Hydrogel and method for inducing lung epithelial cells to differentiate into multidirectional mature lung organs with physiological related functions

Abstract

The invention belongs to the field of cell culture, and particularly relates to hydrogel and a method for inducing lung epithelial cells to be differentiated into multidirectional mature lung organs with physiological related functions. Comprising the following raw materials: a prepolymer solution: a component A: at least one of alginate, hyaluronic acid, gelatin, polyethylene glycol, polyacrylic acid, sericin, polyvinyl alcohol and collagen; the second cross-linking agent is a solution containing divalent or trivalent cations; the content of the second cross-linking agent is greater than or equal to 0; the hydrogel induces the pulmonary epithelial cells to spontaneously assemble to form the organoid with a three-dimensional structure, and promotes the differentiation of pulmonary airway epithelial cells and pulmonary alveolar epithelial cells at the same time, so that the ratio of the obtained pulmonary epithelial cells with a mature function is far higher than that of the organoid in an original culture mode; the method is of great significance to research of mature pulmonary epithelial cell related physiological processes and disease types.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell culture, in particular to a hydrogel and method for inducing lung epithelial cells to differentiate into multi-directionally mature lung organoids with physiological related functions. BACKGROUND

[0002] The lung is an important respiratory organ in the human body. The proximal airway of the lung is divided into bronchi and bronchioles, and the epithelial cells on the side of conducting air include ciliated cells, goblet cells, secretory club cells, and basal progenitor cells. There are fewer cell types including neuroendocrine cells, ion cells, and tuft cells. Gas exchange in the human body occurs in the distal lung, and the airway terminates in respiratory bronchioles, alveolar ducts, and alveolar sacs surrounded by a capillary bed. The distal lung epithelial cells include type 1 and type 2 alveolar epithelial cells. Type 1 alveolar epithelial cells (AT1) are flat squamous cells, accounting for 95% of the alveolar epithelial surface area, and play an important role in gas exchange. Type 2 alveolar epithelial cells (AT2) produce surfactant to maintain the fluid balance of the alveolar unit and serve as progenitor cells for AT1 cells. To further study the treatment methods for human lung diseases, it is essential to reconstruct the extracellular matrix (ECM) environment and its dynamic environment in which lung cells survive in vitro. Traditional research methods mainly rely on animal models and two-dimensional culture bottles, but both of these methods have obvious defects. When using animal models for research, there are many problems such as species differences, ethical controversies, limited experimental throughput, and high financial costs. These problems make it difficult for animal models to become a routine preclinical model for studying human lung diseases and their drug treatments. Two-dimensional culture bottles alleviate some of the economic pressures associated with using animal models, but they cannot effectively simulate the dynamic microenvironment of the lung, such as three-dimensional extracellular matrix, respiratory pressure changes, and blood flow shear effects. This leads to significant differences in gene expression, epigenetics, and cell function between cells cultured in two-dimensional culture bottles and actual in vivo conditions. In addition, the species differences of animal models and the inability of two-dimensional culture bottles to simulate dynamic microenvironments often result in the failure of new drug research in the clinical translation stage, thereby significantly increasing drug development costs. In summary, establishing a three-dimensional lung organ model that highly mimics the extracellular matrix environment, cell types, and mechanical environment is of great significance for the study of lung diseases and the evaluation of therapeutic drugs.

[0003] Organoids and organ-on-a-chip are emerging technologies for constructing three-dimensional lung microenvironments, which can more accurately simulate human physiological characteristics and functions at different scales from molecules to cells, tissues, and organs. Compared with organ-on-a-chip technology, organoid technology is more mature. In the early stage of constructing a three-dimensional dynamic lung microenvironment, lung organoids were relied on. To date, lung organoids have been widely used in the study of regulatory mechanisms of lung epithelial differentiation, lung diseases, and new drug development. In 1987, Jennings et al. first cultured lung organoids that could maintain the properties and morphology of alveolar epithelial type II (AT2) cells in vitro, and the daughter cells exhibited the morphological characteristics of primitive cells. In 2009, Rock et al. found that in vitro basal cells could be cultured to differentiate into tubular lung airway organoids, which had self-renewal and differentiation capabilities and could differentiate into ciliated cells, thereby identifying basal cells as stem cells in mice and humans. By 2014, the culture technology for differentiating lung organoids from human pluripotent stem cells (hPSCs) had become relatively mature, but compared with lung organoids, there were still problems such as a low proportion of functional cells and a long culture period. After 2020, the development of lung organoids tended to simulate the branching structure of the lung. Ana et al. found that lung cells could spontaneously assemble into coral-like cell clusters with a certain spatial distribution structure. Ivana et al. found that the lung-like branching structure formed by the spontaneous assembly of organoids in long-term culture (more than 100 days) simulated the process of embryonic lung development, and the progressive formation of the distal lung was accompanied by the degradation of the endoderm and mesoderm. The culture and induction differentiation methods of lung organoids have gradually become mature, but the culture of organoids with a three-dimensional lung-like branching structure is still in the development stage, and the culture method is still complex.

[0004] From the development history of organoids, it usually takes a lot of culture time to form a three-dimensional microenvironment with a lung-like structure by spontaneous assembly of organoids. This is obviously not conducive to quickly and efficiently constructing a three-dimensional lung model.

[0005] Matrigel is the most widely used product in the culture of three-dimensional lung organoids. It is extracted from mouse sarcoma and is a natural extracellular matrix (ECM) with complex components that can regulate cell growth and differentiation. However, organoids wrapped in Matrigel have the following defects:

[0006] Matrigel-wrapped cells often excessively maintain cell stemness, and the obtained lung epithelial cell types are fewer and have poor cell maturity;

[0007] Matrigel-wrapped cells are isolated from the external environment, which makes it difficult for pathogens and cells to interact, thereby interfering with subsequent research;

[0008] The immune-related functions in the cells wrapped by Matrigel are not activated, which is not conducive to the research on the physiological process of immune function activation. SUMMARY

[0009] Therefore, the technical problem to be solved by the present application is to provide a hydrogel and a method for inducing lung epithelial cells to differentiate into multi-directionally mature lung organoids with physiological-related functions. The hydrogel is used to induce lung epithelial cells to spontaneously assemble on the surface thereof to form three-dimensional shapes such as airway-like, alveolar-like, or lung branch structures. This process can promote the differentiation of lung epithelial cells, and the proportion of mature lung epithelial cells in the finally obtained organoids is much higher than that in the traditional culture mode. Not only can the drug development cycle be effectively shortened and the drug development cost be reduced, but also more reliable preclinical test data can be provided.

[0010] To this end, the present application provides the following technical solutions:

[0011] The present application provides a hydrogel, which comprises the following raw materials:

[0012] The prepolymer solution comprises component A, which is prepared from at least one of alginate, hyaluronic acid, gelatin, polyethylene glycol, polyacrylic acid, sericin, polyvinyl alcohol, and collagen;

[0013] The second crosslinking agent is a solution containing divalent or trivalent cations, and the content of the second crosslinking agent is greater than or equal to 0.

