEM high glucose medium (Thermo Fisher), STEMdiff TM Brain organoid kit (containing EB formation medium, EB induction medium, expansion medium, maturation medium), Matrigel.

[0115] The term: In this application, organic amine refers to an organic amine containing a phenol group, and a representative example is tyramine.

[0116] Preparation Example 1: Preparation of tyramine-modified carboxylated silk protein

[0117] Dissolve 500 mg of carboxylated silk protein powder (synthesized according to Example 1 of CN 115433369 A, except that the reaction time is 3 hours) in 25 mL of 1x 2-(N-morpholino)ethanesulfonic acid solution. After complete dissolution, add 103.5 mg of N-hydroxysuccinimide and 172.5 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide, shake and mix for 30 minutes, then add 78.5 mg of tyramine hydrochloride, and shake and react for two hours. Then transfer the solution to a dialysis bag and dialyze in 0.5x PBS for 36 hours, changing the PBS solution every 12 hours. Then centrifuge the resulting solution at 9000 rpm, and take the supernatant. Finally, freeze-dry to obtain tyramine-modified carboxylated silk protein powder.

[0118] The resulting tyramine-modified carboxylated silk protein was measured by nuclear magnetic hydrogen spectrum using a 600 MHz liquid nuclear magnetic resonance spectrometer (ultra-low temperature probe) (Bruker AVANCE NEO, Bruker Corporation).

[0119] Method: Dissolve 10-20 mg of tyramine-modified carboxylated silk protein in 0.5 mL of 1.0 M DMSO-d6 / LiCl. 1 The H NMR nuclear magnetic resonance spectrum has a width of 0 ppm to 13 ppm, and is scanned 16 times. The results are shown in Figure 2.

[0120] The modification ratio of tyramine was calculated as follows: the peaks at 6.79 and 6.96 ppm in the 1H NMR spectrum correspond to the four hydrogen atoms at the ortho and meta positions of the phenolic hydroxyl group of tyramine, and the peak at 1.26 ppm corresponds to the three hydrogen atoms of the methyl group of alanine in silk protein. The modification ratio of tyramine was calculated by comparing the peak areas.

[0121] The content of phenolic groups in tyramine-modified carboxylated silk protein was calculated according to the following formula:

[0122] P(phenol) represents the total content of phenolic groups in tyramine-modified carboxylated silk protein;

[0123] P(Ala) represents the molar ratio of alanine in silk protein (28.6 mol%);

[0124] S SA-TA-phenolic represents the area integral of the hydrogen of the benzene ring in tyramine and tyrosine residues;

[0125] S SA-TA-Ala represents the area integral of the methyl hydrogen in alanine residues.

[0126] The total content of phenolic hydroxyl groups in tyramine-modified carboxylated silk protein was calculated to be 7.1 mol%.

[0127] Example 1: Preparation of tyramine-modified carboxylated silk protein hydrogel

[0128] Tyramine-modified carboxylated silk protein was dissolved in water to obtain a tyramine-modified carboxylated silk protein solution with a concentration of 25 mg / mL. The protein solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.01 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed in equal volumes at 37°C to form a hydrogel.

[0129] Example 2: Preparation of tyramine-modified carboxylated silk protein hydrogel

[0130] Tyramine-modified carboxylated silk protein was dissolved in water to obtain a tyramine-modified carboxylated silk protein solution with a concentration of 25 mg / mL. The protein solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.02 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed in equal volumes at 37°C to form a hydrogel.

[0131] Example 3: Preparation of tyramine-modified carboxylated silk protein hydrogel

[0132] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 25 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.04 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0133] Example 4: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0134] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 25 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.06 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0135] Example 5: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0136] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 25 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.08 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0137] Example 6: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0138] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 50 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.04 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0139] Example 7: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0140] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a tyramine-modified carboxylated silk fibroin solution of 50 mg / mL. The protein solution was divided into two parts, one was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.08 wt%), and the other was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0141] Example 8: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0142] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a 50 mg / mL tyramine-modified carboxylated silk fibroin solution. The solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.12 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0143] Example 9: Preparation of tyramine-modified carboxylated silk fibroin hydrogel

[0144] Tyramine-modified carboxylated silk fibroin was dissolved in water to obtain a 50 mg / mL tyramine-modified carboxylated silk fibroin solution. The solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (hydrogen peroxide concentration 0.12 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. Solution A and solution B were mixed at 37°C to form a hydrogel.

