Organ-like culture medium, organ-like culture method and application thereof

Abstract

The invention provides an organoid culture medium, an organoid culture method and application of the organoid culture medium, the organoid culture medium comprises a first-stage culture medium, the first-stage culture medium comprises a first-stage basic culture medium and first-stage addition factors, and the first-stage addition factors comprise N-acetylcysteine, nicotinamide, a ROCK inhibitor, Noggin, R-spondin, EGF, HGF, Gastrin, FGF10, Blebbistatin, Forskolin, A83-01, LDN193189, DAPT, TGF-alpha and UDAC. By using the organoid culture medium, the liver organoid and the fatty liver organoid can be effectively obtained, and the obtained fatty liver organoid has obvious fat deposition, is sensitive to fatty liver treatment drugs, highly restores the physiological status when human body fatty liver diseases occur, and has high application value.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and more particularly to an organoid culture medium, an organoid culture method, and their applications. Background Technology

[0002] The liver, a vital organ, is susceptible to metabolic disorders that can lead to various liver diseases. These diseases can progress to hepatitis, cirrhosis, and liver cancer, and may also cause complications in other systems. Currently, mortality rates associated with metabolic liver diseases are increasing dramatically worldwide, with non-alcoholic fatty liver disease alone affecting a quarter of the global adult population. The lack of clear therapeutic targets and drug screening models are major reasons for the scarcity of clinical drugs for metabolic liver diseases, and the proportion of drug-induced liver injury in global deaths is also gradually increasing. Therefore, developing in vitro models that can effectively capture the causes of the disease and its evolution from functional impairment to advanced stages is crucial for breakthroughs in the clinical treatment of metabolic liver diseases. Furthermore, highly efficient and sensitive in vitro drug screening models can detect potential hepatotoxicity of drugs, providing guidance for clinical medication.

[0003] Organoids are cultures formed by seeding stem cells or progenitor cells into 3D matrix gel for in vitro culture. Through cell self-proliferation, directed differentiation, lineage determination, and self-assembly, they possess organ-specific cellular composition, key structures, and functional characteristics. Organoids can partially or even completely replicate the cellular composition and structural functions of in vivo organs, maintaining genetic and phenotypic stability during long-term expansion. This overcomes the difficulty of traditional biological models in simulating tissues and organs, shifting in vitro tissue and organ research from the two-dimensional cellular level to the three-dimensional tissue level. Due to their high tissue memory in replicating in vivo tissues and organs and their powerful self-assembly capabilities, this technology shows significant application prospects in disease models, drug development, toxicology, gene editing, and regenerative medicine. Summary of the Invention

[0004] This application is based on the inventor's discoveries and understanding of the following facts and problems: This invention aims to address the lack of models for the pathological effects of the liver and metabolic liver diseases on the human body, as well as for clinical drug testing. It utilizes organoid technology to construct human liver organoids, and based on this model, uses an inducer to construct a fatty liver disease model. The biomimetic ability and drug sensitivity of the model are verified through the detection of fatty liver pathological characteristics and drug sensitivity testing, providing a breakthrough in modeling for pathological research and drug testing of the liver and fatty liver.

[0005] Therefore, in a first aspect, the present invention provides an organoid culture medium. According to an embodiment of the invention, the culture medium comprises a first-stage culture medium, wherein the first-stage culture medium includes a first-stage basal culture medium and a first-stage additive factor, the first-stage additive factor comprising N-acetylcysteine, nicotinamide, ROCK inhibitor, Noggin, R-spondin, EGF, HGF, Gastrino, FGF10, Blebbistatin, Forskolin, A83-01, LDN193189, DAPT, TGF-α, and UDAC. The culture medium according to embodiments of the present invention can effectively culture liver organoids and fatty liver organoids, and the successfully constructed organoids are spherical in shape with a tightly packed internal tissue structure rather than being hollow.

[0006] According to embodiments of the present invention, the organoid culture medium may further include at least one of the following additional technical features: According to an embodiment of the present invention, the concentration of N-acetylcysteine ​​is 0.5~1.5 mM. Exemplarily, the concentration of N-acetylcysteine ​​is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 mM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of N-acetylcysteine ​​is preferably 0.8~1.3 mM, more preferably 1.25 mM.

[0007] According to an embodiment of the present invention, the concentration of nicotinamide is 5-20 mM. Exemplarily, the concentration of nicotinamide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of nicotinamide is preferably 8-15 mM, more preferably 10 mM.