[0014] In some embodiments, the concentration of component A in the prepolymer solution is 1wt%-5wt%. Further, in some embodiments, the concentration of component A can be any one of 1wt%, 1.2wt%, 1.5wt%, 1.8wt%, 2wt%, 2.2wt%, 2.5wt%, 2.8wt%, 3wt%, 3.2wt%, 3.5wt%, 3.8wt%, 4wt%, 4.2wt%, 4.5wt%, 4.8wt%, 5wt% or a range value between any two of them.

[0015] In some embodiments, the concentration of divalent or trivalent cations ranges from 0.1 to 1 mol / mL. Further, the concentration of divalent or trivalent cations contained in the second crosslinking agent can be any one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mol / mL or a range value between any two of them.

[0016] In some embodiments, the volume ratio of the prepolymer solution and the second crosslinking agent is 1-10:1. Further, in some embodiments, the volume ratio of the prepolymer solution and the second crosslinking agent can be any one of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or a range between any two of them.

[0017] In some embodiments, the second crosslinking agent is a solution containing divalent or trivalent metal ions; preferably, the divalent or trivalent cations contained in the second crosslinking agent are at least one of Fe 3+ , Mg 2+ , Al 3+ , Ca 2+ , Sr 2+ , Ba 2+ .

[0018] In some embodiments, the alginate includes but is not limited to at least one of sodium alginate, potassium alginate, calcium alginate, ammonium alginate, barium alginate or ferric alginate.

[0019] In some embodiments, the gelatin includes but is not limited to RGD short peptide modified acrylated gelatin.

[0020] Optionally, the prepolymer solution further includes at least one of mouse tail type I collagen, fibronectin or laminin to adjust the adhesion ability of the hydrogel to cells.

[0021] In some embodiments, the molecular weight of the polyethylene glycol ranges from 1000 to 20000 Da.

[0022] In some embodiments, the prepolymer solution further includes at least one of fibronectin or laminin.

[0023] In some embodiments, the hydrogel further includes the following raw materials:

[0024] initiator; the initiator includes a thermal initiator or a photo initiator;

[0025] and, the prepolymer solution further includes the following group of components:

[0026] acrylamide monomer;

[0027] first crosslinking agent: bisacrylamide crosslinking monomer;

[0028] catalyst: tetramethyl ethylenediamine.

[0029] In some embodiments, when the content of the second crosslinking agent is 0, the hydrogel includes the following raw materials:

[0030] The prepolymer solution is prepared by mixing component A, acrylamide monomer, first crosslinking agent and catalyst;

[0031] initiator.

[0032] In some embodiments, the first crosslinking agent is a common crosslinking agent for hydrogel, and the double acrylamide crosslinking monomer includes but is not limited to methylene bisacrylamide.

[0033] In some embodiments, the initiator is any one of common initiators for hydrogel, such as thermal initiator or photoinitiator. The photoinitiator can be ketoglutaric acid, and the thermal initiator includes persulfate, which can be selected from ammonium persulfate or potassium persulfate.

[0034] In some embodiments, the mass ratio of the acrylamide monomer, component A, first crosslinking agent and catalyst is 1:0.04-0.5:0.0006-0.06:0.001-0.003.

[0035] In some embodiments, when the initiator includes ketoglutaric acid, the concentration of ketoglutaric acid is 0.5-10wt%. Further, in some embodiments, the concentration of ketoglutaric acid can be any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10wt% or a range value between any two of them.

[0036] In some embodiments, when the initiator includes persulfate, the concentration of persulfate is 0.1-10wt%. Further, in some embodiments, the concentration of persulfate can be any one of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10wt% or a range value between any two of them.

[0037] In some embodiments, the volume ratio of the prepolymer solution and initiator is 100-30:1. Further, in some embodiments, the volume ratio of the prepolymer solution and initiator can be any one of 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1 or a range value between any two of them.

[0038] The preparation method of the hydrogel is provided in the embodiments of the present application, which includes the following steps:

[0039] The component A is taken to prepare the prepolymer solution;

[0040] The second cross-linking agent is added to the prepolymer solution to cross-link, and a hydrogel is obtained.

[0041] In some embodiments, when the content of the second cross-linking agent is >0, the method further comprises the following step:

[0042] The components A, acrylamide monomers, the first cross-linking agent, and the catalyst are mixed to prepare a prepolymer solution;

[0043] The initiator is added to the prepolymer solution to polymerize, and a first composite cross-linking product is obtained;

[0044] The first composite cross-linking product is added to the second cross-linking agent to cross-link, and a hydrogel is obtained.

[0045] In some embodiments, the polymerization comprises ultraviolet polymerization or heat polymerization; when the initiator contains only ketoglutaric acid, the polymerization is ultraviolet polymerization; when the initiator contains ammonium persulfate or a mixture of ammonium persulfate and ketoglutaric acid, the polymerization is ultraviolet polymerization or heat polymerization.

[0046] In some embodiments, the ultraviolet polymerization is performed under the following conditions: the ultraviolet wavelength is 250-400 nm, and the irradiation time is 1-5 h. Further, the ultraviolet wavelength can be any one of 250, 251, 252, 253, 253.7, 254, 255, 260, 265, 270, 280, 285, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 nm or a range between any two of them. Further, the irradiation time can be any one of 1, 2, 3, 4, 5 h or a range between any two of them.

[0047] In some embodiments, the heat polymerization is performed under the following conditions: heating at 60-80℃ for 2-6 h. Further, the heating temperature can be any one of 60, 65, 70, 75, 80℃ or a range between any two of them. Further, the heating time can be any one of 2, 3, 4, 5, 6 h or a range between any two of them.

[0048] In some embodiments, the first composite cross-linking product is added to the second cross-linking agent to cross-link under the following conditions: the soaking time at room temperature is 3-12 h. Further, the soaking time can be any one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 h or a range between any two of them. The temperature range of room temperature is 10-30℃, which can be any one of 10, 15, 20, 25, 30℃ or a range between any two of them.

[0049] In some embodiments, when the content of the second crosslinking agent is 0, the method comprises the following steps:

[0050] The components A, acrylamide monomer, first crosslinking agent and catalyst are mixed to prepare a prepolymer solution;

[0051] The initiator is added to the prepolymer solution, and polymerization is performed to obtain a hydrogel.

[0052] In some embodiments, the polymerization comprises ultraviolet polymerization or heat polymerization; when the initiator only contains ketoglutaric acid, the polymerization is ultraviolet polymerization; when the initiator contains ammonium persulfate or a mixture of ammonium persulfate and ketoglutaric acid, the polymerization is ultraviolet polymerization or heat polymerization.