[0145] Test Example 1:

[0146] The gelation process of tyramine-modified carboxylated silk fibroin in Example 4 was scanned by ARES-G2 rotational rheometer. The test conditions were: 1% strain, frequency 1 Hz, scanning time 1500 seconds, and temperature 37°C. The results are shown in Figure 4. The figure shows the increase in storage modulus of tyramine-modified carboxylated silk fibroin during the transition from solution to gel state.

[0147] Test Example 2:

[0148] The UV spectra of tyramine-modified carboxylated silk fibroin before and after gelation in Example 4 were measured by UV-visible spectrophotometer. The results are shown in Figure 5. The results in the figure show that a new absorption peak appears at 335 nm after crosslinking of tyramine-modified carboxylated silk fibroin.

[0149] Test Example 3:

[0150] The absorption at 335 nm during the gelation process of tyramine-modified carboxylated silk fibroin in Example 4 was measured by UV-visible spectrophotometer. The results are shown in Figure 6. The results in the figure show that under the conditions of 10 U / mL HRP, 0.03 wt% H2O2, and temperature 37°C, tyramine-modified carboxylated silk fibroin can reach the platform of crosslinking reaction in 136 s.

[0151] Test Example 4:

[0152] The hydrogels in Examples 1-5 were tested for storage modulus using an ARES-G2 rotational rheometer, 1% strain, frequency 10 Hz, and the results are shown in Figure 7 (Note: the H2O2 concentrations shown in the figure are the concentrations in the hydrogels). The results show that for tyramide-modified carboxylated silk fibroin at 25 mg / mL, the optimal storage modulus is achieved at a H2O2 concentration of 0.03 wt% under the conditions of 10 U / mL HRP, temperature 37 °C, and H2O2 concentration range 0.005-0.04 wt%.

[0153] Test Example 5:

[0154] The hydrogels in Examples 1-5 were tested for compression modulus using a CellScale Univert mechanical tester, 30% strain, compression speed 0.667% s -1 , and the results are shown in Figure 8 (Note: the H2O2 concentrations shown in the figure are the concentrations in the hydrogels). The results show that for tyramide-modified carboxylated silk fibroin at 25 mg / mL, the optimal compression modulus is achieved at a H2O2 concentration of 0.03 wt% under the conditions of 10 U / mL HRP, temperature 37 °C, and H2O2 concentration range 0.005-0.04 wt%.

[0155] Test Example 6:

[0156] The hydrogels in Example 4 were stored in PBS and placed in a 37 °C incubator, and compression modulus tests were performed at days 0, 7, 14, 21, and 28, respectively, and the results are shown in Figure 9. The results show that the mechanical properties of the tyramide-modified carboxylated silk fibroin hydrogels remained stable for 28 days.

[0157] Test Example 7:

[0158] The hydrogels in Example 4 were stored in PBS and placed in a 37 °C incubator, and were freeze-dried at days 0, 7, 14, 21, and 28, respectively, and infrared spectroscopy was performed, and the spectra were recorded by 64 scans, and the spectral range was 400 to 4000 cm -1 , and the resolution was 4.0 cm -1 . The data analysis results show that the peaks at 1620 and 1698 cm -1 are β-sheet structure, and the peaks at 1645 and 1685 cm -1 are random coil / helix and β-turn structure, respectively.

[0159] The infrared spectroscopy peak separation results are shown in Figure 10. The results show that the β-sheet content in the tyramide-modified carboxylated silk fibroin hydrogels increased slightly but remained at a relatively low level (below 5%) for 28 days.