[0008] According to an embodiment of the present invention, the concentration of the ROCK inhibitor is 5~20 μM. Exemplarily, the concentration of the ROCK inhibitor is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of the ROCK inhibitor is 8~15 μM, more preferably 10 μM.

[0009] According to an embodiment of the present invention, the concentration of Noggin is 50~200 ng / mL. Exemplarily, the concentration of Noggin is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of Noggin is preferably 80~120 ng / mL, more preferably 100 ng / mL.

[0010] According to an embodiment of the present invention, the concentration of R-spondin is 100~1000 ng / mL. Exemplarily, the concentration of R-spondin is 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of R-spondin is 300~800 ng / mL, more preferably 500 ng / mL.

[0011] According to an embodiment of the present invention, the concentration of EGF is 25~100 ng / mL. Exemplarily, the concentration of EGF is 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of EGF is preferably 40~60 ng / mL, more preferably 50 ng / mL.

[0012] According to an embodiment of the present invention, the concentration of HGF is 25~100 ng / mL. Exemplarily, the concentration of HGF is 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of HGF is preferably 40~60 ng / mL, more preferably 50 ng / mL.

[0013] According to an embodiment of the present invention, the concentration of Gastriin is 5~20 nM. Exemplarily, the concentration of Gastriin is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of Gastriin is preferably 8~15 nM, more preferably 10 nM.

[0014] According to an embodiment of the present invention, the concentration of FGF10 is 50~200 ng / mL. Exemplarily, the concentration of FGF10 is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of FGF10 is preferably 80~120 ng / mL, more preferably 100 ng / mL.

[0015] According to an embodiment of the present invention, the concentration of Blebbistatin is 5~20 μM. Exemplarily, the concentration of Blebbistatin is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of Blebbistatin is preferably 8~15 μM, more preferably 10 μM.

[0016] According to an embodiment of the present invention, the concentration of Forskolin is 5~20 μM. Exemplarily, the concentration of Forskolin is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of Forskolin is preferably 8~15 μM, more preferably 10 μM.

[0017] According to an embodiment of the present invention, the concentration of A83-01 is 0.5-2 μM. Exemplarily, the concentration of A83-01 is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 μM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of A83-01 is preferably 1-1.5 μM, more preferably 1 μM.

[0018] According to an embodiment of the present invention, the concentration of LDN193189 is 0.1-0.5 μM. For example, the concentration of LDN193189 is 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 or 0.50 μM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of LDN193189 is preferably 0.1-0.2 μM, more preferably 0.1 μM.

[0019] According to an embodiment of the present invention, the concentration of DAPT is 0.5-2 μM. Exemplarily, the concentration of DAPT is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 μM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of DAPT is preferably 1-1.5 μM, more preferably 1 μM.

[0020] According to an embodiment of the present invention, the concentration of TGF-α is 5-20 ng / mL. Exemplarily, the concentration of TGF-α is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of TGF-α is preferably 10-15 ng / mL, more preferably 10 ng / mL.

[0021] According to an embodiment of the present invention, the concentration of the UDAC is 0.5-2 μM. Exemplarily, the concentration of the UDAC is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 μM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of the UDAC is preferably 1-1.5 μM, more preferably 1 μM.

[0022] According to an embodiment of the present invention, in the above-mentioned organoid culture medium, A8301 can inhibit the transcription induced by TGF-β receptor ALK4 / 5 / 7, thereby preventing the inhibitory effect of TGF-β production on cell proliferation; LDN193189 can inhibit the transcriptional activity of BMP-I receptor ALK2 / 3, thereby preventing the inhibitory effect of related pathways on cell proliferation; DAPT can inhibit the NotchI signaling pathway and induce stem cells to differentiate into liver cells; TGF-α can effectively promote stem cell proliferation; UDAC, or ursodeoxycholic acid, is extremely important for lipid metabolism. The addition of UDAC can further enhance the ability of liver and fatty liver organoids to simulate the condition of organs in vivo. The present invention obtains the above-mentioned components and their optimized concentration ranges through optimization.

[0023] According to an embodiment of the present invention, the ROCK inhibitor includes Y27632.

[0024] According to an embodiment of the present invention, the first-stage added factors further include HEPES, L-glutamine, and Bplus.

