Collagen-based hydrogel and preparation method thereof, in vitro three-dimensional model and culture method thereof, and drug detection method

Abstract

The present invention proposes a collagen-based hydrogel and a preparation method thereof, an in vitro three-dimensional model and a culture method thereof, and a method for drug detection, which belong to the field of tissue engineering technology. The preparation method comprises: dissolving type I collagen in an acid solution, adjusting the pH to neutral, and forming a neutral collagen solution; adding a metal ligand complex photoinitiator and a sodium persulfate photoinitiator to the neutral collagen solution, and forming a hydrogel prepolymer solution after light protection treatment; the hydrogel prepolymer solution is photocrosslinked to form a collagen-based hydrogel. The present invention adopts a photocuring method to form a collagen-based hydrogel with a dense structure. The preparation method is simple, the raw materials are easily available, the cost is low, and the formed hydrogel is transparent, the composition is clear, and there is no cytotoxicity. The biological activity and mechanical properties are good, which meets the needs of three-dimensional cell culture and monitoring of cell growth status.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering, and specifically relates to a collagen-based hydrogel and a preparation method thereof, an in vitro three-dimensional model and a cultivation method thereof, and a drug detection method. Background Art

[0002] Organoids are a type of three-dimensional cell culture that highly simulates human organs in structure and function. They can simulate spatial structures similar to organs and differentiate into corresponding functions. They have the characteristics of cell proliferation and differentiation, self-renewal, self-assembly, long-term culture, and genetic stability. They provide a valuable tissue source for a variety of biomedical applications such as drug screening and toxicology research.

[0003] During the culture process of organoids, the extracellular matrix (ECM) plays an important role in the biomechanical properties of tissues and affects cell behavior and fate. Among them, collagen, as the main component of the extracellular matrix, has the functions of promoting cell growth, differentiation, adhesion and migration.

[0004] However, the heterogeneity of BME components limits the precise regulation of its physical and biochemical properties. In addition, BME has weak mechanical properties, which has intrinsic limitations in recapitulating tumor tissue mechanics and is difficult to adapt to various unique tumor microenvironments.

[0005] In addition, decellularized matrices and decellularized matrices from corresponding organs of organoids have similar matrix material components and factors. However, the composition of decellularized matrices is unclear, and it is impossible to control the setting of experimental parameters and experimental analysis. Matrigel is currently used in in vitro three-dimensional organ culture. However, due to the unclear composition of Matrigel and the instability between batches, its use in large-scale and standardized organoid culture and organoid drug sensitivity screening is greatly limited.

[0006] Synthetic hydrogels can solve problems such as unclear composition of natural matrix gel, animal origin, and unstable batches. However, the synthesis process is relatively complicated, and pure PIC hydrogels have poor biocompatibility, and generally require grafting to promote cell adhesion. The application of collagen is limited by its poor mechanical properties and temperature effects on its concentration, which is insufficient to support three-dimensional cell culture. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art, and provides a collagen-based hydrogel and a preparation method thereof, an in vitro three-dimensional model and a culture method thereof, and a drug detection method.

[0008] In one aspect of the present invention, a method for preparing a collagen-based hydrogel is provided, the preparation method comprising:

[0009] Dissolving type I collagen in an acid solution and adjusting the pH to neutral to form a neutral collagen solution;

[0010] Adding a metal ligand complex photoinitiator and a sodium persulfate photoinitiator to the neutral collagen solution, and performing light-proof treatment to form a hydrogel prepolymer solution;

[0011] The hydrogel prepolymerization solution is photo-crosslinked to form a collagen-based hydrogel.

[0012] Optionally, the metal-ligand complex photoinitiator is tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate.

[0013] Optionally, in the hydrogel prepolymerization solution, the concentration of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate ranges from 0.0005 to 0.005 mol / L; and / or,

[0014] The concentration of the sodium persulfate is 0.005-0.05 mol / L; and / or,

[0015] The concentration of type I collagen is 1-10 mg / mL.

[0016] Optionally, the light-shielding treatment time ranges from 15 to 60 minutes; and / or,

[0017] The time range of the photocrosslinking is 0.5 to 5 minutes.

[0018] Optionally, the hydrogel prepolymer solution is photo-crosslinked and further comprises:

[0019] A thermosensitive material is added to the hydrogel prepolymer solution, the type I collagen is photocrosslinked to form a first network structure, and the thermosensitive material is thermally crosslinked to form a second network structure, and the first network structure and the second network structure are interlaced and interpenetrated.

[0020] Optionally, the hydrogel prepolymer solution is photo-crosslinked and further comprises:

[0021] A photosensitive material and any one of the photoinitiators selected from the group consisting of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone are added to the hydrogel prepolymer solution, and the type I collagen and the photosensitive material are cross-linked through light to form a first network structure and a third network structure that are interpenetrating with each other.

[0022] Another aspect of the present invention provides a collagen-based hydrogel, which is prepared by the preparation method described above.

[0023] Another aspect of the present invention provides a collagen-based hydrogel, comprising a first network structure formed by photocrosslinking of type I collagen under the action of a metal ligand complex photoinitiator and a sodium persulfate photoinitiator; and

[0024] A second network structure formed by thermal crosslinking of a temperature-sensitive material; and / or,

[0025] A third network structure formed by photosensitive materials being cross-linked by light under the action of any one of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2,4,6-trimethylbenzoyl phenyl phosphonic acid ethyl ester, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone;

[0026] The second network structure and / or the third network structure interweave with the first network structure.