[0053] In some embodiments, the ultraviolet polymerization is performed under the following conditions: ultraviolet wavelength is 250-400 nm, and irradiation time is 1-5 h. Further, the ultraviolet wavelength can be any one of 250, 251, 252, 253, 253.7, 254, 255, 260, 265, 270, 280, 285, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 nm or a range value between any two of them. Further, the irradiation time can be any one of 1, 2, 3, 4, 5 h or a range value between any two of them.

[0054] In some embodiments, the heat polymerization is performed under the following conditions: heating at 60-80℃ for 2-6 h. Further, the heating temperature can be any one of 60, 65, 70, 75, 80℃ or a range value between any two of them. Further, the heating time can be any one of 2, 3, 4, 5, 6 h or a range value between any two of them.

[0055] The hydrogel described above is also provided for use in inducing differentiation of lung epithelial cells in the embodiments of the present application.

[0056] The embodiments of the present application provide a method for inducing differentiation of lung epithelial cells, which comprises culturing lung epithelial cells with the hydrogel or the hydrogel prepared by the method described above to induce differentiation of lung epithelial cells.

[0057] In some embodiments, the density of the lung epithelial cells seeded on the surface of the hydrogel is 1-8×10 6 / mL;

[0058] And / or, the culture medium for culturing the lung epithelial cells on the hydrogel is organoid culture medium.

[0059] And / or, the lung epithelial cells are cultured in the hydrogel for 10-30 days, and further, the culturing time can be any one of 10, 13, 15, 17, 18, 20, 23, 25, 28, 30 days or a range between any two of them.

[0060] In some embodiments, before culturing the lung epithelial cells in the hydrogel, the method further comprises at least one of the following steps: lung epithelial cell acquisition, lung organoid acquisition, single cell suspension acquisition.

[0061] In some embodiments, the step of lung epithelial cell acquisition comprises: digesting lung tissue, filtering, collecting cells, removing red blood cells, obtaining lung epithelial cells and tissue clusters;

[0062] And / or, the step of lung organoid acquisition comprises: adding Matrigel to the lung epithelial cells and tissue clusters, resuspending, inoculating, solidifying, culturing in lung epithelial organoid culture medium, and obtaining lung organoids when the density of the organoids in the Matrigel reaches more than 80%.

[0063] And / or, the step of single cell suspension acquisition comprises: removing Matrigel from the lung organoids and digesting.

[0064] The embodiments of the present application provide a lung organoid model obtained by the method of inducing differentiation of lung epithelial cells.

[0065] The lung organoid model provided by the present application has any one of the following uses:

[0066] (1) Use in the study of the interaction between pathogens and lung cells in the lung organ of a human or non-human mammal;

[0067] (2) Use in the study of the activation of the immune function of lung epithelial cells in a human or non-human mammal;

[0068] (3) Use in the study or screening of drugs for preventing, adjuvant therapy or treating lung diseases in humans;

[0069] (4) Use in the study of lung diseases in a human or non-human mammal.

[0070] The technical solution of the present application has the following advantages:

[0071] 1. A hydrogel provided by the present application, comprising the following raw materials: a prepolymer solution: component A: at least one of alginate, hyaluronic acid, gelatin, polyethylene glycol, polyacrylic acid, sericin, polyvinyl alcohol and collagen; a second crosslinking agent: a solution containing divalent or trivalent cations; the content of the second crosslinking agent is > 0; in the above scheme, the hydrogel prepared by selecting component A can induce lung epithelial cells to spontaneously assemble into three-dimensional shapes such as airway-like, alveolar or lung branch structures, promote the differentiation of cells into various mature lung epithelial cells such as AT1 cells, AT2 cells, rod-shaped cells, goblet cells and ciliated cells, and obtain lung organoids with a much higher proportion of mature functional lung epithelial cells than the original culture method; in particular, compared with the lung organoids obtained by the existing Matrigel wrapping culture method, the morphology of the cells is more prone to ingrowth to form spheres, and the morphology of the AT1 cells in the lung alveolar region is quite different from that of the human body; the lung organoids obtained above are multi-directionally mature and have physiological related functions, which are of great significance for studying the physiological processes and disease types related to mature lung epithelial cells;

[0072] Further, compared with the existing Matrigel-wrapped lung organoid model cells, which are isolated from the external environment and are not conducive to the study of the interaction between pathogens and apical layer cells, the lung organoids obtained by the hydrogel culture of the present application do not require Matrigel culture, and the apical surface of the lung organoid cells is exposed to the culture solution environment, allowing the cells to directly interact with the added pathogens or molecules, which is conducive to the study of the interaction between pathogens and apical layer cells;

[0073] Further, compared with the existing Matrigel-wrapped lung organoids, the immune-related functions of which are not activated, the lung organoids obtained by the hydrogel culture of the present application are identified to have enhanced lung epithelial cell-related immune functions, as shown by the up-regulation of the expression of innate immune molecule-related genes, the significant increase in mucus secretion, and the enhanced ability to resist viral invasion, indicating that the lung organoids obtained by the method of the present application can activate the innate immune functions of lung epithelial cells, providing a research reference for the activation of human physiological processes related to immune functions;

[0074] The culture method of the present application uses clinical sample-derived normal lung tissue adjacent to cancer, which can be cultured after simple processing, reducing the use of expensive reagents and lowering the experimental cost;

[0075] In summary, the lung organoids obtained by the present application have significant advantages over the prior art in improving the yield of mature functional lung epithelial cells, being closer to the AT1 morphology of the alveolar region in the human body, improving the limitations of the culture method, facilitating the study of the interaction between pathogens and apical layer cells, activating the immune function of lung epithelial cells, and reducing experimental costs. BRIEF DESCRIPTION OF DRAWINGS

[0076] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0077] Figure 1 is a real object picture of the composite gel of sodium alginate and acrylamide obtained in Example 2 with ketoglutaric acid as an initiator;

[0078] Figure 2 is a real object picture of the composite gel of sodium alginate and acrylamide obtained in Example 3 with ammonium persulfate as an initiator;

[0079] Figure 3 is a real object picture of the polyacrylic acid gel in Example 5;

[0080] Figure 4 is a real object picture of the silk fibroin gel in Example 6;

[0081] Figure 5 is a real object picture of the polyvinyl alcohol in Example 7;

[0082] Figure 6 is a microscope observation result of the spherical organoids obtained by surface culture of the methacrylated gelatin gel in Example 8;

[0083] Figure 7 is a real object picture of the sodium alginate gel crosslinked by the Ca 2+ crosslinking agent solution in Example 9;

[0084] Figure 8 is a microscope observation result of the spherical organoids obtained by surface culture of the calcium alginate gel in Example 9;

[0085] Figure 9 is a microscope observation result of the lung epithelial cells in the water gel in the experimental group of Example 1 in the experimental example of the present application, which can spontaneously assemble into a three-dimensional structure (the scale is 200 μm);

[0086] Figure 10is the microscope observation result of lung epithelial cells in the control group in the experimental example of the present application after culture in Matrigel (the scale is 200 μm);