[0025] According to an embodiment of the present invention, the volume fraction of HEPES is 1% to 3%. For example, the volume fraction of HEPES is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the volume fraction of HEPES is preferably 1% to 2%, more preferably 1%.

[0026] According to an embodiment of the present invention, the volume fraction of L-glutamine is 1% to 3%. Exemplarily, the volume fraction of L-glutamine is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the volume fraction of L-glutamine is preferably 1% to 2%, more preferably 1%.

[0027] According to an embodiment of the present invention, the volume fraction of B plus is 2% to 6%. Exemplarily, the volume fraction of B plus is 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, or 6.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the volume fraction of B plus is preferably 2% to 4%, more preferably 2%.

[0028] According to an embodiment of the present invention, the first-stage additive factor further includes an antibacterial component, wherein the antibacterial component includes primary antibiotics and penicillin-streptomycin bispecific antibodies.

[0029] According to an embodiment of the present invention, the volume fraction of the antibacterial component is 1% to 5%. Exemplarily, the volume fraction of the antibacterial component is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or 5.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the volume fraction of the antibacterial component is preferably 1% to 3%, more preferably 1% to 2%.

[0030] According to an embodiment of the present invention, the volume fraction of the primary antibiotic is 0.5% to 3%. Exemplarily, the volume fraction of the primary antibiotic is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the volume fraction of the primary antibiotic is preferably 0.5% to 2%, more preferably 0.5% to 1%.

[0031] According to an embodiment of the present invention, the volume fraction of the penicillin-streptomycin bispecific antibody is 0.5% to 3%. Exemplarily, the volume fraction of the penicillin-streptomycin bispecific antibody is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the volume fraction of the penicillin-streptomycin bispecific antibody is preferably 0.5% to 2%, more preferably 0.5% to 1%.

[0032] According to an embodiment of the present invention, the culture medium includes a second-stage culture medium, wherein the second-stage culture medium comprises a first-stage culture medium and a second-stage additive factor, and the second-stage additive factor comprises Dexamethasone and TNF-α.

[0033] In the above culture medium, Dexamethasone can regulate the secretion of various hormones in cells or in vivo, and regulate OSM protein to promote liver development. TNF-α can effectively improve passage efficiency in liver organoid culture.

[0034] According to an embodiment of the present invention, the concentration of dexamethasone is 1-5 μM. Exemplarily, the concentration of dexamethasone is 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 μM, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of dexamethasone is preferably 2-4 μM, more preferably 3 μM.

[0035] According to an embodiment of the present invention, the concentration of TNF-α is 5-20 ng / mL. Exemplarily, the concentration of TNF-α is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ng / mL, or a range between any two of the above values. According to a preferred embodiment of the present invention, the concentration of TNF-α is preferably 10-15 ng / mL, more preferably 10 ng / mL.

[0036] In a second aspect, the present invention provides the use of the organoid culture medium described in the first aspect in culturing liver organoids and / or fatty liver organoids. As previously stated, the culture medium described in the first aspect can effectively culture liver organoids and fatty liver organoids, and the successfully constructed organoids are spherical in shape with a tightly packed internal tissue structure rather than being hollow.

[0037] In a third aspect, the present invention provides a method for culturing liver organoids. According to an embodiment of the invention, the method includes: performing a first-stage culture and a second-stage culture of liver stem cells using the organoid culture medium described in the first aspect to obtain liver organoids. The method according to the embodiment of the invention can rapidly and effectively obtain fatty liver organoids, and the successfully constructed organoids are spherical in shape with a tightly packed internal tissue structure rather than being hollow.

[0038] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features: According to an embodiment of the present invention, the first stage of culture involves culturing the liver stem cells for 3 to 8 days using the first stage culture medium in the organoid culture medium.

[0039] According to an embodiment of the present invention, the second stage culture involves culturing the first stage culture product for at least 3 to 7 days using the second stage culture medium in the organoid culture medium.

[0040] According to an embodiment of the present invention, the conditions for the first stage of cultivation are 35℃~37℃ and 4%~6% CO2, and / or the conditions for the second stage of cultivation are 35℃~37℃ and 4%~6% CO2.

[0041] According to a preferred embodiment of the present invention, the conditions for the first stage of cultivation are 37°C and 5% CO2, and / or the conditions for the second stage of cultivation are 37°C and 5% CO2.