[0027] Another aspect of the present invention provides a method for culturing an in vitro three-dimensional model, the method comprising:

[0028] Forming a cell suspension for a three-dimensional model to be cultured; the cells in the cell suspension include cell lines, primary cells, and stem cells;

[0029] Add the hydrogel prepolymer solution before cross-linking of the collagen-based hydrogel described above,

[0030] Mixing the hydrogel prepolymer solution with the cell suspension, adding an appropriate amount of the mixture into a culture chamber or a culture well for culturing the cells, and forming a cell gel through photocrosslinking;

[0031] A culture medium is added to the culture cavity or culture well containing the cell gel, and an in vitro three-dimensional model is obtained through culture.

[0032] Another aspect of the present invention provides a method for culturing an in vitro three-dimensional model, the method comprising:

[0033] Forming a cell or cell cluster suspension of an organoid to be cultured; the cells or cell clusters in the cell or cell cluster suspension are obtained by digesting, separating and resuspending tumor tissue or non-tumor tissue, or by digesting, separating and resuspending tumor organoids or non-tumor organoids to be passaged;

[0034] Obtaining a hydrogel prepolymerization solution, wherein the hydrogel prepolymerization solution comprises type I collagen, a metal ligand complex photoinitiator, and a sodium persulfate photoinitiator;

[0035] Mixing the hydrogel prepolymer solution with the cell or cell cluster suspension, adding an appropriate amount of the mixture into a culture chamber or culture well for culturing the organoid, and forming an organoid gel through photocrosslinking;

[0036] A culture medium is added to the culture chamber or culture well containing the organoid gel, and an in vitro three-dimensional model of a tumor organoid or a non-tumor organoid is obtained through culture.

[0037] Another aspect of the present invention provides an in vitro three-dimensional model, which is formed by culturing using the culturing method described above.

[0038] Another aspect of the present invention provides a method for drug detection using the above-mentioned three-dimensional in vitro model, the drug detection method comprising:

[0039] Introducing the drug to be tested into the in vitro three-dimensional model;

[0040] Obtain the effect of the drug to be tested on the in vitro three-dimensional model.

[0041] The present invention proposes a collagen-based hydrogel and a preparation method thereof, an in vitro three-dimensional model and a culture method thereof, and a method for drug detection. The preparation method comprises: dissolving type I collagen in an acid solution, adjusting the pH to neutral, and forming a neutral collagen solution; adding a metal ligand complex photoinitiator and a sodium persulfate photoinitiator to the neutral collagen solution, and performing a light-proof treatment to form a hydrogel prepolymer solution; and the hydrogel prepolymer solution is subjected to photocrosslinking to form a collagen-based hydrogel. The present invention adopts a photocuring method to form a collagen-based hydrogel with a dense structure. The preparation method is simple, the raw materials are easily available, the cost is low, and the formed hydrogel is transparent, has clear ingredients, is non-cytotoxic, has good biological activity and mechanical properties, and meets the needs of three-dimensional cell culture and monitoring of cell growth status. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a flowchart of a method for preparing a collagen-based hydrogel according to an embodiment of the present invention;

[0043] Figure 2 A flowchart of a method for cultivating an in vitro three-dimensional model according to another embodiment of the present invention;

[0044] Figure 3 A flowchart of a method for cultivating an in vitro three-dimensional model according to another embodiment of the present invention;

[0045] Figure 4 A flowchart of a drug detection method according to another embodiment of the present invention;

[0046] Figure 5 Schematic diagram of the structure of the self-made collagen-based hydrogel of Example 1 of the present invention and the hydrogel of the control group; wherein, Figure 5 (A) is an electron microscopic image of the hydrogel in the control group at 1000x. Figure 5 (B)

[0047] This is an electron micrograph of a homemade collagen-based hydrogel at 1000x;

[0048] Figure 6 Schematic diagram of the structure of the self-made collagen-based hydrogel of Example 2 of the present invention; wherein, Figure 6 (A) is an electron micrograph of a homemade collagen-based hydrogel at a collagen concentration of 1 mg / mL at 1000x. Figure 6 (B) is an electron micrograph of a homemade collagen-based hydrogel at a collagen concentration of 2 mg / mL at 1000x. Figure 6 (C) is an electron micrograph of a homemade collagen-based hydrogel at a collagen concentration of 10 mg / mL at 1000x;

[0049] Figure 7 Schematic diagram of the structure of the homemade collagen-based hydrogel of Example 3 of the present invention and the hydrogel of the control group; wherein, Figure 7 (A) is an electron microscopic image of the hydrogel in the control group at 1000x. Figure 7 (B)

[0050] This is an electron micrograph of a homemade collagen-based hydrogel at 1000x;

[0051] Figure 8 Schematic diagram of the structure of the self-made collagen-based hydrogel of Example 4 of the present invention and the hydrogel of the control group; wherein, Figure 8 (A) is an electron microscopic image of the hydrogel in the control group at 1000x. Figure 8 (B)

[0052] This is an electron micrograph of a homemade collagen-based hydrogel at 1000x;

[0053] Fig. 9 Microscopic observation of the homemade collagen-based hydrogel of Example 5 of the present invention and the control group hydrogel used to culture pancreatic cancer organoids. DETAILED DESCRIPTION

[0054] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0055] like Figure 1 As shown, in one aspect of the present invention, a method S100 for preparing a collagen-based hydrogel is provided, comprising steps S110 to S130:

[0056] S110, dissolving type I collagen in an acid solution, adjusting the pH to neutral, and forming a neutral collagen solution.

[0057] Specifically, type I collagen was dissolved in 0.02 M acetic acid solution, NaOH was added to adjust the pH to neutral, and the mixture was mixed evenly to obtain a neutral collagen solution.

[0058] In some preferred embodiments, the collagen is naturally extracted animal collagen or recombinant collagen prepared by a microbial method.