[0087] Figure 11 is the observation diagram of immunofluorescence labeling of various lung epithelial cell specific markers in the organoid cells obtained from the experimental group and the control group of Example 1 in the experimental example of the present application (the scale is 100 μm); A is the experimental group, and B is the control group;

[0088] Figure 12 is the observation diagram of organoid cells obtained from the experimental group of Example 1 in the experimental example of the present application (the scale is 100 μm); A is the thin wall of the organoid; AQP5 is positive around the three-dimensional structure of the organoid;

[0089] Figure 13 is the characterization result of organoid cells obtained from the experimental group and the control group of Example 1 in the experimental example of the present application (the scale is 100 μm); A is the experimental group, and B is the control group; SPB represents AT2 cells, CC10 represents club cells, and MUC5AC represents goblet cells;

[0090] Figure 14 is the observation result of the organoid cells of the experimental group and the control group of Example 1 in the experimental example of the present application after continuous culture for 30 days; Example 1 experimental group: A is optical microscope shooting; B is scanning electron microscope shooting (the scale is 4 μm); Control group: A is optical microscope shooting; B is scanning electron microscope shooting (the scale is 4 μm);

[0091] Figure 15 is the transcriptome sequencing result of the organoid cells obtained from the experimental group and the control group of Example 1 in the experimental example of the present application; in the figure, APM is the experimental group, and Matrigel is the control group;

[0092] Figure 16 is the qPCR result of the complement of the organoid cells obtained from the experimental group and the control group of Example 1 in the experimental example of the present application; in the figure, APM is the experimental group, and Matrigel is the control group;

[0093] Figure 17 is the qPCR result of the mucus secretion related genes of the organoid cells obtained from the experimental group and the control group of Example 1 in the experimental example of the present application; in the figure, APM is the experimental group, and Matrigel is the control group;

[0094] Figure 18is the result of the class organ cell obtained from the experimental group and the control group of Example 1 in the experimental example of the present application after being infected by adenovirus; A is the result of the infected virus of the experimental group and the control group of Example 1 (green is adenovirus positive); B is the result of flow analysis; C is the statistical result of the proportion of infected virus cells of the experimental group and the control group by flow analysis comparison; APM in the figure is the experimental group of Example 1, and Matrigel is the control group. DETAILED DESCRIPTION

[0095] The following examples are provided to better further understand the present application and are not limited to the best mode, and do not constitute a limitation on the content and scope of protection of the present application, and any person under the inspiration of the present application or the combination of the present application with other prior art features will fall within the scope of protection of the present application.

[0096] The specific experimental steps or conditions are not indicated in the examples, which can be carried out according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not indicated by the manufacturer, which are conventional reagent products that can be obtained by market purchase.

[0097] In the following examples, the main experimental reagents involved are as follows:

[0098] Table 1, main reagents

[0099]

[0100] The lung epithelial cell organoid subculture medium involved in the following examples mainly includes Advanced DMEM / F12, GlutaMAX, HEPES buffer, penicillin / streptomycin double antibiotic, Primocin primary cell antibiotic, Noggin, R-spondin 1, Nicotinamide, Nac, B27 additive, N2 additive, FGF7, FGF10, Y-27632, A83-01, SB202190, Forskolin. Among them, the Advanced DMEM / F12 culture medium is a mother liquor, and the use concentration of other components is shown in the following table:

[0101] Table 2, lung epithelial cell organoid subculture medium formula

[0102] Reagent name Working concentration (preferred) Advanced DMEM / F12 N / A GlutaMAX 1-3 mM Primocin 30-60 ng / mL P / S double antibiotic 100-200 fold dilution HEPES 5-15 mM N2 supplement 0.5-2 mM B27 supplement 1-3 mM Noggin 50-200 ng / mL R-Spondin 1 300-800 ng / mL FGF10 20-300 ng / mL FGF7 5-50 ng / mL Y-27632 2-20 μΜ A83-01 300-1000 nM SB202190 3-10 μΜ Forskolin 3-30 μΜ Nac 0.5-10 mM Nicotinamide 1-20 mM

[0103] DPBS: Dulbecco's phosphate buffer;

[0104] Cell Recovery Solution is purchased from Corning Company;

[0105] Tryple digestion solution was purchased from Gibco company.

[0106] Example 1 hydrogel

[0107] The present example provides a hydrogel and a preparation method thereof, and the raw material composition of the hydrogel is as follows:

[0108] Prepolymer solution: sodium alginate and acrylamide monomer, methylene bisacrylamide, tetramethyl ethylenediamine are mixed to prepare a 10 mL solution; wherein the added mass of acrylamide monomer is 1200 mg, the added mass of the first crosslinking agent methylene bisacrylamide is 7.2 mg, the added mass of the catalyst tetramethyl ethylenediamine is 3 mg, and the added mass of sodium alginate is 300 mg.

[0109] Initiator: oxoglutaric acid and ammonium persulfate are mixed to prepare a 10 mL solution; wherein the added mass of oxoglutaric acid is 200 mg, and the added mass of ammonium persulfate is 100 mg.

[0110] Second crosslinking agent: aqueous solution containing Fe 3+ , concentration is 1 mol / mL, and the amount used is 10 mL.

[0111] The volume ratio of the prepolymer solution to the initiator is 52:1; and the volume ratio of the prepolymer solution to the second crosslinking agent is 1:1.

[0112] The hydrogel is prepared by using the above raw material composition, including the following steps:

[0113] (1) The prepolymer solution is prepared according to the prepolymer solution formula;

[0114] (2) The obtained prepolymer solution is introduced into a container, and then the initiator is added; the volume ratio of the prepolymer solution to the initiator is 52:1; then irradiation is performed under a UV lamp (253.7 nm) for 3 h to initiate polymerization, and a first composite crosslinking product is obtained;

[0115] (3) The first composite crosslinking product is taken out and then soaked in the second crosslinking agent; the volume ratio of the prepolymer solution to the second crosslinking agent is 1:1, and the soaking time is 3 h at 25°C, and the required hydrogel is obtained.

[0116] Example 2

[0117] The present example provides a hydrogel and a preparation method thereof, and the raw material composition of the hydrogel is as follows:

[0118] Prepolymer solution: sodium alginate and acrylamide monomer, methylene bisacrylamide, tetramethyl ethylenediamine are mixed to prepare a 10 mL solution; wherein the acrylamide monomer is added in a mass of 200 mg, the first crosslinking agent methylene bisacrylamide is added in a mass of 12 mg, the catalyst tetramethyl ethylenediamine is added in a mass of 0.6 mg, and the sodium alginate is added in a mass of 100 mg.

[0119] Initiator: prepare a 10 mL solution of ketoglutaric acid, and add 1000 mg of ketoglutaric acid.

[0120] Second crosslinking agent: an aqueous solution containing Al 3+ with a concentration of 0.5 mol / mL, and a usage of 10 mL.