[0042] In a fourth aspect, the present invention provides a method for culturing fatty liver organoids. According to an embodiment of the invention, the method includes: inducing liver organoids obtained using the method described in the third aspect with an inducing agent to obtain the fatty liver organoids.

[0043] According to embodiments of the present invention, the above-described method for culturing fatty liver organoids may further include at least one of the following additional technical features: According to an embodiment of the invention, the inducing agent includes at least one of sodium oleate and sodium palmitate.

[0044] According to an embodiment of the present invention, the concentration of the inducer in the culture medium is 200-1000 μM, preferably 500 μM.

[0045] According to an embodiment of the present invention, the conditions for the induction treatment are 37°C and 5% CO2, and the induction treatment time is 2 to 4 days.

[0046] In a fifth aspect, the present invention provides a method for screening drugs. According to an embodiment of the invention, the method includes: contacting the drug to be screened with liver organoids cultured using the method described in the third aspect or fatty liver organoids cultured using the method described in the fourth aspect to obtain the target drug.

[0047] According to an embodiment of the present invention, the target drug is a drug that can significantly reduce the oil droplet component in the fatty liver organoid after immunofluorescence staining.

[0048] According to an embodiment of the present invention, the contact time is 5 to 10 days, preferably 7 days.

[0049] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0050] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1The images show the bright-field growth status of normal human liver organoids on days 1, 3, 5, and 7 of liver stem cell culture in Example 1 of the present invention (scale bar: 100 μm). Figure 2 The images show bright-field growth status of liver organoids (BSA group), FFA-induced fatty liver organoids (Ctrl group), and organoids after drug addition (2.5 µM and 5 µM) in Example 2 of the present invention (scale bar: 100 μm). Figure 3 The images show immunofluorescence staining images of liver organoids (BSA group), FFA-induced fatty liver organoids (Ctrl group), and fatty liver organoids treated with the drug Denifanstat in Example 2 of the present invention (scale bar: 100 μm). Figure 4 The statistical data of average fluorescence intensity of immunofluorescence staining of liver organoids, FFA-induced fatty liver organoids, and organoids treated with the drug Denifanstat are shown in Example 2 of the present invention. Detailed Implementation

[0051] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0052] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0053] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0054] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0055] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0056] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0057] For ease of understanding, the following is an overview of this application: This invention primarily utilizes liver stem cells to construct 3D liver organoids through in vitro culture, highly replicating the functional gene expression and metabolic stability of in vivo liver cells. After constructing the 3D spherical liver organoids, the organoids are subjected to FFA induction to construct fatty liver organoids in vitro. For this model, intracellular lipid deposition, expression of metabolism-related genes, and expression of functional genes are detected to clarify model characteristics. Furthermore, combined with nanoparticle delivery, immunofluorescence staining, and other techniques, a drug screening system capable of efficiently and sensitively evaluating drug efficacy is further developed. The human fatty liver organoids constructed using this model have moderate cell number and aggregation, drug penetration capacity comparable to the in vivo environment, and easily detectable drug sensitivity. They are also suitable for high-volume scaling and can be integrated with high-throughput drug screening platforms to create efficient drug screening systems.

[0058] The following will provide a detailed description of the embodiments. Unless otherwise specified, the reagents, sequences, software, and instruments involved in the following embodiments are all conventional commercially available products or open-source products.

[0059] Example 1: Construction of an organoid disease model for fatty liver Human liver organoids can be constructed from primary embryonic liver tissue samples. Prepare human liver organoid MP medium (Maturation promotion (MP) medium) according to the formula in Table 1, and prepare human liver organoid MP-Plus medium according to the formula in Table 2.

[0060] Table 1

[0061] Table 2

[0062] The method for constructing primary liver organoids includes the following steps: Human liver organoids and fatty liver organoids were cultured using the above-mentioned culture medium. The specific culture method is as follows: 1. Commercially purchased human liver stem cells (purchased from Celprogen, Cat# 36098-25) were thoroughly mixed with DMEM / F12 basal medium. The precipitate was seeded into ultra-low adhesion 96-well plates at a density of 3000-5000 cells / well. 200 μL of liver organoid MP medium as shown in Table 1 was added to each well. The plates were centrifuged at 4°C and 500xg for 5 min.

[0063] 2. The above-mentioned well plates were placed in a 37°C, 5% CO2 incubator for 7 days and sealed with DPBS containing 1% penicillin and streptomycin. The MP medium for the liver organoids was replaced every 2 days for each droplet. After 7 days of culture, the medium was replaced with the MP-Plus medium shown in Table 2 and cultured for another 7 days to obtain human liver organoids.