[0059] S120, adding a metal ligand complex photoinitiator and a sodium persulfate photoinitiator to a neutral collagen solution, and subjecting the solution to light-proof immersion treatment so that the photoinitiator penetrates the colloid to obtain a hydrogel prepolymer solution.

[0060] In some preferred embodiments, the metal-ligand complex photoinitiator is tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate.

[0061] In other preferred embodiments, in the hydrogel prepolymerization solution, the concentration of type I collagen is 1-10 mg / mL, the concentration of tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator is 0.0005-0.005 mol / L, and the concentration of sodium persulfate photoinitiator is 0.005-0.05 mol / L. The above preferred concentrations of type I collagen are beneficial to improving the mechanical strength and biological activity of the hydrogel. When the collagen concentration is high, the hydrogel remains transparent, which is conducive to cell culture observation; at the preferred concentrations of the above photoinitiators, the reaction speed is faster, the gelation speed is faster, the efficiency of organoid culture is effectively improved, and the toxicity is lower, further improving the success rate of organoid culture.

[0062] In other preferred embodiments, the time range of the light-proof immersion treatment is 15 to 60 minutes, which is conducive to the penetration of the photoinitiator into the colloid.

[0063] In other preferred embodiments, the hydrogel prepolymer solution can be filtered through a filter membrane to obtain a sterile solution for cell culture, for example, filtered through a 0.22 micron filter membrane.

[0064] S130, cross-linking the hydrogel prepolymer solution for a certain period of time under irradiation with light at 240 to 500 nm to form a collagen-based hydrogel with a dense structure, wherein the collagen-based hydrogel has a first network structure.

[0065] In some preferred embodiments, the time range of photocrosslinking is 0.5 to 5 minutes.

[0066] In this embodiment, by selecting a suitable photoinitiator, type I collagen is photocured to form a dense collagen-based hydrogel, which effectively improves the cross-linking density. The preparation method is simple, the raw materials are easily available, the cost is low, and the formed hydrogel is transparent, with clear ingredients, no cytotoxicity, good biological activity and mechanical properties, which meets the needs of three-dimensional cell culture and monitoring of cell growth status.

[0067] Furthermore, in this embodiment, in addition to forming a single-component collagen-based hydrogel, type I collagen can also be compounded with other materials to form an interpenetrating network collagen-based hydrogel. For example, a thermosensitive material can be compounded with type I collagen, or a photosensitive material can be compounded with type I collagen. Of course, a thermosensitive material, a photosensitive material and type I collagen can also be compounded at the same time to form a collagen-based hydrogel.

[0068] Exemplarily, the thermosensitive material is compounded with type I collagen to form an interpenetrating network collagen-based hydrogel, which specifically includes the following steps:

[0069] S1. Dissolve type I collagen in 0.02 M acetic acid solution, add NaOH to adjust the pH to neutral, and obtain a neutral collagen solution.

[0070] In some preferred embodiments, the collagen is naturally extracted animal collagen or recombinant collagen prepared by a microbial method.

[0071] S2. Add tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate solution photoinitiator to the neutral collagen solution, mix evenly in the dark, and form a hydrogel prepolymer solution.

[0072] In some preferred embodiments, in the hydrogel prepolymerization solution, the concentration of type I collagen is 0.5-10 mg / mL, the concentration of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate is 0.0005-0.005 mol / L, and the concentration of sodium persulfate is 0.005-0.05 mol / L.

[0073] S3. Add thermosensitive material to the hydrogel prepolymer solution, first crosslink it under light with a wavelength of 240-500nm for a certain period of time, so that type I collagen forms a first network structure through photocrosslinking under the action of photoinitiator, and then crosslink it at a water bath temperature of 25-37℃ for a certain period of time, so that the thermosensitive material forms a second network structure through thermal crosslinking. The first network structure and the second network structure are intertwined to obtain an interpenetrating network with a dense structure of collagen-based hydrogel, which is used to simulate the spatial structure similar to organs.

[0074] In other preferred embodiments, the concentration of the temperature-sensitive material is 0.1% to 30% (mass-to-volume ratio). When the concentration is too low, gelation cannot be achieved. When the concentration is too high, the viscosity of the solution is too high, making operation difficult.

[0075] In some preferred embodiments, the temperature-sensitive material is one of polyisocyanate polypeptide (PIC) and polyoxyethylene polyoxypropylene ether (Pluronic F-127). The preferred concentration of polyoxyethylene polyoxypropylene ether (Pluronic F-127) is 10%-30% (mass volume ratio); the preferred concentration of polyisocyanate polypeptide (PIC) is 0.1%-1% (mass volume ratio).

[0076] In other preferred embodiments, the light cross-linking time ranges from 0.5 to 5 min; the heat cross-linking time ranges from 15 to 60 min.

[0077] It should be noted that when the hydrogel of this embodiment is used for cell culture, the above-mentioned thermal cross-linking process can be a cell culture process. For example, the above-mentioned hydrogel is placed in an incubator with 95% humidity, 5% CO2, and 37°C for cell culture, and the thermal cross-linking process of the thermosensitive material is simultaneously realized to achieve double cross-linking, effectively improve the cross-linking density, and enhance the mechanical properties. There is no need to add additional cross-linking steps. Only one step of photo-cross-linking is required to obtain the interpenetrating network hydrogel, and the preparation process is simple.

[0078] In this embodiment, when the thermosensitive material is compounded with type I collagen, under light initiation conditions, the tyrosine residues on type I collagen undergo an oxidative coupling reaction to form a first network structure, and under heat initiation conditions, the thermosensitive material forms a second network structure, and the second network structure and the first network form a two-layer interpenetrating network hydrogel. For example, the thermosensitive material is any one of polyisocyanurate polypeptide (PIC) and polyoxyethylene polyoxypropylene ether (Pluronic F-127), and its hydrophobic chain ends are aggregated to form a second network structure. Due to polar groups, hydrogen bonds are formed between the networks to form a two-layer interpenetrating network hydrogel with the first network structure.