[0121] The volume ratio of the prepolymer solution to the initiator is 100:1; and the volume ratio of the prepolymer solution to the second crosslinking agent is 10:1.

[0122] The hydrogel is prepared by using the above raw material composition, including the following steps:

[0123] (1) The prepolymer solution is prepared according to the prepolymer solution formula;

[0124] (2) The obtained prepolymer solution is introduced into a container, and then the initiator is added; the volume ratio of the prepolymer solution to the initiator is 100:1; then irradiation under a UV lamp (253.7 nm) for 3 h initiates polymerization to obtain a first composite crosslinking product;

[0125] (3) The first composite crosslinking product is taken out and then soaked in the second crosslinking agent; the volume ratio of the prepolymer solution to the second crosslinking agent is 10:1, and the soaking time is 3 h at 25℃ to obtain the required hydrogel, as shown in Figure 1 .

[0126] Example 3

[0127] The present embodiment provides a hydrogel and a preparation method thereof, and the raw material composition of the hydrogel is as follows:

[0128] Prepolymer solution: sodium alginate and acrylamide monomer, methylene bisacrylamide, tetramethyl ethylenediamine are mixed to prepare a 10 mL solution; wherein the acrylamide monomer is added in a mass of 2000 mg, the first crosslinking agent methylene bisacrylamide is added in a mass of 1.2 mg, the catalyst tetramethyl ethylenediamine is added in a mass of 2 mg, and the sodium alginate is added in a mass of 500 mg.

[0129] Initiator: prepare a 10 mL solution of ammonium persulfate, and add 500 mg of ammonium persulfate.

[0130] Second crosslinking agent: an aqueous solution containing Mg 2+Aqueous solution of 0.1 mol / mL, 10 mL was used.

[0131] The volume ratio of the prepolymer solution to the initiator was 30:1, and the volume ratio of the prepolymer solution to the second crosslinking agent was 5:1.

[0132] The hydrogel was prepared by using the above raw material composition, including the following steps:

[0133] (1) The prepolymer solution was prepared according to the prepolymer solution formula;

[0134] (2) The obtained prepolymer solution was introduced into a container, and then the initiator was added, the volume ratio of the prepolymer solution to the initiator was 30:1; then the polymerization was initiated by heating at 80°C for 3h, and the first composite crosslinking product was obtained;

[0135] (3) The first composite crosslinking product was taken out and then soaked in the second crosslinking agent, the volume ratio of the prepolymer solution to the second crosslinking agent was 5:1, and the soaking time was 12h at 25°C, and the desired hydrogel was obtained, as shown in Figure 2 .

[0136] Example 4

[0137] The difference between this example and Example 1 is that the content of polyethylene glycol or the like is replaced by sodium alginate in the raw material composition of the hydrogel, and the molecular weight of the polyethylene glycol is 10000 Da; at the same time, the second crosslinking agent is omitted in the raw material of the hydrogel. In the preparation method of the hydrogel, the second crosslinking agent is not added directly.

[0138] Example 5

[0139] The difference between this example and Example 1 is that the content of polyacrylic acid or the like is replaced by sodium alginate in the raw material composition of the hydrogel, and the second crosslinking agent is omitted. The hydrogel was prepared according to the raw material composition of the hydrogel, including the following steps:

[0140] (1) The prepolymer solution was prepared according to the prepolymer solution formula;

[0141] (2) The obtained prepolymer solution was introduced into a container, and then the initiator was added; the volume ratio of the prepolymer solution to the initiator was 52:1; then the polymerization was initiated by irradiation under a UV lamp (253.7 nm) for 3h, and the hydrogel was obtained, as shown in Figure 3 .

[0142] Example 6

[0143] The difference between this example and Example 1 is that the content of silk fibroin or the like is replaced by sodium alginate in the raw material composition of the hydrogel, and the second crosslinking agent is omitted. The hydrogel was prepared according to the raw material composition, including the following steps:

[0144] (1) Preparation of a prepolymer solution according to the prepolymer solution formula;

[0145] (2) The obtained prepolymer solution was introduced into a container, and then an initiator was added; the volume ratio of the prepolymer solution to the initiator was 52:1; then polymerization was initiated under the irradiation of a UV lamp (253.7 nm) for 3 h to obtain a hydrogel, as shown in Figure 4 .

[0146] Example 7

[0147] The difference between this example and Example 1 is that the content of polyvinyl alcohol in the raw material composition of the hydrogel is replaced by sodium alginate, and the second crosslinking agent is omitted. The hydrogel is prepared according to the raw material composition, including the following steps:

[0148] (1) Preparation of a prepolymer solution according to the prepolymer solution formula;

[0149] (2) The obtained prepolymer solution was introduced into a container, and then an initiator was added; the volume ratio of the prepolymer solution to the initiator was 52:1; then polymerization was initiated under the irradiation of a UV lamp (253.7 nm) for 3 h to obtain a hydrogel, as shown in Figure 5 .

[0150] Example 8

[0151] The difference between this example and Example 1 is that the content of methacrylated gelatin in the raw material composition of the hydrogel is replaced by sodium alginate, and the second crosslinking agent is omitted. The hydrogel is prepared according to the raw material composition, including the following steps:

[0152] (1) Preparation of a prepolymer solution according to the prepolymer solution formula;

[0153] (2) The obtained prepolymer solution was introduced into a container, and then an initiator was added; the volume ratio of the prepolymer solution to the initiator was 52:1; then polymerization was initiated under the irradiation of a UV lamp (253.7 nm) for 3 h to obtain a hydrogel.

[0154] The hydrogel prepared based on this example induces lung epithelial cell differentiation, including the following steps:

[0155] (1) The cancer-adjacent lung normal tissue from a clinical sample was digested by a lung tissue digestion solution (1 mg / mL collagenase type I, 0.1-0.5 mg / mL DNase I, 1% penicillin / streptomycin double antibody) at 37°C for 1 hour, then filtered by a 70 μm cell screen and centrifuged, the cell precipitate was collected, and red blood cells were further removed by a red blood cell lysis solution to obtain relatively pure lung epithelial cells and tissue clusters;

[0156] (2), wash the lung epithelial cells and tissue mass with DPBS for 2-3 times, add appropriate amount of Matrigel, resuspend the lung epithelial cells, and inoculate in 24-well plates, after solidification for 15-20 minutes in a cell incubator, add lung epithelial organoid culture medium (Table 2), and place in a cell culture incubator for culture, replace the medium every 5 days during the culture process, when the density of the organoids in the Matrigel reaches 80%, the organoids can be subcultured or transferred to the surface of the gel for culture to promote differentiation;

[0157] (3), after the lung epithelial organoids are removed from the Matrigel by Cell Recovery Solution, digestion with Tryple is used to disperse into a single cell suspension, inoculate on the surface of the hydrogel in this embodiment at a density of 1x10 6 / mL (the culture medium is the organoid culture medium (Table 2)), and culture to the third day, at which time the cells can spontaneously form spherical organoids, and the results are shown in Figure 6 From Figure 6 it can be seen that the cells spontaneously form spherical organoid cells, indicating that the hydrogel in this embodiment can induce lung epithelial cells to spontaneously assemble into a three-dimensional shape of alveoli, and only needs to be cultured for 3 days, which is shorter than the 15 days required for Matrigel wrapping culture, and is lower in cost.