[0064] like Figure 1 As shown, the first day of organoid culture using MP-Plus medium is marked as day-7, and the seventh day of culture is marked as day-1. Using the medium and culture method of this embodiment, cells can be observed to aggregate into spheres after 24 hours of culture, and spheres with clear boundaries are formed after 72 hours of culture.

[0065] 3. Based on the results in Part 2, well-defined spheres can be formed after 72 hours of culture. For the above-mentioned liver organoids, induction should begin on the 7th day of culture in MP-Plus medium. After a series of preliminary experiments, this application found that adding 500 μM of free fatty acid FFA (sodium palmitate and sodium oleate in a volume ratio of 1:1) to MP-Plus medium and incubating in a 37°C, 5% CO2 incubator for 3-7 days can successfully obtain fatty liver organoids. Therefore, in order to reduce time costs, the induction time of the following examples in this application is 3 days.

[0066] Example 2: Drug Addition Test and Pathological Feature Detection of Fatty Liver Organoids This embodiment uses the liver organoids constructed in Example 1 to induce the construction of fatty liver organoids, and performs drug addition tests on the model and detects changes in pathological characteristics. Specifically, this embodiment sets up four groups: the liver organoids induced and treated with 500 μM BSA (BSA group), the liver organoids induced and treated with 500 μM FFA (i.e., fatty liver organoid group, FFA-Ctrl group), the fatty liver organoids constructed with FFA induced by FFA treated with 2.5 μM Denifanstat (purchased from MedChemExpress (MCE), HY-112829) (FFA-2.5 μM Drug group), and the fatty liver organoids constructed with FFA induced by FFA treated with 5 μM Denifanstat (FFA-5 μM Drug group). The specific experimental procedures are as follows: In Example 1, liver organoids cultured in MP-Plus medium for 7 days were induced by adding 500 μM free fatty acid FFA (sodium palmitate and sodium oleate in a 1:1 ratio) or BSA to the culture medium. The culture was then kept at 37°C and 5% CO2. The organs were incubated in an incubator for 3 days to obtain successfully induced fatty liver organoids and control organoids. Subsequently, the successfully induced fatty liver organoids were treated with the aforementioned FFA, BSA, 2.5 μM Denifanstat, or 5 μM Denifanstat for 7 days, with 50%–60% of the culture medium replaced every 2 days during the maintenance period. On day 1 (labeled day 4) to day 7 (labeled day 10) of drug treatment, the organoids were observed under a microscope and subjected to immunofluorescence staining. The experimental procedures were standard techniques in the field and will not be described in detail here. Human liver organoids, fatty liver organoids, and fatty liver organoids treated with different concentrations of drugs were collected from the above groups. They were fixed in 4% formaldehyde at room temperature for 30–60 min, washed 2–3 times with PBS, stained with 0.5 μg / mL Nile Red and 1 μg / mL DAPI at room temperature for 20–30 min, washed 2–3 times with PBS, and then mounted for imaging to observe the organoid morphology. The experimental results are as follows: Figure 2 , 3 As shown in Figure 4, it should be noted that... Figure 2 and Figure 3 The methods of marking time differ between China and other countries. Figure 2 The time markers for inducing liver organoids into fatty liver organoids are Day 0 to Day 3, and the time markers for drug treatment are Day 4 to Day 10. Figure 3The time shown indicates the specific number of days of treatment. The 10d BSA group refers to inducing liver organoids with BSA for 3 days followed by 7 days of treatment. The 3+7d FFA-Ctrl group, 3+7d FFA-2.5 μM Denifanstat group, and 3+7d FFA-5 μM Denifanstat group all refer to inducing liver organoids with the aforementioned FFA for 3 days followed by 7 days of treatment with FFA, 2.5 μM Denifanstat, or 5 μM Denifanstat. Overall, compared to liver organoids, fatty liver organoids without Denifanstat treatment showed a significant increase in fatty acid content, consistent with clinical fatty liver pathological characteristics. Conversely, fatty acid content in organoids treated with the drug was significantly decreased, exhibiting a clear dose-dependent effect. This demonstrates that the liver and fatty liver organoids constructed in this application can highly replicate the physiological state of the human liver and fatty liver, and can be used for drug screening.