[0079] Furthermore, the photosensitive material is compounded with type I collagen to form an interpenetrating network collagen-based hydrogel, which specifically includes the following steps:

[0080] S1. Dissolve type I collagen in 0.02 M acetic acid solution, add NaOH to adjust the pH to neutral, and obtain a neutral collagen solution.

[0081] S2. Add tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate solution photoinitiator to the neutral collagen solution, mix evenly in the dark, and form a hydrogel prepolymer solution.

[0082] S3. Add photosensitive material and a certain amount of photoinitiator to the hydrogel prepolymer solution, and cross-link under irradiation of 240-500nm, so that type I collagen and the photosensitive material form an interpenetrating first network structure and a third network structure respectively under the action of their respective corresponding photoinitiators, so as to obtain an interpenetrating network collagen-based hydrogel for simulating an organ-like spatial structure.

[0083] In some preferred embodiments, the photosensitive material is one of methacryloyl gelatin (GelMA), methacryloyl hyaluronic acid (HAMA), methacryloyl sodium alginate (AlgMA), methacryloyl chitosan (CSMA), methacryloyl silk fibroin (SilMA), methacryloyl chondroitin sulfate (ChSMA), and methacryloyl heparin (HepMA);

[0084] In other preferred embodiments, the concentration of the photosensitive material is 0.5-20% (mass-to-volume ratio). When the concentration is lower than 0.5%, the hydrogel is soft or cannot gel, and cannot support the three-dimensional growth of cells. When the concentration is higher than 20%, the hydrogel becomes hard and has poor compatibility with cells.

[0085] In other preferred embodiments, the photoinitiator of the photosensitive material in the above step S3 is any one of phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), 2,4,6-trimethylbenzoylphenyl phosphonic acid ethyl ester (TPO), and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (2959).

[0086] In this embodiment, when the photosensitive material is compounded with type I collagen, under light-initiated conditions, the tyrosine residues on the type I collagen undergo an oxidative coupling reaction to form a first network structure, and the double bonds on the photosensitive material undergo a polymerization reaction to form a third network structure. The two-layer network structure is connected through the carboxyl groups on the collagen and the amino groups on the partially double-bonded photosensitive material to form a two-layer interpenetrating network hydrogel.

[0087] Furthermore, the thermosensitive material, the photosensitive material and type I collagen are compounded to form an interpenetrating network collagen-based hydrogel, which specifically includes the following steps:

[0088] S1. Dissolve type I collagen in 0.02 M acetic acid solution, add NaOH to adjust the pH to neutral, and obtain a neutral collagen solution.

[0089] S2. Add tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate solution photoinitiator to the neutral collagen solution, mix evenly in the dark, and form a hydrogel prepolymer solution.

[0090] S3. Add photosensitive material, photoinitiator and thermosensitive material to the hydrogel prepolymer solution respectively, first cross-link them under 240-500nm light irradiation for a certain time, so that type I collagen and photosensitive material form a first network structure and a third network structure respectively under the action of their respective corresponding photoinitiators, then cross-link them in a 25-37℃ water bath for a certain time, so that the thermosensitive material forms a second network structure through thermal cross-linking, thus obtaining a collagen-based hydrogel with an interlaced three-layer network structure, which is used to simulate an organ-like spatial structure.

[0091] It should be noted that, in the above preparation process, the temperature-sensitive material and its concentration, the photosensitive material and its concentration, the photoinitiator in step S3, and the time of thermal crosslinking and photocrosslinking are the same as those described above.

[0092] In this embodiment, when the photosensitive material and the thermosensitive material are simultaneously compounded with type I collagen, under light-initiated conditions, the tyrosine residues on the type I collagen undergo an oxidative coupling reaction to form a first network structure, and the double bonds on the photosensitive material undergo a polymerization reaction to form a third network structure. Under thermal-initiated conditions, the thermosensitive material, such as the hydrophobic chain ends, aggregate to form a second network structure. Due to the polar groups, hydrogen bonds are formed between the networks to form a three-layer interpenetrating network hydrogel.

[0093] Another aspect of the present invention provides a collagen-based hydrogel, which is formed by the method described above. Please refer to the above description for the specific preparation process, which will not be repeated here.

[0094] Specifically, the collagen-based hydrogel includes a first network structure formed by photocrosslinking of type I collagen under the action of a metal ligand complex photoinitiator and a sodium persulfate photoinitiator, wherein the metal ligand complex photoinitiator is preferably tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate.

[0095] Furthermore, the collagen-based hydrogel may also include a first network structure and a second network structure that are interlaced and interpenetrating, wherein the second network structure is formed by thermal crosslinking of a thermosensitive material, and the thermosensitive material is preferably one of polyisocyanurate polypeptide (PIC) and polyoxyethylene polyoxypropylene ether (Pluronic F-127).

[0096] Furthermore, the collagen-based hydrogel can also include an interlaced first network structure and a third network structure, wherein the third network structure is formed by photo-crosslinking of a photosensitive material and any one of the photoinitiators selected from the group consisting of phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), 2,4,6-trimethylbenzoyl phenyl phosphonate (TPO), and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (2959).

[0097] In some preferred embodiments, the photosensitive material is one of methacryloyl gelatin (GelMA), methacryloyl hyaluronic acid (HAMA), methacryloyl sodium alginate (AlgMA), methacryloyl chitosan (CSMA), methacryloyl silk fibroin (SilMA), methacryloyl chondroitin sulfate (ChSMA), and methacryloyl heparin (HepMA).