[0160] Example 10: Tyramide-modified carboxylated silk fibroin hydrogels for cell surface culture

[0161] Adherent NIH / 3T3 cells were digested, centrifuged, supernatant removed, and resuspended to obtain a cell suspension. Tyramide-modified carboxylated silk protein powder was dissolved in DMEM medium to form a protein solution of 25 mg / mL and filtered to remove bacteria. The protein solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (concentration 0.06 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) to prepare solution B. 50 μL of solution A and 50 μL of solution B were added to a 96-well plate, mixed uniformly to form a hydrogel, and placed in an incubator at 37°C for 1 minute. After complete gelation, 5000 cells were added to each well, 100 μL of medium was added to cover the surface of the hydrogel, and the plate was placed in a 37°C incubator for culture. On the first and third days, cell viability was determined using Alamar Blue reagent, and the results are shown in Figure 11. As can be seen in the figure, NIH / 3T3 cells can proliferate on the surface of tyramide-modified carboxylated silk protein hydrogel.

[0162] Example 11: Tyramide-modified carboxylated silk protein hydrogel encapsulating cells

[0163] Adherent NIH / 3T3 cells were digested, centrifuged, supernatant removed, and resuspended to obtain a cell suspension. Tyramide-modified carboxylated silk protein powder was dissolved in DMEM medium to form a protein solution of 25 mg / mL and filtered to remove bacteria. The protein solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (concentration 0.06 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) and added with the cell suspension (final concentration 1*10 6 Adherent NIH / 3T3 cells were digested, centrifuged, supernatant removed, and resuspended to obtain a cell suspension. Tyramide-modified carboxylated silk protein powder was dissolved in DMEM medium to form a protein solution of 25 mg / mL and filtered to remove bacteria. The protein solution was divided into two parts, one part was added with hydrogen peroxide to prepare solution A (concentration 0.06 wt%), and the other part was added with horseradish peroxidase solution (concentration 20 U / mL) and added with the cell suspension (final concentration 1*10

[0164] Example 12: Tyramide-modified carboxylated silk protein hydrogel for brain organoid culture

[0165] According to the STEMdiff TMThe brain organoid kit operation instruction, hiPSCs were cultured in ultra-low adsorption plate to form embryoid bodies. Observation on the 9th day, the size of the embryoid body was 400-600 μm, with smooth edges and formed an optically translucent edge. Dissolve tyramide-modified carboxylated silk protein powder with expansion medium to form a 25 mg / mL solution, mix 20 μg / mL of laminin, or 100 μg / mL of collagen, or 40 μg / mL of fibronectin, or a mixture of the three proteins in it, and filter sterilization, divide the protein solution into two parts, one part add hydrogen peroxide to prepare solution A (concentration 0.04wt%), one part add horseradish peroxidase solution (concentration 10 U / mL) to prepare solution B. Laying the sealing film, gently pressing with tweezers to make a small groove on the sealing film, ultraviolet sterilization for 60 minutes. Use a cut-off dropper to transfer the embryoid bodies to the groove on the sealing film, suck out the original induction medium, add 10 μL of solution A and 10 μL of solution B in turn, and let the embryoid bodies be embedded in the hydrogel after one minute. Put the hydrogel with embryoid bodies into a 24-well plate, 500 μL of expansion medium per well. Put into the incubator at 37℃ for 3 days of shaking culture. The surface of the embedded embryoid bodies will develop sprouts. Suck out the original expansion medium, add 500 μL of maturation medium per well. Put into the incubator at 37℃ for shaking culture, change the medium every 3 days. (Note: The medium replacement in brain organoid culture refers to STEMdiff TM Brain organoid kit operation instruction)

[0166] The embryoid bodies will continue to grow and eventually develop into brain organoids. The culture process is shown in Figure 13. The growth and development of the organoids are shown in Figure 14. On the 15th day and the 40th day, the brain organoids were fixed, paraffin-embedded and sectioned, and immunofluorescence staining was performed on specific markers SOX2 (stem cell marker), Nestin (neural precursor cell marker), Tuj-1 (neural class III beta tubulin), PAX6 (glial cell marker), FOXG1 (forebrain marker), DCX (doublecortin), MAP2 (microtubule-associated protein 2), CTIP (cortical plate neuron marker), GFAP (astrocyte marker), NeuN (neuronal nuclear marker), respectively. The results are shown in Figures 15 (15th day) and 16 (40th day), respectively. The staining results show that on the 15th day, the organoids appear neural differentiation cells and specific "rosette" structures, and on the 40th day, the organoids appear more mature neural differentiation cells.