[0158] Example 9

[0159] This embodiment provides a hydrogel and a preparation method thereof, and the raw material composition of the hydrogel is as follows:

[0160] Prepolymer solution: sodium alginate is prepared into a 10 mL solution, and the added amount of sodium alginate is 200 mg;

[0161] Second crosslinking agent: an aqueous solution containing Ca 2+ , with a concentration of 1.0 mol / mL, and a usage amount of 10 mL.

[0162] The volume ratio of the prepolymer solution to the second crosslinking agent is 1:1.

[0163] The hydrogel is prepared by using the above formulation, including the following steps:

[0164] The prepolymer solution is prepared according to the prepolymer solution formulation;

[0165] The obtained prepolymer solution is introduced into a container, and the second crosslinking agent is added, the volume ratio of the prepolymer solution to the second crosslinking agent is 1:1, and the soaking time is 3 h at 25℃, to obtain the required hydrogel, as shown in Figure 7 .

[0166] The hydrogel prepared based on this embodiment induces lung epithelial cell differentiation, including the following steps:

[0167] (1), using clinical sample-derived normal lung tissue adjacent to cancer, after 1 hour of digestion at 37°C using lung tissue digestion solution (1 mg / mL collagenase type I, 0.1-0.5 mg / mL DNase I, 1% penicillin / streptomycin double antibody), 70 μm cell screen filtration, centrifugation, collection of cell precipitate, and further removal of red blood cells using red blood cell lysis solution, to obtain relatively pure lung epithelial cells and tissue clusters;

[0168] (2), washing the lung epithelial cells and tissue clusters 2-3 times with DPBS, adding an appropriate amount of Matrigel, resuspending the lung epithelial cells, and inoculating in a 24-well plate, after 15-20 minutes of solidification in a cell incubator, adding lung epithelial organoid culture medium (Table 2), and placing in a cell culture incubator for culture, with medium replacement every 5 days during the culture process, and when the density of the organoids in the Matrigel reaches 80%, the organoids can be subcultured or transferred to the surface of the gel for culture to promote differentiation;

[0169] (3), after removing the Matrigel from the lung epithelial organoids using Cell Recovery Solution, dispersing the organoids into a single cell suspension using Tryple, inoculating the single cell suspension onto the surface of the hydrogel of this example at a density of 1 x 10 6 Figure 8

[0170] Example 10

[0171] The difference between this example and Example 1 is that the content of hyaluronic acid is replaced with sodium alginate in the raw material composition of the hydrogel, and the second crosslinking agent is omitted. The hydrogel is prepared according to the raw material composition, including the following steps:

[0172] (1), the prepolymer solution is prepared according to the prepolymer solution formula;

[0173] (2), the obtained prepolymer solution is introduced into a container, then an initiator is added; the volume ratio of the prepolymer solution to the initiator is 52:1; then the polymerization is initiated under ultraviolet light (253.7 nm) for 3 h to obtain the hydrogel.

[0174] Example 11

[0175] ​​The difference between this embodiment and embodiment 1 is that the content of collagen and the like in the raw material composition of the hydrogel is replaced by sodium alginate, and the second crosslinking agent is omitted. The hydrogel is prepared according to the raw material composition, including the following steps:

[0176] (1) A prepolymer solution is prepared according to the prepolymer solution formula;

[0177] (2) The obtained prepolymer solution is introduced into a container, and then an initiator is added; the volume ratio of the prepolymer solution to the initiator is 52:1; then polymerization is initiated by irradiation under a UV lamp (253.7 nm) for 3 h to obtain the hydrogel.

[0178] Embodiment 12: Method for inducing differentiation of lung epithelial cells

[0179] This embodiment provides a method for inducing differentiation of lung epithelial cells, including the following steps:

[0180] (1) The cancer-adjacent normal lung tissue from a clinical sample source is digested by lung tissue digestive juice (1 mg / mL collagenase type I, 0.1-0.5 mg / mL DNase I, 1% penicillin / streptomycin double antibody) at 37°C for 1 h, then filtered by a 70 μm cell screen and centrifuged, the cell precipitate is collected, and red blood cells are further removed by red blood cell lysis solution to obtain relatively pure lung epithelial cells and tissue clusters;

[0181] (2) The lung epithelial cells and tissue clusters are washed with DPBS for 2-3 times, an appropriate amount of Matrigel is added, the lung epithelial cells are resuspended, and inoculated in a 24-well plate, after solidification for 15-20 min in a cell incubator, lung epithelial organoid culture medium (Table 2) is added, and placed in a cell incubator for culture, the medium is changed every 5 days during the culture process, when the density of the organoids in the Matrigel reaches 80%, the organoids can be subcultured or transferred to the surface of the gel for culture to promote differentiation;

[0182] (3) After the lung epithelial organoids are removed from the Matrigel by Cell Recovery Solution, the Tryple is used to digest and disperse the lung epithelial organoids into a single cell suspension, which is inoculated on the surface of the hydrogel in embodiment 1 at a density of 4×10 6 / mL (the culture medium is the organoid culture medium (Table 2)), and cultured to the 15th day.

[0183] Embodiment 13: Method for inducing differentiation of lung epithelial cells

[0184] The difference between this embodiment and embodiment 12 is that in step (3), the single cells are inoculated on the surface of the hydrogel in embodiment 1 at a density of 1×10 6 / mL (the culture medium is the organoid culture medium (Table 2)), and cultured to the 15th day.

[0185] Example 14 Method for inducing lung epithelial cell differentiation

[0186] The difference between this example and Example 12 is that in step (3), the single cells are inoculated onto the surface of the hydrogel prepared in Example 1 at a density of 8 x 10 6 / mL (culture medium is organoid culture medium (Table 2)) and cultured to day 15.

[0187] Experimental Example

[0188] The hydrogel prepared in Example 1 is used to perform Example 12, and the lung organoids obtained at the end are identified. Example 1 is used as the experimental group. At the same time, a control group is set up, and the difference between the control group and Example 12 is that in step (3), the same culture medium is used to culture the same batch of cells, and the organoids cultured by wrapping with Matrigel (performed according to step (2) in Example 12) are used as the control group. The experimental group and the control group are each tested in triplicate.