[0098] Furthermore, the collagen-based hydrogel can also include an interlaced and interpenetrating first network structure, a second network structure and a third network structure, wherein the second network structure is formed by thermal crosslinking of a thermosensitive material, and the third network structure is formed by photocrosslinking of a photosensitive material and a corresponding photoinitiator, that is, a collagen-based hydrogel with a three-layer network structure is formed.

[0099] The present invention mainly uses type I collagen as a component to form a collagen-based hydrogel with a variety of interpenetrating network structures, which can simulate similar spatial structures of different organs, is conducive to three-dimensional cell culture, has a microenvironment that promotes cell adhesion, survival, and proliferation, and meets cell culture needs.

[0100] like Figure 2 As shown, another aspect of the present invention provides a method S400 for cultivating an in vitro three-dimensional model, comprising S410 to S440:

[0101] S410, forming a cell suspension for a three-dimensional model to be cultured; the cells in the cell suspension include cell lines, primary cells, and stem cells.

[0102] S420, adding a hydrogel prepolymer solution before cross-linking of the collagen-based hydrogel. The formation process of the hydrogel prepolymer solution can refer to the above description.

[0103] It should be understood that the gel prepolymerization solution in step S420 may be a multi-component collagen-based hydrogel prepolymerization solution in addition to a single-component collagen-based hydrogel prepolymerization solution.

[0104] In some preferred embodiments, the gel prepolymerization solution includes type I collagen, a metal ligand complex photoinitiator, a sodium persulfate photoinitiator, and a temperature-sensitive material.

[0105] In other preferred embodiments, the gel prepolymerization solution includes type I collagen, a metal ligand complex photoinitiator, a sodium persulfate photoinitiator, and a photosensitive material and any one of the photoinitiators selected from phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2,4,6-trimethylbenzoylphenylphosphonic acid ethyl ester, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone.

[0106] In other preferred embodiments, the gel prepolymerization solution includes type I collagen, a metal ligand complex photoinitiator, a sodium persulfate photoinitiator, a photosensitive material and any one of the photoinitiators selected from phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2,4,6-trimethylbenzoylphenylphosphonic acid ethyl ester, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, and a thermosensitive material.

[0107] S430, mixing the hydrogel prepolymerization solution with the cell suspension, taking an appropriate amount of the mixed solution and adding it into a culture chamber or culture well for culturing the cells, and forming a cell gel through photocrosslinking.

[0108] S440, adding culture medium to the culture chamber or culture well containing the cell gel, and culturing to obtain an in vitro three-dimensional model.

[0109] like Figure 3 As shown, another aspect of the present invention provides a method S200 for cultivating an in vitro three-dimensional model, comprising steps S210 to S240:

[0110] S210, forming a cell or cell cluster suspension for organoids to be cultured; the cells or cell clusters in the cell or cell cluster suspension are separated by digestion of tumor tissue or non-tumor tissue, or are separated by digestion of tumor organoids or non-tumor organoids to be passaged.

[0111] It should be noted that the organoids to be cultured can be tumor organoids, such as liver cancer organoids, colorectal cancer organoids, lung cancer organoids, pancreatic cancer organoids, etc.; they can also be non-tumor organoids, such as colorectal organoids, lung organoids, liver organoids, pancreatic organoids, etc.

[0112] S220, obtaining a gel prepolymerization solution, wherein the gel prepolymerization solution comprises type I collagen, a metal ligand complex photoinitiator, and a sodium persulfate photoinitiator.

[0113] It should be understood that the gel prepolymerization solution in step S220 may be a multi-component collagen-based hydrogel prepolymerization solution in addition to a single-component collagen-based hydrogel prepolymerization solution.

[0114] In some preferred embodiments, the gel prepolymerization solution includes type I collagen, a metal ligand complex photoinitiator, a sodium persulfate photoinitiator, and a temperature-sensitive material.

[0115] In other preferred embodiments, the gel prepolymerization solution includes type I collagen, a metal ligand complex photoinitiator, a sodium persulfate photoinitiator, and a photosensitive material and any one of the photoinitiators selected from phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2,4,6-trimethylbenzoylphenylphosphonic acid ethyl ester, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone.

[0116] In other preferred embodiments, the gel prepolymerization solution includes type I collagen, a metal ligand complex photoinitiator, a sodium persulfate photoinitiator, a photosensitive material and any one of the photoinitiators selected from phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2,4,6-trimethylbenzoylphenylphosphonic acid ethyl ester, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, and a thermosensitive material.

[0117] S230, mixing the gel prepolymerization solution with the cell suspension, taking an appropriate amount of the mixed solution and adding it to the culture chamber or culture well for culturing organoids, and cross-linking under 240-500nm light irradiation to form an organoid gel.

[0118] It should be noted that when the gel prepolymerization solution in step S220 includes a thermosensitive material, it is also required to undergo thermal cross-linking to form an organoid hydrogel. Of course, the above-mentioned thermal cross-linking process can be carried out simultaneously with the cell culture process, and there is no need to add additional cross-linking steps to meet the cell culture requirements, thereby simplifying the preparation process.

[0119] S240. Add culture medium to the culture chamber or culture well containing the organoid gel, and culture to obtain an in vitro three-dimensional model of tumor organoids or non-tumor organoids.

[0120] When the collagen-based hydrogel is used for three-dimensional cell culture, a reliable in vitro organoid model is formed, which meets the mechanical strength and biological activity required by the organoid, and the culture method is simple and easy to operate.