[0067] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0068] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An organoid culture medium, characterized in that, The culture medium comprises a first stage culture medium, wherein the first stage culture medium comprises a first stage basal medium and a first stage additive, the first stage additive comprises N-acetyl cysteine, nicotinamide, ROCK inhibitor, Noggin, R-spondin, EGF, HGF, Gastrin, FGF10, Blebbistatin, Forskolin, A83-01, LDN193189, DAPT, TGF-α and UDAC.

2. The organoid culture medium of claim 1, wherein, The concentration of the N-acetyl cysteine is 0.5-1.5 mM, preferably 0.8-1.3 mM, more preferably 1.25 mM; and / or The concentration of the nicotinamide is 5-20 mM; and / or The concentration of the ROCK inhibitor is 5-20 μM; and / or The concentration of the Noggin is 50-200 ng / mL; and / or The concentration of the R-spondin is 100-1000 ng / mL; and / or The concentration of the EGF is 25-100 ng / mL; and / or The concentration of the HGF is 25-100 ng / mL; and / or The concentration of the Gastrin is 5-20 nM; and / or The concentration of the FGF10 is 50-200 ng / mL; and / or The concentration of the Blebbistatin is 5-20 μM; and / or The concentration of the Forskolin is 5-20 μM; and / or The concentration of the A83-01 is 0.5-2 μM; and / or The concentration of the LDN193189 is 0.1-0.5 μM; and / or The concentration of the DAPT is 0.5-2 μM; and / or The concentration of the TGF-α is 5-20 ng / mL; and / or The concentration of the UDAC is 0.5-2 μM.

3. The organoid culture medium of claim 1, wherein, The first stage additive further comprises HEPES, L-glutamine, B plus; Optionally, the volume fraction of the HEPES is 1%-3%; and / or The volume fraction of the L-glutamine is 1%-3%; and / or The volume fraction of the B plus is 2%-6%.

4. The organoid culture medium of claim 1, wherein, The first stage additive further comprises an antibacterial component, wherein the antibacterial component comprises primary antibiotics, cefazolin; Optionally, the volume fraction of the antibacterial component is 1%-5%, preferably 1%-3%, more preferably 1%-2%; The volume fraction of the primary antibiotics is 1%-3%; and / or The volume fraction of the cefazolin is 1%-3%.

5. The organoid culture medium of claim 1, wherein, The organoid culture medium further comprises a second stage culture medium, the second stage culture medium comprises a first stage culture medium and a second stage additive, the second stage additive comprises Dexamethasone and TNF-α; Optionally, the concentration of the Dexamethasone is 1-5 μM; and / or The concentration of the TNF-α is 5-20 ng / mL; Optionally, the basal medium is selected from any one of Advanced DMEM / F12, Ham’s F-12, SILAC Advanced DMEM / F-12 Flex, DMEM / F-12 and William’s E medium.

6. Use of the organoid culture medium of any one of claims 1-5 in culturing a liver organoid and / or a fatty liver organoid.

7. A method of culturing a liver organoid, characterized by, The method comprises: culturing the liver stem cells in the first stage and the second stage using the organoid culture medium of any one of claims 1-5 to obtain a liver organoid.

8. The method of claim 7, wherein, The first stage culture is culturing the liver stem cells in the first stage culture medium in the organoid culture medium for 3-8 days; and / or The second stage culture is culturing the first stage culture product in the second stage culture medium in the organoid culture medium for at least 3-7 days; Optionally, the condition of the first stage culture is 37℃, 5% CO2, and / or the condition of the second stage culture is 37℃, 5% CO2.

9. A method of culturing a fatty liver organoid, characterized by, The method comprises: inducing the liver organoid obtained by the method of any one of claims 7-8 using an inducing agent to obtain the fatty liver organoid.

10. The method of claim 9, wherein, The inducing agent comprises free fatty acid; Optionally, the inducing agent comprises at least one of sodium oleate and sodium palmitate; Optionally, the concentration of the inducing agent in the culture medium is 200-1000 μM; Optionally, the condition of the inducing treatment is 37℃, 5% CO2, and the time of the inducing treatment is 2-7 days.

11. A method of screening for a drug, characterized by, The method comprises: contacting a drug to be screened with the liver organoid obtained by the method of any one of claims 7-8 or the fatty liver organoid obtained by the method of any one of claims 8-9 to obtain a target drug.

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