[0189] Data processing: t-test is used for data statistics, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001;

[0190] The identification results are as follows:

[0191] (1) The results of the lung epithelial cell differentiation induced by the hydrogel corresponding to Example 1 are as follows:

[0192] After 15 days of culture, the lung epithelial cells in Example 1 can spontaneously assemble into three-dimensional structures of vacuole structures or branched tooth-like structures Figure 9 on the surface of the hydrogel. As can be seen from Figure 9 , the lung epithelial cells can spontaneously assemble into three-dimensional structures of vacuole structures and branched tooth-like structures on the surface of the hydrogel, indicating that the lung epithelial cells induced by the hydrogel containing the special group obtained by the method of the present application can spontaneously assemble into three-dimensional shapes such as airway-like, alveolar or lung branch structures, and mature lung organoid tissues can be obtained. The control group is Figure 10 , and as can be seen from Figure 10 , no branched tooth-like structures are present. Therefore, it can be shown that the method of the present application can induce lung epithelial cells to spontaneously assemble into three-dimensional shapes such as airway-like, alveolar or lung branch structures.

[0193] After 15 days of culture, the organoid cells obtained in the experimental group (APM) and the control group (Matrigel) are fixed and labeled with immunofluorescence for various lung epithelial cell-specific markers, and the results are shown in Figure 11 , as shown in A of Figure 11 , compared with the traditional Matrigel-wrapped organoids Figure 11Compared to group B, the APM group had a higher proportion of positive areas for the AT1 marker AQP5, as shown in the statistical results. Figure 12 As shown in C, the cells exhibit a single-layer thin-walled morphology, clustered on the outer side of the cell spheroid, as... Figure 12 As shown in Figures A and B, the AT1 cells in the experimental group exhibit a single-layer thin-walled morphology, clustered on the outer side of the cell spheres. This morphology is not observed in the conventional Matrigel-encapsulated organoid culture of the control group. The lung organoids obtained by APM hydrogel culture of this invention have an ultra-thin AT1 cell morphology and a very large spreading area, which is quite similar to the AT1 morphology in the alveolar region of human lungs.

[0194] After 15 days of culture, the organoid cells obtained from the experimental and control groups in Example 1 were characterized using immunofluorescence staining. The results are as follows: Figure 13 As shown, SPB represents AT2 cells, CC10 represents rod cells, and MUC5AC represents goblet cells. Characterization revealed that compared to traditional Matrigel-encapsulated organoid culture, organoids cultured on hydrogel surfaces exhibited a higher proportion of AT2 cells, rod cells, and goblet cells. Figure 13 (A, B, and C) as follows: Figure 13 In the staining diagrams of the first column (from left to right) of A and B, orange immunofluorescence represents SPB staining, indicating AT2 cells, and blue immunofluorescence represents DAPI staining, indicating cell nuclei. (For comparison...) Figure 13 The SPB / DAPI staining plots in the first column of A and B show that... Figure 13 The orange area in A is significant, while Figure 13 There is almost no orange area in B, which is consistent with Figure 13 The quantitative statistical results of the SPB staining region in C are consistent. For example... Figure 13 In the second column (from left to right) of staining images A and B, purple immunofluorescence represents CC10 staining, indicating rod-shaped cells with visible tooth-like branching structures; blue immunofluorescence represents DAPI staining, indicating the cell nucleus. (For comparison...) Figure 13 In the second column (from left to right) of A and B in the image, the CC10 / DAPI staining patterns show that the purple immunofluorescence area is more prominent in image A, while the purple immunofluorescence area is less prominent in image B. This is because... Figure 13 The quantitative statistical results of the CC10 staining region in C are consistent. For example... Figure 13 In the third column (from left to right) of the staining diagrams in sections A and B, red immunofluorescence represents MUC5AC staining, indicating goblet cells, and blue immunofluorescence represents DAPI staining, indicating cell nuclei. (For comparison...) Figure 13 The MUC5AC / DAPI staining plots in the third column of columns A and B show that... Figure 13 The red immunofluorescence region in A is significant, while the red immunofluorescence region in B is less pronounced. This is related to... Figure 14The quantitative results of the MUC5AC staining area of C are consistent. The cells in the experimental group and the control group of Example 1 were continuously cultured for 30 days, and the organoids obtained by culturing on the hydrogel surface of the experimental group of Example 1 can differentiate into clustered cilia with outward swinging cilia Figure 14 A and B). As shown in C and D of Figure 14 , by comparing A and C in Figure 14 , it can be seen that the organoids obtained by culturing on the hydrogel surface of the experimental group differentiate into outward cilia structures, while the control group does not form mature outward cilia structures. By comparing B and D in Figure 15 , it can be seen that the experimental group has a light blue immunofluorescence staining area representing cilia, while the control group has almost none. Therefore, it can be shown that the method of the present application promotes the differentiation of cells into AT1 cells, AT2 cells, club cells, goblet cells, ciliated cells and other mature lung epithelial cells, and the proportion of lung epithelial cells with mature functions obtained by the method is much higher than that of the control group.

[0195] After 15 days of culture, the RNA of the organoid cells obtained in the experimental group and the control group was extracted, and the expression of innate immune molecules was identified by transcriptome sequencing and fluorescence quantitative polymerase chain reaction (qPCR). The transcriptome sequencing results are shown in Figure 16 , and the results show that the expression of immune molecule genes related to the culture of cells on the surface of the hydrogel material in the experimental group is up-regulated compared with the control group, indicating that the culture of the hydrogel (APM) can activate the gene expression of the innate immune molecules of lung epithelial cells, and the culture of the hydrogel of the present application can activate the innate immune function of lung epithelial cells, providing a research reference for the activation of human related immune function physiological process. Further, the qPCR results are shown in Figure 17 and Figure 16 , the expression of complement in the cultured cells of the experimental group to resist the invasion of pathogens is up-regulated Figure 17 , and the expression of mucus molecules is up-regulated Figure 18 . Therefore, it can be concluded that the lung organoids obtained by the present application have up-regulated expression of innate immune molecule related genes and significantly increased mucus secretion, which indirectly indicates that they have anti-virus ability.

[0196] After 15 days of culture, the organoid cells in the experimental group and the control group were infected with adenovirus of the same titer (MOI = 1000) for 48 h, Figure 18 A, the first row corresponds to the results of the Matrigel group, and the second row corresponds to the results of the experimental group (APM), by comparing the second column (from left to right) in Figure 18 , it can be seen that the area of the APM group infected with virus (green fluorescence part) is less than that of the Matrigel group, and further by flow identification, the proportion of virus infected cells (ADV +The proportion of cells in the lung organ is 54.04% in the Matrigel group and 14.26% in the APM group, which is higher than that in the APM group, which is consistent with the quantitative statistical results of the three groups Figure 18 The infection efficiency of the experimental group is lower (C in the three groups (A, B and C). ​ Thus, the lung organ obtained by the application has significant antiviral ability and is more conducive to experimental research.

[0197] Obviously, the above embodiments are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the application.