[0121] In another aspect of the present invention, an in vitro three-dimensional model is provided. A variety of in vitro three-dimensional organoid models can be cultured using the culture method described above. For specific culture methods, please refer to the above description and will not be repeated here.

[0122] Specifically, the in vitro three-dimensional models include non-tumor organoid in vitro three-dimensional models and tumor organoid in vitro three-dimensional models. Among them, the non-tumor organoid in vitro three-dimensional models include pancreatic organoids, colorectal organoids, lung organoids, liver organoids and other in vitro three-dimensional models; the tumor organoid in vitro three-dimensional models include pancreatic cancer organoids, colorectal cancer organoids, lung cancer organoids, liver cancer organoids and other in vitro three-dimensional models.

[0123] like Figure 4 As shown, another aspect of the present invention provides a method S300 for drug detection using an in vitro three-dimensional tumor model, comprising steps S310 to S320:

[0124] S310, introducing the drug to be tested into the in vitro three-dimensional tumor model;

[0125] S320, obtaining the effect results of the drug to be tested on the in vitro three-dimensional tumor model.

[0126] In this embodiment, based on the above-mentioned in vitro tumor three-dimensional model, such as the in vitro tumor organoid model, the complexity of the human body-specific microenvironment, extracellular matrix, etc. can be simulated to achieve drug screening.

[0127] Specifically, a conventional drug sensitivity test for an in vitro tumor three-dimensional model on a well plate may include the following steps:

[0128] (1) Prepare a drug-containing culture medium of appropriate concentration,

[0129] (2) The in vitro tumor 3D model is placed in a culture well in a non-dynamic culture environment, such as a culture well in a 96-well plate;

[0130] (4) Observe and record the growth status of the in vitro tumor three-dimensional model, remove the culture medium in the culture wells, add 100 μL of culture medium containing drug solution to each well, and place it in an incubator at 37°C and 5% CO2 for culture;

[0131] (4) Observe and record the growth status of the in vitro tumor 3D model on days 3-5;

[0132] (5) Use CTG detection kit to detect activity. Add 100 μL CTG detection reagent to each well, shake it for 5 minutes using a shaker, and incubate it at room temperature for 25 minutes. After incubation, use a chemiluminescence enzyme reader to detect it.

[0133] Furthermore, conventional drug sensitivity testing of the in vitro tumor three-dimensional model under dynamic culture may include the following steps:

[0134] (1) Prepare drug-containing culture medium of appropriate concentration:

[0135] (2) The in vitro tumor three-dimensional model formed by culture is placed in a culture chamber of a dynamic culture environment, for example, a chip with three interconnected chambers is used, and the in vitro tumor three-dimensional model is placed in the middle chamber;

[0136] (3) Observe and record the growth status of the in vitro tumor three-dimensional model. Remove the culture medium from the chip and add culture medium containing the drug solution. Add 50 μL of culture medium to the wells on the left and right sides of the chip and 30 μL of culture medium to the middle well;

[0137] (4) After adding the drug, the chip was placed in a rocking perfusion apparatus for flow culture at 3° / 120 min;

[0138] (5) On days 3 to 5, observe and record the growth status of the in vitro tumor three-dimensional model, and add 30 μL of drug-containing culture medium to the middle well;

[0139] (6) Use the CTG detection kit to perform activity detection. Discard all the culture medium on both sides of the wells, add 20 μL CTG diluent (1:1 dilution of CTG reagent stock solution and culture medium), add 50 μL CTG stock solution, pipette to mix, shake on a shaker for 5 minutes, incubate at room temperature for 25 minutes, remove all the lysate, and place it in a 96-well plate with completely opaque sides and bottom. Use a chemiluminescence enzyme reader for detection.

[0140] The preparation method and specific application of collagen-based hydrogel will be further described below in combination with several specific examples:

[0141] Example 1

[0142] This embodiment provides a method for preparing a single-component collagen-based hydrogel, comprising:

[0143] S1. Dissolve rat tail type I collagen in 0.02 M acetic acid solution, add NaOH to adjust the pH to neutral, mix well, and obtain a neutral collagen solution with a final concentration of 2 mg / mL.

[0144] S2. Add tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and sodium persulfate photoinitiator to the neutral collagen solution, immerse in light-proof for 30 minutes, so that the photoinitiator penetrates the colloid, and obtain a hydrogel prepolymer solution, wherein the concentration of tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator is 0.015%, and the concentration of sodium persulfate is 0.005%.

[0145] S3. Cross-link the hydrogel prepolymer solution under 405 nm wavelength irradiation for 1 min to obtain a collagen-based hydrogel with a dense structure.

[0146] It should be noted that when the collagen-based hydrogel obtained in step S3 is used for cell culture, the type I collagen can be thermosensitively solidified as required to further increase the cross-linking density and mechanical strength.

[0147] like Figure 5 As shown in (A), the control group is a single component 2 mg / mL collagen, and the hydrogel structure is prepared by curing at 37°C for 30 min. After freeze-drying the control group and the two hydrogels of this example, a scanning electron microscope was used to observe, as shown in FIG. Figure 5 As shown in (A) and (B), the collagen-based hydrogel of this embodiment has a porous structure, which is conducive to supporting the three-dimensional growth of cells.

[0148] Example 2

[0149] The preparation method of the collagen-based hydrogel in this embodiment is the same as that in Example 1, except that the concentration of the neutral collagen solution, the concentration of the tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator, and the concentration of the sodium persulfate photoinitiator are changed, as follows:

[0150] Figure 6 (A) shows the electron microscopy results of the hydrogel formed when the concentration of the neutral collagen solution is 1 mg / mL, the concentration of the tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator is 0.0005 mol / L, and the concentration of the sodium persulfate photoinitiator is 0.005 mol / L.