[0167] Comparative example: brain organoid culture with matrigel

[0168] According to STEMdiff TMBrain organoid kit operation instruction, hiPSCs were cultured in ultra-low adsorption plate to form embryoid bodies. Observation on the 9th day, the size of the embryoid body was 400-600 μm, with smooth edges and formed an optically translucent edge. The embryoid body was taken out with a cut-off pipette, and the residual medium was sucked off. Matrigel was thawed in advance in the 4-degree refrigerator, and 15 μL of matrigel was added to the embryoid body with a pre-cooled gun head, so that it was completely coated. The matrigel-coated embryoid body was placed in a 37°C incubator for half an hour, and then the expansion medium was added and placed in a 37°C incubator for 3 days. The surface of the embedded embryoid body developed sprouts. The original expansion medium was sucked off, and 500 mL of maturation medium was added to each well. Place in a 37°C incubator and shake culture, and replace the culture medium every 3 days. The embryoid body will continue to grow and eventually develop into a brain organoid. On the 15th and 40th days, the brain organoids were fixed, paraffin-embedded and sectioned, and specific markers SOX2, Nestin, Tuj-1, PAX6, FOXG1, DCX, MAP2, CTIP, GFAP, and NeuN were immunofluorescently stained. The results are shown in Figures 14, 15, and 16.

[0169] The results of Example 12 and the comparative example show that the tyramide-modified carboxylated silk fibroin hydrogel can support the growth and development of brain organoids, and the size is close. The sections of brain organoids in matrigel and tyramide-modified carboxylated silk fibroin hydrogel on the 15th day have a characteristic "rosette" structure, and have SOX2, Nestin, Tuj-1, PAX6, and FOXG1 protein expression; the 40th day section staining shows that FOXG1, DCX, MAP2, CTIP, GFAP, and NeuN proteins are expressed.

[0170] Example 13: Carboxylated silk fibroin hydrogel for brain organoid culture

[0171] According to STEMdiff TMThe brain-like organ kit operation instruction is that the hiPSCs are cultured in an ultra-low adsorption plate to form a blastoid. On the 9th day of observation, the blastoid is 400-600 μm in size, has a smooth edge and forms an optically translucent edge. Carboxylated silk fibroin (synthesized in Example 1 of CN 115433369 A, except that the silk fibroin degumming time is 2 hours and the carboxylation reaction time is 30 minutes) is dissolved in an expansion culture medium to form a solution of 20 mg / mL, 40 μg / mL of laminin, or 20 μg / mL of collagen, or 10 μg / mL of fibronectin, or 0.2 mg / mL of hyaluronic acid, or four ingredients are simultaneously added according to the above concentrations are mixed, and sterilized by filtration. The carboxylated silk fibroin solution is incubated in a 37°C incubator in advance to form a hydrogel. The parafilm is laid flat, and a small groove is formed on the parafilm by gently pressing it with tweezers. Ultraviolet sterilization is performed for 60 minutes. The blastoid is transferred to the groove on the parafilm using a cut-off dropper, the original induction culture medium is aspirated, 20 μL of carboxylated silk fibroin hydrogel is added, and the blastoid is embedded in the hydrogel after being placed in a 37°C incubator for 2 hours. The blastoid-embedded hydrogel is placed in a 24-well plate, and 500 μL of expansion culture medium is added to each well. The plate is placed in a 37°C incubator and shaken for 3 days. The original expansion culture medium is aspirated, and 500 μL of maturation culture medium is added to each well. The plate is placed in a 37°C incubator and shaken, and the culture medium is replaced every 3 days. (Note: The culture medium replacement in the brain-like organ culture refers to STEMdiff TM Brain-like organ kit operation instruction)

[0172] The blastoid will continue to grow and eventually develop into a brain-like organ. The culture process is shown in FIG. 17. The brain-like organ is peeled off from the carboxylated silk fibroin hydrogel on the 15th day and the 40th day, fixed, frozen sectioned, and subjected to immunofluorescence staining for specific markers SOX2, Nestin, Tuj-1, PAX6, DCX, MAP2, CTIP, GFAP, NeuN, CD31 (platelet endothelial cell marker). Among them, FIG. 18 (15th day) shows the results of using Matrigel, carboxylated silk fibroin hydrogel, and carboxylated silk fibroin hydrogel with the addition of laminin, or fibronectin, or hyaluronic acid at the above concentrations, or the addition of collagen, laminin, fibronectin and hyaluronic acid for organoid culture, and FIG. 19 (40th day) shows the results of using Matrigel and carboxylated silk fibroin hydrogel for organoid culture. The staining results show that on the 15th day, the organoid appears neural differentiation cells and specific "rosette" structures, and on the 40th day, the organoid appears more mature neural differentiation cells.