Claims

1. A hydrogel, characterized in that, Including the following raw materials: A prepolymer solution, comprising component A, wherein component A comprises at least one of: alginate, hyaluronic acid, gelatin, polyethylene glycol, polyacrylic acid, sericin, polyvinyl alcohol, and collagen; Second crosslinking agent: is a solution containing divalent or trivalent cations; the content of the second crosslinking agent is ≥0.

2. The hydrogel according to claim 1, characterized in that, In the prepolymer solution, the concentration of component A is 1wt%-5wt%; And / or, the second crosslinking agent contains divalent or trivalent cations in the range of 0.1 to 1 mol / mL; And / or, when the content of the second crosslinking agent is >0, the volume ratio of the prepolymer solution to the second crosslinking agent is 1 to 10:

1.

3. The hydrogel according to claim 1 or 2, characterized in that, The second crosslinking agent is a solution containing divalent or trivalent metal ions; optionally, the divalent or trivalent cation contained in the second crosslinking agent is Fe. 3+ Mg 2+ Al 3+ Ca 2+ 、Sr 2+ and Ba 2+ At least one of them; And / or, the alginate includes, but is not limited to, at least one of sodium alginate, potassium alginate, calcium alginate, ammonium alginate, barium alginate or iron alginate; And / or, the gelatin includes, but is not limited to, acrylated gelatin modified with RGD short peptides; And / or, the molecular weight range of the polyethylene glycol is 1000 to 20000 Da; And / or, the collagen includes, but is not limited to, rat tail type I collagen; And / or, the prepolymer solution may also include at least one of fibronectin or laminin.

4. The hydrogel according to any one of claims 1-3, characterized in that, It also includes the following raw materials: initiator; the initiator includes thermal initiator or photoinitiator; The prepolymer solution also includes the following components: Acrylamide monomer; First crosslinking agent: bisacrylamide crosslinking monomer; Catalyst: Tetramethylethylenediamine.

5. The hydrogel according to claim 1, characterized in that, When the content of the second crosslinking agent is 0, the hydrogel comprises the following raw materials: The prepolymer solution is prepared by including component A, acrylamide monomer, first crosslinking agent, and catalyst; Initiator.

6. The hydrogel according to claim 4 or 5, characterized in that, The mass ratio of the acrylamide monomer, component A, first crosslinking agent, and catalyst is 1:0.04-0.5:0.0006-0.06:0.001-0.

003.

7. The hydrogel according to any one of claims 4-6, characterized in that, The photoinitiator includes ketoglutaric acid; the concentration of ketoglutaric acid is 0.5–10 wt%. And / or, the thermal initiator comprises persulfate; the concentration of the persulfate is 0.1–10 wt%; And / or, the volume ratio of the prepolymer solution to the initiator is 100 to 30:

1.

8. A method for preparing a hydrogel according to any one of claims 1-7, characterized in that, The steps include the following: Take component A and prepare a prepolymer solution; A second crosslinking agent is added to the prepolymer solution to crosslink it, resulting in a hydrogel.

9. The method for preparing the hydrogel according to claim 8, characterized in that, When the content of the second crosslinking agent is >0, the following steps are included: Component A, acrylamide monomer, first crosslinking agent and catalyst are mixed to prepare a prepolymer solution; An initiator was added to the prepolymer solution, and polymerization was carried out to obtain the first composite crosslinked product. The first composite crosslinking product is added to the second crosslinking agent for crosslinking to obtain a hydrogel. Optionally, the polymerization includes ultraviolet polymerization or heating polymerization; when the initiator contains only ketoglutaric acid, the polymerization is ultraviolet polymerization. When the initiator contains ammonium persulfate or a mixture of ammonium persulfate and ketoglutaric acid, the polymerization is either ultraviolet polymerization or heating polymerization. Optionally, the conditions for the ultraviolet polymerization are: ultraviolet wavelength of 250-400 nm, irradiation for 1-5 hours; Optionally, the heating polymerization conditions are: heating at 60-80℃ for 2-6 hours; Optionally, the cross-linking conditions for adding the first composite cross-linking product to the second cross-linking agent are: soaking time of 3 to 12 hours at room temperature.

10. The method for preparing the hydrogel according to claim 8, characterized in that, When the content of the second crosslinking agent is 0, the following steps are included: Component A, acrylamide monomer, first crosslinking agent and catalyst are mixed to prepare a prepolymer solution; An initiator is added to the prepolymer solution, and polymerization is carried out to obtain a hydrogel. Optionally, the polymerization includes ultraviolet polymerization or heating polymerization; when the initiator contains only ketoglutaric acid, the polymerization is ultraviolet polymerization; when the initiator contains ammonium persulfate or a mixture of ammonium persulfate and ketoglutaric acid, the polymerization is ultraviolet polymerization or heating polymerization. Optionally, the conditions for the ultraviolet polymerization are: ultraviolet wavelength of 250-400 nm, irradiation for 1-5 hours; Optionally, the heating polymerization conditions are: heating at 60-80℃ for 2-6 hours.

11. A method for inducing lung epithelial cell differentiation, characterized in that, This includes culturing lung epithelial cells in hydrogels prepared using the hydrogels described in any one of claims 1-7 or the hydrogel preparation methods described in any one of claims 8-10, in order to induce lung epithelial cell differentiation.

12. The method for inducing lung epithelial cell differentiation according to claim 11, characterized in that, The density of the lung epithelial cells seeded onto the hydrogel surface is 1–8 × 10⁻⁶. 6 cells / mL; And / or, the culture medium in which the lung epithelial cells are cultured on the hydrogel is an organoid culture medium; And / or, the lung epithelial cells are cultured in the hydrogel for 10 to 30 days.

13. The method for inducing lung epithelial cell differentiation according to claim 11 or 12, characterized in that, Before hydrogel culture, the lung epithelial cells include at least one of the following steps: lung epithelial cell acquisition, lung organoid acquisition, and single-cell suspension acquisition.

14. The method for inducing lung epithelial cell differentiation according to claim 13, characterized in that, The steps for obtaining lung epithelial cells include: digesting lung tissue, filtering, collecting cells, removing red blood cells, and obtaining lung epithelial cells and tissue masses. And / or, the steps for obtaining lung organoids include: adding matrix gel to lung epithelial cells and tissue aggregates, resuspending, inoculating, coagulating, adding lung epithelial organoid culture medium for culture, and obtaining lung organoids when the organoid density in the matrix gel reaches more than 80%. And / or, the steps for obtaining the single-cell suspension include: removing matrix gel from lung organoids and digesting them.

15. A lung organoid model obtained by the method for inducing lung epithelial cell differentiation according to any one of claims 11-14.

16. The lung organoid model of claim 15 has any of the following uses: (1) Use in the study of pathogen-lung cell interactions in the lungs of humans or non-human mammals; (2) Use in the study of the physiological process of immune function activation in lung epithelial cells of human or non-human mammals; (3) Use in drug research and screening for the prevention, adjuvant treatment or treatment of human lung diseases; (4) Use in the study of lung diseases in humans or non-human mammals.

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