[0151] Figure 6 (B) shows the electron microscopy results of the hydrogel formed when the concentration of the neutral collagen solution is 2 mg / mL, the concentration of the tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator is 0.001 mol / L, and the concentration of the sodium persulfate photoinitiator is 0.01 mol / L.

[0152] Figure 6 (C) shows the electron microscopy results of the hydrogel formed when the concentration of the neutral collagen solution is 10 mg / mL, the concentration of the tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator is 0.005 mol / L, and the concentration of the sodium persulfate photoinitiator is 0.05 mol / L.

[0153] In summary, if Figure 6 As shown, after freeze-drying, each hydrogel formed in this embodiment was observed using a scanning electron microscope. The collagen-based hydrogel in this embodiment has a porous structure, which is conducive to supporting the three-dimensional growth of cells.

[0154] Example 3

[0155] This embodiment provides a method for preparing a composite collagen-based hydrogel, comprising:

[0156] S1. Dissolve recombinant type I collagen in 0.02 M acetic acid solution, add NaOH to adjust the pH to neutral, mix well, and obtain a 2 mg / mL neutral collagen solution;

[0157] S2. Add 0.001 mol / L tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and 0.01 mol / L sodium persulfate photoinitiator to the neutral collagen solution, and mix them evenly in the dark to form a hydrogel prepolymer solution.

[0158] S3. Add 10% Pluronic F-127 solution into the hydrogel prepolymerization solution. The mixed solution is first cross-linked under 405 nm wavelength irradiation for 1 min, and then thermally cross-linked in a 37° C. water bath for 30 min to obtain a collagen-based hydrogel with a dense structure.

[0159] like Figure 7 As shown in (A), the control group is a single component 2 mg / mL collagen, 0.015% tris(2,2'-bipyridyl)ruthenium(II) hexahydrate photoinitiator and 0.005% sodium persulfate photoinitiator are added, and cross-linked for 1 min under 405 nm wavelength irradiation to prepare a collagen-based hydrogel with a dense structure. After freeze-drying the control group and the two hydrogels of this example, a scanning electron microscope was used to observe, as shown in FIG. Figure 7As shown in (A) and (B) in the figure, the self-made hydrogel in this example has a porous structure with dense pores, which can support the three-dimensional growth of cells.

[0160] Example 4

[0161] This embodiment provides a method for preparing a composite collagen-based hydrogel, comprising:

[0162] S1. Dissolve pig skin type I collagen in 0.02 M acetic acid solution, add NaOH to adjust the pH to neutral, mix well, and obtain a 2 mg / mL neutral collagen solution.

[0163] S2. Add 0.001 mol / L tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and 0.01 mol / L sodium persulfate photoinitiator to the neutral collagen solution, mix evenly in the dark, and obtain a hydrogel prepolymer solution.

[0164] S3. Add 2% GelMA solution and 0.1% LAP photoinitiator to the hydrogel prepolymer solution; and add 0.2% polyisocyanurate polypeptide (PIC) solution to the above mixed solution. Then, crosslink for 1 minute under 405nm wavelength irradiation, and then place in a 95% humidity, 5% CO2, 37°C incubator for 30 minutes to obtain a collagen-based hydrogel that is double crosslinked by light curing and temperature curing.

[0165] like Figure 8 As shown in (A), the control group is a single component 2 mg / mL collagen, 0.015% tris(2,2'-bipyridyl)ruthenium(II) hexahydrate photoinitiator and 0.005% sodium persulfate photoinitiator are added, and cross-linked for 1 min under 405 nm wavelength irradiation to prepare a collagen-based hydrogel. After freeze-drying the control group and the two hydrogels of this example, a scanning electron microscope was used to observe, as shown in FIG. Figure 8 As shown in (A) and (B) in the figure, the self-made hydrogel in this example has a porous structure with dense pores, which can support the three-dimensional growth of cells.

[0166] Example 5

[0167] In this embodiment, collagen-based hydrogel is used for pancreatic cancer organoid culture, and the culture method includes:

[0168] S1. Dissolve rat tail type I collagen in 0.02 M acetic acid solution, add NaOH to adjust the pH to neutral, mix well, and obtain a 2 mg / mL neutral collagen solution.

[0169] S2. Add 0.001 mol / L tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate photoinitiator and 0.01 mol / L sodium persulfate photoinitiator to the neutral collagen solution, mix evenly in the dark, then add 2% (mass volume ratio) GelMA solution and 0.1% (mass volume ratio) LAP photoinitiator to the mixed solution to form a hydrogel prepolymer solution.

[0170] S3. Obtaining a cell suspension of organoids, specifically comprising: selecting pancreatic cancer organoids to be passaged, adding pre-cooled PBS to a 24-well plate, adding 1 mL of PBS to each well, collecting the cells into a centrifuge tube, refrigerating at 4°C for 15 min, and then centrifuging (1000 rpm, 5 min) to remove the supernatant to obtain an organoid precipitate; adding Tryple E for enzymatic hydrolysis for 1 min, and then centrifuging (1000 rpm, 5 min) to remove the supernatant to obtain a precipitate; adding PBS and centrifuging again (1000 rpm, 5 min) to remove excess enzyme solution to obtain a precipitate, and then resuspending the precipitate to obtain a cell suspension.

[0171] S4. Mix the hydrogel prepolymer solution and the cell suspension at a ratio of 85:15 (v / v), drip gel in a 24-well plate, 30 μl per well, solidify at 405 nm for 15 min to form a gel, add pancreatic cancer culture medium for culture and observation; in addition, the control group is a commercial matrix gel Matrigel and the precipitated cell suspension are evenly mixed, drip gel in a 24-well plate, 30 μl per well, solidify at 37°C for 30 min to form a gel, add pancreatic cancer culture medium for culture and observation.