Claims

1. A method of making an organic amine-modified carboxylated silk protein, the method comprising: carboxylated silk fibroin is subjected to amidation reaction with an organic amine to obtain an organic amine modified carboxylated silk fibroin, wherein, The carboxylated silk protein is a silk protein having a side chain containing the following structure: wherein R represents a C2-C6 alkylene group, represents the connection to the side chain from this position; the organic amine carries a -NH2 and a phenol group, and the amidation reaction is carried out between the -NH2 and the carboxyl group of the carboxylated silk fibroin, preferably, in the carboxylated silk fibroin, Structure the modification rate of serine is between 20-90%, R represents ethylene or propylene; The organic amine is represented by the structure: wherein R1 represents C1-C6 alkylene, preferably, R1 represents methylene, ethylene, propylene or butylene, preferably, in the organic amine, R1 is connected to the para or meta position of the hydroxyl group, in particular the para position, in particular, the organic amine is tyramine.

2. The method according to claim 1, which is carried out as follows: S1: preparing a carboxylated silk fibroin solution; S2: adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the carboxylated silk fibroin solution obtained in S1 to activate the carboxylated silk fibroin; S3: adding tyramine hydrochloride to the activated carboxylated silk fibroin in S2 to carry out the reaction to obtain tyramine modified carboxylated silk fibroin, preferably, the carboxylated silk fibroin solution is prepared by dissolving carboxylated silk fibroin powder in an aqueous solution of 2-(N-morpholino) ethanesulfonic acid, the concentration of carboxylated silk fibroin in the carboxylated silk fibroin solution is 1-100 mg / mL, and the concentration of 2-(N-morpholino) ethanesulfonic acid is 5-100 mM, preferably, in S2, the amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide added is 172.5-1725 mg, preferably 345 mg, and the amount of N-hydroxysuccinimide added is 103.5-1035 mg, preferably 207 mg, relative to 1 g of carboxylated silk fibroin, preferably, in S3, the amount of tyramine hydrochloride added is 31.4-314 mg, the reaction temperature is 18-25°C, the reaction time is 1-24 hours, preferably 2 hours, and the pH of the reaction system is 6, and the reaction is carried out under shaking at a rotation speed of 25-100 rpm.

3. An organic amine modified carboxylated silk protein comprising wherein R and R1are each as defined in claim 1, represents the connection to the side chain from this position; preferably, the total content of phenol groups in the organic amine modified carboxylated silk fibroin is 4.6 mol% to 10 mol%, for example 7.1 mol%, based on the total moles of amino acid units in the silk fibroin.

4. A method for preparing an organic amine modified carboxylated silk fibroin hydrogel, the method comprising: S1-1: dissolving the organic amine modified carboxylated silk fibroin according to claim 3 in water to obtain an aqueous solution of organic amine modified carboxylated silk fibroin; S1-2: dividing the solution obtained in S1-1 into two equal parts, adding hydrogen peroxide to one part to prepare solution A, and adding horseradish peroxidase (HRP) to the other part to prepare solution B; S1-3: mixing equal volumes of solution A and solution B to obtain an organic amine modified carboxylated silk fibroin hydrogel, preferably, in S1-1, the concentration of organic amine modified carboxylated silk fibroin in the organic amine modified carboxylated silk fibroin solution is 25-50 mg / mL. In S1-2, the concentration of hydrogen peroxide in solution A is 0.01wt%-0.16wt%, for example, 0.06wt%; the concentration of horseradish peroxidase in solution B is 1-20U / mL; In S1-3, the mixing temperature of solution A and solution B is 20-55℃, for example, at 37℃.

5. An organic amine modified carboxylated silk fibroin hydrogel prepared by the method of claim 4.

6. A method for preparing a carboxylated silk fibroin hydrogel, the method comprising: standing the aqueous solution of carboxylated silk fibroin at room temperature or under heating to prepare a carboxylated silk fibroin hydrogel, wherein the carboxylated silk fibroin is as defined in claim 1.

7. A carboxylated silk fibroin hydrogel prepared by the method of claim 6.

8. A Matrigel for cell or organoid culture, comprising at least: the organic amine modified carboxylated silk fibroin hydrogel of claim 5 or the carboxylated silk fibroin hydrogel of claim 7, and optionally, a natural material selected from the group consisting of laminin, collagen, fibronectin, gelatin, serum protein, milk protein, hyaluronic acid, sodium alginate, chitosan, dextran, chondroitin sulfate and carboxymethyl cellulose.

9. Use of the organic amine modified carboxylated silk fibroin of claim 3, the organic amine modified carboxylated silk fibroin hydrogel of claim 5 or the carboxylated silk fibroin hydrogel of claim 7 in the preparation of a biomaterial, preferably, the biomaterial is used for organoid culture, cell delivery, biological 3D printing, tissue filling, tissue regeneration, drug release.

10. A kit for cell and organoid culture, the kit comprising: the organic amine modified carboxylated silk fibroin of claim 3; hydrogen peroxide solution and horseradish peroxidase; and optionally, a natural material for promoting the interaction between cells and materials, improving cell adhesion and promoting cell differentiation; or the carboxylated silk fibroin as defined in claim 1; and optionally, a natural material for promoting the interaction between cells and materials, improving cell adhesion and promoting cell differentiation, preferably, the natural material is selected from the group consisting of laminin, collagen, fibronectin, gelatin, serum protein, milk protein, hyaluronic acid, sodium alginate, chitosan, dextran, chondroitin sulfate and carboxymethyl cellulose, more preferably, the natural material is selected from the group consisting of laminin, collagen, fibronectin.

11. A method for three-dimensional culture of cells, the method comprising: S2-1: dissolving the organic amine modified carboxylated silk fibroin of claim 3 in DMEM medium, and dividing the solution into two parts, one part adding hydrogen peroxide to obtain solution A', and the other part adding horseradish peroxidase to obtain solution B'; S2-2: digesting, collecting, centrifuging and resuspending adherent cells to prepare a cell suspension; S2-3: adding the cell suspension to solution B' of step S2-1, and then mixing solutions A' and B' in equal volumes, and incubating in a 37℃ incubator for 1 minute. S2-4: After the gel of S2-3 is completely formed into a hydrogel, add culture medium to cover the hydrogel, and place in a 37℃ incubator for three-dimensional culture of cells.

12. A three-dimensional culture method of an organoid, the method being one of the following methods: Method (i): The organic amine-modified carboxylated silk fibroin of claim 3 is dissolved in expansion medium, blended with natural materials, and the solution is divided into two parts, one part is added with hydrogen peroxide to obtain solution A", and the other part is added with horseradish peroxidase to obtain solution B"; An equal volume of solution A" and B" is simultaneously added dropwise on the embryoid bodies (EB) formed by induced culture of human induced pluripotent stem cells, incubated in a 37℃ incubator for 1-20 minutes to obtain EBs encapsulated by hydrogel, and then expansion medium is added and placed in a 37℃ shaker for shaking culture; starting from the 11th day, the medium is replaced with induced brain organoid maturation medium for subsequent culture, Method (ii) The carboxylated silk fibroin as defined in claim 1 is dissolved in expansion medium, blended with natural materials, and incubated in a 37℃ incubator to form a carboxylated silk fibroin hydrogel; The carboxylated silk fibroin hydrogel is added dropwise on the embryoid bodies (EB) formed by induced culture of human induced pluripotent stem cells, incubated in a 37℃ incubator for 60-180 minutes to obtain EBs encapsulated by hydrogel, and then expansion medium is added and placed in a 37℃ shaker for shaking culture; starting from the 11th day, the medium is replaced with induced brain organoid maturation medium for subsequent culture.

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