[0172] like Fig. 9 As shown, compared with the control group, the homemade hydrogel cultured pancreatic cancer organoids were in better condition and showed an increasing trend with time, indicating that the homemade hydrogel in this example has the ability to culture organoids in three-dimensional cells.

[0173] The present invention provides a collagen-based hydrogel and a preparation method thereof, an in vitro three-dimensional model and a culture method thereof, and a drug detection method, which have the following beneficial effects:

[0174] First, the preparation method of the present invention is simple, the materials are easily available, the preparation cost is low, and the formed collagen-based hydrogel is transparent, has clear ingredients, is non-cytotoxic, has good mechanical properties and biological activity, has a microenvironment that promotes cell adhesion, survival, and proliferation, can support three-dimensional cell growth, and is suitable for application in cell culture observation, non-tumor organoid or tumor organoid culture, and drug screening;

[0175] Second, the present invention compounds photosensitive materials and / or thermosensitive materials with type I collagen to form a collagen-based hydrogel with an interpenetrating network structure. The double cross-linking method doubles the cross-linking effect, improves the mechanical properties, and does not add additional cross-linking steps to meet cell culture requirements. The preparation method is simple.

[0176] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a collagen-based hydrogel, characterized in that: The preparation method comprises: Dissolving type I collagen in an acid solution and adjusting the pH to neutral to form a neutral collagen solution; Adding a metal ligand complex photoinitiator and a sodium persulfate photoinitiator to the neutral collagen solution, and performing light-proof treatment to form a hydrogel prepolymer solution; A thermosensitive material and / or a photosensitive material and any one of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, ethyl 2,4,6-trimethylbenzoyl phenylphosphonate, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone are added to the hydrogel prepolymer solution, and cross-linked under light of 240 to 500 nm to form a transparent collagen-based hydrogel; wherein, The type I collagen forms a first network structure through photo-crosslinking, the thermosensitive material forms a second network structure through thermal crosslinking, and the photosensitive material forms a third network structure through photo-crosslinking. The first network structure is interlaced with the second network structure and / or the third network structure.

2. The preparation method according to claim 1, characterized in that: The metal ligand complex photoinitiator adopts tri(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate.

3. The preparation method according to claim 2, characterized in that: In the hydrogel prepolymerization solution, the concentration of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate is in the range of 0.0005 to 0.005 mol / L; and / or, The concentration of the sodium persulfate is 0.005-0.05 mol / L; and / or, The concentration of type I collagen is 1 to 10 mg / mL.

4. The preparation method according to claim 1, characterized in that: The time range of the light-shielding treatment is 15 to 60 minutes; and / or, The time range of the photocrosslinking is 0.5 to 5 minutes.

5. A collagen-based hydrogel, characterized in that: The collagen-based hydrogel is prepared by the preparation method according to any one of claims 1 to 4.

6. A collagen-based hydrogel, characterized in that: It includes a first network structure formed by cross-linking type I collagen under the action of a metal ligand complex photoinitiator and a sodium persulfate photoinitiator under 240-500nm light irradiation; and A second network structure formed by thermal crosslinking of a temperature-sensitive material; and / or, A third network structure formed by cross-linking of a photosensitive material under 240-500 nm light irradiation under the action of any one of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2,4,6-trimethylbenzoyl phenylphosphonic acid ethyl ester, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone; The second network structure and / or the third network structure interweave with the first network structure.

7. A method for cultivating an in vitro three-dimensional model, characterized in that: The culture method comprises: Forming a cell suspension for a three-dimensional model to be cultured; the cells in the cell suspension include cell lines, primary cells, and stem cells; Adding the hydrogel prepolymer solution before cross-linking of the collagen-based hydrogel according to any one of claims 1 to 4, Mixing the hydrogel prepolymer solution with the cell suspension, adding an appropriate amount of the mixture into a culture chamber or a culture well for culturing the cells, and forming a cell gel through photocrosslinking; A culture medium is added to the culture cavity or culture well containing the cell gel, and an in vitro three-dimensional model is obtained through culture.

8. A method for cultivating an in vitro three-dimensional model, characterized in that: The culture method comprises: Forming a cell or cell cluster suspension of an organoid to be cultured; the cells or cell clusters in the cell or cell cluster suspension are obtained by digesting, separating and resuspending tumor tissue or non-tumor tissue, or by digesting, separating and resuspending tumor organoids or non-tumor organoids to be passaged; Obtaining a hydrogel prepolymerization solution, wherein the hydrogel prepolymerization solution comprises type I collagen, a metal ligand complex photoinitiator and a sodium persulfate photoinitiator, a thermosensitive material and / or a photosensitive material, and any one of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, ethyl 2,4,6-trimethylbenzoyl phenylphosphonate, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone; The hydrogel prepolymer solution is mixed with the cell or cell cluster suspension, and an appropriate amount of the mixed solution is added to a culture chamber or culture well for culturing the organoid, and cross-linked under light of 240 to 500 nm to form an organoid gel; A culture medium is added to the culture chamber or culture well containing the organoid gel, and an in vitro three-dimensional model of a tumor organoid or a non-tumor organoid is obtained through culture.

9. An in vitro three-dimensional model, characterized in that The in vitro three-dimensional model is formed by culture using the culture method described in claim 7 or 8.

10. A method for drug detection using the in vitro three-dimensional model as claimed in claim 9, characterized in that: The method for drug detection comprises: Introducing the drug to be tested into the in vitro three-dimensional model; Obtain the effect of the drug to be tested on the in vitro three-dimensional model.

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